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Publication numberCN105796602 A
Publication typeApplication
Application numberCN 201610172204
Publication date27 Jul 2016
Filing date20 Aug 2009
Priority date20 Aug 2008
Also published asCA2734237A1, CN102186971A, EP2329012A1, US8828376, US20100047351, US20140341867, WO2010021715A1
Publication number201610172204.2, CN 105796602 A, CN 105796602A, CN 201610172204, CN-A-105796602, CN105796602 A, CN105796602A, CN201610172204, CN201610172204.2
Inventors安迪查特林, 阿贾伊帕尔
Applicant人类起源公司
Export CitationBiBTeX, EndNote, RefMan
External Links: SIPO, Espacenet
Treatment of stroke using isolated placental cells
CN 105796602 A
Abstract
Provided herein are methods for the treatment of stroke comprising administering to a stroke victim placental stem cells, populations of cells comprising placental stem cells, and/or compositions comprising placental stem cells. The invention also provides the application of isolated human adherent placental cells in the production for the treatment of an individual having a disruption in the flow of blood in or around the brain.
Claims(53)  translated from Chinese
1. 治疗有效量的分离的人贴壁胎盘细胞在制备用于治疗脑中或脑周围血流破坏的个体的药物中的应用,其中,所述分离的人贴壁胎盘细胞为CD73+、CD105+和HLA-G+。 1. therapeutically effective amount of isolated human adherent placental cells are subject application drug treatment or brain blood flow around the brain damage in the preparation for, wherein said isolated human adherent placental cells are CD73 +, CD105 +, and HLA-G +.
2. 治疗有效量的分离的人贴壁胎盘细胞在制备用于治疗脑中或脑周围血流破坏的个体的药物中的应用,其中所述分离的人贴壁胎盘细胞为⑶1〇+、⑶34'⑶105+和⑶200+;并且其中所述细胞以比骨髓来源的间充质干细胞可检测的更高水平表达一种或多种基因, 其中所述一种或多种基因为以下中的一种或多种:ACTG2、ADARB1、AMIG02、ARTS-1、 B4GALT6、BCHE、Cllorf9、CD200、raL4Al、C0L4A2、CPA4、DMD、DSC3、DSG2、EL0VL2、F2RLl、 FLJ10781、GATA6、GPR126、GPRC5B、ICAM1、IER3、IGFBP7、IL1A、IL6、IL18、KRT18、KRT8、LIPG、 LRAP、MATN2、MEST、NFE2L3、NUAK1、PCDH7、PDLIM3、PKP2、RTN1、SERPINB9、ST3GAL6、 ST6GALNAC5、SLC12A8、TCF21、TGFB2、VTN 和ZC3H12A,并且其中所述骨髓来源的间充质干细胞已经进行了与所述胎盘细胞相同传代次数的传代培养。 2. therapeutically effective amount of isolated human adherent placental cells are applied to the preparation of a medicament for the treatment of an individual brain damage or brain blood flow in the surrounding, wherein said isolated human adherent placental cells are ⑶1〇 +, ⑶34 '⑶105 + and ⑶200 +; and wherein the ratio between the cells are bone marrow-derived mesenchymal stem cells detectably higher levels of expression of one or more genes, wherein the one or more genes of one or more of the following species: ACTG2, ADARB1, AMIG02, ARTS-1, B4GALT6, BCHE, Cllorf9, CD200, raL4Al, C0L4A2, CPA4, DMD, DSC3, DSG2, EL0VL2, F2RLl, FLJ10781, GATA6, GPR126, GPRC5B, ICAM1, IER3, IGFBP7, IL1A, IL6, IL18, KRT18, KRT8, LIPG, LRAP, MATN2, MEST, NFE2L3, NUAK1, PCDH7, PDLIM3, PKP2, RTN1, SERPINB9, ST3GAL6, ST6GALNAC5, SLC12A8, TCF21, TGFB2, VTN and ZC3H12A, and wherein said bone marrow-derived mesenchymal stem cells have been carried out the number of passages said placental cells subcultured same.
3. 权利要求1的应用,其中,所述CD73+、CD105+、HLA-G+细胞还为CD200+。 Application of claim 1, wherein said CD73 +, CD105 +, HLA-G + cells are additionally CD200 +.
4. 权利要求1的应用,其中,所述CD73+、CD 105+、HLA-G+细胞还为CD34-、CD45-和0CT-4+。 Application of claim 1, wherein said CD73 +, CD 105 +, HLA-G + cells were also CD34-, CD45-, and 0CT-4 +.
5. 权利要求1的应用,其中,所述CD73+、CD105+、HLA-G+细胞还为CD34-、CD45-、0CT-4+和CD200+〇 Application of claim 1, wherein said CD73 +, CD105 +, HLA-G + cells were also CD34-, CD45-, 0CT-4 + and CD200 + square
6. 权利要求2的应用,其中,所述CD10+、CD34-、CD105+、CD200+细胞还为CD45-或CD90+。 Application according to claim 2, wherein said CD10 +, CD34-, CD105 +, CD200 + cells were also CD45- or CD90 +.
7. 权利要求2的应用,其中,所述CD10+、CD34-、CD105+、CD200+细胞还为CD45-和CD90+。 Application according to claim 2, wherein said CD10 +, CD34-, CD105 +, CD200 + cells were also CD45- and CD90 +.
8. 权利要求1或2的应用,其中,所述治疗有效量为使所述个体表现出的脑中或脑周围血流破坏消除、可检测的改善、严重程度减轻、或一种或多种症状发展变缓的所述细胞的量。 Application 1 or claim 2, wherein said therapeutically effective amount is such that the subject exhibits damage the brain or the cerebral blood flow around the elimination, can improve the detection of, reduce the severity of, or one or more the amount of the cells of slow development of symptoms.
9. 权利要求8的应用,其中,所述症状为偏瘫或轻偏瘫。 9. The use as claimed in claim 8, wherein said pathological condition is hemiparesis or hemiplegia.
10. 权利要求8的应用,其中,所述症状为面部肌肉虚弱;麻木;感觉下降;嗅觉、味觉、听力或视力改变;嗅觉、味觉、听力或视力丧失;眼皮耷拉(下垂症);可检测的眼肌肉虚弱;呕吐反射降低;吞咽能力降低;瞳孔光反应性降低;面部知觉降低;平衡降低;眼震症;呼吸速率改变;心率改变;锁骨乳突肌虚弱,转动头至一侧的能力降低或不能;舌虚弱;失语症(不能说或理解语言);失用症(改变的自觉运动);视野缺陷;记忆力缺损;半侧忽视或半侧空间忽视(对病变相对的视野另一侧空间的关注缺乏);思维混乱;意识模糊;纵欲姿态的发展; 疾病失认症(持续否认缺陷的存在);难以行走;运动协同作用改变;眩晕;不平衡;意识丧失;头痛或呕吐,其中所述症状由脑中或脑周围血流破坏引起。 10. The use as claimed in claim 8, wherein the symptom is a facial muscle weakness; numbness; decreased feeling; smell, taste, hearing, or changes in vision; smell, taste, hearing, or vision loss; drooping eyelids (ptosis); detectable eye muscle weakness; decreased gag reflex; decreased ability to swallow; pupillary light reduced reactivity; perceived reduction portion; Ping Heng decreased; nystagmus disease; respiration rate change; change in heart rate; subclavian papillary muscles weakness, the ability of the rotary head to one side reduced or not; tongue weakness; aphasia (not speak or understand language); apraxia (altered consciousness movement); View defects; memory impairment; hemi hemi-spatial neglect or ignored (the other side of the visual field opposite the lesion concern lack of space); confusion; confusion; development indulgence attitude; disease agnosia (continued denying the existence of the defect); difficulty walking; motion synergistic effect change; dizziness; imbalance; loss of consciousness; headache or vomiting, which the symptoms of brain damage caused by cerebral or peripheral blood flow.
11. 权利要求1或2的应用,其中,所述分离的人贴壁胎盘细胞包含在细胞群中,所述细胞群中至少80 %为分离的人贴壁胎盘细胞。 11. The use as claimed in claim 1 or 2, wherein said isolated human adherent placental cells are contained in a cell population, said cell population is at least 80% of the isolated human adherent placental cells.
12. 权利要求1或2的应用,其中,所述分离的人贴壁胎盘细胞包含在细胞群中,所述细胞群中至少90 %为所述分离的人贴壁胎盘细胞。 12. The application of claim 1 or claim 2, wherein said isolated human adherent placental cells are contained in a cell population, said cell population is at least 90% of said isolated human adherent placental cells.
13. 权利要求1或2的应用,其中,所述血流破坏为中风。 13. The application of claim 1 or claim 2, wherein said stroke is a disruption of blood flow.
14. 权利要求13的应用,其中,所述中风为缺血性中风。 14. Application as claimed in claim 13, wherein said stroke is ischemic stroke.
15. 权利要求13的应用,其中,所述中风为出血性中风。 15. Use as claimed in claim 13, wherein said stroke is hemorrhagic stroke.
16. 权利要求1或2的应用,其中,所述血流破坏为血肿。 16. The application of claim 1 or claim 2, wherein the disruption of blood flow is a hematoma.
17. 权利要求16的应用,其中,所述血肿为硬脑膜血肿、硬膜下血肿或蛛网膜下血肿。 17. Application as claimed in claim 16, wherein the hematoma is a dural hematoma, a subdural hematoma, or subarachnoid hematoma.
18. 权利要求1或2的应用,其中,所述血流破坏为血管痉挛。 18. The application of claim 1 or claim 2, wherein the disruption of blood flow is a vasospasm.
19. 权利要求1或2的应用,其中,所述分离的人贴壁胎盘细胞通过大丸注射给予。 19. The use of claim 1 or 2, wherein said isolated human adherent placental cells are administered by bolus injection.
20. 权利要求1或2的应用,其中,所述分离的人贴壁胎盘细胞通过静脉内输注给予。 20. The use of claim 1 or claim 2, wherein said isolated human adherent placental cells are administered by intravenous infusion.
21. 权利要求1或2的应用,其中,所述分离的人贴壁胎盘细胞通过头颅内给予。 21. The application of claim 1 or claim 2, wherein said isolated human adherent placental cells are administered by intracranial.
22. 权利要求21的应用,其中,所述分离的人贴壁胎盘细胞在局部缺血区域内给予。 22. Application as claimed in claim 21, wherein said isolated human adherent placental cells are administered within the ischemic area.
23. 权利要求21的应用,其中,所述分离的人贴壁胎盘细胞在所述局部缺血区域周围给予。 23. The application of claim 21, wherein said isolated human adherent placental cells are administered around the ischemic region.
24. 权利要求1或2的应用,其中,所述分离的人贴壁胎盘细胞经腹膜、肌内、真皮内或眼内给予。 24. The application of claim 1 or claim 2, wherein said isolated human adherent placental, intramuscular, intradermal, intraocular administration or cells intraperitoneally.
25. 权利要求1或2的应用,其中,所述分离的人贴壁胎盘细胞通过手术植入包含所述分离的人贴壁胎盘细胞的组合物而给予。 25. The application of claim 1 or claim 2, wherein said isolated human adherent placental cells by surgical implantation composition comprising said isolated human adherent placental cells are being administered.
26. 权利要求25的应用,其中,所述组合物为基质或支架。 26. The use as claimed in claim 25, wherein the composition is a matrix or scaffold.
27. 权利要求26的应用,其中,所述基质或支架为水凝胶。 27. Application as claimed in claim 26, wherein said matrix or scaffold is a hydrogel.
28. 权利要求26的应用,其中,所述基质或支架为脱细胞组织。 28. Application as claimed in claim 26, wherein said matrix or scaffold for the tissue acellular.
29. 权利要求26的应用,其中,所述基质或支架为合成的生物可降解组合物。 29. Application as claimed in claim 26, wherein said matrix or scaffold is a synthetic biodegradable composition.
30. 权利要求1或2的应用,其中,所述分离的人贴壁胎盘细胞一次给予所述个体。 30. The application of claim 1 or claim 2, wherein said isolated human adherent placental cells are administered once to said individual.
31. 权利要求1或2的应用,其中,所述分离的人贴壁胎盘细胞分多次给予所述个体。 31. The application of claim 1 or claim 2, wherein said isolated human adherent placental cells are administered to said individual a plurality of times.
32. 权利要求1或2的应用,其中,所述应用包括给予所述个体每千克约1 X 104至1 X 106 个分离的人贴壁胎盘细胞。 32. The application of claim 1 or claim 2, wherein said application comprises administering to said subject from about 1 X 104 to 1 X 106 separate human adherent placental cells per kilogram.
33. 权利要求1或2的应用,其中,所述应用包括给予所述个体每千克约1 X 105至1 X 106 个分离的人贴壁胎盘细胞。 33. The application of claim 1 or claim 2, wherein said application comprises administering to said subject from about 1 X 105 to 1 X 106 separate human adherent placental cells per kilogram.
34. 权利要求1或2的应用,其中,所述应用包括给予所述个体每千克约1 X 106至1 X 107 个分离的人贴壁胎盘细胞。 34. The application of claim 1 or claim 2, wherein said application comprises administering to said subject from about 1 X 106 to 1 X 107 separate human adherent placental cells per kilogram.
35. 权利要求1或2的应用,其中,所述应用包括给予所述个体每千克约1 X 107至1 X 108 个分离的人贴壁胎盘细胞。 35. The application of claim 1 or claim 2, wherein said application comprises administering to said subject from about 1 X 107 to 1 X 108 separate human adherent placental cells per kilogram.
36. 权利要求1或2的应用,其中,所述应用包括静脉内给予约5X107至3X 109个分离的人贴壁胎盘细胞。 36. The application of claim 1 or claim 2, wherein said application comprises administering from about 5X107 3X 109 to separate human adherent placental cells intravenously.
37. 权利要求36的应用,其中,所述应用包括给予约9 X 108个分离的人贴壁胎盘细胞。 37. The use as claimed in claim 36, wherein said application comprises administering about 9 X 108 separate human adherent placental cells.
38. 权利要求36的应用,其中,所述应用包括给予约1.8X 109个分离的人贴壁胎盘细胞。 38. The use as claimed in claim 36, wherein said application comprises administering to about 1.8X 109 separate human adherent placental cells.
39. 权利要求1或2的应用,其中,所述应用包括头颅内给予约5 X 107至1 X 108个分离的人贴壁胎盘细胞。 39. The application of claim 1 or claim 2, wherein said application administration of about 5 X 107 to 1 X 108 separate human adherent placental cells comprising the head.
40. 权利要求39的应用,其中,所述应用包括给予约9 X 107个分离的人贴壁胎盘细胞。 40. The use as claimed in claim 39, wherein said application comprises administering about 9 X 107 separate human adherent placental cells.
41. 权利要求1或2的应用,包括给予所述个体第二种治疗剂。 41. The application of claim 1 or claim 2, comprising administering to the subject a second therapeutic agent.
42. 权利要求41的应用,其中,所述第二种治疗剂为神经保护剂。 42. Application as claimed in claim 41, wherein said second therapeutic agent is a neuroprotective agent.
43. 权利要求41的应用,其中,所述第二种治疗剂为NXY-059(苯基丁基硝酮的二磺酰基衍生物)。 43. The use as claimed in claim 41, wherein said second therapeutic agent is NXY-059 (phenylbutyl nitrone two sulfonyl derivative).
44. 权利要求41的应用,其中,所述第二种治疗剂为血栓溶解剂。 44. Application as claimed in claim 41, wherein said second therapeutic agent is a thrombolytic agent.
45. 权利要求44的应用,其中,所述血栓溶解剂为组织纤溶酶原激活物(tPA)。 45. Application as claimed in claim 44, wherein the thrombolytic agent is tissue plasminogen activator (tPA).
46. 权利要求1或2的应用,其中,所述分离的人贴壁胎盘细胞在所述个体脑中或脑周围血流破坏的一种或多种症状发展的48小时内给予所述个体。 46. The application of claim 1 or claim 2, wherein said isolated human adherent placental cells are administered to said individual within 48 hours of the subject or brain damage in cerebral blood flow around one or more symptoms of the development.
47. 权利要求1或2的应用,其中,所述分离的人贴壁胎盘细胞在所述个体脑中或脑周围血流破坏的一种或多种症状发展的24小时内给予所述个体。 47. Application of 1 or claim 2, wherein said isolated human adherent placental cells are administered to the subject within 24 hours the brains of individuals around the damaged cerebral blood flow or one or more symptoms of the development.
48. 权利要求1或2的应用,其中,所述分离的人贴壁胎盘细胞在所述个体脑中或脑周围血流破坏的一种或多种症状发展的12小时内给予所述个体。 48. The application of claim 1 or claim 2, wherein said isolated human adherent placental cells are administered to said individual within 12 hours of the individual cerebral blood flow around the brain or one or more symptoms of damage development.
49. 权利要求1或2的应用,其中,所述分离的人贴壁胎盘细胞在所述个体脑中或脑周围血流破坏的一种或多种症状发展的3小时内给予所述个体。 49. The application of claim 1 or claim 2, wherein said isolated human adherent placental cells are administered to said individual within 3 hours of the individual cerebral blood flow around the brain or one or more symptoms of damage development.
50. 权利要求1或2的应用,其中,所述分离的人贴壁胎盘细胞在所述给予之前冷冻保存。 50. The application of claim 1 or claim 2, wherein said isolated human adherent placental cells are cryopreserved prior to said administering.
51. 权利要求1或2的应用,其中,所述分离的人贴壁胎盘细胞获自胎盘干细胞库。 51. The application of claim 1 or claim 2, wherein said isolated human adherent placental cells are obtained from a placental stem cell bank.
52. 权利要求2的应用,其中,所述分离的人贴壁胎盘细胞在培养基中培养约3至约35个群倍增时表达所述一种或多种基因,所述培养基包括60 %DMEM-LG和40 %MCDB-201; 2 %胎牛血清;1 X胰岛素-转铁蛋白-硒(ITS); 1 X亚油酸-牛血清白蛋白(LA-BSA); 10、地塞米松;10-4Μ抗坏血酸2-磷酸盐;表皮生长因子10ng/mL和血小板来源的生长因子(PDGF-BB) 10ng/mL 〇 52. Application as claimed in claim 2, wherein said isolated human adherent placental cell culture expression of a gene when said one or more population doublings of about 3 to about 35 in a medium, said medium comprising 60% DMEM-LG and 40% MCDB-201; 2% fetal calf serum; 1 X insulin - transferrin - selenium (ITS); 1 X linoleic acid - bovine serum albumin (LA-BSA); 10, dexamethasone ; 10-4Μ ascorbic acid 2-phosphate; epidermal growth factor 10ng / mL and platelet-derived growth factor (PDGF-BB) 10ng / mL billion
53. 权利要求2的应用,其中,所述分离的人贴壁胎盘细胞在培养基中培养约3至约35个群加倍增时表达所述一种或多种基因,所述培养基包含60%DMEM-LG(Gibco)和40%M⑶B-201(Sigma) ;2%胎牛血清(Hyclone Labs.) ;1 X胰岛素-转铁蛋白-硒(ITS); 1 X亚油酸-牛血清白蛋白(LA-BSA); 10-9Μ地塞米松(Sigma); 10-4Μ抗坏血酸2-磷酸盐(Sigma);表皮生长因子10ng/mL(R&D Systems);和血小板来源的生长因子(PDGF_BB)10ng/mL(R&D Systems)。 53. Application as claimed in claim 2, wherein said isolated human adherent placental cell culture expression of the one or more genes overtime doubled from about 3 to about 35 groups in a culture medium, said medium comprising 60 % DMEM-LG (Gibco) and 40% M⑶B-201 (Sigma); 2% fetal bovine serum (Hyclone Labs.); 1 X insulin - transferrin - selenium (ITS); 1 X linoleic acid - bovine serum albumin protein (LA-BSA); 10-9Μ dexamethasone (Sigma); 10-4Μ ascorbic acid 2-phosphate (Sigma); epidermal growth factor 10ng / mL (R & D Systems); and platelet-derived growth factor (PDGF_BB) 10ng / mL (R & D Systems).
Description  translated from Chinese

利用分离的胎盘细胞治疗中风 Treatment of stroke using the isolated placental cells

[0001] 本申请是申请日为2009年8月20日,申请号为200980141316.X,名称为"利用分离的胎盘细胞治疗中风"的中国发明专利申请的分案申请。 [0001] This application is filed August 20, 2009, Application No. 200980141316.X, entitled divisional applications "isolated placental cells using treating stroke" Chinese invention patent application. 本申请要求2008年8月20日提交的美国临时专利申请号61/090,565的优先权,其在此通过引用将全文纳入本文。 This application claims priority to US Provisional Patent Application No. 61 / 090,565 of August 20, 2008 filed, which is hereby incorporated herein by reference. 发明领域 Field of the Invention

[0002] 本发明提供利用分离的胎盘细胞,例如胎盘多能细胞、所述分离的胎盘细胞群、 和/或包含所述细胞的组合物治疗具有低氧性损伤、或脑内或脑周围血流破坏的个体的方法,所述个体具有例如归因于脑内或脑周围血流破坏的神经性缺损的症状或缺陷。 [0002] The present invention provides the use of isolated placental cells, e.g., placental multipotent cells, said isolated placental cells, and / or therapeutic compositions comprising said cells having hypoxic injury, cerebral or peripheral blood or brain the method of destruction of individual streams, the individual has symptoms due to defects such as the brain or the cerebral blood flow or peripheral nerve damage defects.

[0003] 发明背景 [0003] Background of the Invention

[0004] 中风亦称1亩中风"、脑血管损伤(CVA)或急性局部缺血脑血管综合症,为大脑功能的丧失,通常发展迅速,其病因是供给血液至脑或脑干的血管素乱。所述素乱可W是由例如血栓或栓塞形成引起的局部缺血(缺乏血液)或由于溢血。根据世界卫生组织,中风为持续超过24小时或在24小时内死亡而中断的脑血管神经性缺损病因。症状持续超过24小时将中风和瞬时的局部缺血损伤(TIA)区分开,后者症状持续少于24小时。 [0004] 1 mu stroke, also known as stroke ", cerebrovascular injury (CVA) or acute ischemic cerebrovascular syndrome, loss of function of the brain, usually develops rapidly, is supplied which cause blood vessels to the brain or brain stem of prime arbitrary. the element may be W is a disorder due to ischemia (lack of blood) is formed by a thrombus or embolus, for example, or due to hemorrhage. persistent death in more than 24 hours or 24 hours according to the world Health organization, stroke, cerebrovascular interrupted cause neurological deficit. the symptoms last more than 24 hours of stroke and transient ischemic damage (TIA) distinguish which symptoms last less than 24 hours.

[0005] 目前,局部缺血性中风的治疗通常包括抗血小板药物例如阿斯匹林、氯化格雷、双喀达莫或抗凝剂药物,例如丙酬节径香豆素,W降低或减轻引起局部缺血的障碍。 [0005] Currently, treatment of ischemic stroke typically include antiplatelet agents, aspirin, Gray dichloride, bis Kada Mo or anticoagulant drugs such as, for example, prop-paid pitch diameter coumarin, W reduce or mitigate causing obstacles ischemia. 此外,尽量保持正常的血糖水平,并且提供给中风患者充足的氧气和静脉注射液。 In addition, try to maintain normal blood sugar levels, and to provide sufficient stroke patients oxygen and intravenous fluids. 出血性中风的治疗通常包括给予一种或多种降压药、除了非酱体类抗炎药(NSAID)外的疼痛药、巧离子通道阻断剂(例如尼莫地平)、W及如果需要,进行手术W修复导致出血的血管裂口。 Hemorrhagic stroke generally comprise administering one or more antihypertensive agents, in addition to the non-body butter anti-inflammatory drug (NSAID) drugs pain, Qiao ion channel blocker (e.g. nimodipine), W, and if desired , W surgery to repair cracks lead to vascular hemorrhage.

[0006] 然而,所述治疗仅试图减轻进行性的神经损伤,而无助于恢复已丧失的功能。 [0006] However, the treatment only attempt to mitigate the progressive nerve damage, and does not contribute to restore lost function. 已检测了许多非细胞神经保护剂治疗中风的功效,但都失败了,包括N-甲基-D-天冬氨酸受体括抗剂、纳美芬、芦贝鲁挫、氯美嚷挫、巧离子通道阻断剂(包括α-氨基-3-径基-5-甲基异挫- 4-丙酸括抗剂、5-径色胺激动剂(例如瑞匹洛坦),W及跨膜钟通道调节剂)、替拉扎特、抗- ICAM-I抗体、人抗白细胞抗体化U23F2G)、抗血小板抗体(例如阿昔单抗)、胞憐胆碱(胞巧- 5'-二憐酸胆碱的外源形式)和碱性成纤维细胞生长因子。 Efficacy has been detected neuroprotective agents for the treatment of many non-stroke cells, but failed, include N- methyl -D- aspartate receptor antagonist including, Na Meifen, Lo Bayrou setback, US-chloro setback cried , Qiao ion channel blockers (including α- amino-3-isobutyl-5-methyl-diameter fell --4- acid antagonist including, serotonin agonists diameter 5- (e.g. horses Luotan Switzerland), W and transmembrane clock channel modulators), tirilazad, anti - ICAM-I antibody, anti-human leukocyte antibodies of U23F2G), anti-platelet antibodies (e.g., abciximab), choline pity cell (cell Qiao - 5'- exogenous choline acid forms two pity) and basic fibroblast growth factor.

[0007] 对于中风没有有效的疗法。 [0007] there is no effective treatment for stroke. 因此,需要不仅能减轻任何原因导致的神经损伤,且能改善神经功能和预后的疗法。 Therefore, not only can reduce nerve damage from any cause, and can improve nerve function and prognosis of therapy.

[000引发明概述 [000 triggered Ming Overview

[0009 ] -方面,本发明提供了利用分离的胎盘细胞、分离的胎盘细胞群、包含分离的胎盘干细胞的细胞群和包含分离的胎盘细胞的组合物在用于治疗个体中枢神经系统(CNS)例如脑内或脊髓内或者脑和脊髓周围血流破坏的个体的方法。 [0009] - aspect, the present invention provides the use of isolated placental cells, the isolated placental cells, populations of cells comprising isolated placental stem cells and a composition comprising isolated placental cells in an individual for the treatment of central nervous system (CNS) the method of the subject, for example, the brain or spinal cord or around the spinal cord and brain disruption of blood flow. 所述方法包括,例如治疗个体由于脑内或脑周围血流破坏而导致的神经性缺损症状的治疗,例如低氧损伤、缺氧损伤、中风(例如局部缺血或出血性中风)、非中风性出血或ΤΙΑ。 The method includes, for example, therapeutic treatment of neurological deficit symptoms because individual disruption of blood flow around the brain or brain caused, e.g. hypoxic injury, hypoxic injury, stroke (e.g., ischemic or hemorrhagic stroke), non-stroke bleeding or ΤΙΑ. 如此处考虑的,治疗个体脑内或脑周围血流破坏导致的神经性缺损症状包括治疗个体脑内或脑周围血流破坏所伴随的再灌注损伤导致的神经性缺损症状。 So at the consideration of the treatment of neurological deficit symptoms neurological defect symptom of cerebral blood flow around the brain or damage resulting from the treatment of individuals, including disruption of blood flow around the brain or the brain associated with reperfusion injury caused. 本发明对缺血性中风(例如,缺氧损伤或低氧损伤)的成功治疗已在被接收的动物中风模型中被验证。 The present invention is ischemic stroke (e.g., hypoxic injury or anoxic injury) successful treatment has been validated in animal models of stroke was received. 参见实施例1和实施例2。 Example 1 and Example 2 Referring to FIG.

[0010] 在一个方面,本发明提供了治疗脑内或脑周围血流破坏的个体的方法,例如个体脑或中枢神经系统(CNS)内或周围血流破坏导致的神经性缺损症状,所述方法包括将治疗有效量的分离的塑料贴壁组织培养人胎盘细胞给予所述个体,其中所述分离的胎盘细胞具有多能细胞或干细胞的特性。 [0010] In one aspect, the present invention provides a method of treating an individual brain damage or brain blood flow around, for example, in individual brain or central nervous system (CNS) or peripheral neurological deficit symptoms of disruption of blood flow caused by the comprising a therapeutically effective amount of isolated tissue culture plastic-adherent human placental cells are administered to said individual, wherein said isolated placental cells have characteristics of multipotent cells or stem cells. 在某些实施方案中,血流破坏导致个体脑或CNS的缺氧损伤或低氧损伤。 In certain embodiments, the disruption of blood flow resulting in anoxic injury or hypoxic brain injury or CNS of an individual.

[0011] 在某些实施方案中,所述分离的胎盘细胞为分离的胎盘干细胞。 [0011] In certain embodiments, the isolated placental cells are isolated placental stem cells. 在某些其它的实施方案中,所述分离的胎盘细胞为分离的胎盘多能细胞。 In certain other embodiments, the isolated placental cells are isolated placental multipotent cells. 在一个具体的实施方案中,如流式细胞计检测的,所述分离的胎盘细胞为CD34-、CD10+和CD105+的。 In a specific embodiment, as detected by flow cytometry, the isolated placental cells are CD34-, CD10 + and CD105 + a. 在更具体的实施方案中,所述分离的CD34-、CD 10+、CD 105+胎盘细胞为胎盘干细胞。 In a more specific embodiment, the isolated CD34-, CD 10 +, CD 105+ placental cells are placental stem cells. 在另外更具体的实施方案中,所述分离的CD34-、CD10+、CD105+胎盘细胞为多能胎盘细胞。 In another more specific embodiment, the isolated CD34-, CD10 +, CD105 + placental cells are multipotent placental cells. 在另外的具体实施方案中,所述分离的CD34-、CD10+、CD105+胎盘细胞具有分化为成骨表型细胞或成软骨表型细胞的潜能。 In a further specific embodiment, the isolated CD34-, CD10 +, CD105 + placental cells differentiate into osteoblast phenotype cells, or to the potential of the cartilage phenotype. 在另一个实施方案中,所述分离的CD34-、CD10+、CD105+胎盘细胞具有分化为神经表型细胞的潜能。 In another embodiment, the isolated CD34-, CD10 +, CD105 + placental cells differentiate into neural phenotype potential. 在更具体的实施方案中,所述分离的CD34-、CD10+、CD105+胎盘细胞还为CD200+。 In a more specific embodiment, the isolated CD34-, CD10 +, CD105 + placental cells are additionally CD200 +. 在另外更具体的实施方案中,如流式细胞计检测的,所述分离的CD34-、CD10+、CD105+胎盘细胞还为CD90 +或CD45-的。 In another more specific embodiment, as detected by flow cytometry, the isolated CD34-, CD10 +, CD105 + placental cells are additionally CD90 + or CD45- of. 在另外更具体的实施方案中,如流式细胞计检测的,所述分离的CD34-、CD10\ CD105+胎盘细胞还为CD90+或CD45-的。 In another more specific embodiment, as detected by flow cytometry, the isolated CD34-, CD10 \ CD105 + placental cells are additionally CD90 + or CD45- of. 在更具体的实施方案中,如流式细胞计检测的,所述CD34-、CD10\CD105\CD200+胎盘细胞还为CD90+或CD45-的。 In a more specific embodiment, as detected by flow cytometry, the CD34-, CD10 \ CD105 \ CD200 + placental cells are additionally CD90 + or CD45- of. 在另外更具体的实施方案中,如流式细胞计检测的,所述分离的CD34-、CD10\CD105\CD200+细胞还为CD90+和CD45-的。 In another more specific embodiment, as detected by flow cytometry, the isolated CD34-, CD10 \ CD105 \ CD200 + cells are additionally CD90 + and CD45- of. 在另外更具体的实施方案中,如流式细胞计检测的,所述分离的CD%-、CD10\CD105+、CD200\ CD90+、CD45-细胞还为CD8〇-和CD8扩的。 In another more specific embodiment, as detected by flow cytometry, the isolated CD% -, CD10 \ CD105 +, CD200 \ CD90 +, CD45- cells and CD8 CD8〇- also flared.

[0012] 在更具体的实施方案中,所述CD:34-、CD10\CD105+细胞还为CD29+、CD38-、CD44\ CD54\CD8〇-、CD86-、SH3+或細4+中的一种或多种。 [0012] In a more specific embodiment, the CD: 34-, CD10 \ CD105 + cells further into a CD29 +, CD38-, CD44 \ CD54 \ CD8〇-, CD86-, SH3 + or fine or 4+ variety. 在更具体的实施方案中,所述分离的CD:34-、CD10+、CD105+胎盘细胞还为CD44+。 In a more specific embodiment, the isolated CD: 34-, CD10 +, CD105 + placental cells are additionally CD44 +. 在另外的具体实施方案中,所述CD:34-、CD10+、 CD105+胎盘细胞还为CD 13+、CD29+、CD33+、CD38-、CD44+、CD45-、CD54+、CD62E-、CD62L-、CD62P-、 S册+(CD73+)、SH4+ (CD73+)、CD8〇-、CD86-、CD90+、SH2+(CD 105+)、CD 106/VCAM+、CD 11 厂、CD 144/ VE-巧粘蛋白1。 In a further particular embodiment, the CD: 34-, CD10 +, CD105 + placental cells are additionally CD 13 +, CD29 +, CD33 +, CD38-, CD44 +, CD45-, CD54 +, CD62E-, CD62L-, CD62P-, S Volume + (CD73 +), SH4 + (CD73 +), CD8〇-, CD86-, CD90 +, SH2 + (CD 105 +), CD 106 / VCAM +, CD 11 plants, CD 144 / VE- Qiao mucin 1. \〔0184八乂〔34-、〔0200\〔0133-、0(:1'-4\85643-、55644-、48(:-口\1(0尺- (VEGFR2-)、HLA-A、B、C+、HLA-DP、DQ、DR-、HLA-G+或程序性死亡-1 配体(PDL1) + 中的一种或多种,或它们的任何组合。在更具体实施方案中,所述CD34-、CD10+、CD105+胎盘细胞还为CD13 +、CD29+、CD33+、CD38-、CD44+、CD45-、CD54/1CAM+、CD6沈-、CD62L-、CD62P-、細3+(CD73+)、SH4+ (CD73+)、CD8〇-、CD86-、CD90+、S肥+(CD 105+)、CD 106/VCAM+、CD 11 厂、CD 144/VE-巧粘蛋白1™、 CD 184/CXCR4-、CD200+、CD 133-、0CT-4+、S 沈A3-、SSEA4-、ABC-P+、邸R- (VEGFR2-)、HLA-A、B、C+、 化八-0?、09、0扩、化4-6+和程序性死亡-1配体。化1) +。 \ [0184 eight qe [34 -, [0200 \ [0133-, 0 (: 1'-4 \ 85643-, 55644-, 48 (: - port \ 1 (scale 0 - (VEGFR2 -), HLA-A, + one or more of, or any combination thereof, B, C +, HLA-DP, DQ, DR-, HLA-G + or programmed death 1 ligand (PDL1). in a more specific embodiment, the said CD34-, CD10 +, CD105 + the placental cells are additionally CD13 +, CD29 +, CD33 +, CD38-, CD44 +, CD45-, CD54 / 1CAM +, CD6 sink -, CD62L-, CD62P-, fine 3+ (CD73 +), SH4 + (CD73 + ), CD8〇-, CD86-, CD90 +, S fertilizer + (CD 105 +), CD 106 / VCAM +, CD 11 plant, CD 144 / VE- coincidence mucin 1 ™, CD 184 / CXCR4-, CD200 +, CD 133 -, 0CT-4 +, S Shen A3-, SSEA4-, ABC-P +, Di R- (VEGFR2 -), HLA-A, B, C +, of eight -0, 09,0 expansion, of 4-6? + and programmed death- 1 ligand. of 1) +.

[0013] 在其它的实施方案中,所述分离的胎盘细胞为CD200+和化A-G+;CD73+、CD105+和CD200+; CD200+和0CT-4+; CD73+、CD105+和HLA-G+; CD73+和CD105% 并且当所述群在允许形成胚样体的条件下培养时,有助于在包含所述分离胎盘细胞的胎盘细胞群中形成一种或多种胚样体;或0CT-4+,并且当所述群在允许形成胚样体的条件下培养时,有助于在包含所述分离胎盘细胞的胎盘细胞群中形成一种或多种胚样体;或它们的任何组合。 [0013] In other embodiments, the isolated placental cells are CD200 + and of the A-G +; CD73 +, CD105 + and CD200 +; CD200 + and 0CT-4 +; CD73 +, CD105 +, and HLA-G +; CD73 + and CD105% and when the population is cultured under conditions that allow formation of embryoid-like bodies, contribute to the formation in a population of placental cells comprising the isolated placental cells are one or more embryoid-like bodies; or 0CT-4 +, and when the when cultured under conditions that allow formation of said group of embryoid-like bodies, contribute to the formation of one or more embryoid-like bodies in a population of placental cells comprising the isolated placental cells; or any combination thereof. 其它的实施方案中,所述分离的胎盘细胞为〔034-、〔010\〔0105+,且还为〔0200+和化4-6+;〔073+、〔0105+和CD200+; CD200+和0CT-4+; CD73+、CD105+和HLA-G+; CD73+和CD105% 并且当所述群在允许形成胚样体的条件下培养时有助于在包含所述分离胎盘细胞的胎盘细胞群中形成一种或多种胚样体;或0CT-4%并且当所述群在允许形成胚样体的条件下培养时有助于在包含所述分离胎盘细胞的胎盘细胞群中形成一种或多种胚样体;或它们的任何组合。 In other embodiments, the isolated placental cells [034--, [010 \ [0105+ and 0200+ and also of [4-6 +; 073 [+ [0105+ and CD200 +; CD200 + and 0CT -4+; CD73 +, CD105 +, and HLA-G +; CD73 + and CD105% and contributes, when cultured under conditions that allow formation of embryoid-like bodies in a population to form a population of placental cells comprising the isolated placental cells or more embryoid-like bodies; or 0CT-4% and contributes, when cultured under conditions allowing formation of embryoid-like bodies of the group form one or more embryos in a population of placental cells comprising the isolated placental cells like bodies; or any combination thereof.

[0014] 在一个具体的实施方案中,所述CD200+、HLA-G+胎盘细胞为CD34-、CD38-、CD45-、 CD73+和CD105+。 [0014] In one particular embodiment, the CD200 +, HLA-G + placental cells are CD34-, CD38-, CD45-, CD73 + and CD105 +. 在另外具体的实施方案中,所述分离的CD73+、CD105+和CD200+胎盘细胞为0034-、〔038-、〔045-、〔073+和化4-6+。 In another specific embodiment, said isolated CD73 +, CD105 + and CD200 + placental cells 0034--, [038--, [045--, and of [4-6 + 073+. 在另外具体的实施方案中,所述〔0200\0(:1'-4+干细胞为〔034-、〔038-、〔045-、〔073+、〔0105+和化4-6+。在另外具体的实施方案中,所述分离的〔073 +、CD105+和HLA-G+胎盘细胞为CD34-、CD45-、0CT-4+和CD200+。在另外具体的实施方案中,所述分离的CD73+和CD105+胎盘细胞为0CT-4+、CD34-、CD38-和CD45-。在另外具体的实施方案中,所述细胞为CD73+、CD105+、CD200+、CD34-、CD38-和CD45-。 In another particular embodiment, the [0200 \ 0 (: 1'-4 + stem cells [034--, [038--, [045--, [073 + + [0105+ and of 4-6 in. another specific embodiment, said isolated [073 +, CD105 +, and HLA-G + placental cells are CD34-, CD45-, 0CT-4 + and CD200 +. in another specific embodiment, said isolated CD73 + and placental cells are CD105 + 0CT-4 +, CD34-, CD38- and CD45-. in another specific embodiment, said cells are CD73 +, CD105 +, CD200 +, CD34-, CD38- and CD45-.

[0015] 在某些实施方案中,所述分离的胎盘细胞为CD10+、CD29+、CD34-、CD38-、CD44+、 CD45-、CD54+、CD90+、甜2+、S册+、SH4+、SSEA3-、S沈A4-、0CT-4+、MHC-r或ABC-P+中的一种或多种,其中ABC-P为胎盘-特异性ABC转运蛋白(亦称乳腺癌耐受性蛋白(BCRP)和米托蔥酿耐受性蛋白(MXR))。 [0015] In certain embodiments, the isolated placental cells are CD10 +, CD29 +, CD34-, CD38-, CD44 +, CD45-, CD54 +, CD90 +, sweet 2 +, S books +, SH4 +, SSEA3-, S Shen A4-, 0CT-4 +, MHC-r or ABC-P + one or more, wherein the ABC-P placenta - specific ABC transporter protein (also known as breast cancer resistance protein (of BCRP) and m Torr onion stuffed resistance protein (MXR)). 在一个具体的实施方案中,所述分离的胎盘细胞为CD10+、CD29\CD%-、 CD38-、CD44+、CD45-、CD54+、CD90+、S肥+、S册+、SH4+、S沈A3-、S沈A4-和0CT-4+。 In a specific embodiment, the isolated placental cells are CD10 +, CD29 \ CD% -, CD38-, CD44 +, CD45-, CD54 +, CD90 +, S fertilizer +, S books +, SH4 +, S Shen A3-, Shen S A4- and 0CT-4 +. 在另一个实施方案中,所述分离的胎盘细胞为〔010+,〔029+、〔034-、〔038-、〔045-、〔054+、細2+、5册+和甜4+。 In another embodiment, the isolated placental cells are [010, 029 [+ [034--, [038--, [045--, [054+ fine 2 + 4 + 5 + and sweet. 在另一个实施方案中,所述分离的胎盘细胞为CD10+,CD29+、CD34-、CD38-、CD45-、CD54+、W2\ SH3+、細4+和0CT-4+。 In another embodiment, the isolated placental cells are CD10 +, CD29 +, CD34-, CD38-, CD45-, CD54 +, W2 \ SH3 +, 4+, and fine 0CT-4 +. 在另一个实施方案中,所述分离的胎盘细胞为CD10+,CD29+、CD%-、 〔038-、〔044\〔045-、〔054\〔090\化4-1 +、甜2+、5册\甜4+。 In another embodiment, the isolated placental cells are CD10 +, CD29 +, CD% -, [038--, [044 \ [045--, [054 \ [090 \ of 4-1 +, 2 + sweet, 5 book \ sweet 4+. 在另一个实施方案中,所述分离的胎盘细胞为0CT-4+和ABC-P+。 In another embodiment, the isolated placental cells are 0CT-4 + and ABC-P +. 在另一个实施方案中,所述分离的胎盘细胞为細2+、SH3+、 SH4+和0CT-4+。 In another embodiment, the isolated placental cells are thin 2 +, SH3 +, SH4 + and 0CT-4 +. 在另一个实施方案中,所述分离的胎盘细胞为0CT-4\CD34-、SSEA3-和SSEA4-。 In another embodiment, the isolated placental cells are 0CT-4 \ CD34-, SSEA3- and SSEA4-. 在一个具体的实施方案中,所述0CT-4+、CD34-、SSEA3-和SSEA4-细胞还为CD10+、CD29 +、〔034-、〔044\〔045-、〔054+、〔090+、甜2+、甜3+和細4+。 In a specific embodiment, the 0CT-4 +, CD34-, SSEA3- and SSEA4- cells are additionally CD10 +, CD29 +, [034--, [044 \ [045--, [+ 054, [090+, 2+ sweet, sweet and fine 3+ 4+. 在另一个实施方案中,所述分离的胎盘细胞为0CT-4+和CD%-,并且細3+或細4+。 In another embodiment, the isolated placental cells are 0CT-4 + and CD% -, and fine or fine 3+ 4+. 在另一个实施方案中,所述分离的胎盘细胞为CD34-,并且CD10\CD29+、CD44\CD54\CD90+、0CT-4+中任一种。 In another embodiment, the isolated placental cells are CD34-, and CD10 \ CD29 +, CD44 \ CD54 \ CD90 +, 0CT-4 + any one. 在某些实施方案中,所述分离的胎盘细胞为CD10+、CD34-、CD105+和CD200+。 In certain embodiments, the isolated placental cells are CD10 +, CD34-, CD105 + and CD200 +.

[0016] 在另一个实施方案中,可用于此处所述治疗方法的分离的胎盘细胞为CD10+、 CD29-、CD44+、CD45-、CD54/ICANT、CD62-E-、CD62-L-、CD62-p-、CD8〇-、CD86-、CD 103-、CD 104-、CD105+、CD 106/VCAM+、CD144/VE-巧粘蛋白l。 [0016] In another embodiment, be used to separate the therapeutic methods described herein placental cells are CD10 +, CD29-, CD44 +, CD45-, CD54 / ICANT, CD62-E-, CD62-L-, CD62- p-, CD8〇-, CD86-, CD 103-, CD 104-, CD105 +, CD 106 / VCAM +, CD144 / VE- Qiao mucin l. '^CD184/CXCR4-、β2-微球蛋白l。 '^ CD184 / CXCR4-, β2- microglobulin l. '^HLA- Il。 '^ HLA- Il. \HLA-Π -、HLA-Gl™和/或PDLll™中的一种或多种。 \ HLA-Π -, HLA-Gl ™ and / or one or more of PDLll ™. 在一个具体的实施方案中,所述分离的胎盘细胞至少为CD29-和CD54-。 In a specific embodiment, the isolated placental cells are at least CD29- and CD54-. 在另外具体的实施方案中,所述分离的胎盘细胞至少为CD44+和CD106+。 In another specific embodiment, the isolated placental cells are at least CD44 + and CD106 +. 在另外具体的实施方案中,所述分离的胎盘细胞至少为CD29+。 In another specific embodiment, the isolated placental cells are at least CD29 +.

[0017] 在另外具体的实施方案中,所述分离的胎盘细胞W比等量骨髓来源的间充质干细胞可检测的更高水平表达一种或多种基因,其中所述一种或多种基因为ACTG2、ADARB1、 AMIG02、ARTS-I、B4GALT6、BOffi、ClIorf9、CD200、C0L4Al、C0L4A2、CPA4、DMD、DSC3、DSG2、 EL(WL2、F2I?L1、F1J10781、GATA6、GPR126、GPRC5B、ICAM1、IER3、IGFBP7、ILIA、IL6、IL18、 KRT18、KRT8、LIPG、LRAP、MATN2、MEST、NFE^3、NUAK1、PCDH7、PDLIM3、PKP2、RTN1、WRPINB9、 ST3GAL6、ST6GALNAC5、SLCl 2A8、TCF21、TGFB2、VTN 和ZC3H12A中的一种或多种,并且其中所述骨髓来源的间充质干细胞已经历与所述分离的胎盘细胞相同数量的传代培养。在更具体的实施方案中,所述分离的胎盘细胞在包含60%DMEM-LG(例如来自Gibco)和40%MCDB-201 (例如来自Sigma);2%胎牛血清(例如来自HycloneLabs.);lX膜岛素-转铁蛋白-砸(ITS); 1 X亚油酸-牛血清白蛋白(LA-BSA); 10-9M地塞米 [0017] In another specific embodiment, said isolated placental cells W between higher levels than the equivalent amount of bone marrow-derived mesenchymal stem cells may express one or more detectable genes, wherein the one or more gene ACTG2, ADARB1, AMIG02, ARTS-I, B4GALT6, BOffi, ClIorf9, CD200, C0L4Al, C0L4A2, CPA4, DMD, DSC3, DSG2, EL (WL2, F2I? L1, F1J10781, GATA6, GPR126, GPRC5B, ICAM1, IER3, IGFBP7, ILIA, IL6, IL18, KRT18, KRT8, LIPG, LRAP, MATN2, MEST, NFE ^ 3, NUAK1, PCDH7, PDLIM3, PKP2, RTN1, WRPINB9, ST3GAL6, ST6GALNAC5, SLCl 2A8, TCF21, TGFB2, VTN and ZC3H12A one or more, and wherein said inter-bone marrow-derived mesenchymal stem cells have undergone the same number of isolated placental cells were subcultured. in a more specific embodiment, said isolated placental cells comprising 60% DMEM-LG (e.g., from Gibco) and 40% MCDB-201 (e.g. from Sigma); 2% fetal calf serum (e.g. from HycloneLabs.); lX membrane Insulin - transferrin - drop (the ITS); 1 X linoleic acid - bovine serum albumin (LA-BSA); 10-9M dexamethasone (例如来自Sigma); 10-4M抗坏血酸2-憐酸盐(例如来自Sigma);表皮生长因子lOng/血(例如来自R&D Systems);和血小板来源的生长因子(PDGF-BB)10ng/mL(例如来自R&D Systems)的培养基中培养约3至约35个群倍增时表达所述一种或多种基因。在更具体的实施方案中,所述分离的胎盘细胞在包含60%016]-1^;(例如来自6化(3〇)和40%]\:08-201(例如来自51旨1113);2%胎牛血清(例如来自Hyclone Labs.); 1 X膜岛素-转铁蛋白-砸(ITS); 1 X亚油酸-牛血清白蛋白(LA-BSA); 10-9M 地塞米松(例如来自Sigma); 10-4Μ抗坏血酸2-憐酸盐(例如来自Sigma);表皮生长因子long/ mL(例如来自R&D SySterns);和血小板来源的生长因子(PDGF-BB) 1 Ong/mL(例如来自R&D Systems)的培养基中培养约3至约35个群倍增时表达所述一种或多种基因。 (E.g., from Sigma); 10-4M ascorbic acid 2-pity salt (e.g. from Sigma); epidermal growth factor lOng / blood (e.g., from R & D Systems); and platelet-derived growth factor (PDGF-BB) 10ng / mL (e.g. media from R & D Systems) were cultured for about 3 to one or more genes expressing the population doubling time of about 35 in a more specific embodiment, said isolated placental cells comprising 60% 016] - ^ 1; (e.g., of from 6 (3〇) and 40%] \ : 08-201 (1113 aims e.g. from 51); 2% fetal calf serum (e.g., from Hyclone Labs); 1 X film Insulin - turn. ferritin - hit (ITS); 1 X linoleic acid - bovine serum albumin (LA-BSA); 10-9M dexamethasone (e.g., from Sigma); 10-4Μ ascorbic acid 2-pity salt (e.g. from Sigma) ; epidermal growth factor long / mL (e.g., from R & D SySterns); and platelet-derived growth factor (PDGF-BB) 1 Ong / mL (e.g., from R & D Systems) medium for about expression when population doublings 3 to about 35 culture the one or more genes.

[0018] 在所述治疗方法的另外具体的实施方案中,所述胎盘干细胞表达神经营养性生长因子胶质细胞来源的神经营养因子(GDNF)、脑来源的神经营养因子(抓NF)、肝细胞生长因子(服巧、胎盘生长因子(PG巧和血管内皮细胞生长因子(VEG巧。 [0018] In another specific embodiment of the method of treatment, said placental stem cells express the neurotrophic growth factors glial cell line-derived neurotrophic factor (of GDNF), brain-derived neurotrophic factor (grasping of NF), liver cell growth factor (Qiao clothes, placental growth factor (PG clever and vascular endothelial growth factor (VEG clever.

[0019] 在另外具体的实施方案,所述分离的胎盘细胞包含在细胞群中,其中至少50%的细胞为所述分离的胎盘细胞。 [0019] In another specific embodiment, said isolated placental cells are contained within a population of cells, wherein at least 50% of the cells of the isolated placental cells. 在另外具体的实施方案中,所述分离的胎盘细胞包含在细胞群中,其中至少70%的细胞为所述分离的胎盘细胞。 In another specific embodiment, said isolated placental cells are contained within a population of cells, wherein at least 70% of the cells of the isolated placental cells. 在另外具体的实施方案中,所述分离的胎盘细胞包含在细胞群中,其中至少80%的细胞为所述分离的胎盘细胞。 In another specific embodiment, said isolated placental cells are contained within a population of cells wherein at least 80% of the cells of the isolated placental cells. 在另外具体的实施方案中,所述分离的胎盘细胞包含在细胞群中,其中至少90%的细胞为所述分离的胎盘细胞。 In another specific embodiment, said isolated placental cells are contained within a population of cells, wherein at least 90% of the cells of the isolated placental cells. 在某些其它的实施方案中,所述细胞群中的胎盘细胞基本上不包括具有母系基因型的细胞;例如所述群中至少40%、45%、50%、55%、60%、65%、70%、75%、80%、85%、 90%、95%、98%或99%的胎盘细胞具有胎儿基因型,即为胎儿来源的。 In certain other embodiments, the cell population comprises placental cells having substantially no maternal cells genotype; e.g. at least 40% of the population, 45%, 50%, 55%, 60%, 65 %, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of the placental cells have a fetal genotype, i.e. fetal origin. 在某些其它的实施方案中,包含胎盘细胞的所述细胞群基本上不包括具有母系基因型的细胞;例如所述群中至少40%、45%、50%、55%、60%、65%、70%、75%、80%、85%、90%、95%、98% 或99% 的细胞具有胎儿基因型,即为胎儿来源的。 In certain other embodiments, the cell population comprises placental cells that do not substantially include cell maternal genotype; e.g. at least 40% of the population, 45%, 50%, 55%, 60%, 65 %, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of the cells have a fetal genotype, i.e. fetal origin.

[0020] 在某些实施方案中,任何胎盘细胞,例如此处所述的胎盘干细胞或胎盘多能细胞对受者而言是自体同源的,例如受者为已患有中风或中风症状的个体。 [0020] In certain embodiments, any of the placental cells, e.g., placental stem cells described herein, or placental multipotent cells of the recipient is autologous in terms of, for example, recipients have suffered a stroke or stroke of symptoms individual. 在某些其它的实施方案中,任何胎盘细胞,例如此处所述的胎盘干细胞或胎盘多能细胞对受者而言是异源的, 例如受者为已患有中风或中风症状的个体。 In certain other embodiments, any of the placental cells, e.g., placental stem cells described herein, or placental multipotent cells is heterologous to the recipient, for example, the individual recipients have suffered a stroke or stroke symptoms.

[0021] 在所述治疗方法的另外具体的实施方案中,所述分离的胎盘细胞在给予之前冷藏。 [0021] In another specific embodiment of the method of treatment, said isolated placental cells are frozen prior to administration. 在另外具体的实施方案中,所述分离的胎盘细胞获自胎盘干细胞库。 In another specific embodiment, said isolated placental cells are obtained from a placental stem cell bank.

[0022] 在任何上述分离的胎盘细胞的实施方案中,所述分离的胎盘细胞在生长培养基(即配制成促进增殖的培养基,例如在生长培养基中增殖期间促进增殖)中培养期间通常不分化。 During [0022] In any embodiment of the above isolated placental cells, said isolated placental cells in growth medium (i.e., medium formulated to promote proliferation, e.g., proliferation during proliferation in growth medium) in the culture is usually undifferentiated. 在另外具体的实施方案中,所述分离的胎盘细胞不需要饲养层来增殖。 In another specific embodiment, said isolated placental cells do not require a feeder layer in order to proliferate. 在另外具体的实施方案中,由于在不存在饲养细胞层时培养,所述分离的胎盘细胞在培养时不分化。 In another specific embodiment, since the feeder cell layer cultured in the absence of said isolated placental cells do not differentiate in culture.

[0023] 在另外更具体的实施方案中,所述分离的胎盘细胞通过灌注产后胎盘获得,其中所述胎盘已被排干血并被灌注W除去残余血液;或者所述胎盘已被排干血但未经灌注W除去残余血液;或者所述胎盘既不没有被排干血也未经灌注W除去残余血液。 [0023] In another more specific embodiment, said isolated placental cells obtained by perfusion postpartum placenta, wherein said placenta has been exsanguinated and perfused to remove residual blood W; or the placenta has been exsanguinated W but not perfused to remove residual blood; neither placenta or the blood can not be drained without removing the residual blood perfusion W. 在另外更具体的实施方案中,所述分离的胎盘细胞通过物理和/或酶促破坏胎盘组织获得。 In another more specific embodiment, said isolated placental cells are obtained by physical destruction of placental tissue and / or enzymatically.

[0024] 用于此处提供的方法的胎盘细胞的细胞表面、分子和遗传标记在W下5.4.2章节详细描述。 [0024] surface of the cell used in the methods provided herein placental cells, molecular and genetic markers are described in Section 5.4.2 at W detail.

[0025] 在另外具体的实施方案中,所述血流破坏为中风。 [0025] In another specific embodiment, the disruption of blood flow to stroke. 在更具体的实施方案中,所述中风为局部缺血性中风。 In a more specific embodiment, the stroke is ischemic stroke. 在另外更具体的实施方案中,所述中风为出血性中风,例如烦内脑溢血或自发蛛网膜下出血。 In another more specific embodiment, said stroke is hemorrhagic stroke such as cerebral hemorrhage or subarachnoid hemorrhage spontaneously trouble. 在另外具体的实施方案中,所述破坏为血肿。 In another specific embodiment, said disruption is a hematoma. 在更具体的实施方案中,所述血肿为硬脑膜血肿、硬膜下血肿或蛛网膜下血肿。 In a more specific embodiment, the hematoma is a dural hematoma, a subdural hematoma, or subarachnoid hematoma. 在另外具体的实施方案中,所述血肿由对头骨的外部力量引起,例如头损伤。 In another specific embodiment, the skull hematoma caused by external forces, such as head trauma. 在另外具体的实施方案中,所述破坏为瞬时的局部缺血损伤(TIA),例如周期性TIA。 In another specific embodiment, said disruption is a transient ischemic injury (the TIA), e.g. periodic TIA. 在另外具体的实施方案中,所述破坏为血管疫李,例如伴随出血性中风的血管疫李。 In another specific embodiment, said disruption is a vascular P. Lee, e.g. hemorrhagic stroke vascular accompanying Phytophthora Li.

[0026] 在所述方法另外具体的实施方案中,所述治疗有效量为使得所述个体由于脑或CNS内或周围血流破坏的一种或多种症状或血流破坏所引起的神经性缺损(例如缺氧损伤或低氧损伤)消除、可检测的改善、严重程度的减轻、或者发展变缓的分离的胎盘细胞的量。 [0026] In another specific embodiment of the method, said therapeutically effective amount is such that since the individual neurological or around the brain or CNS disruption of blood flow, or one or more symptoms of disruption of blood flow caused by defects (e.g., hypoxic injury or anoxic injury) elimination, detectable improvement, reduce the severity of, or slowing the development of an amount of isolated placental cells. 在另外具体的实施方案中,所述治疗有效量的分离的胎盘细胞被预防性地给予个体,例如W降低或消除由血流破坏后第二或后续的脑或CNS内或周围血流破坏引起的神经性损伤。 In another specific embodiment, the therapeutically effective amount of isolated placental cells is administered prophylactically to an individual, such as W is reduced or eliminated in the second or subsequent brain or CNS damage to surrounding blood or blood flow due to the damage caused by neurological damage.

[0027] 在另外具体的实施方案中,所述脑内或脑周围血流破坏的症状例如中风、缺氧损伤或低氧性损伤为W下病症中的一种或多种:偏擁(身体一侧麻搏);轻偏擁(身体一侧虚弱);面部肌肉虚弱;麻木;感觉下降;嗅觉、味觉、听力或视力改变;嗅觉、味觉、听力或视力丧失;眼皮茸拉(下垂症);可检测的眼肌肉虚弱;呕吐反射降低;吞咽能力降低;瞳孔光反应性降低;面部知觉降低;平衡降低;眼震症;呼吸速率改变;屯、率改变;锁骨乳突肌虚弱,转动头至一侧的能力降低或不能;舌虚弱;失语症(不能说或理解语言);失用症(改变的自觉运动);视野缺陷;记忆力缺损;半侧忽视或半侧空间忽视(对病变相对的视野另一侧空间的关注缺乏);思维混乱;意识模糊;纵欲姿态的发展;疾病失认症(持续否认缺陷的存在);难W 行走;运动协同作用改变;眩晕;不平衡;意识丧失; [0027] In another specific embodiment, the brain or the cerebral blood flow around the symptoms of damage such as stroke, anoxic injury or hypoxic injury W is one or more of the conditions: Partial Yong (body Ma side stroke); light partial owner (weak side of the body); facial muscle weakness; numbness; feeling of decline; smell, taste, hearing or visual changes; smell, taste, hearing or vision loss; Velvet pull the eyelid (ptosis) ; detectable eye muscle weakness; decreased gag reflex; decreased ability to swallow; pupillary light reduced reactivity; perceived reduction portion; Ping Heng decreased; nystagmus disease; respiratory rate change; Tun, rate of change; subclavian papillary muscles weakness, rotary head decreased ability or inability to side; tongue weakness; aphasia (not speak or understand language); apraxia (altered consciousness movement); View defects; memory impairment; hemi hemi-spatial neglect or ignore (relative lesions concerned about the lack of space on the other side vision); confusion; confusion; development indulgence attitude; disease agnosia (continued denying the existence of the defect); W difficulty walking; motion synergistic effect change; dizziness; imbalance; loss of consciousness ; 痛或呕吐。 Pain or vomiting.

[0028] 在如上所述治疗方法的另外具体的实施方案中,所述分离的胎盘细胞通过弹丸注射给予。 [0028] In another specific embodiment of the method of treatment described above, said isolated placental cells are administered by bolus injection. 在另外具体的实施方案中,所述分离的胎盘细胞通过静脉注射注入给予。 In another specific embodiment, said isolated placental cells are administered by intravenous injection. 在一个具体的实施方案中,所述静脉注射注入超过约1至约8个小时。 In a specific embodiment, the intravenously infused over about 1 to about 8 hours. 在另外具体的实施方案中,所述分离的胎盘细胞头烦内给予。 In another specific embodiment, said isolated placental cells are administered within the first trouble. 在另外具体的实施方案中,所述分离的胎盘细胞经腹膜给予。 In another specific embodiment, said isolated placental cells are administered intraperitoneally. 在另外具体的实施方案中,所述分离的胎盘细胞动脉内给予。 In another specific embodiment, said isolated placental cells are administered arteries. 在更具体的实施方案中,所述分离的胎盘细胞在局部缺血区域内给予。 In a more specific embodiment, said isolated placental cells are administered within the ischemic area. 在另外更具体的实施方案中,所述分离的胎盘细胞在局部缺血外周区域内给予。 In another more specific embodiment, said isolated placental cells are administered in the outer peripheral region of ischemia. 在所述治疗方法另外具体的实施方案中,所述分离的胎盘细胞为肌内、真皮内、皮下或眼内给予。 In the method of treatment further specific embodiment, the isolated placental cells are intramuscular, intradermal, subcutaneous or intraocular administration.

[0029] 在如上所述治疗方法的另一实施方案中,所述分离的胎盘细胞通过包含所述分离胎盘细胞的组合物物质手术移植入所述个体而给予。 [0029] In another embodiment the method of treatment described above, said isolated placental cells comprising said isolated placental cells by a surgically implanted into a composition of matter of the subject and administered. 在更具体的实施方案中,所述物质组合物为基质或支架。 In a more specific embodiment, the composition of matter is a matrix or scaffold. 在另外更具体的实施方案中,所述基质或支架为水凝胶。 In another more specific embodiment, said matrix or scaffold is a hydrogel. 在另外更具体的实施方案中,所述基质或支架为脱细胞组织。 In another more specific embodiment, said matrix or scaffold is a decellularized tissue. 在另外更具体的实施方案中,所述基质或支架为合成的生物可降解组合物。 In another more specific embodiment, said matrix or scaffold is a synthetic biodegradable composition. 在另外更具体的实施方案中,所述基质或支架为泡沫。 In another more specific embodiment, said matrix or scaffold is a foam.

[0030] 在如上所述治疗方法的另外具体的实施方案中,所述分离的胎盘细胞一次性给予所述个体。 [0030] In another specific embodiment of the method of treatment described above, said isolated placental cells are administered once to said individual. 在另外具体的实施方案中,所述分离的胎盘细胞分两次或两次W上单独给予所述个体。 In another specific embodiment, said isolated placental cells are administered to the subject alone divided on two or more times W. 在另外具体的实施方案中,所述给予包括给予每千克所述个体约1X 1〇4至1X 1〇5个分离的胎盘细胞,例如胎盘干细胞。 In another specific embodiment, said administering comprises administering to said subject per kilogram to about 1X 1X 1〇5 1〇4 isolated placental cells, e.g., placental stem cells. 在另外具体的实施方案中,所述给予包括给予每千克所述个体约1 X 105至1 X 106个分离的胎盘细胞。 In another specific embodiment, said administering comprises administering to said subject per kilogram of from about 1 X 105 to 1 X 106 isolated placental cells. 在另外具体的实施方案中,所述给予包括给予每千克所述个体约1X 1〇6至1X 1〇7个分离的胎盘细胞。 In another specific embodiment, said administering comprises administering to said subject per kilogram to about 1X 1X 1〇7 1〇6 isolated placental cells. 在另外具体的实施方案中,所述给予包括给予每千克所述个体约1X 1〇7至1X 1〇8个分离的胎盘细胞。 In another specific embodiment, said administering comprises administering to said subject per kilogram to about 1X 1X 1〇8 1〇7 isolated placental cells. 在其它具体的实施方案中, 所述给予包括给予每千克所述个体约1 X 1〇6至约2 X 106个分离的胎盘细胞;每千克所述个体约2 X 10哇约3 X 106个分离的胎盘细胞;每千克所述个体约3 X 10哇约4 X 106个分离的胎盘细胞;每千克所述个体约4 X 106至约5 X 106个分离的胎盘细胞;每千克所述个体约5 X 106至约6 X 106个分离的胎盘细胞;每千克所述个体约6 X 106至约7 X 106个分离的胎盘细胞;每千克所述个体约7 X 106至约8 X 106个分离的胎盘细胞;每千克所述个体约8 X 106至约9 X 106个分离的胎盘细胞;每千克所述个体约9 X 106至约1 X 107个分离的胎盘细胞。 In other specific embodiments, said administering comprises administering to said subject per kilogram of from about 1 X 2 X 106 to about 1〇6 isolated placental cells; per kilogram of said individual to about 2 X 10 wow about 3 X 106 th isolated placental cells; per kilogram of said individual to about 3 X 10 wow about 4 X 106 isolated placental cells; per kilogram of said individual to about 4 X 106 and about 5 X 106 isolated placental cells; per kilogram of said individual about 5 X 106 to about 6 X 106 isolated placental cells; per kg of the subject from about 6 X 106 to about 7 X 106 isolated placental cells; per kilogram of said individual to about 7 X 106 to about 8 X 106 th isolated placental cells; per kg of the subject from about 8 X 106 9 X 106 to about isolated placental cells; per kg of the subject from about 9 X 106 to about 1 X 107 isolated placental cells. 在另外具体的实施方案中,所述给予包括给予个体每千克所述个体约1 X 1〇7至约2 X 107个分离的胎盘细胞。 In another specific embodiment, said administering comprises administering to said subject per kilogram of subject to about 1 X 2 X 107 to about 1〇7 isolated placental cells. 在另外具体的实施方案中,所述给予包括给予个体每千克所述个体约1.3 X 1〇7 至约1.5 X 107个分离的胎盘细胞。 In another specific embodiment, said administering comprises administering to said subject per kilogram of subject to about 1.3 X 1.5 X 107 to about 1〇7 isolated placental cells. 在另外具体的实施方案中,所述给予包括给予个体每千克所述个体上至约3 X107个分离的胎盘细胞。 In another specific embodiment, said administering comprises administering to the subject the subject per kilogram to about 3 X107 isolated placental cells. 在一个具体的实施方案中,所述给予包括给予个体约5 X 106至约2 X 107个分离的胎盘细胞。 In a specific embodiment, said administering comprises administering to a subject of about 5 X 106 to about 2 X 107 isolated placental cells. 在另外具体的实施方案中,所述给予包括给予个体约20毫升溶液中的150 X 106个分离的胎盘细胞。 In another specific embodiment, said administering comprises administering to a subject about 20 ml of solution of 150 X 106 isolated placental cells.

[0031 ]在一个具体的实施方案中,所述给予包括给予所述个体约5 X 106至约2 X 107个分离的胎盘细胞,其中所述细胞包含于包含10%葡聚糖(例如葡聚糖-40)、5%人血清白蛋白和任选的免疫抑制剂的溶液中。 [0031] In a specific embodiment, said administering comprises administering to said subject from about 5 X 106 to about 2 X 107 isolated placental cells, wherein said cell comprises containing 10% dextran (e.g. dextran sugar -40), a 5% solution of human serum albumin and optionally the immunosuppressant.

[0032] 在另外具体的实施方案中,所述给予包括静脉内给予约5 X107至3 X109个分离的胎盘细胞。 [0032] In another specific embodiment, said administering comprises administering from about 5 X107 to 3 X109 separate placental cells intravenously. 在更具体的实施方案中,所述给予包括静脉内给予约9X108个分离的胎盘细胞或约1.8X 109个分离的胎盘细胞。 In a more specific embodiment, the administration of about 9X108 administered isolated placental cells, or about 1.8X 109 comprises isolated placental cells intravenously. 在另外具体的实施方案中,所述给予包括头烦内给予约5 X107至IX 108个分离的胎盘细胞。 In another specific embodiment, said administering comprises administering to the head bother to about 5 X107 IX 108 isolated placental cells. 在更具体的实施方案中,所述给予包括头烦内给予约9X 1〇7个分离的胎盘细胞。 In a more specific embodiment, said administering comprises administering about 9X inner head trouble 1〇7 isolated placental cells.

[0033] 在另外具体的实施方案中,如上所述的治疗方法包括给予第二种治疗剂至所述个体。 [0033] In another specific embodiment, the method of treatment described above comprising administering a second therapeutic agent to the subject. 在更具体的实施方案中,所述第二种治疗剂为神经保护剂。 In a more specific embodiment, the second therapeutic agent is a neuroprotective agent. 在更具体的实施方案中,所述第二种治疗剂为NXY-059(苯基下基硝酬的二横酷基衍生物:二钢4-((叔-下基亚胺基)- 甲基)苯-1,3-二横酸盐N-氧化物,或二钢4-((氧化-叔-下基-二甲基亚氮鐵基)甲基)苯-1, 3-二横酸盐;亦称二硫酪)。 In a more specific embodiment, the second therapeutic agent is NXY-059 (two horizontal cool phenyl derivatives of the pay nitro group: two steel 4 - ((tert - the imino) - A yl) benzene-1,3-dicarboxylic acid cross-N- oxide, or titanium steel 4 - ((oxidation - t - the group - N and Fe dimethylsilylene-yl) methyl) benzene-1, 3-horizontal acid; also known as disulfide casein). 在另外更具体的实施方案中,第二种治疗剂为血栓溶解剂。 In another more specific embodiment, the second therapeutic agent is a thrombolytic agent. 在更具体的实施方案中,所述血栓溶解剂为组织纤溶酶原激活物(tPA)。 In a more specific embodiment, the thrombolytic agent is tissue plasminogen activator (tPA). 在其中脑内或脑周围血流破坏为出血的实施方案中,第二种治疗剂可W为抗高血压药物,例如郎且断剂或利尿药物,利尿药物和滞钟利尿药物的组合,郎且断剂和利尿药物的组合,血管紧张素-转化酶(ACE)抑制剂和利尿剂的组合,血管紧张素-Π 括抗剂和利尿药物,和/或巧离子通道阻断剂和ACE抑制剂。 Or in which the brain damage to cerebral blood flow around the bleeding embodiment, the second therapeutic agent may be an anti-hypertensive drugs W, for example, breaking or a combination of Lang and diuretic drugs, diuretic drugs, and slow clock diuretic drugs, Lang and the combination of the breaking agent and diuretic drugs, angiotensin - converting enzyme (ACE) inhibitor and a diuretic combinations, including angiotensin -Π antagonist and diuretic drugs, and / or ion channel blockers, and clever ACE inhibition agents. 在另外更具体的实施方案中,第二种治疗剂为巧离子通道阻断剂、谷氨酸括抗剂、丫氨基下酸(GABA)激动剂、抗氧化剂或自由基清除剂。 In another more specific embodiment, the second therapeutic agent is a clever ion channel blockers, including glutamate antagonist, Ah amino acid (GABA) agonist, an anti-oxidant or radical scavengers.

[0034] 在所述治疗方法另外具体的实施方案中,所述分离的胎盘细胞在所述个体脑内或脑周围血流破坏的一种或多种症状形成的21-30天内,例如第21天,例如中风、缺氧损伤或低氧损伤症状形成的21-30天内,例如第21天,给予所述个体。 [0034] 21-30 days another specific embodiment, said isolated placental cells are one or more symptoms of the subject around the brain or brain blood flow in a disruption of the method of treatment, 21 of e.g. days, for example 21-30 days, stroke, hypoxic injury or anoxic injury symptoms formed, for example, on day 21, the administration subject. 在所述治疗方法另外具体的实施方案中,所述分离的胎盘细胞在所述个体脑内或脑周围血流破坏的一种或多种症状形成的14天内给予所述个体。 14 days of administering to said subject another specific embodiment, said isolated placental cells in the brain or the cerebral blood flow around the subject of one or more symptoms of a disruption of the treatment method. 在所述治疗方法另外具体的实施方案中,所述分离的胎盘细胞在所述个体脑内或脑周围血流破坏的一种或多种症状形成的7天内给予所述个体。 Another specific embodiment, the subject is administered within 7 days of the isolated placental cells in the brain or the cerebral blood flow around the subject of one or more symptoms of a disruption of the treatment method. 在所述治疗方法另外具体的实施方案中,所述分离的胎盘细胞在所述个体脑内或脑周围血流破坏的一种或多种症状形成的48小时内给予所述个体。 Another specific embodiment, said isolated placental cells are administered to said individual within 48 hours of the brain or the cerebral blood flow around the individual one or more symptoms of damage formed in the method of treatment. 在另外具体的实施方案中,所述分离的胎盘细胞在所述个体脑内或脑周围血流破坏的一种或多种症状形成的24小时内给予所述个体。 In another specific embodiment, said isolated placental cells are administered to said individual within 24 hours of the brain or the cerebral blood flow around the individual one or more symptoms of damage formation. 在另外具体的实施方案中,所述分离的胎盘细胞在所述个体脑内或脑周围血流破坏的一种或多种症状形成的12小时内给予所述个体。 In another specific embodiment, said isolated placental cells are administered to said individual within 12 hours of the brain or the cerebral blood flow around the individual one or more symptoms of damage formation. 在另外具体的实施方案中,所述分离的胎盘细胞在所述个体脑内或脑周围血流破坏的一种或多种症状形成的3小时内给予所述个体。 In another specific embodiment, said isolated placental cells are administered to said individual within 3 hours of the brain or the cerebral blood flow around the individual one or more symptoms of damage formation.

[0035] 3.1 定义 [0035] 3.1 Definitions

[0036] 如此处所用的,术语"约"当设及数值时,表示所述数值的20%内的值。 [0036] As used herein, the term "about" when the set value and indicates a value within 20% of the stated value.

[0037] 如此处所用的,术语"缺氧损伤"指损伤(例如由脑或CNS区域氧的整体缺乏)引起的神经学损伤或症状。 [0037] As used herein, the term "hypoxia" refers to injury (e.g. brain or overall lack of oxygen CNS regions) neurological damage or symptoms caused.

[0038] 如此处所用的,术语"低氧损伤"指损伤(例如由脑或CNS区域氧的部分缺乏)引起的神经学损伤或症状。 [0038] As used herein, the term "hypoxic injury" means injury (e.g. brain or part by a lack of oxygen in the CNS regions) neurological damage or symptoms caused.

[0039] 如此处所用的,术语"甜2"指结合细胞标记CD105上表位的抗体。 [0039] As used herein, the term "sweet 2" refers to a cell marker CD105 antibody binding epitope. 因此,被称为甜2+ 的细胞为CD105+。 Thus, known sweet 2+ cells are CD105 +.

[0040] 如此处所用的,术语"SH3和細4"指结合呈现在细胞标记CD73上的表位的抗体。 [0040] As used herein, the term "fine SH3 and 4" refers to an antibody binding epitopes presented on the cell marker is CD73. 因此,被称为甜3+和/或甜4+的细胞为CD73+。 Thus, it is known sweet 3+ / 4+ sweet or cells and to CD73 +.

[0041] 胎盘具有在其内发育的胎儿的基因型,但在妊娠期间其也与母系组织密切接触。 [0041] The genotype of the fetus in the placenta having its inner development, but also in intimate contact with the mother during pregnancy tissue. 因而如此处所用的,相对于携带胎儿的母体的基因型,术语"胎儿基因型"指胎儿的基因型, 例如与胎盘有关的胎儿基因型,如此处所述,特定分离的胎盘细胞获自所述胎盘。 Thus as used herein, with respect to the carrying fetal maternal genotype, the term "fetal genotype" refers to the genotype of the fetus, placenta e.g. fetal genotype-related, as described herein, the particular isolated placental cells are obtained from the said placenta. 如此处所用的,术语"母系基因型"指携带胎儿,例如与胎盘有关的胎儿的母体的基因型,如此处所述,特定分离的胎盘细胞获自所述胎盘。 As used herein, the term "maternal genotype" refers to carrying fetus, placenta genotype e.g. fetal maternal related, as described herein, the particular isolated placental cells are obtained from the placenta.

[0042] 如此处所用的,术语"分离的细胞",例如"分离的干细胞"指基本上与其它组织(例如所述干细胞来源的胎盘)的不同细胞分开的细胞。 [0042] As used herein, the term "isolated cell", such as "isolated stem cell" means substantially separated from other tissues (e.g., the placenta-derived stem cells) in different cells. 如果至少50%、60%、70%、80%、90%、 95%或至少99%与干细胞存在天然关联的细胞例如非-干细胞、或显示不同标记谱的干细胞在干细胞采集和/或培养期间从所述干细胞中除去,则所述干细胞为"分离的"。 During the stem cells, or show different marker profile of stem cells in the stem cell collection and / or culture - if at least 50%, 60%, 70%, 80%, 90%, 95%, or at least 99% of the stem cell is naturally associated cells, such as non-existent removed from the stem cells, the stem cells are then "isolated."

[0043] 如此处所用的,"多能"当设及细胞时,指细胞具有分化为一些而不必然是全部体细胞类型,或分化为具有一些而不是全部体细胞类型特性的细胞的能力。 When [0043] As used herein, "pluripotent" and set when the cells differentiate into several cell means and not necessarily all, somatic cell types, or differentiate into some but not all cell types characteristic of the ability of somatic cells. 在某些实施方案中,例如具有分化为具有神经形成、软骨形成和/或成骨细胞特性的细胞之能力的分离的胎盘细胞为多能。 In certain embodiments, for example, a neural differentiation having formed, cartilage formation and / or the ability to separate cells of the bone cell characteristics multipotent placental cells.

[0044] 如此处所用的,术语"分离的细胞群"指基本上与其它组织细胞(例如所述细胞群来源的胎盘)分开的细胞群。 [0044] As used herein, the term "isolated population of cells" means substantially separated from other tissue cells (e.g., the placental cell population derived) cell population.

[0045] 如此处所用的,不考虑形态、细胞表面标记或原代培养后传代数,术语"胎盘干细胞"指源自哺乳动物胎盘的干细胞或祖细胞。 [0045] As used herein, regardless of morphology, cell surface markers or the number of passages after a primary culture, the term "placental stem cell" refers to mammalian placenta derived stem cells or progenitor cells. 然而,如此处所用的术语"胎盘干细胞"不指W 下细胞,即胎盘干细胞不是滋养层细胞、成血管细胞、造血成血管细胞、胚胎生殖细胞、胚胎干细胞、获自胚囊内细胞团的细胞、或获自晚期胚胎生殖腺脊的细胞,例如生殖细胞。 However, as used herein, the term "placental stem cells" does not refer to the cell W, i.e., the placental stem cells are not trophoblasts, into vascular cells, hematopoietic hemangioblasts, embryonic germ cells, embryonic stem cells, obtained from the cells in the embryo sac cell mass or obtained from the late embryonic gonadal ridge cells, such as germ cells. 如果细胞显示干细胞属性,例如与一种或多种干细胞类型有关的标记或基因表达谱;培养时复制至少10-40次的能力,W及分化为呈现Ξ个胚层中一层或多层的已分化细胞之特性的细胞的能力,则认为所述细胞为"干细胞"。 If the cells display stem cell properties, for example, with one or more stem cell marker or gene expression profile associated types; the ability to replicate at least 10-40 times in culture when, W, and differentiation of germ layers present Ξ one or more layers have been the ability of cells to differentiated cells characteristic of, the cell is considered "stem cells." 术语"胎盘干细胞"和"胎盘来源的干细胞"可互换使用。 The term "placental stem cell" and "placenta-derived stem cell" are used interchangeably. 此处除非另作说明,术语"胎盘"包括厮带。 Unless otherwise specified herein, the term "placenta" band comprises a servant. 在某些实施方案中,此处公开的分离的胎盘细胞在分化环境下体外分化、体内分化或体外体内都分化。 In certain embodiments, disclosed herein isolated placental cells in vitro in differentiation environment, both in vivo or in vitro differentiation in vivo differentiation.

[0046] 如此处所用的,当标记可检测地大于背景时,胎盘细胞就是该特定标记为"阳性" 的。 [0046] As used herein, detectably labeled when greater than the background, the specific placental cells is labeled "positive". 例如,由于CD73在胎盘干细胞上的量可检测地大于背景(例如,与同型对照相比),因此胎盘干细胞是CD73阳性的。 For example, since the amount of CD73 on placental stem cells detectably greater than background (e.g., as compared to isotype control), thus placental stem cells are CD73 positive. 当标记可用于将所述细胞与至少一种其它细胞类型区分开时, 或当所述标记在细胞中存在或表达时,该标记可用于选择或分离所述细胞,则该细胞也是所述标记阳性的。 When the flag may be used to distinguish the cell with at least one other cell type zone, or when the marker is present or expressed in the cells, the marker may be used to select or isolate the cell, the cell marker is the positive. 在例如抗体介导的检测中,当存在特定细胞表面标记时,"阳性"指所述标记可利用抗体检测,例如对该标记特异的巧光-标记抗体;"阳性"也指运样的细胞,其表现出的所述标记的量例如在细胞计数器中产生的信号可检测地大于背景。 In the detection of antibody-mediated example, when there are certain cell surface markers, "positive" means that the marker can be detected using antibodies, for example, the marker specific for clever light - labeled antibody; "positive" also refers to a cell sample transport , the amount of the marker which exhibit, for example, signals generated in the cell counter detectably greater than background. 例如,细胞为乂D200"',其中该细胞可用CD200特异性抗体可检测地标记,并且来自该抗体的信号可检测地高于对照(例如背景或同型对照)。相反,相同情景下,"阴性"指相比对照(例如背景或同型对照),利用所述标记特异性抗体检测不到细胞表面标记。例如,细胞为"CD3r",指相比对照(例如背景或同型对照),所述细胞不能W更大程度可重复检测地用CD34特异性抗体标记。利用合适的对照,W类似的方式来确定抗体所不能检测到的标记为阳性或阴性的。例如,通过检测RNA的方法如RT-PCR、槽斑点等等所测定的,如果来自细胞或细胞群RNA中检测的0CT-4RNA的量可检测地大于背景,则所述细胞或细胞群被认为是0CT-4+。此处除非另作说明,利用抗体检测分化("CD")标记簇。在某些实施方案中,如果利用RT-PCR可检测0CT-4 时,则确定0CT-4存在,并且细胞为"0CT-4+"。 For example, cells qe D200 " ', wherein the CD200 cells can be detectably labeled specific antibody, and the signal from the antibody is detectably higher than control (e.g., background or an isotype control). In contrast, under the same situation," negative "means compared to a control (e.g., background or an isotype control), with the labeled antibody not specific for cell surface markers. For example, a cell is" CD3r ", means compared to a control (e.g., background or an isotype control), the cells W can not be repeated detectably greater degree of CD34-specific antibody labeled with a with appropriate controls, W similar manner the labeled antibody is determined to not be detected as positive or negative. for example, detection of RNA by a method as RT- the PCR, the measured groove spots and the like, if the amount of 0CT-4RNA RNA from cells or cell populations can be detected in detectably greater than background, then the cell or cell population is considered 0CT-4 +. here unless otherwise for illustration, antibody detection using differentiation ( "CD") clusters of markers. in certain embodiments, if detectable by RT-PCR 0CT-4, it is determined 0CT-4 is present, and the cell is "0CT-4 +" .

[0047] 如此处所用的,"治疗"包含疾病、素乱或病情或其任何参数或症状的治愈、治疗、 改善、严重程度减轻、病程缩短。 [0047] As used herein, "treatment" includes a disease, disorder, or cure the disease hormone or any parameter or symptom, treat, ameliorate, reduce the severity, duration shortened.

[004引附图简述 [BRIEF DESCRIPTION primer 004

[0049] 图1:提高身体摆动检测结果。 [0049] FIG. 1: improved physical wobble detection result. 纵轴:摆动活力百分比偏差。 Vertical axis: percentage deviation swing activity. 横轴:评估摆动活力的天数。 Horizontal axis: number of days to assess the vitality of swing. 基线为诱导局部缺血之前摆动活力的百分比偏差。 Percentage induction baseline deviation swing activity prior to ischemia. 在梗塞后第2天头烦内给予分离的胎盘细胞之前评估了百分比偏差摆动活力,并且在梗塞后第7和14天再次评估。 Evaluation of the percentage deviation swing activity prior to administration of isolated placental cells in the first two days after infarction head trouble, and 7 and 14 day evaluation again after infarction. 有活力的400K: 4 X 105个有活力的分离的胎盘细胞。 Dynamic 400K: 4 X 105 th viable isolated placental cells. 无活力的:无活力的胎盘干细胞。 Nonviable: nonviable placental stem cells. CsA:环胞素A。 CsA: cyclosporine A.

[0050] 图2 :Bederson检测结果。 [0050] FIG. 2: Bederson detection result. 纵轴:神经性缺损平均得分。 Vertical axis: average score of neurological deficit. 横轴:评估神经性缺损的天数。 Abscissa: days of assessment of neurological deficit. 基线为诱导局部缺血之前的神经性缺损;0表示不缺损。 Baseline before inducing ischemic neurological deficit; 0 means no defects. 梗塞后第2天给予分离的胎盘细胞时评估平均神经性活力,并且在梗塞后第7和14天再次评估。 The average viability assessment of neuropathic the first two days after administration of isolated placental cells infarction, 7 and 14 days and assessed again after infarction. 有活力的400Κ:4Χ105个有活力的分离的胎盘细胞。 Dynamic 400Κ: 4Χ105 a viable isolated placental cell. 无活力的:无活力的胎盘干细胞。 Nonviable: nonviable placental stem cells. CsA:环胞素Α。 CsA: cyclosporine Α.

[0051] 图3:提高身体摆动检测结果。 [0051] FIG. 3: improved physical wobble detection result. 纵轴:摆动活力百分比偏差。 Vertical axis: percentage deviation swing activity. 横轴:评估摆动活力的天数。 Horizontal axis: number of days to assess the vitality of swing. 基线为诱导局部缺血之前摆动活力的百分比偏差。 Percentage induction baseline deviation swing activity prior to ischemia. 在梗塞后第2天给予分离的胎盘细胞静脉内时评估百分比偏差摆动活力,并且在梗塞后第7和14天再次评估。 When evaluating the isolated placental cells are administered intravenously on day 2 post-infarct percentage deviation swing activity, and re-evaluated 7 and 14 days after infarction. 无活力的:无活力的胎盘干细胞。 Nonviable: nonviable placental stem cells. 给予4 X 105、1 X 106、4 X 106或8 X 106个有活力的分离的胎盘细胞(图例)。 Administration of 4 X 105,1 X 106,4 X 106 or 8 X 106 th viable isolated placental cells (legend).

[0052] 图4: Bederson检测结果。 [0052] FIG. 4: Bederson detection result. 纵轴:神经性缺损平均得分。 Vertical axis: average score of neurological deficit. 横轴:评估神经性缺损的天数。 Abscissa: days of assessment of neurological deficit. 基线为诱导局部缺血之前的神经性缺损;0表示不缺损。 Baseline before inducing ischemic neurological deficit; 0 means no defects. 在梗塞后第2天给予分离的胎盘细胞静脉内时评估平均神经性活力,并且在梗塞后第7和14天再次评估。 Assessing the viability average neuropathic isolated placental cells intravenously administered on day 2 post-infarct, and 7 and 14 day evaluation again after infarction. 无活力的:无活力的胎盘干细胞。 Nonviable: nonviable placental stem cells. 给予4 X 105、1 X 106、4 X 106或8 X 106个有活力的分离的胎盘细胞(图例)。 Administration of 4 X 105,1 X 106,4 X 106 or 8 X 106 th viable isolated placental cells (legend).

[0053] 图5:改进的神经严重程度得分检测结果。 [0053] FIG. 5: neurological severity score improved detection results. Υ轴:得分(参见表1计分方法)"X轴:大脑中动脉闭塞(MCA0)手术后的天数。PDA:胎盘干细胞;FBC-对照:成纤维细胞对照;葡聚糖,无细胞对照;1PDA,1 X 106个细胞;4PDA,4 X 106个细胞;8PDA,8 X 106个细胞。Rx:给予细胞或葡聚糖。 Υ axes: score (see Table 1 scoring) "X axis: cerebral artery occlusion (MCA0) .PDA days after surgery: placental stem cells; FBC- Control: fibroblast control; glucan, a cell-free control; 1PDA, 1 X 106 cells; 4PDA, 4 X 106 cells; 8PDA, 8 X 106 cells .Rx: administering to a cell or dextran.

[0054] 图6:粘贴物移除体感检测结果。 [0054] FIG. 6: sticker removal somatosensory test results. Y轴:移除粘贴测试物的秒数。 Y-axis: number of seconds the test adhesive was removed. X轴:大脑中动脉闭塞(MCA0)手术后的天数。 X-axis: cerebral artery occlusion (MCA0) days after surgery. PDA:胎盘干细胞;FBC-对照:成纤维细胞对照;葡聚糖,无细胞对照;1PDA,1 X 106个细胞;4PDA,4 X 106个细胞;8PDA,8 X 106个细胞。 A PDA: Placental stem cells; FBC- Control: fibroblast control; glucan, a cell-free control; 1PDA, 1 X 106 cells; 4PDA, 4 X 106 cells; 8PDA, 8 X 106 cells. Rx:给予细胞或葡聚糖。 Rx: administering to a cell or dextran.

[0055] 图7:足失误检测结果。 [0055] Figure 7: foot fault detection result. Y轴:在金属网格上100步中的足失误百分比。 Y axis: percentage of the metal mesh 100 in the foot fault step. X轴:进行足失误检测的治疗后天数(Rx)。 X-axis: number of treatment (Rx) foot fault detection acquired. *:接受4X106个胎盘干细胞(PDA-4M)的动物对比赋形剂对照动物,足失误检测显著改善(P<〇.〇5)。 *: Receiving 4X106 placental stem cells (PDA-4M) vehicle control animals Animals contrast, foot fault detection significantly improved (P <〇.〇5). #:接受4X106个胎盘干细胞(PDA-4M)的动物对比成纤维细胞对照的动物,足失误检测显著改善(P<〇.05)。 #: Receiving 4X106 placental stem cells (PDA-4M) in contrast to animal cell control animal fibers, foot fault detection significantly improved (P <〇.05).

[0056] 图8:大脑中动脉闭塞后治疗56天后血管生成的测定。 [0056] FIG. 8: Determination of the brain 56 days after the treatment of angiogenesis after arterial occlusion. 胎盘干细胞治疗显著提高内皮细胞增殖W及局部缺血边界区(IBZ)中的血管密度和血管周长。 Placental stem cell treatment significantly increased endothelial cell proliferation and W ischemic border zone (IBZ) vascular density and vascular Zhou Chang. N=10/组。 N = 10 / group. 图8A:石蜡脑切片的抗-Br加抗体染色检测。 FIG 8A: Paraffin brain sections plus anti -Br antibody staining. Y轴:局部缺血病变边界中的化加-阳性内皮细胞化C)百分比;X轴:实验条件(MCAo-Dex:大脑中动脉闭塞-葡聚糖(最初的病情预处理);细胞-对照:给予成纤维细胞;PDA-4M:给予4X106个胎盘干细胞)d#:PDA-4M条件下对比成纤维细胞对照, 内皮细胞增殖显著增加(P<〇.〇5)d*:PDA-4M条件下对比葡聚糖(赋形剂)对照,内皮细胞增殖的显著增加(P<〇.05)。 The Y-axis: the ischemic lesion boundaries of plus - positive endothelial cells of C) a percentage; X-axis: experimental conditions (MCAo-Dex: Middle Cerebral Artery Occlusion - dextran (initial condition pre-treatment); Cell - control : administration of fibroblasts; PDA-4M: administration 4X106 placental stem cells) d #: comparison fibroblasts control, endothelial cell proliferation was significantly increased (P <〇.〇5) D the PDA-4M condition *: PDA-4M condition Comparative dextran (vehicle) control was significantly increased (P <〇.05) endothelial cell proliferation. 图8B:用胎盘干细胞治疗后局部缺血边界区的血管密度。 FIG. 8B: vascular density in the ischemic boundary zone after treatment with placental stem cells. Y轴:每mm2血管数;X轴:实验条件(MCAo-Dex:大脑中动脉闭塞-葡聚糖(最初的病情预处理);细胞- 对照:给予成纤维细胞;PDA-4M:给予4 X 106个胎盘干细胞)。 Y-axis: number of vessels per mm2; X-axis: experimental conditions (MCAo-Dex: Middle Cerebral Artery Occlusion - dextran (initial condition pre-treatment); Cell - control: administration of fibroblasts; PDA-4M: administration of 4 X 106 placental stem cells). #: PDA-4M条件下对比成纤维细胞对照,血管密度显著增加(P<〇.〇5)d*:PDA-4M条件下对比葡聚糖(赋形剂)对照,血管密度显著增加(P<〇.05)。 #: Comparison of the PDA-4M condition control fibroblasts, vascular density was significantly increased (P <〇.〇5) d *: Comparative under PDA-4M condition dextran (vehicle) control, vascular density was significantly increased (P <〇.05). 图8C:局部缺血边界区周围血管周长的增加。 FIG 8C: increase in the ischemic border zone around the Zhou Chang of the vessel. Y轴:血管周长的毫米长度;X轴:实验条件(MCAo-Dex:大脑中动脉闭塞-葡聚糖(最初的病情预处理);细胞-对照:给予成纤维细胞;PDA-4M:给予4X106个胎盘干细胞)d#:PDA-4M条件下对比成纤维细胞对照, 血管周长长度显著增加(P<〇.〇5)"*:PDA-4M条件下对比葡聚糖(赋形剂)对照,血管周长长度显著增加(P<〇.〇5)。 Y-axis: the vascular perimeter mm in length; X-axis: experimental conditions (MCAo-Dex: Middle Cerebral Artery Occlusion - dextran (initial condition pre-treatment); Cell - control: administration of fibroblasts; PDA-4M: administration 4X106 placental stem cells) d #: comparison of the PDA-4M condition control fibroblasts, vascular length of the Zhou Chang was significantly increased (P <〇.〇5) "*: dextran (vehicle) control the conditions of Comparative PDA-4M vascular Zhou Chang length significantly increased (P <〇.〇5).

[0057] 图9:胎盘干细胞治疗显著提高局部缺血脑的局部缺血边界中突触素的表达。 [0057] Figure 9: Placental stem cell treatment significantly increased expression of ischemic cerebral ischemic boundary synaptophysin. N= 10/组。 N = 10 / group. Y轴:检测石蜡脑切片中检查区域的突触素% ;X轴:实验条件。 Y-axis: detecting Paraffin brain sections of the examination region synaptophysin%; X-axis: experimental conditions. MCAo:大脑中动脉闭塞时突触素的表达;细胞-对照:给予成纤维细胞;PDA-4M:给予4X106个胎盘干细胞。 Of MCAo: Expression of middle cerebral artery occlusion when synaptophysin; Cell - control: administration of fibroblasts; PDA-4M: administration 4X106 placental stem cells. *:PDA- 4M对比成纤维细胞对照,突触素表达区域的显著增加。 *: PDA- 4M Comparative fibroblast controls, a significant increase in synaptophysin expression region.

[0化引发明详述 [0 initiators described in detail next

[0059] 5.1脑内和脑周围血流破坏的治疗 [0059] 5.1 treatment of disruption of blood flow in the brain and around the brain

[0060] 此处提供用于治疗脑内或脑周围血流破坏的个体的方法,例如对个体脑内或脑周围血流破坏的一种或多种症状或血流破坏所引起的神经性缺损的治疗,包括给予个体治疗有效量的分离的塑料贴壁组织培养人胎盘细胞,其中所述分离的胎盘细胞具有多能细胞或干细胞的特性,并且其中所述分离的胎盘细胞不是骨髓来源的间充质细胞、脂肪来源的间充质干细胞或者获自厮带血、胎盘血或外周血的间充质细胞。 Neurological deficit [0060] A method provided herein for treating a subject around the brain or the cerebral blood flow failure, e.g. caused by the individual brain damage or brain blood flow around or one or more symptoms of disruption of blood flow treatment, comprising administering a therapeutically effective amount of isolated tissue culture plastic-adherent human placental cells, wherein said isolated placental cells have characteristics of multipotent cells or stem cells, and wherein between said isolated placental cells are not bone marrow-derived mesenchymal cells, adipose-derived mesenchymal stem cells obtained from a servant or blood, placental blood, or peripheral blood between mesenchymal cells. 在某些实施方案中,所述损伤为低氧损伤或缺氧损伤。 In certain embodiments, the injury is hypoxic injury or anoxic injury. 在此处提供的某些实施方案中,所述治疗有效量为使所述个体表现出的脑内或脑周围血流破坏的一种或多种症状或血流破坏所引起的神经性缺损消除、可检测的改善、严重程度减轻、或者发展变缓的量。 In certain embodiments provided herein, the therapeutically effective amount is the subject neurological deficit exhibited cerebral blood flow around the brain or one or more symptoms or damage caused by the disruption of blood flow to eliminate can improve the detection, reduce the severity of, or slowing down the development of the amount.

[0061] 在一个具体的实施方案中,一种或多种症状或神经性缺损,例如中风症状、低氧损伤或缺氧损伤,至少在某种程度上或全部归因于血流破坏后的再灌注损伤。 [0061] In a particular embodiment, the one or more symptoms or neurological deficit, symptoms such as stroke, hypoxic injury or anoxic injury, at least in part or all of the disruption of blood flow due to reperfusion injury. 如此处所用的, "再灌注损伤"指在局部缺血一段时期中断血液供给后恢复组织血液供给时引起的组织损伤。 As used herein, "reperfusion injury" tissue damage caused when blood supply to tissue recovery after ischemia refers to a period of interrupted blood supply. 局部缺血期间血源性氧和营养缺乏产生W下情形,其中循环的恢复通过引起氧化应激而不是通过恢复正常功能而引起炎症和氧化损伤。 Blood-borne oxygen partial ischemia and nutritional deficiencies during the generation situation W, wherein the recovery cycle by not cause oxidative stress and inflammation caused by oxidative damage by restoring normal function.

[0062] 如此处所用的,所述治疗血流破坏的内容中,术语"脑内或脑周围血流的破坏"包括治疗具有血流所述破坏的个体表现出的一种或多种症状W及血流破坏所引起的个体神经性缺损,例如一种或多种中风症状、低氧损伤或缺氧损伤。 Individual [0062] As used herein, the treatment of disruption of blood flow in the content, the term "cerebral blood flow around the brain or destruction" includes treating the blood having disrupted exhibits one or more symptoms of W and disruption of blood flow caused by the individual neurological deficit, such as one or more symptoms of stroke, hypoxic injury or anoxic injury. 在某些实施方案中,所述一种或多种症状为主要或全部归因于局部缺血本身的一种或多种症状。 In certain embodiments, the one or more symptoms attributed mainly or entirely of one or more symptoms of ischemia itself. 在某些其它的实施方案中,所述一种或多种症状为主要或全部归因于例如与局部缺血有关的再灌注损伤。 In certain other embodiments, the one or more or all of the primary symptoms due to e.g. reperfusion injury associated with ischemia.

[0063] 5.2 中风 [0063] 5.2 stroke

[0064] 可通过此处提供的方法治疗的脑内或脑周围血流的破坏可W是在受影响的个体中导致一种或多种可检测症状或神经性缺损的任何血流破坏(见下文)。 [0064] The breakable cerebral blood flow around the brain or by therapeutic methods provided herein may be W is affected individuals results in any of one or more detectable blood neurological deficit symptoms or damage (see below). 在某些实施方案中,所述血流破坏为中风,例如局部缺血性中风或出血性中风,例如烦内脑溢血。 In certain embodiments, the disruption of blood flow is a stroke, e.g. ischemic stroke or hemorrhagic stroke, cerebral hemorrhage, for example, the trouble. 在某些其它的实施方案中,所述血流的破坏为脑外出血,例如硬脑膜血肿、硬膜下血肿或蛛网膜下血月中。 In certain other embodiments, the disruption of blood flow to the brain hemorrhage, subdural hematoma e.g., subdural hematoma, or subarachnoid blood months. 在某些其它的实施方案中,所述血流破坏为瞬时的,例如瞬时局部缺血损伤(TIA)。 In certain other embodiments, the disruption of blood flow is a transient, such as transient ischemic injury (TIA). 在某些其它的实施方案中,所述血流破坏为血管疫李。 In certain other embodiments, the disruption of blood flow is a vascular P. Lee. 在一个具体的实施方案中,所述脑中的血流破坏存在于大脑中。 In a specific embodiment, the disruption of blood flow to the brain is present in the brain. 在更具体的实施方案中,所述破坏存在于大脑顶叶、额叶、颠叶或枕叶。 In a more specific embodiment, the damage in the brain parietal, frontal, or occipital lobe Britain. 在另外具体的实施方案中,所述破坏存在于小脑中。 In another specific embodiment, said damage is present in the cerebellum. 在另外具体的实施方案中,所述破坏存在于脑干或脊柱中。 In another specific embodiment, said damage is present in the brain stem or spinal column. 在某些实施方案中,所述破坏引起低氧损伤或缺氧损伤。 In certain embodiments, the damage caused hypoxic injury or anoxic injury.

[0065] 在一个实施方案中,此处提供了治疗患有低氧损伤或缺氧损伤例如中风(例如中风患者)个体的方法,包括给予所述个体例如W下5.4.2节所述的治疗有效量的分离的塑料贴壁组织培养人胎盘细胞。 [0065] In one embodiment, provided herein is a method of treating hypoxic injury or anoxic injury such as stroke (e.g. stroke patients) an individual, comprising administering to said subject a therapeutically in Section 5.4.2, the W e.g. effective amount of isolated tissue culture plastic-adherent human placental cells. 在一个具体的实施方案中,所述治疗有效量为使所述个体表现出的中风的一种或多种症状消除、可检测的改善、严重程度减轻或者发展变缓的量。 In a specific embodiment, the therapeutically effective amount is such that the subject exhibits one or more symptoms of stroke eliminate detectable improvement or reduce the severity of an amount of slow development. 所述分离的胎盘细胞,例如大量分离的胎盘细胞或其分离的群可W是例如W下5.4.2节所述的任何分离的胎盘细胞。 The isolated placental cells, for example, a large number of isolated placental cells or populations may be isolated, for example, W is any isolated placental cells described in Section 5.4.2 of the W.

[0066] 根据此处提供的方法能治疗的中风可W是任何病因引起的中风。 [0066] The methods provided herein can treat stroke can be caused by a stroke W of any etiology. 在一个具体的实施方案中,所述中风为局部缺血性中风。 In a specific embodiment, the stroke is ischemic stroke. 在更具体的实施方案中,所述局部缺血性中风为血栓形成引起的中风或栓塞中风。 In more specific embodiments, the ischemic stroke is formed due to stroke or embolism stroke is thrombotic. 在另外具体的实施方案中,所述中风是由于全身的灌注不足,即流至身体所有部位的血流减少;或静脉血栓形成引起的。 In another specific embodiment, the stroke is due to lack of systemic perfusion, i.e., to reduce blood flow to all parts of the body; or venous thrombosis caused. 在其它具体的实施方案中, 所述局部缺血性中风由W下病因引起:屯、纤维性颤动,例如屯、房纤维性颤动;突发性屯、房纤维性颤动;风湿疾病;二尖或主动脉瓣疾病;人造屯、脏瓣膜;屯、房或屯、室的屯、血栓;病窦综合症;持续的屯、房扑动;屯、肌梗塞;射血分数少于28%的慢性屯、肌梗塞;射血分数少于30%的有症状的充血性屯、力衰竭;屯、肌病;屯、内膜炎,例如Libman-Sacks屯、内膜炎、消耗性屯、内膜炎或感染性屯、内膜炎;乳头状弹力纤维瘤;左房粘液瘤;冠状动脉旁路术移植手术;二尖瓣环巧化;卵圆孔未闭;房隔膜动脉瘤、无血栓的左屯、室动脉瘤、超声屯、动图显示的无二尖狭窄或屯、房颤动的单独左房"雾化";和/或升主动脉或主动脉弓近端的复杂动脉粥样化。 In other specific embodiments, the ischemic stroke caused by the cause of W: Tun, fibrillation, e.g. Tun, atrial fibrillation; Tun sudden, atrial fibrillation; rheumatic diseases; mitral or aortic valve disease; artificial Tun, dirty valve; Tun Tun, village or room, chamber, thrombosis; sick sinus syndrome; Tun sustained atrial flutter; Tun, myocardial infarction; ejection fraction less than 28% chronic Tun, myocardial infarction; ejection fraction less than 30% of the symptomatic congestive Tun, power failure; Tun, myopathy; Tun, endometritis, e.g. Libman-Sacks Tun, endometritis, expendable Tun, the Tun or infectious meningitis, endometritis; papilloma fibroelastoma; left atrial myxoma; coronary artery bypass graft surgery; Qiao of the mitral valve annulus; patent foramen ovale; atrial septum aneurysm without thrombus Tun left ventricular aneurysm, Tun ultrasound, the moving map display goes narrow tip or village, a separate room left atrial fibrillation "fog"; and / or proximal end of the ascending aorta or the aortic arch complex atheroma.

[0067] 在另外具体的实施方案中,所述中风为出血性中风。 [0067] In another specific embodiment, said stroke is hemorrhagic stroke. 在更具体的实施方案中,所述出血性中风由轴内出血(脑内部血液渗漏)引起。 In more specific embodiments, the hemorrhagic stroke is caused by bleeding shaft (internal cerebral blood leakage). 在另外更具体的实施方案中,所述出血性中风由轴外出血(脑外部头骨内部的血液渗漏)引起。 In further more specific embodiments, the hemorrhagic stroke by the outer shaft hemorrhage (cerebral internal leakage of blood outside the skull) caused. 在更具体的实施方案中,所述中风由薄壁组织内出血、屯、室内出血(屯、室系统中的血液)、硬膜外血肿(硬脑膜和头骨之间流血)、 硬膜下血肿(硬膜下空隙流血)或蛛网膜下出血(蜘蛛膜和软脑膜之间)引起。 In a more specific embodiment, the stroke by the parenchymal hemorrhage, village, bleeding chamber (Tun chamber system blood), epidural hematoma (bleeding between the dura and skull), subdural hematoma ( void subdural bleeding) or subarachnoid hemorrhage (between the arachnoid and pia mater) caused. 大多数出血性中风综合症具有特定的症状(例如头痛、先前的头部损伤)。 Most hemorrhagic stroke syndromes have specific symptoms (eg headache, previous head injury). 在其它更具体的实施方案中,所述出血性中风由高血压、创伤、流血素乱、淀粉样蛋白血管病、违禁药品的使用(例如安非他明或可卡因)或血管崎形引起或与之有关。 In other more specific embodiments, the hemorrhagic stroke, trauma use, blood hormone disorder, amyloid angiopathy, illicit drugs (e.g., amphetamine or cocaine) caused by vascular hypertension or shape or Kawasaki the related.

[006引在另一个实施方案中,此处提供治疗脑或CNS中或周围血流破坏的个体的方法,例如治疗个体脑或CNS中或周围血流破坏的一种或多种症状或血流破坏引起的神经性缺损, 包括给予所述个体治疗有效量的分离的胎盘细胞,例如胎盘干细胞或胎盘多能细胞,其中所述血流破坏的直接病因不是中风,例如闭合的头损伤或非-冲击-相关的血肿。 [006] In another embodiment cited embodiment, there is provided a method of treating the brain or CNS of an individual or of disruption of blood flow around here, for example in the brain or CNS of treating peripheral or one or more symptoms of disruption of blood flow or blood flow neurological deficit caused by damage, comprising administering to the subject a therapeutically effective amount of isolated placental cells, e.g., placental stem cells or placental multipotent cells, wherein said direct cause disruption of blood flow is not a stroke, for example, or a closed head trauma - impact - related hematoma. 在一个具体的实施方案中,所述治疗有效量为使所述个体表现出的所述血流破坏的一种或多种症状消除、可检测的改善、严重程度减轻、或者发展变缓的量。 In a specific embodiment, the therapeutically effective amount is such that the subject exhibits the disruption of blood flow to eliminate one or more symptoms, improve the detection of, reduce the severity of, or slowing the development of an amount . 如中风一样,分离的胎盘细胞可W 是例如W下5.4.2节所述的任何胎盘细胞,例如胎盘干细胞或胎盘多能细胞。 Such as stroke, isolated placental cells can be, for example, W is the W section 5.4.2 of any of the placental cells, e.g., placental stem cells or placental multipotent cells.

[0069] 如上所述,在某些实施方案中,此处提供的治疗方法使脑或CNS中或周围血流破坏的一种或多种症状或血流破坏引起的神经性缺损(例如中风、血肿引起的例如缺氧损伤或低氧损伤)消除、可检测的改善、严重程度减轻、或者发展变缓。 [0069] As described above, in certain embodiments, methods of treatment provided herein the brain or CNS of disruption of blood flow around or one or more symptoms or neurological deficit due to disruption of blood flow (e.g. stroke, For example anoxic injury or hypoxic injury) eliminate hematoma caused a detectable improvement, reduce the severity of, or slow development. 在具体的实施方案中,所述症状或神经性缺损包括偏擁(身体一侧麻搏);轻偏擁(身体一侧虚弱);面部肌肉虚弱;麻木;感觉下降;嗅觉、味觉、听力或视力改变;嗅觉、味觉、听力或视力丧失;眼皮茸拉(下垂症);眼肌肉虚弱;呕吐反射降低;吞咽能力降低;瞳孔光反应性降低;面部知觉降低;平衡降低;眼震症;呼吸速率改变;屯、率改变;不能转动头至一侧的锁骨乳突肌虚弱,或能力降低; 舌虚弱;失语症(不能说或理解语言);失用症(改变的自觉运动);视野缺陷;记忆力缺损;半侧忽视或半侧空间忽视(对病变相对的视野另一侧空间的关注缺乏);混乱思维;意识模糊; 纵欲姿态的发展;疾病失认症(持续否认缺陷的存在);难W行走;运动协同动作改变;眩晕; 不平衡;意识丧失;头痛和/或呕吐。 In a specific embodiment, said symptom or neurological deficit comprising vinylidene owner (Ma stroke side of the body); Yong bias light (weak side of the body); facial muscle weakness; numbness; decreased feeling; smell, taste, hearing, or changes in vision; smell, taste, hearing or vision loss; eyelids Velvet pull (ptosis); eye muscle weakness; vomiting reflex reduce; reduce the ability to swallow; pupillary light response decreased; facial perception reduced; Ping Heng reduced; nystagmus disease; breathing rate changes; Tun, rate of change; not rotating head to the side of the clavicle papillary muscles weakness, or reduce the ability; tongue weakness; aphasia (not speak or understand language); apraxia (altered conscious movement); View defects ; memory impairment; hemi-neglect or unilateral spatial neglect (focus on the opposite side of the lesion space vision deficiency); confusion thinking; confusion; development indulgence attitude; disease agnosia (continued denying the existence of the defect); W difficulty walking; motion coordinated action to change; dizziness; imbalance; loss of consciousness; headache and / or vomiting.

[0070] 脑或CNS中或周围血流破坏的严重程度例如中风或中风症状和/或中风引起的神经性缺损的严重程度可W利用一种或多种普遍-接受的神经学功能评分来评估。 [0070] in or around the brain or CNS disruption of blood flow such as stroke or stroke severity of symptoms and / or severity of neurological deficit may be caused by stroke W with one or more generally - accepted neurological function score assessed .

[0071] 例如,在一个实施方案中,此处提供治疗脑或CNS中或周围血流破坏的个体的方法,例如患有个体脑内或脑周围血流破坏引起的症状或神经性缺损,例如低氧损伤或缺氧损伤的个体,包括给予所述个体治疗有效量的分离的人贴壁胎盘细胞,其中所述治疗有效量为足W引起个体神经学功能可检测的改善或可检测和持续的改善的胎盘细胞量,所述神经学功能通过一种或多种改进的Rankin评分、NIH中风评分、Canadian神经病学评分(CNS)、 昏迷评分(GCS)、化mpis地eric中风评分、化nt&胎SS评分、Mathew中风评分、细微精神状态检查(MMSE)、0rgogozo中风评分、Oxfordshire Community中风计划分类(Bamford)、 Scandinavian中风评分、日本昏迷评分(JCS)、Ba;rthel指数和/或日本中风评分(JSS)评估。 [0071] For example, in one embodiment, provided herein is a method of treating brain or CNS disruption of blood flow in or around the subject, e.g. an individual suffering from brain damage or brain blood flow caused around the symptoms or neurological deficit, e.g. hypoxic injury or anoxic injury human individual, comprising administering to the subject a therapeutically effective amount of an isolated adherent placental cells, wherein said therapeutically effective amount is sufficient to cause W ameliorating neurologic detectable or detectable and sustained placental cells, the amount of improvement of the neurological function by one or more modified Rankin Scale, NIH stroke Scale, Canadian neurological score (the CNS), coma score (GCS), of the mpis eric stroke Scale, of nt & tire SS score, Mathew stroke Scale, fine-mental state examination (MMSE), 0rgogozo stroke Scale, Oxfordshire Community stroke Program Category (Bamford), Scandinavian stroke Scale, Japan coma Scale (JCS), Ba; rthel index and / or Japan stroke Scale (JSS) assessment. 在具体的实施方案中,所述改善在最初的评估W及在一次或多次给予分离的胎盘细胞后的1、2、3、4、5或6天内,或1、2、3、4、5、6、8、9、10、11或12周内是可检测到的。 In a specific embodiment, the improvement in the five or six days after the initial assessment and W administered in one or more isolated placental cells, or 1,2,3,4, 5,6,8,9,10,11 or 12 weeks are detectable. 在其它具体的实施方案中,所述最初的评估在一次或多次中风症状形成后的1、2、3、4、5、6、7、8、9、10、11、12、 13、14、15、16、17、18、19、20、21、22或23小时内,或1、2、3、4或5天内进行。 In the other particular embodiments, the initial assessment in one or more symptoms of stroke formed 1,2,3,4,5,6,7,8,9,10,11,12, 13, 14 performed, the 15,16,17,18,19,20,21,22 or 23 hours, or 4 or 5 days. 在其它实施方案中,无论是否测定,所述改善持续例如超过至少1、2、3、4、5、6、7、8、9、10、11或12个月,或1、 2、3、4、5年或W上。 In other embodiments, whether measured, for example, a sustained improvement over 1,2,3,4,5,6,7,8,9,10,11, or at least 12 months, or 1, 2, 3, 4, 5, or on W.

[0072] 5.3给予分离的胎盘细胞 [0072] The isolated placental cells are administered 5.3

[0073] 此处提供的治疗方法中使用的分离的胎盘细胞可W通过任何医学上可接受的方法或途径给予表现出脑内或脑周围血流破坏引起的或缘自所述破坏的一种或多种症状或神经性缺损的个体。 [0073] The isolated placental cells described herein provides a method of treating may be used in administering W exhibit damage from the edge or one or brain damage caused by cerebral blood flow around by any method or medically acceptable route or more symptoms or neurological deficit in an individual. 在一个实施方案中,所述治疗有效量的分离的胎盘细胞经头烦内给予所述个体,例如给至个体受影响的脑或CNS中的局部缺血或出血性部位。 In one embodiment, the therapeutically effective amount of the placental cells isolated by first administering to the individual trouble, for example to a site of ischemic or hemorrhagic brain of affected individuals or the CNS. 对于烦内给予,所述中风位置(例如受影响区域)可W例如通过CT扫描、核磁共振(MRI)(例如Τ1-、Τ2-、扩散- 和/或灌注-加权MR)、钻-55正电子放射层析X线断层造影或类似技术显现。 For administration of the trouble, the stroke position (e.g., affected area) W can be, for example, by CT scan, magnetic resonance (the MRI) (e.g. Τ1-, Τ2-, diffusion - and / or perfusion - weighting the MR), the drill -55 n X-ray electron emission tomography chromatography or similar techniques appear. 在另一个实施方案中,所述分离的胎盘细胞通过静脉内或动脉内给予所述个体。 In another embodiment, the isolated placental cells are administered to the subject by intravenous or intraarterial. 在另外的实施方案中,所述治疗有效量的分离的胎盘细胞通过肌内、腹膜内、真皮内、皮下、眼内或肠胃外给予所述个体。 In further embodiments, the therapeutically effective amount of isolated placental cells by intramuscular, intraperitoneal, intradermal, subcutaneous, intraocular or parenterally administered to said individual. 所述分离的胎盘细胞可通过弹丸注射或静脉注射注入给予。 The isolated placental cells can be bolus injection or administered by intravenous injection. 在一个具体的实施方案中, 所述静脉注射注入为超过约1至约8个小时的静脉注射注入。 In a specific embodiment, the intravenous injection is intravenously over about 1 to about 8 hours of injection. 在某些实施方案中,所述分离的胎盘细胞通过组合途径,例如头烦内W及通过静脉注射注入给予所述个体。 In certain embodiments, the isolated placental cells by a combination of routes, such as the head W and the trouble administering to the subject intravenously injected.

[0074] 所述治疗有效量的分离的胎盘细胞可根据个体年龄和/或体重W及局部缺血区域的大体体积而不同。 [0074] The therapeutically effective amount of isolated placental cells may vary depending on the individual volumes substantially age and / or weight W and ischemic regions. 局部缺血区域的大概体积和位置可例如通过连续核磁共振图像或计算机X线断层造影(CT)扫描估计。 Approximate size and location of the ischemic region may be estimated, for example, by a continuous scan or magnetic resonance image of X-ray computer tomography (CT).

[0075] 例如单次剂量中给予的分离的胎盘细胞数,可每次给予约为或至少,或超过例如1 X1〇5、5X1〇5、1X1〇6、5X1〇6、1X1〇7、5X1〇7、1X1〇8、5X1〇8、1X1〇9、5X1〇9、1X1〇i〇、5X l〇w、1 X 1〇11或5 X 1〇11个分离的胎盘细胞。 [0075] For example the number of isolated placental cells is administered in a single dose, or at least can each administration is about, or more than 1 X1〇5,5X1〇5,1X1〇6,5X1〇6,1X1〇7,5X1 e.g. 〇7,1X1〇8,5X1〇8,1X1〇9,5X1〇9,1X1〇i〇, 5X l〇w, 1 X 5 X 1〇11 1〇11 or isolated placental cells. Z具体的实施方案中,患有脑内或脑周围血流的破坏的个体,例如患有中风、缺氧损伤或低氧损伤的个体可静脉内给予约5 X107至约3 Xl〇9 个分离的胎盘细胞。 Z specific embodiment, the subject suffers from brain damage or brain blood flow around, e.g. suffered a stroke, hypoxia, or administered from about 3 to about 5 X107 Xl〇9 separate the hypoxic injury in an individual intravenously placental cells. 在更具体的实施方案中,患有脑内或脑周围血流的破坏的个体,例如患有中风、低氧损伤或缺氧损伤的个体可静脉内给予约9 X 107个分离的胎盘细胞、约3.6 X 1〇8 个分离的胎盘细胞、约9 X 108个分离的胎盘细胞或约1.8 X 109个分离的胎盘细胞。 In more specific embodiments, an individual suffering from brain damage or brain blood flow around, e.g. suffered a stroke, hypoxic injury or anoxic injury within an individual intravenously administered from about 9 X 107 isolated placental cells, about 3.6 X 1〇8 isolated placental cells, about 9 X 108 isolated placental cells, or from about 1.8 X 109 isolated placental cells. 在另外具体的实施方案中,个体静脉内给予一次剂量约2 X108个分离的胎盘细胞。 In another specific embodiment, a dose of about 2 X108 administered isolated placental cells intravenously in an individual. 在另外具体的实施方案中,个体静脉内给予一次剂量约8X108个分离的胎盘细胞。 In another specific embodiment, one dose of approximately 8X108 administered isolated placental cells intravenously in an individual. 在另外具体的实施方案中,个体静脉内给予两次剂量,每次包括约2 X108个分离的胎盘细胞。 In another specific embodiment, a subject intravenously administered two doses, each comprising about 2 X108 isolated placental cells. 在另外具体的实施方案中,个体静脉内给予两次剂量,每次包括约8X108个分离的胎盘细胞。 In another specific embodiment, a subject intravenously administered two doses, each comprising about 8X108 isolated placental cells. 在其它更具体的实施方案中,患有脑或CNS中或周围血流破坏的个体,例如患有中风、低氧损伤或缺氧损伤的个体可头烦内给予约5 X 107至约1 X 108个分离的胎盘细胞。 In other more specific embodiments, with the brain or CNS or disruption of blood flow around the subject, e.g. suffered a stroke, administration of about 5 X 107 to about 1 X within a head trouble hypoxic injury or anoxic injury individuals 108 isolated placental cells. 在更具体的实施方案中,所述个体头烦内给予约9 X 107个分离的胎盘细胞。 In a more specific embodiment, the subject is administered from about 9 X 107 isolated placental cells within the first trouble.

[0076] 可在疗程中给予患有脑或CNS中或周围血流破坏、所述破坏的症状和/或所述破坏引起的神经性缺损的个体一次或一次W上,例如两次或更多次的分离的胎盘细胞。 [0076] In the course of treatment can be administered to the brain or CNS with disruption of blood flow in or around the primary symptom of the subject to damage and / or destruction of the neurological deficit caused by the first or W, for example, two or more times isolated placental cells. 所述分离的胎盘细胞可W用适合烦内给予的合适体积给予,例如约10化L、20化U30化L、40化L、 500yL、600yL、700yL、800yL、900yL、1000化、1.5血、2mL、2.5mL、3血、3.5血、4mL、4.5血、5mL、 5.5mL、6.OmL、6.5mL、7mL、7.5mL、8mL、8.5mL、9mL、9.5mL、1OmL、1ImL、12mL、13mL、14mL、 15mL>16mL>17mL>18mL>19mL>20mL>21mL>22mL>23mL>24mL>25mL>26mL>27mL>28mL>29mL^ 约30mL药学可接受溶液。 The isolated placental cells can be administered with a suitable volume W trouble for the administration, for example, of about 10 L, 20 of U30 of L, 40 of the L, 500yL, 600yL, 700yL, 800yL, 900yL, 1000 of 1.5 blood, 2mL, 2.5mL, 3 blood, blood 3.5, 4mL, 4.5 blood, 5mL, 5.5mL, 6.OmL, 6.5mL, 7mL, 7.5mL, 8mL, 8.5mL, 9mL, 9.5mL, 1OmL, 1ImL, 12mL, 13mL , 14mL, 15mL> 16mL> 17mL> 18mL> 19mL> 20mL> 21mL> 22mL> 23mL> 24mL> 25mL> 26mL> 27mL> 28mL> 29mL ^ about 30mL pharmaceutically acceptable solution. 对于静脉注射给予,大量分离的胎盘细胞(例如约1X105、5X105、1 X1〇6、5X1〇6、1X1〇7、5X1〇7、1X1〇8、5X1〇8、1X1〇9、5X1〇9、1X1〇i〇、5X1〇i〇、1X1〇ii 或5 Xl〇ii)可W例如约或不超过100mL、150mL、200mL、250mL、300mL、350mL、400mL、450mL、 500血、550血、600血、650血、700血、750血、800血、850血、900血、950mL、lOOOmL、1. IL、1.化、 1.3L、1.化或1.化,例如通过静脉注射注入。 For intravenous administration, a large number of isolated placental cells (e.g., about 1X105,5X105,1 X1〇6,5X1〇6,1X1〇7,5X1〇7,1X1〇8,5X1〇8,1X1〇9,5X1〇9, 1X1〇i〇, 5X1〇i〇, 1X1〇ii Xl〇ii or 5) may be, for example, W or no more than about 100mL, 150mL, 200mL, 250mL, 300mL, 350mL, 400mL, 450mL, 500 blood, blood 550, 600 blood , blood 650, 700 blood, blood 750, 800 blood, blood 850, 900 blood, 950mL, lOOOmL, 1. IL, 1. technology, 1.3L, 1. 1 or, for example by intravenous injection.

[0077] 在其他的实施方案中,给予所述个体约每千克个体(例如具有脑或CNS中或周围血流破坏引起的或缘于所述破坏或再灌注损伤的一种或多种症状或神经性缺损的个体)1X 10哇约2 X 106个分离的胎盘细胞;每千克个体约2 X 10哇约3 X 106个分离的胎盘细胞;每千克个体约3 X 106至约4 X 106个分离的胎盘细胞;每千克个体约4 X Χίο6至约5 X 106个分离的胎盘细胞;每千克个体约5X10哇约6 Χ106个分离的胎盘细胞;每千克个体约6X10哇约7 X 106个分离的胎盘细胞;每千克个体约7 X 106至约8 X 106个分离的胎盘细胞;每千克个体约8 X 10哇约9 X 106个分离的胎盘细胞;每千克个体约9 X 10哇约1 X 107个分离的胎盘细胞。 [0077] In other embodiments, the subject is administered approximately every kilogram of subject (e.g., having the blood flow around the brain or CNS damage caused by or one or more symptoms or damage due to the reperfusion injury or or individual neurological defects) 1X 10 wow about 2 X 106 isolated placental cells; kg of body about 3 X 106 isolated placental cells to about 2 X 10 wow; kg individual to about 3 X 106 to about 4 X 106 th isolated placental cells; kg of body about 4 X Χίο6 to about 5 X 106 isolated placental cells; kg of body about 5X10 wow to about 6 Χ106 isolated placental cells; kg of body about 6X10 wow to about 7 X 106 isolates placental cells; kg individual to about 7 X 106 to about 8 X 106 isolated placental cells; kg individual to about 8 X 10 wow from about 9 X 106 isolated placental cells; kg individual to about 9 X 10 wow about 1 X 107 isolated placental cells. 在另外具体的实施方案中,所述给予包括给予个体每千克所述个体约1 X 1〇7至约2 X 1〇7 个分离的胎盘细胞。 In another specific embodiment, said administering comprises administering to said subject per kilogram of subject to about 1 X 2 X 1〇7 about 1〇7 isolated placental cells. 在另外具体的实施方案中,所述给予包括给予个体每千克所述个体约1.3 X 107至约1.5 X 107个分离的胎盘细胞。 In another specific embodiment, said administering comprises administering to said subject per kilogram of subject to about 1.3 X 107 and about 1.5 X 107 isolated placental cells. 在另外具体的实施方案中,所述给予包括给予个体每千克所述个体总共约3 X 107个分离的胎盘细胞。 In another specific embodiment, said administering comprises administering to said subject an individual per kilogram total of about 3 X 107 isolated placental cells. 在另外具体的实施方案中,所述给予包括给予个体约20毫升溶液中的15Χ 107个分离的胎盘细胞。 In another specific embodiment, said administering comprises administering to a subject of about 20 ml solution 15Χ 107 isolated placental cells. 在一个优选的实施方案中,分离的胎盘细胞的给予包括给予不超过约1升溶液中的、每千克受者不超过7.5 Χ106个分离的胎盘细胞。 In a preferred embodiment, the isolated placental cells comprising administering administering not more than about 1 liter of solution per kilogram does not exceed 7.5 Χ106 isolated placental cells. 在一个具体的实施方案中,所述给予包括例如头烦内给予个体约5X 106至约2 X 1〇7个分离的胎盘细胞。 In a specific embodiment, said administering comprises administering, for example, from about 5X 106 to about 2 X 1〇7 individual isolated placental cells within the first trouble. 在一个具体的实施方案中,所述给予包括头烦内给予所述个体每千克约5Χ106至约3Χ107个分离的胎盘细胞,其中所述细胞包含于含有10%葡聚糖,5%人血清白蛋白和任选的免疫抑制剂,例如环胞素A的溶液中。 In a specific embodiment, said administering comprises administering to the subject within the first bother about 5Χ106 per kilogram to about 3Χ107 isolated placental cells, wherein said cell comprises containing 10% dextran and 5% human serum albumin protein and optionally an immunosuppressant, e.g. cyclosporine a at room temperature. 在另外具体的实施方案中,所述给予包括给予所述个体约IX 1〇9至约3 X107个胎盘多能细胞,其中所述细胞包含于含有10%葡聚糖,5%人血清白蛋白和任选的免疫抑制剂,例如环胞素A的溶液中。 In another specific embodiment, said administering comprises administering to said subject from about IX to about 3 X107 1〇9 placental multipotent cells, wherein said cell comprises containing 10% dextran and 5% human serum albumin and optionally an immunosuppressant, such as cyclosporin a in the solution. 在另外具体的实施方案中,所述给予包括给予个体约20毫升溶液中的25 X107个分离的胎盘细胞。 In another specific embodiment, said administering comprises administering to a subject about 20 ml of solution of 25 X107 isolated placental cells. 在任何上述实施方案中,所述分离的胎盘细胞可静脉内或头烦内给予,例如作为丸剂或滴剂。 In any of the above embodiments, the isolated placental cells intravenously or administered within the first trouble, such as pills or drops. 又一个具体的实施方案中,所述给予包括给予个体约20毫升溶液中的2 X108个分离的胎盘细胞。 Yet another specific embodiment, said administering comprises administering to a subject about 20 ml of solution of 2 X108 isolated placental cells.

[0078] 所述分离的胎盘细胞可输注任何医学上可接受一段时间。 [0078] The isolated placental cells can be infused in any medically acceptable period of time. 在不同的实施方案中, 例如可给予如上所述数目的分离的胎盘细胞,例如在15、20、25、30、35、40、45、50或55分钟内或不超过上述时间,或者在1小时或1.5、2、2.5、3、3.5、4、4.5、5、5.5或6小时内或不超过上述时间,通过静脉内或头烦内输注。 In various embodiments, for example, can be administered as described above, the number of isolated placental cells, e.g. in 15,20,25,30,35,40,45,50 or 55 minutes, or no longer than this, or 1 1.5,2,2.5,3,3.5,4,4.5,5,5.5 or hours, or 6 hours, or not more than the above time, by intravenous or infusion head trouble.

[0079] 分离的胎盘细胞可在例如低氧损伤或缺氧损伤引起的一种或多种症状或神经性缺损形成后的任何时候给予脑或CNS中或周围血流破坏的个体。 [0079] The isolated placental cells can be administered to an individual in or around the brain or CNS disruption of blood flow at any time after hypoxic injury or anoxic injury, for example caused by one or more symptoms or neurological deficit formed. 在不同的实施方案中,分离的胎盘细胞在个体表现出第一种症状或神经性缺损形成后的第21、20、19、18、17、16、15、 14、13、12、11、10、9、8、7、6、5、4、3或2天内给予;优选分离的胎盘细胞在所述个体中第一种可检测症状或神经性缺损形成后的第48、47、46、45、44、43、42、41、40、39、38、37、36、35、:34、 33、32、31、30、29、28、27、26、25、24、23、22、21、20、19、18、17、16、15、14、13、12、11、10、9、8、 7、6、5、4、3或2小时内给予,或所述个体中第一种可检测症状或神经性缺损形成后的第一个小时内给予。 In various embodiments, the isolated placental cells in an individual exhibiting a first symptom or neurological deficit after the formation of 21,20,19,18,17,16,15, 14,13,12,11,10 administered 9,8,7,6,5,4,3 or 2 days; 48,47,46,45 of the isolated placental cells is preferably a first detectable symptom or neurological deficit in the individual formed , 44,43,42,41,40,39,38,37,36,35,: 34, 33,32,31,30,29,28,27,26,25,24,23,22,21, 20,19,18,17,16,15,14,13,12,11,10,9,8, administered within 2 hours, or 7,6,5,4,3, or the individual may first administered within the first hour after the formation of the defect detection or neurological symptoms.

[0080] 在其他的实施方案中,所述分离的胎盘细胞例如在脑或CNS中或周围血流破坏的一种或多种症状、中风症状、低氧损伤或缺氧损伤显现或可检测之前预防性地给予。 Before [0080] In other embodiments, the isolated placental cells, for example in the brain or surrounding the CNS or one or more symptoms of disruption of blood flow, symptoms of stroke, hypoxic injury or anoxic injury visualized or detected administered prophylactically.

[0081] 对具有脑或CNS中或周围血流破坏的个体的治疗,包括给予所述个体分离的胎盘细胞,可进一步包括给予所述个体一种或多种第二种治疗剂。 [0081] in the brain or CNS of an individual having a disruption of blood flow in or around the treatment, comprising administering to the individual isolated placental cells, can further comprise administering to the subject one or more second therapeutic agents. 所述第二种治疗剂可在给予分离的胎盘细胞之前、给予所述分离的胎盘细胞期间或给予所述分离的胎盘细胞之后给予。 Administration or after administration of the isolated placental cells are placental cells during the second therapeutic agent may be administered before the isolated placental cells, said isolated administered. 所述第二种治疗剂给予的次数可W少于、多于或等于所给予分离的胎盘细胞的次数。 The number of second therapeutic agent can be administered less than W, or greater than the number of isolated placental cells administered is equal.

[0082] 第二种治疗剂可W是对患有脑或CNS中或周围血流破坏的个体具有治疗益处的任何制剂。 [0082] The second therapeutic agent can be any formulation W therapeutic benefit to individuals suffering from in or around the brain or CNS disruption of blood flow. 在一个实施方案中,所述治疗剂为制剂,例如药物,其用于治疗中风、低氧损伤或缺氧损伤。 In one embodiment, the formulation is a therapeutic agent such as a drug for the treatment of stroke, hypoxic injury or anoxic injury. 在一个具体的实施方案中,第二种治疗剂为神经保护剂。 In a specific embodiment, the second therapeutic agent is a neuroprotective agent. 在一个具体的实施方案中,所述神经保护剂为二硫酪钢(NXY-059;苯基下基硝酬的二横酷基衍生物),其结构如下显不: In a specific embodiment, the neuroprotective agent is casein steel disulfide (NXY-059; two cross-phenyl derivative under cool remuneration nitro group), the following structure is not obvious:

[0083] [0083]

Figure CN105796602AD00181

[0084] 在另一个实施方案中,所述个体患有出血性中风,第二种治疗剂为降低所述个体血压的治疗剂。 [0084] In another embodiment, the subject has hemorrhagic stroke, the second therapeutic agent is a therapeutic agent to reduce the blood pressure of the subject. 在另一个实施方案中,第二种治疗剂为血栓溶解剂。 In another embodiment, the second therapeutic agent is a thrombolytic agent. 在一个具体的实施方案中,所述血栓溶解剂为组织纤溶酶原激活物(tPA)。 In a specific embodiment, the thrombolytic agent is tissue plasminogen activator (tPA). 巧A可W是天然来源的或重组的。 A clever W may be naturally derived or recombinant. 在更具体的实施方案中,tPA在所述个体中一种或多种症状或神经性缺损形成后的Ξ个小时内给予。 In a more specific embodiment, the tPA Ξ hours after one or more symptoms or neurological deficit in the individual administration form. 在另外更具体的实施方案中,tPA在中风患者中中风的一种或多种症状形成Ξ个小时后给予。 In another more specific embodiment, tPA one or more symptoms of a stroke forming Ξ hours after administration of stroke patients. 在某些实施方案中,禁忌使用血栓溶解剂,例如当中风患者具有头部损伤或当中风是由头部损伤引起时。 In certain embodiments, contraindications to thrombolytic agents, for example a patient which air which has a head injury or head injury is caused by the wind. 在另外具体的实施方案中,第二种治疗剂为抗凝血剂或抗血小板剂。 In another specific embodiment, the second therapeutic agent is an anticoagulant or antiplatelet agent. 在脑或CNS中或周围血流破坏为出血的实施方案中,第二种治疗剂可W为抗高血压药物,例如0阻断剂或利尿药物、利尿药物和滞钟利尿药物的组合、郞且断剂和利尿药物的组合,血管紧张素-转化酶(ACE)抑制剂和利尿剂的组合、血管紧张素-II括抗剂和利尿药物,和/或巧离子通道阻断剂和ACE抑制剂。 In the brain or CNS is a disruption of blood flow in or around the bleeding embodiment, the second therapeutic agent may be an anti-hypertensive drugs W, for example, 0-blockers or combination diuretic drugs, diuretic drugs, and slow clock diuretic drugs, Lang and the combination of the breaking agent and diuretic drugs, angiotensin - converting enzyme (ACE) inhibitor and a diuretic combinations, including antagonist and angiotensin -II diuretic drugs, and / or ion channel blockers, and clever ACE inhibition agents. 在另一个实施方案中,给予第二种治疗剂W降低烦内压。 In another embodiment, the second therapeutic agent is administered to reduce the annoying W pressure. 在更具体的实施方案中,第二种治疗剂为利尿剂。 In a more specific embodiment, the second therapeutic agent is a diuretic.

[0085] 在给予的分离的胎盘细胞与患有脑或CNS中或周围血流破坏个体不是自体同源的实施方案中,第二种治疗剂可W是免疫抑制剂。 [0085] The administration of isolated placental cells and brain or CNS with disruption of blood flow in or around the individual is not self-autologous embodiment, the second therapeutic agent can be an immunosuppressant W. 免疫抑制剂为本领域熟知并且包括,例如抗-T细胞受体抗体(单克隆或多克隆的,或其抗体片段或衍生物,例如莫罗单抗-〔03)、抗- 比-被体抗体(例如己利昔单抗(SIMULECT)或达珠单抗(ZENAPAX))、咪挫硫嚷岭、皮质酱类、环胞素、他克莫司、麦考酪酸吗嘟乙醋、西罗莫司、巧调神经憐酸酶抑制剂等等。 Immunosuppressive agents are well known art and include, for example, anti -T cell receptor antibodies (monoclonal or polyclonal, or an antibody fragment or derivative thereof, e.g. muromonab - [03), anti - than - the body antibodies (e.g., infliximab hexyl (SIMULECT) or daclizumab (ZENAPAX) ), cried microphone setback sulfur Ridge, cortical sauce, cyclosporine, tacrolimus, mycophenolate butyric acid ethyl ester beep it, sirolimus, clever modulation nerve Rei acid inhibitor like. 在一个具体的实施方案中,所述免疫抑制剂为巨隧细胞炎性蛋白(ΜΙΡ)-Ια或ΜΙΡ-1β的中和抗体。 In a particular embodiment, the immunosuppressant is Giant tunneling inflammatory protein (ΜΙΡ) -Ια or neutralizing antibodies of ΜΙΡ-1β. 优选的,给予的所述抗-ΜΙΡ-1α或ΜΙΡ-1β抗体的量例如足W引起所述个体中ΜΙΡ- 化和/或ΜΙΡ-1β的量可检测地降低。 Preferably, -ΜΙΡ-1α or amount of the anti-ΜΙΡ-1β antibody administered, for example, sufficient to cause the individual ΜΙΡ- W and / or amount of ΜΙΡ-1β detectably reduced.

[0086] 5.4分离的胎盘细胞和分离的胎盘细胞群 [0086] The isolated placental cells, and 5.4 isolated placental cells

[0087] 可用于治疗患有脑或CNS中或周围血流破坏,包括其引起的症状和神经性缺损的个体的分离的胎盘细胞为从胎盘或其部分获得的细胞,其粘附(即,贴壁)于组织培养底物并且具有多能细胞或干细胞的特性,但不是滋养层细胞。 [0087] can be used for treating patients in or around the brain or CNS disruption of blood flow, including its symptoms and neurological deficit resulting individual isolated placental cells are obtained from the placenta, or a portion of the cells, their adhesion (i.e., adherent) to the tissue culture substrate and have characteristics of multipotent cells or stem cells, but the cells are not trophoblasts. 在某些实施方案中,可用于此处公开的方法的分离的胎盘细胞具有分化为非胎盘细胞类型的能力。 In certain embodiments, the method can be used to isolate placental cells disclosed herein have the ability to differentiate non-placental cell types. 可用于此处所公开方法的分离的胎盘细胞可W是胎儿或母体来源的(即,可W分别具有胎儿或母体的基因型)。 Isolated placental cells can be used in methods disclosed herein may be W fetal or maternal in origin (i.e., W may each have the genotype of the mother or fetus). 优选的,所述分离的胎盘细胞和分离的胎盘细胞群为胎儿来源的。 Preferably, the isolated placental cells and populations of isolated placental cells of fetal origin. 如此处所用的,短语"胎儿来源的"或"非母体来源的"表示所述分离的胎盘细胞或分离的胎盘细胞群获自与胎儿有关的厮带或胎盘结构,即具有胎儿基因型。 As used herein, or "non-maternal in origin" phrases "fetal origin" indicates that the isolated placental cells or isolated placental cells obtained from a fetus or a placenta associated with a servant structure, i.e., have the fetal genotype. 如此处所用的,短语"母体来源的"表示所述细胞或细胞群获自与母体有关的胎盘结构,例如其具有母体基因型。 As used herein, the phrase "maternal origin" indicates that the cell or population of cells obtained from a placental structures associated with the mother, for example, having a maternal genotype. 分离的胎盘细胞或包含分离的胎盘细胞的细胞群可包括仅仅为胎儿或母体来源的分离的胎盘细胞,或可包括胎儿和母体来源的混合的分离胎盘细胞群。 Isolated placental cells or isolated placental cells comprising a cell population may comprise merely isolated placental cells or fetal maternal in origin, or can comprise a mixed population of isolated placental cells of both fetal and maternal origin. 所述分离的胎盘细胞和包含分离的胎盘细胞的细胞群可通过下述形态学、标记和培养特性进行鉴定和选择。 And said isolated placental cells comprising the isolated placental cells populations can be identified and selected by the following morphological, marker, and culture characteristics. 在某些实施方案中,任何胎盘细胞,例如此处所述的胎盘干细胞或胎盘多能细胞为受者自体同源的,例如受者为已患有中风或患有中风症状的个体。 In certain embodiments, any of the placental cells, e.g., placental stem cells described herein, or placental multipotent cells are autologous to the recipient, such as recipients or an individual having suffered a stroke is stroke symptoms. 在某些其他的实施方案中,任何胎盘细胞,例如此处所述的胎盘干细胞或胎盘多能细胞为受者异源的,例如受者为已患有中风或患有中风症状的个体。 In certain other embodiments, any of the placental cells, e.g., placental stem cells described herein, or placental multipotent is heterologous to the recipient cell, for example, an individual recipient, or to have suffered a stroke having a stroke symptoms.

[0088] 5.4.1物理和形态学特性 [0088] 5.4.1 Physical and Morphological Characteristics

[0089] 此处所述的分离的胎盘细胞,当在原代培养或细胞培养时,粘附于组织培养底物, 例如组织培养容器表面(例如组织培养塑料制品),或粘附于涂有细胞外基质或配体如层粘连蛋白、胶原(例如天然的或变性的)、明胶、纤连蛋白、鸟氨酸、玻连蛋白和胞外膜蛋白(例如MAT民iGEL⑩(BD Discove;ryLabware,Be壯ord,Mass))的组织培养表面。 [0089] The isolated placental cells described herein, when cultured in primary cultures or cell adhesion to the tissue culture substrate, e.g., tissue culture container surface (e.g., tissue culture plastic), coated with or adhered to the cells extracellular matrix or ligands such as laminin, collagen (e.g., native or denatured), gelatin, fibronectin, ornithine, vitronectin, and extracellular membrane protein (e.g. MAT China iGEL⑩ (BD Discove; ryLabware, Be Zhuang ord, Mass)) tissue culture surface. 培养中的所述分离的胎盘细胞呈现常见的成纤维样、星状外形,具有自中屯、细胞体延伸的许多胞质突起。 The culture of isolated placental cells exhibit a common fibroblastoid, stellate appearance, with the self-Tun, many cytoplasmic organelles extending protrusions. 然而,所述细胞在形态上与相同条件下培养的成纤维细胞可区分,因为所述分离的胎盘细胞具有比成纤维细胞更多的所述突起。 However, the cells are cultured under the same conditions morphologically distinguishable from fibroblasts, because the isolated placental cells than the fibroblasts more protrusions. 分离的胎盘细胞在形态上还可与造血干细胞区分,造血干细胞通常在培养中呈现更圆或碟卵石状的形态。 Isolated placental cells in morphology of hematopoietic stem cells may differentiate, hematopoietic stem cells typically exhibit a more rounded pebbles or disc-shaped form in culture.

[0090] 在某些实施方案中,可用于此处公开的治疗方法的所述分离的胎盘细胞,当在生长培养基中培养时,形成胚样体。 The isolated placental cells [0090] In certain embodiments, the method can be used for the treatment disclosed herein, when cultured in a growth medium, the formation of embryoid-like bodies. 胚样体为可在增殖的分离的胎盘细胞贴壁层上生长的非汇合细胞块。 Non-confluent cell mass embryoid-like bodies to be attached to the wall layer on the proliferation of isolated placental cells growth. 使用术语"胚一样"是因为所述细胞块类似胚体,其为生长自胚胎干细胞培养物的细胞块。 The term "embryo as" cell mass is similar because the embryo, its dry cell mass cell culture is grown from the embryo. 可供胚样体在增殖的分离的胎盘细胞培养物中发育的生长培养基包含例如DMEM-LG(例如来自Gibco) ;2%胎牛血清(例如来自HycloneLabs.); 1 X膜岛素-转铁蛋白- 砸(ITS); 1 X亚油酸-牛血清白蛋白(LA-BSA); 10-9M地塞米松(例如来自Sigma); 10-4M抗坏血酸巧溝酸盐(例如来自Sigma);表皮生长因子lOng/mL(例如来自R&D Systems);和血小板来源的生长因子(PDGF-BB)10ng/mL(例如来自R&D Systems)。 For the development of embryoid-like bodies in culture of isolated placental cells proliferation in growth medium containing, for example, DMEM-LG (e.g., from Gibco); 2% fetal calf serum (e.g. from HycloneLabs.); 1 X film Insulin - turn ferritin - hit (ITS); 1 X linoleic acid - bovine serum albumin (LA-BSA); 10-9M dexamethasone (e.g., from Sigma); 10-4M ascorbic acid Qiao groove (e.g., from Sigma); epidermal growth factor lOng / mL (e.g., from R & D Systems); and platelet-derived growth factor (PDGF-BB) 10ng / mL (e.g., from R & D Systems).

[0091] 5.4.2细胞表面的分子和遗传标记 [0091] 5.4.2 cell surface molecules, and genetic markers

[0092] 可用于此处公开的治疗方法的所述分离的胎盘细胞,例如多能细胞或干细胞W及分离的胎盘细胞群,为塑料贴壁组织培养人胎盘细胞,其具有多能细胞干细胞的特性,并且表达可用于鉴定和/或分离所述细胞或包括所述干细胞的细胞群的多种标记。 [0092] The isolated placental cells can be used in disclosed methods of treatment herein, for example, pluripotent cells or stem cells W and the isolated placental cells, plastic adherent tissue cultured human placental cells with pluripotent stem cells of characteristics, and expression may be used to identify and / or isolate the cell markers comprise one or more of the population of stem cells. 此处所述的分离的胎盘细胞和胎盘细胞群(即两种或多种分离的胎盘细胞)包括胎盘细胞和含有直接获自胎盘或其任何部分(例如羊膜、绒毛膜、胎盘绒毛叶等等)的细胞群的胎盘细胞。 Herein isolated placental cells and placental cell populations (i.e., two or more isolated placental cells) include placental cells obtained directly from placental containing or any part thereof (e.g., amnion, chorion, placental cotyledons, etc. ) population of placental cells. 分离的胎盘细胞群还包括培养中的分离的胎盘细胞群(即两种或多种),和容器例如袋中的细胞群。 Isolated placental cells comprising the isolated placental further population of cells in culture (i.e. two or more), and the container bag e.g. cell population. 此处所述的分离的胎盘细胞不是骨髓来源的间充质细胞、脂肪来源的间充质干细胞或获自厮带血、胎盘血或外周血的间充质细胞。 Inter isolated placental cells described herein are not bone marrow-derived mesenchymal cells, adipose-derived mesenchymal stem cells obtained from a servant or blood, placental blood, or peripheral blood between mesenchymal cells.

[0093] 在某些实施方案中,所述分离的胎盘细胞为分离的胎盘干细胞。 [0093] In certain embodiments, the isolated placental cells are isolated placental stem cells. 在某些其他的实施方案中,所述分离的胎盘细胞为分离的胎盘多能细胞。 In certain other embodiments, the isolated placental cells are isolated placental multipotent cells. 在一个实施方案中,所述分离的胎盘细胞为如流式细胞计检测的CD%-、CD10+和CD105+。 In one embodiment, the isolated placental cells are counted as detected by flow cytometry CD% -, CD10 + and CD105 +. 在具体的实施方案中,所述分离的CD34-、CD10+、CD105+胎盘细胞为胎盘干细胞。 In a specific embodiment, the isolated CD34-, CD10 +, CD105 + placental cells are placental stem cells. 在另外具体的实施方案中,所述分离的CD34-、 CD10+、CD105+胎盘细胞为多能胎盘细胞。 In another specific embodiment, the isolated CD34-, CD10 +, CD105 + placental cells are multipotent placental cells. 在另外具体的实施方案中,所述分离的CD34-、CD10 +、CD105+胎盘细胞具有分化为神经表型细胞、成骨表型细胞和/或成软骨表型细胞的潜能。 In another specific embodiment, the isolated CD34-, CD10 +, CD105 + placental cells differentiate into neural phenotype cells, osteoblast phenotype cells and / or potential of a chondrogenic phenotype. 在另外具体的实施方案中,所述分离的CD34-、CD10+、CD105+胎盘细胞还为CD200+。 In another specific embodiment, the isolated CD34-, CD10 +, CD105 + placental cells are additionally CD200 +. 在另外具体的实施方案中,所述分离的CD34-、CD10+、CD105+胎盘细胞还为CD45-或CD90+。 In another specific embodiment, the isolated CD34-, CD10 +, CD105 + or CD45- placental cells are additionally CD90 +. 在另外具体的实施方案中,如流式细胞计检测的,所述分离的CD34-、CD10+、CD105+胎盘细胞还为CD45- 和CD90+。 In another specific embodiment, as detected by flow cytometry, the isolated CD34-, CD10 +, CD105 + and CD45- placental cells are also CD90 +. 在更具体的实施方案中,如流式细胞计检测的,所述分离的CD34-、CD10+、CD105\ CD200+胎盘细胞还为CD90+或CD45-。 In a more specific embodiment, as detected by flow cytometry, the isolated CD34-, CD10 +, CD105 \ CD200 + placental cells are additionally CD90 + or CD45-. 在另外更具体的实施方案中,如流式细胞计检测的,所述分离的〔034-、〔010+、〔0105+、〔0200+胎盘细胞还为〔090+或〔045-,即所述细胞为〔0:34-、 CD10+、CD45-、CD90+、CD105+和CD200+。 In another more specific embodiment, as detected by flow cytometry, the isolated [034--, [010 + 0105 + [, [[0200+ 090+ placental cells are additionally or [045, i.e., the said cell is [0: 34-, CD10 +, CD45-, CD90 +, CD105 + and CD200 +. 在更具体的实施方案中,所述CD34-、CD10+、CD45-、CD90 +、〔0105、〔0200+细胞还为〔080-和〔086-。 In a more specific embodiment, the CD34-, CD10 +, CD45-, CD90 +, [0105, [0200+ 080 cells and also [[086.

[0094] 在具体的实施方案中,如上所述的任何CD34-、CD10+、CD105+细胞还为CD29+、CD38-、 CD44+、CD54+、SH3+或細4+中的。 [0094] In a specific embodiment, as described above, any CD34-, CD10 +, CD105 + cells are additionally CD29 +, CD38-, CD44 +, CD54 +, SH3 +, or the fine 4+. 在更具体的实施方案中,所述分离的CD34-、CD 10+、CD 105+胎盘细胞还为CD44\W上任何分离的CD34-CD10+、CD105+胎盘细胞另外具体的实施方案中,所述细胞还为CD11厂、CD133-、邸扩(66。32-)、化4-4、8、(:\化4-0?、09、0扩或程序性死亡-1配体(P化1) +中的一种或多种,或任何它们的组合。 In a more specific embodiment, the isolated CD34-, CD 10 +, CD 105+ placental cells are additionally of CD44 \ any CD34-CD10 +, CD105 + placental cells are isolated further specific embodiment of the W, the cell CD11 is also factory, CD133-, Di expansion ( 66.32-), of 4-4,8, (: \ of 4-0, 09,0 expansion or programmed death-1 ligand (P for 1? one or more) of +, or any combination thereof.

[00巧]在另一个实施方案中,所述CD:34-、CD10+、CD105+细胞还为CD13\CD29\CD33+、 CD38-、CD44+、CD45-、CD54+、CD62E-、CD62L-、CD62P-、甜3+(CD73+)、SH4+(CD73+)、CD80-、CD86-、 CD90+、甜化(CD 105+)、CD 106/VCAM+、CD 117-、CD 144/VE-巧粘蛋白1™、CD 184/CXCR4-、CD200+、 CD 133-、0CT-4+、S沈A3-、SSEA4-、ABC-P+、KDR-(VEGFR2-)、HLA-A、B、C\HLA-DP、DQ、DR-、HLA-G+ 或程序性死亡-1配体(PDLir中的一种或多种,或任何它们的组合。其他实施方案中,所述CD:M-、CD10\CD105+细胞还为CD13+、CD29\CD33\CD38-、CD44\CD45-、CD54/ICAM+、CD62E-、 CD6化-、CD62P-、S册+(CD73+)、SH4+(CD73+)、CD8〇-、CD8扩、CD90\SH2+(CD105+)、CD106/VCAM+、 CD 11 厂、CD 144/VE-巧粘蛋白1™、CD 184/CXCR4-、CD200+、CD 133-、0CT-4+、SSEA3-、SSEA4-、ABC- p+、邸R- (VEGFR2-)、HLA-A、B、C+、HLA-DP、DQ、DR-、HLA-G+和程序性死亡-1 配体(PDLl) +。 [Qiao 00] In another embodiment, the CD: 34-, CD10 +, CD105 + cells are additionally CD13 \ CD29 \ CD33 +, CD38-, CD44 +, CD45-, CD54 +, CD62E-, CD62L-, CD62P-, sweet 3+ (CD73 +), SH4 + (CD73 +), CD80-, CD86-, CD90 +, sweetening (CD 105 +), CD 106 / VCAM +, CD 117-, CD 144 / VE- Qiao mucin 1 ™, CD 184 / CXCR4-, CD200 +, CD 133-, 0CT-4 +, S Shen A3-, SSEA4-, ABC-P +, KDR- (VEGFR2 -), HLA-A, B, C \ HLA-DP, DQ, DR-, HLA-G + or programmed death ligand -1 (PDLir one or more, or any combination thereof in other embodiments, the CD:. M-, CD10 \ CD105 + cells are additionally CD13 +, CD29 \ CD33 \ CD38-, CD44 \ CD45-, CD54 / ICAM +, CD62E-, CD6 of -, CD62P-, S books + (CD73 +), SH4 + (CD73 +), CD8〇-, CD8 expansion, CD90 \ SH2 + (CD105 +), CD106 / VCAM +, CD 11 plants, CD 144 / VE- Qiao mucin 1 ™, CD 184 / CXCR4-, CD200 +, CD 133-, 0CT-4 +, SSEA3-, SSEA4-, ABC- p +, Di R- (VEGFR2 -), HLA-A, B, C +, HLA-DP, DQ, DR-, HLA-G + and programmed death 1 ligand (PDLl) +.

[0096] 在某些实施方案中,所述细胞为SSEA3-、SSEA4-或ABC-P+中的一种或多种。 [0096] In certain embodiments, the cell is one or more SSEA3-, SSEA4- or the ABC-P +. 所述分离的胎盘细胞还可W表达HLA-ABC(MHC-I)。 The isolated placental cells can also be the expression W HLA-ABC (MHC-I). 运些标记可任何组合使用,W鉴定所述分离的胎盘细胞,例如,分离的胎盘干细胞或分离的多能细胞,并且区分分离的胎盘细胞和其他细胞类型。 These markers may be transported in any combination, W identify the isolated placental cells, e.g., isolated placental stem cells or isolated multipotent cells and to distinguish the isolated placental cells and other cell types. 由于所述分离的胎盘细胞可表达CD73和CD105,其可具有间充质干细胞样的特性。 Because the isolated placental cells can express CD73 and CD105, which may have characteristics of mesenchymal stem cell-like. 例如不表达CD34、CD38和/或CD45,则鉴定所述分离的胎盘细胞为非造血干细胞。 For example, do not express CD34, CD38 and / or CD45, is identified the isolated placental cells are non-hematopoietic stem cells.

[0097] 此处还提供分离的胎盘细胞群或细胞群,例如包含可用于此处公开的治疗方法的富集的分离胎盘细胞的胎盘细胞群。 [0097] Also provided herein are isolated placental cells or population of cells, comprising e.g. enriched population of placental cells can be used in therapeutic methods disclosed herein are isolated placental cells. 优选包括所述分离的胎盘细胞的细胞群,其中所述细胞群可用于此处公开的治疗方法,所述细胞群包含例如至少10%、15%、20%、25%、30%、 35%、40%、45%、50%、55%、60%、65%、70%、75%、80%、85%、90%、95% 或98% 分离的CD10+、CD105+和CD34-胎盘细胞;即所述群中至少10%、15%、20%、25%、30%、35%、40%、 45%、50%、55%、60%、65%、70%、75%、80%、85%、90%、95%或98%的细胞为分离的CD10+、CD105+和CD34-胎盘细胞。 Preferably the isolated population of cells comprising placental cells, wherein said population of cells may be used for therapeutic methods disclosed herein, for example, the population of cells comprises at least 10%, 15%, 20%, 25%, 30%, 35% , 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% of the isolated CD10 +, CD105 + and CD34- placental cells; i.e., in the group of at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% , 85%, 90%, 95% or 98% of cells are isolated CD10 +, CD105 + and CD34- placental cells. 在具体的实施方案中,所述分离的CD34-、CD10+、CD105+胎盘细胞还为CD200+。 In specific embodiments, the isolated CD34-, CD10 +, CD105 + the placental cells are additionally CD200 +. 在更具体的实施方案中,如流式细胞计检测的,所述分离的CD34-、CD10\ CD105\CD200+胎盘细胞还为CD90+或CD45-。 In a more specific embodiment, as detected by flow cytometry, the isolated CD34-, CD10 \ CD105 \ CD200 + placental cells are additionally CD90 + or CD45-. 在另外更具体的实施方案中,如流式细胞计检测的,所述分离的CD34-、CD10+、CD105+、CD200+胎盘细胞还为CD90+和CD45-。 In another more specific embodiment, as detected by flow cytometry, the isolated CD34-, CD10 +, CD105 +, CD200 + placental cells are additionally CD90 + and CD45-. 在更具体的实施方案中,如上所述的任何分离的CD34-、CD10+、CD105+胎盘细胞还为CD29+、CD38-、CD44+、CD54 +、SH3+或甜4+中的一种或多种。 In a more specific embodiment, as described above, any of the isolated CD34-, CD10 +, CD105 + placental cells are additionally CD29 +, CD38-, CD44 +, CD54 +, SH3 + or one or more of the sweet 4+. 在另外更具体的实施方案中,所述分离的CD34-、CD10+、CD105 +胎盘细胞或分离的CD34-、CD10\CD105\CD200+胎盘细胞还为CD44+。 In another more specific embodiment, the isolated CD34-, CD10 +, CD105 + cells or isolated placental CD34-, CD10 \ CD105 \ CD200 + placental cells are additionally CD44 +. 包含W上分离的CD34-、CD10+、CD105+胎盘细胞的任何细胞群的具体实施方案中,所述分离的胎盘细胞还为CD13\CD29\CD33\CD38-、CD44+、CD45-、CD54\CD62E-、CD62L-、CD62P-、甜3+(CD73+)、甜4+ (CD73+)、CD8〇-、CD86-、CD9〇\ 甜化(〔0105+)、〔0106八〔八1\〔0117-、〔0144八6-巧粘蛋白1。 W comprises the separation CD34-, CD10 +, CD105 + cell population of any of the specific embodiments of the placental cells, said isolated placental cells are additionally CD13 \ CD29 \ CD33 \ CD38-, CD44 +, CD45-, CD54 \ CD62E-, CD62L-, CD62P-, sweet 3+ (CD73 +), sweet 4+ (CD73 +), CD8〇-, CD86-, CD9〇 \ sweetening (0105 [+), [0106 eight eight [1 \ [0117-, [ 0144 eight 6- clever mucin 1. \ CD 184/CXCR4-、CD200+、CD 133-、0CT-4+、S 沈A3-、SSEA4-、ABC-P+、邸R- (VEGFR2-)、HLA-A、B、C+、 化八-0?、09、0扩、化4-6+或程序性死亡-1配体。 \ CD 184 / CXCR4-, CD200 +, CD 133-, 0CT-4 +, S Shen A3-, SSEA4-, ABC-P +, Di R- (VEGFR2 -), HLA-A, B, C +, of eight -0 ?, 09,0 expansion, of 4-6 + or programmed death-1 ligand. 化1) +中的一种或多种,或它们的任何组合。 One or more of 1) + of, or any combination thereof. 在更具体的实施方案中,所述CD34-、CD10+、CD105+细胞还为CD13+、CD29+、CD33+、CD38-、CD44 +、CD45-、CD54/ICAM+、CD62E-、CD6化-、CD62P-、S册+(CD73+)、SH4+ (CD73+)、CD80-、CD86-、CD90 +、SH2+(CD105+)、CD106/VCAM+、CD11厂、CD144/VE-巧粘蛋白i〇w、CD184/CXCR4-、CD200+、 CD 133-、0CT-4+、S沈A3-、SSEA4-、ABC-P+、KDR-(VEGFR2-)、HLA-A、B、C\HLA-DP、DQ、DR-、HLA-G+ 和程序性死亡-1配体(P化1 ) +。 In a more specific embodiment, the CD34-, CD10 +, CD105 + cells are additionally CD13 +, CD29 +, CD33 +, CD38-, CD44 +, CD45-, CD54 / ICAM +, CD62E-, CD6 of -, CD62P-, S Volume + (CD73 +), SH4 + (CD73 +), CD80-, CD86-, CD90 +, SH2 + (CD105 +), CD106 / VCAM +, CD11 plant, CD144 / VE- clever mucin i〇w, CD184 / CXCR4-, CD200 +, CD 133-, 0CT-4 +, S Shen A3-, SSEA4-, ABC-P +, KDR- (VEGFR2 -), HLA-A, B, C \ HLA-DP, DQ, DR-, HLA-G + and procedural death ligand -1 (P of 1) +.

[0098] 在某些实施方案中,可用于此处所述治疗方法的所述分离的胎盘细胞为分离的胎盘细胞,其为CD 10+、CD29+、CD34-、CD38-、CD44+、CD45-、CD54+、CD90+、S 肥+、甜3+、SH4+、SSEA3-、 SSEA4-、0CT-4+和ABC-P+中的一种或多种或全部,其中所述分离的胎盘细胞通过物理和/或酶促破坏胎盘组织获得。 The isolated placental cells [0098] In certain embodiments, methods described herein can be used in the treatment of isolated placental cells, which are CD 10 +, CD29 +, CD34-, CD38-, CD44 +, CD45-, CD54 +, CD90 +, S + fat, sweet 3 +, SH4 +, SSEA3-, SSEA4-, 0CT-4 + and ABC-P + one or more or all, wherein said isolated placental cells by physical and / or enzymatic destruction of placental tissue is obtained. 在具体的实施方案中,所述分离的胎盘细胞为0CT-4+和ABC-P+。 In a specific embodiment, the isolated placental cells are 0CT-4 + and ABC-P +. 在另外具体的实施方案中,所述分离的胎盘细胞为0CT-4+和CD34-,其中所述分离的胎盘细胞具有至少一种下列特性:CD10+、CD29+、CD44+、CD45-、CD54+、CD90+、SH3\細4+、SSEA3-和SSEA4-。 In another specific embodiment, said isolated placental cells are 0CT-4 + and CD34-, wherein said isolated placental cells have at least one of the following characteristics: CD10 +, CD29 +, CD44 +, CD45-, CD54 +, CD90 +, SH3 \ fine 4 +, SSEA3- and SSEA4-. 在另外具体的实施方案中,所述分离的胎盘细胞为0CT-4+、CD34-、CD 10+、CD29+、CD44 +、CD45-、CD54+、CD90+、甜3+、SH4+、SSEA3-和SSEA4-。 In another specific embodiment, said isolated placental cells are 0CT-4 +, CD34-, CD 10 +, CD29 +, CD44 +, CD45-, CD54 +, CD90 +, sweet 3 +, SH4 +, SSEA3- and SSEA4- . 在另一个实施方案中,所述分离的胎盘细胞为0CT-4+、CD34-、S沈A3-和SSEA4-。 In another embodiment, the isolated placental cells are 0CT-4 +, CD34-, S Shen A3- and SSEA4-. 在更具体的实施方案中,所述分离的胎盘细胞为0CT-4+ 和CD34-,并且为甜2+或甜3+。 In a more specific embodiment, said isolated placental cells are 0CT-4 + and CD34-, and is a sweet sweet 2+ or 3+. 在更具体的实施方案中,所述分离的胎盘细胞为0CT-4+、CD34-、 細2+和細3+。 In a more specific embodiment, said isolated placental cells are 0CT-4 +, CD34-, fine and fine 2+ 3+. 在另外更具体的实施方案中,所述分离的胎盘细胞为0CT-4+、CD34-、SSEA3-和SSEA4-,并且为細2+或細3+。 In another more specific embodiment, said isolated placental cells are 0CT-4 +, CD34-, SSEA3- and SSEA4-, thin and fine 2+ or 3+. 在另外更具体的实施方案中,所述分离的胎盘细胞为0CT-4+和CD:M-,和甜2+或甜3+,并且为CD 10+、CD29+、CD44+、CD45-、CD54+、CD90+、SSEA3-或SSEA4-中的至少一种。 In another more specific embodiment, said isolated placental cells are 0CT-4 + and CD: M-, sweet and sweet 2+ or 3+, and is a CD 10 +, CD29 +, CD44 +, CD45-, CD54 +, CD90 +, at least one of SSEA3- or SSEA4-. 在另外更具体的实施方案中,所述分离的胎盘细胞为0CT-4\CD3r、CD10+、CD29\ CD44+、CD45-、CD54+、CD90+、S沈A3-和SSEA4-,并且为甜2+或甜3+。 In another more specific embodiment, said isolated placental cells are 0CT-4 \ CD3r, CD10 +, CD29 \ CD44 +, CD45-, CD54 +, CD90 +, S Shen A3- and SSEA4-, and is a sweet or sweetened 2+ 3+.

[0099] 在另一个实施方案中,可用于此处公开的治疗方法的分离的胎盘细胞为甜2+、SH3 +、SH4+和0CT-4+。 Isolated placental cells [0099] In another embodiment, the disclosed methods of treatment that can be used herein is sweet 2 +, SH3 +, SH4 + and 0CT-4 +. 在更具体的实施方案中,所述分离的胎盘细胞为CD10\CD29+、CD44+、CD54 +、CD90+、CD34-、CD45-、SSEA3-或SSEA4-。 In a more specific embodiment, said isolated placental cells are CD10 \ CD29 +, CD44 +, CD54 +, CD90 +, CD34-, CD45-, SSEA3- or SSEA4-. 在另一个实施方案中,所述分离的胎盘细胞为甜2+、 甜3+、SH4%SSEA3-和SSEA4-。 In another embodiment, the isolated placental cells are 2+ sweet, sweet 3 +, SH4% SSEA3- and SSEA4-. 在更具体的实施方案中,所述分离的胎盘细胞为甜2+、S册+、SH4 +、S沈A3-和S沈A4-、CD 10+、CD29+、CD44+、CD54+、CD90+、0CT-4+、CD34-或CD45-。 In a more specific embodiment, said isolated placental cells are sweet 2 +, S books +, SH4 +, S Shen A3- and S sink A4-, CD 10 +, CD29 +, CD44 +, CD54 +, CD90 +, 0CT- 4 +, CD34- or CD45-.

[0100] 在另一个实施方案中,可用于此处所公开方法的分离的胎盘细胞为CD10+、CD29\ CD34-、CD44+、CD45-、CD54+、CD90+、甜化、SH3+和甜4+;其中所述分离的胎盘细胞还为0CT-4+、 SSEA3-或SSEA4-中的一种或多种。 [0100] In another embodiment, it is used to separate the methods disclosed herein placental cells are CD10 +, CD29 \ CD34-, CD44 +, CD45-, CD54 +, CD90 +, sweetening, SH3 + and sweet 4+; wherein said isolated placental cells are additionally 0CT-4 +, SSEA3- SSEA4- or one or more.

[0101] 在某些实施方案中,可用于此处所公开的治疗例如脑或CNS中或周围血流破坏、中风的方法的分离的胎盘细胞为CD200+或HLA-G+。 [0101] In certain embodiments, useful for the treatment disclosed herein, for example, in or around the brain or CNS disruption of blood flow, stroke method for separating placental cells or CD200 + HLA-G +. 在具体的实施方案中,所述分离的胎盘细胞为CD200+和HLA-G+。 In a specific embodiment, the isolated placental cells are CD200 +, and HLA-G +. 在另外具体的实施方案中,所述分离的胎盘细胞还为CD73+和CD105+。 In another specific embodiment, said isolated placental cells are additionally CD73 + and CD105 +. 在另外具体的实施方案中,所述分离的胎盘细胞还为CD34-、CD38-或CD45-。 In another specific embodiment, said isolated placental cells are additionally CD34-, CD38- or CD45-. 在另外具体的实施方案中,所述分离的胎盘细胞还为CD34-、CD38-和CD45-。 In another specific embodiment, said isolated placental cells are additionally CD34-, CD38- and CD45-. 在另外具体的实施方案中,所述干细胞为〔034-、〔038-、〔045-、〔073+和〔0105+。 In another specific embodiment, said stem cells [034--, [038--, [045--, and [[073+ 0105+. 在另外具体的实施方案中,所述分离的〔0200+或HLA-G+胎盘细胞当在允许形成胚样体的条件下时,有利于在包含所述分离胎盘细胞的胎盘细胞群中形成胚样体。 In another specific embodiment, the isolated [0200+ or HLA-G + placental cells when the conditions allow the formation of embryoid-like bodies when conducive to the formation of embryoid-like in a population of placental cells comprising the isolated placental cells body. 在另外具体的实施方案中,所述分离的胎盘细胞与不为干细胞或多能细胞的胎盘细胞分离。 In another specific embodiment, said isolated placental cells and not stem cells or multipotent cells are isolated placental cells. 在另外具体的实施方案中,所述分离的胎盘细胞与不显示运些标记的胎盘干细胞分离。 In another specific embodiment, said isolated placental cells that do not display these markers transported isolated placental stem cells.

[0102] 在另一个实施方案中,可用于此处所述治疗方法的细胞群为包含例如富集CD200 +、HLA-G+干细胞的细胞群。 Cell population [0102] In another embodiment, the treatment methods described herein may be used for example, comprise enriched CD200 +, HLA-G + stem cell population of cells. 在具体的实施方案中,所述细胞群为胎盘细胞群。 In a specific embodiment, the cell population is a population of placental cells. 在不同的实施方案中,所述细胞群中至少约10%、至少约20%、至少约30%、至少约40%、至少约50%或至少约60%的细胞为分离的CD200+、HLA-G+胎盘细胞。 In various embodiments, the population of cells at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, or at least about 60% of the cells isolated CD200 +, HLA- G + placental cells. 优选的,所述细胞群中至少约70%的细胞为分离的CD200+、HLA-G+胎盘细胞。 Preferably, the cell population of at least about 70% of the cells isolated CD200 +, HLA-G + placental cells. 更优选的,至少约90%、95%或99%的所述细胞为分离的CD200+、HLA-G+胎盘细胞。 More preferably, at least about 90%, 95%, or 99% of the cells are isolated CD200 +, HLA-G + placental cells. 所在述细胞群的具体实施方案中,所述分离的CD200+、HLA-G+胎盘细胞还为CD73+和CD105+。 DETAILED embodiment where said population of cells, said isolated CD200 +, HLA-G + placental cells are additionally CD73 + and CD105 +. 在另外具体的实施方案中,所述分离的CD200+、HLA-G+胎盘细胞还为CD34-、CD38-或CD45-。 In another specific embodiment, said isolated CD200 +, HLA-G + placental cells are additionally CD34-, CD38- or CD45-. 在更具体的实施方案中,所述分离的CD200+、HLA-G+胎盘细胞还为CD34-、CD38-、CD45-、CD73+和CD105+。 In a more specific embodiment, said isolated CD200 +, HLA-G + placental cells are additionally CD34-, CD38-, CD45-, CD73 + and CD105 +. 在另一个实施方案中,当在允许形成胚样体的条件下培养时,所述细胞群产生一个或多个胚样体。 In another embodiment, when cultured under conditions to form embryoid-like bodies in allowing the cell population produces one or more embryoid-like bodies. 在另外具体的实施方案中,所述细胞群与不是干细胞的胎盘细胞分离。 In another specific embodiment, said population of cells and not cells are isolated placental stem cells. 在另外具体的实施方案中,所述分离的CD200+、HLA-G+胎盘细胞与不显示运些标记的胎盘细胞分离。 In another specific embodiment, said isolated CD200 +, HLA-G + placental cells that do not display these markers transport placental cells are isolated.

[0103] 在另一个实施方案中,可用于此处所述治疗方法的分离的胎盘细胞为CD73+、 CD105+和CD200+。 Isolated placental cells [0103] In another embodiment, the treatment methods described herein may be used for CD73 +, CD105 + and CD200 +. 在另外具体的实施方案中,所述分离的胎盘细胞为HLA-G+。 In another specific embodiment, said isolated placental cells are HLA-G +. 在另外具体的实施方案中,所述分离的胎盘细胞还为CD34-、CD38-或CD45-。 In another specific embodiment, said isolated placental cells are additionally CD34-, CD38- or CD45-. 在另外具体的实施方案中,所述分离的胎盘细胞还为CD34-、CD38-和CD45-。 In another specific embodiment, said isolated placental cells are additionally CD34-, CD38- and CD45-. 在更具体的实施方案中,所述分离的胎盘细胞为CD34-、CD38-、CD45-和HLA-G+。 In a more specific embodiment, said isolated placental cells are CD34-, CD38-, CD45- and HLA-G +. 在另外具体的实施方案中,当在允许形成胚样体的条件下培养时,所述分离的CD73+、CD105+和CD200+胎盘细胞促进在包括所述分离的胎盘细胞的胎盘细胞群中形成一个或多个胚样体。 In another specific embodiment, when cultured under conditions that allow formation of embryoid-like bodies, said isolated CD73 +, CD105 + and CD200 + placental cells facilitate the formation of one or more comprising the isolated placental cells in placental cell population an embryo-like bodies. 在另外具体的实施方案中,所述分离的胎盘细胞与非分离的胎盘细胞分离。 In another specific embodiment, said isolated placental cells are isolated and non-isolated placental cells. 在另外具体的实施方案中,所述分离的胎盘细胞与不显示运些标记的胎盘细胞分离。 In another specific embodiment, said isolated placental cells that do not display these markers transport placental cells are isolated.

[0104] 在另一个实施方案中,可用于此处所述治疗方法的细胞群为包含例如富集分离的CD73+、CD105+、CD200+胎盘细胞的细胞群。 [0104] In another embodiment, the methods of treatment may be used herein to include, for example, a cell population enriched isolated CD73 +, CD105 +, CD200 + cell population of placental cells. 在不同的实施方案中,所述细胞群中至少约10%、 至少约20%、至少约30%、至少约40%、至少约50%或至少约60%的细胞为〔073\〔0105+、 CD200+的分离的胎盘细胞。 In various embodiments, the population of cells at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, or at least about 60% of the cells [073 \ [0105+ isolated placental CD200 + cells. 在另一个实施方案中,所述细胞群中至少约70%的细胞为CD73+、 CD105+、CD200+的分离的胎盘细胞。 In another embodiment, the cell population of at least about 70% of the cells are CD73 +, CD105 +, CD200 + isolated placental cells. 在另一个实施方案中,所述细胞群中至少约90%、95%或99%的细胞为CD73\CD105\CD200+的分离的胎盘细胞。 In another embodiment, the population of cells at least about 90%, 95%, or 99% of cells are isolated CD73 \ CD105 \ CD200 + placental cells. 在所述细胞群的具体实施方案中, 所述分离的胎盘细胞为HLA-G+。 In a specific embodiment the population of cells, said isolated placental cells are HLA-G +. 在另外具体的实施方案中,所述分离的胎盘细胞还为CD34\ CD38-或CD45-。 In another specific embodiment, said isolated placental cells are additionally CD34 \ CD38- or CD45-. 在另外具体的实施方案中,所述分离的胎盘细胞还为CD34-、CD38-和CD45-。 In another specific embodiment, said isolated placental cells are additionally CD34-, CD38- and CD45-. 在更具体的实施方案中,所述分离的胎盘细胞还为CD34-、CD38-、CD45-和HLA-G+。 In a more specific embodiment, said isolated placental cells are additionally CD34-, CD38-, CD45- and HLA-G +. 在另外具体的实施方案中,当在允许形成胚样体的条件下培养时,所述细胞群产生一个或多个胚样体。 In another specific embodiment, when cultured under conditions that allow formation of embryoid-like bodies, the cell population produces one or more embryoid-like bodies. 在另外具体的实施方案中,所述胎盘细胞群与非干细胞分离。 In another specific embodiment, said population of placental cells separated from non-stem cells. 在另外具体的实施方案中,所述胎盘细胞群与不显示运些特性的胎盘细胞分离。 In another specific embodiment, said population of placental cells and placental cells that do not display these characteristics separation operation.

[01化]在某些其他的实施方案中,所述分离的胎盘细胞为CD10\CD29+、CD%-、CD38-、 CD44+、CD45-、CD54+、CD90+、SH2+、細3+、SH4+、SSEA3-、S 沈A4-、HLA-G+或ABC-P+ 中的一种或多种。 [01 oriented] In certain other embodiments, the isolated placental cells are CD10 \ CD29 +, CD% -, CD38-, CD44 +, CD45-, CD54 +, CD90 +, SH2 +, fine 3 +, SH4 +, SSEA3- , S Shen A4-, HLA-G + ABC-P + or one or more. 在一个具体的实施方案中,所述分离的胎盘细胞为CD10+、CD29\CD34-、CD38-、CD44+、 CD45-、CD54+、CD90+、甜化、S册+、SH4+、SSEA3-、SSEA4-和0CT-4+。 In a specific embodiment, the isolated placental cells are CD10 +, CD29 \ CD34-, CD38-, CD44 +, CD45-, CD54 +, CD90 +, sweetening, S books +, SH4 +, SSEA3-, SSEA4- and 0CT -4+. 在另外具体的实施方案中,所述分离的胎盘细胞为CD 10% CD29+、CD34-、CD38-、CD45-、CD54+、甜化、S册+和SH4+。 In another specific embodiment, said isolated placental cells are CD 10% CD29 +, CD34-, CD38-, CD45-, CD54 +, sweetening, S + volumes and SH4 +. 在另外具体的实施方案中,所述分离的胎盘细胞为〔010%〔029+、〔034-、〔038-、〔045-、〔054\甜2\5册+、 細4+和0CT-4+。 In another specific embodiment, the isolated placental cells [029 + [010%, [034--, [038--, [045--, [054 \ sweet 2 \ 5 +, 4 +, and fine 0CT- 4+. 在另外具体的实施方案中,所述分离的胎盘细胞为CD10+,CD29 +、CD%-、 CD38-、CD44+、CD45-、CD54+、CD90+、HLA-G+、甜2+、S册+、甜4+。 In another specific embodiment, said isolated placental cells are CD10 +, CD29 +, CD% -, CD38-, CD44 +, CD45-, CD54 +, CD90 +, HLA-G +, sweet 2 +, S books +, sweet 4 +. 在另外具体的实施方案中,所述分离的胎盘细胞为0CT-4+和ABC-P+。 In another specific embodiment, said isolated placental cells are 0CT-4 + and ABC-P +. 在另外具体的实施方案中,所述分离的胎盘细胞为甜2 +、SH3+、甜4+和0CT-4+。 In another specific embodiment, said isolated placental cells are sweet 2 +, SH3 +, + and sweet 0CT-4 +. 在另一个实施方案中,所述分离的胎盘细胞为0CT-4+、CD34-、SSEA3- 和SSEA4-。 In another embodiment, the isolated placental cells are 0CT-4 +, CD34-, SSEA3- and SSEA4-. 在一个具体的实施方案中,所述分离的0CT-4+、CD34-、SSEA3-和SSEA4-胎盘细胞还为〔010+、〔029+、〔0:34-、〔044\〔045-、〔054+、〔090\甜2\5册+和甜4+。 In a specific embodiment, the isolated 0CT-4 +, CD34-, SSEA3- placental cells are additionally SSEA4- and [010, 029 [+, [0:34 -, [044 \ [045, [+ 054, [090 \ sweet 2 \ 4 + 5 + and sweet. 在另一个实施方案中, 所述分离的胎盘细胞为0CT-4+和CD34-,或者S册+或甜4+。 In another embodiment, the isolated placental cells are 0CT-4 + and CD34-, or sweet or S 4+ + book. 在另一个实施方案中,所述分离的胎盘细胞为CD34-,并且CD10+、CD29+、CD44+、CD54+、CD90+或者0CT-4+。 In another embodiment, the isolated placental cells are CD34-, and CD10 +, CD29 +, CD44 +, CD54 +, CD90 + or 0CT-4 +.

[0106] 在另一个实施方案中,可用于此处所述治疗方法的分离的胎盘细胞为CD200+和0CT-4+。 Isolated placental cells [0106] In another embodiment, the methods described herein may be used for the treatment and CD200 + 0CT-4 +. 在具体的实施方案中,所述分离的胎盘细胞为CD73+和CD105+。 In a specific embodiment, the isolated placental cells are CD73 + and CD105 +. 在另外具体的实施方案中,所述分离的胎盘细胞为HLA-护。 In another specific embodiment, said isolated placental cells are HLA- protection. 在另外具体的实施方案中,所述分离的CD200+、0CT-4+ 胎盘细胞为CD34-、CD38-或CD45-。 In another specific embodiment, said isolated CD200 +, 0CT-4 + placental cells are CD34-, CD38- or CD45-. 在另外具体的实施方案中,所述分离的CD200+、0CT-4+胎盘细胞为CD34-、CD38-和CD45-。 In another specific embodiment, said isolated CD200 +, 0CT-4 + placental cells are CD34-, CD38- and CD45-. 在更具体的实施方案中,所述分离的CD200+、0CT-4+胎盘细胞为CD34-、CD38-、CD45-、CD73+、CD105+和HLA-G+。 In a more specific embodiment, said isolated CD200 +, 0CT-4 + placental cells are CD34-, CD38-, CD45-, CD73 +, CD105 +, and HLA-G +. 在另外具体的实施方案中,当所述细胞群在允许形成胚样体的条件下培养时,所述分离的CD200+、0CT-4+胎盘细胞有助于在包含所述分离细胞的胎盘细胞群中形成一个或多个胚样体。 In another specific embodiment, when the population of cells cultured under conditions that allow formation of embryoid-like bodies, said isolated CD200 +, 0CT-4 + cells contribute to placental cell population comprising the isolated placental cells forming one or more embryoid-like bodies. 在另外具体的实施方案中,所述分离的CD200 +、0CT-4+胎盘细胞与非干细胞的胎盘细胞分离。 In another specific embodiment, said isolated CD200 +, 0CT-4 + placental cells and non-stem cell separation. 在另外具体的实施方案中,所述分离的CD200+、0CT-4+胎盘细胞与不显示运些特性的胎盘细胞分离。 In another specific embodiment, said isolated CD200 +, 0CT-4 + placental cells are placental cells that do not display these characteristics of the separation operation.

[0107] 在另一个实施方案中,可用于此处所述治疗方法的细胞群为包含例如富集CD200 +、0CT-4+胎盘细胞的细胞群。 [0107] In another embodiment, the methods of treatment can be used where cell population CD200 +, 0CT-4 + cell population comprises placental cells are enriched, for example. 在不同的实施方案中,所述细胞群中至少约10%、至少约20%、 至少约30%、至少约40%、至少约50%或至少约60%的细胞为分离的CD200+、0CT-4+胎盘细胞。 In various embodiments, the population of cells at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, or at least about 60% of the cells isolated CD200 +, 0CT- 4+ placental cells. 在另一个实施方案中,所述细胞群中至少约70%的细胞为分离的CD200+、0CT-4+胎盘细胞。 In another embodiment, the cell population of at least about 70% of the cells isolated CD200 +, 0CT-4 + placental cells. 在另一个实施方案中,所述细胞群中至少约80 %、90 %、95 %或99 %的细胞为所述分离的CD200+、0CT-4+胎盘细胞。 In another embodiment, the cell population of at least about 80%, 90%, 95%, or 99% of cells are said isolated CD200 +, 0CT-4 + placental cells. 在所述细胞群的具体的实施方案中,所述分离的CD200+、0CT-4+ 胎盘细胞还为CD73+和CD105+。 In a specific embodiment the cell population, said isolated CD200 +, 0CT-4 + CD73 + placental cells are additionally and CD105 +. 在另外具体的实施方案中,所述分离的CD200+、0CT-4+胎盘细胞还为HLA-G+。 In another specific embodiment, said isolated CD200 +, 0CT-4 + placental cells are additionally HLA-G +. 在另外具体的实施方案中,所述分离的CD200+、0CT-4+胎盘细胞还为CD34-、 CD38-和CD45-。 In another specific embodiment, said isolated CD200 +, 0CT-4 + placental cells are additionally CD34-, CD38- and CD45-. 在更具体的实施方案中,所述分离的CD200+、0CT-4+胎盘细胞还为CD:34-、 CD38-、CD45-、CD73+、CD105+和HLA-G+。 In a more specific embodiment, said isolated CD200 +, 0CT-4 + placental cells are additionally CD: 34-, CD38-, CD45-, CD73 +, CD105 +, and HLA-G +. 在另外具体的实施方案中,当在允许形成胚样体的条件下培养时,所述细胞群产生一个或多个胚样体。 In another specific embodiment, when cultured under conditions that allow formation of embryoid-like bodies, the cell population produces one or more embryoid-like bodies. 在另外具体的实施方案中,所述细胞群与非分离的CD200+、0CT-4+胎盘细胞的胎盘细胞分离。 In another specific embodiment, said population of cells with a non-isolated CD200 +, 0CT-4 + placental cells isolated. 在另外具体的实施方案中,所述细胞群与不显示运些标记的胎盘细胞分离。 In another specific embodiment, said population of cells that do not display these markers transport placental cells are isolated.

[0108] 在另一个实施方案中,可用于此处所述治疗方法的分离的胎盘细胞为CD73+、 CD105+和HLA-G+。 [0108] In another embodiment, it is used to separate the therapeutic methods described herein placental cells are CD73 +, CD105 +, and HLA-G +. 在另外具体的实施方案中,所述分离的CD73+、CD105+和HLA-G+胎盘细胞还为CD34-、CD38-或CD45-。 In another specific embodiment, said isolated CD73 +, CD105 +, and HLA-G + placental cells are additionally CD34-, CD38- or CD45-. 在另外具体的实施方案中,所述分离的CD73+、CD105+、HLA-G+胎盘细胞还为CD34-、CD38-和CD45-。 In another specific embodiment, said isolated CD73 +, CD105 +, HLA-G + placental cells are additionally CD34-, CD38- and CD45-. 在另外具体的实施方案中,所述分离的CD73+、CD105+、HLA-G+胎盘细胞还为0CT-4+。 In another specific embodiment, said isolated CD73 +, CD105 +, HLA-G + placental cells are additionally 0CT-4 +. 在另外具体的实施方案中,所述分离的CD73\CD105+、HLA-G+胎盘细胞还为CD200+。 In another specific embodiment, said isolated CD73 \ CD105 +, HLA-G + placental cells are additionally CD200 +. 在更具体的实施方案中,所述分离的CD73+、CD 105+、HLA-G+胎盘细胞还为CD34-、 CD38-、CD45-、0CT-4+和CD200+。 In a more specific embodiment, said isolated CD73 +, CD 105 +, HLA-G + placental cells are additionally CD34-, CD38-, CD45-, 0CT-4 + and CD200 +. 在另外具体的实施方案中,当所述群在允许形成胚样体的条件下培养时,所述分离的CD73+、CD105+、HLA-G+胎盘细胞有助于在包含所述分离胎盘细胞的胎盘细胞群中形成胚样体。 In another specific embodiment, said population when cultured under conditions that allow formation of embryoid-like bodies, said isolated CD73 +, CD105 +, HLA-G + placental cells facilitate the isolated placental cells comprising placental cells group forming embryoid-like bodies. 在另外具体的实施方案中,所述分离的CD73\CD105+、HLA-G+胎盘细胞与非分离的CD73+、CD105+、HLA-G+胎盘细胞的胎盘细胞分离。 In another specific embodiment, the isolated CD73 \ CD105 +, HLA-G + isolated cells and non-placental CD73 +, CD105 +, HLA-G + placental cells isolated. 在另外具体的实施方案中,所述分离的CD73+、CD105+、HLA-G+胎盘细胞与不显示运些标记的胎盘细胞分离。 In another specific embodiment, said isolated CD73 +, CD105 +, HLA-G + placental cells that do not display these markers transport placental cells are isolated.

[0109] 在另一个实施方案中,可用于此处所述治疗方法的细胞群为包含例如富集分离的CD73\CD105+和化A-G+胎盘细胞的细胞群。 [0109] In another embodiment, a cell population useful for the methods of treatment described herein include, for example separation and enrichment of CD73 \ CD105 + and A-G + cells of placental cells. 在不同的实施方案中,所述细胞群中至少约10%、至少约20 %、至少约30 %、至少约40 %、至少约50 %或至少约60 %的细胞为分离的CD73+、CD105+、HLA-G+胎盘细胞。 In various embodiments, the population of cells at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, or at least about 60% of cells are isolated CD73 +, CD105 +, HLA-G + placental cells. 在另一个实施方案中,所述细胞群中至少约70%的细胞为分离的CD73+、CD105+、HLA-G+胎盘细胞。 In another embodiment, the cell population of at least about 70% of the cells isolated CD73 +, CD105 +, HLA-G + placental cells. 在另一个实施方案中,所述细胞群中至少约90%、 95%或99%的细胞为分离的CD73+、CD105+、HLA-G+胎盘细胞。 In another embodiment, the population of cells at least about 90%, 95%, or 99% of cells are isolated CD73 +, CD105 +, HLA-G + placental cells. 上述细胞群的具体的实施方案中,所述分离的CD73+、CD105+、HLA-G+胎盘细胞还为CD34-、CD38-或CD45-。 Specific embodiments of the above cell populations, said isolated CD73 +, CD105 +, HLA-G + placental cells are additionally CD34-, CD38- or CD45-. 在另外具体的实施方案中,所述分离的CD73\CD105+、HLA-G+胎盘细胞还为CD34-、CD38-和CD45-。 In another specific embodiment, said isolated CD73 \ CD105 +, HLA-G + placental cells are additionally CD34-, CD38- and CD45-. 在另外具体的实施方案中,所述分离的CD73\CD105+、HLA-G+胎盘细胞还为0CT-4+。 In another specific embodiment, said isolated CD73 \ CD105 +, HLA-G + placental cells are additionally 0CT-4 +. 在另外具体的实施方案中,所述分离的CD73+、CD105+、HLA-G+胎盘细胞还为CD200+。 In another specific embodiment, said isolated CD73 +, CD105 +, HLA-G + placental cells are additionally CD200 +. 在更具体的实施方案中,所述分离的CD73+、CD105+、HLA-G+胎盘细胞还为0034-、〔038-、〔045-、0(:1'-4+和〔0200+。在另外具体的实施方案中,所述细胞群与非CD73+、CD105+、HLA-G+胎盘细胞的胎盘细胞分离。另外具体的实施方案中,所述细胞群与不显示运些标记的胎盘细胞分离。 In a more specific embodiment, said isolated CD73 +, CD105 +, HLA-G + placental cells are additionally 0034--, [038--, [045, 0 (: [1'-4 +, and in particular additional 0200+. embodiment, the cell population is non-CD73 +, CD105 +, HLA-G + placental cells is isolated. in another specific embodiment, the population of cells that do not display these markers transport placental cells are isolated.

[0110] 在另一个实施方案中,可用于此处所述治疗方法的分离的胎盘细胞为CD73 +和CD105+,并且当所述细胞群在允许形成胚样体的条件下培养时,有助于在包含所述分离胎盘细胞的胎盘细胞群中形成一个或多个胚样体。 [0110] In another embodiment, described herein can be used to separate the treatment process and placental cells are CD73 + CD105 +, and when the cell population is cultured under conditions that allow formation of embryoid-like bodies, help forming one or more embryoid-like bodies in a population of placental cells comprising the isolated placental cells. 在另外具体的实施方案中,所述分离的CD73+、CD105+胎盘细胞还为CD:34-、CD38-或CD45-。 In another specific embodiment, said isolated CD73 +, CD105 + placental cells are additionally CD: 34-, CD38- or CD45-. 在另外具体的实施方案中,所述分离的CD73+、CD105+胎盘细胞还为CD:34-、CD38-和CD45-。 In another specific embodiment, said isolated CD73 +, CD105 + placental cells are additionally CD: 34-, CD38- and CD45-. 在另外具体的实施方案中,所述分离的CD73+、CD105+胎盘细胞还为0CT-4+。 In another specific embodiment, the isolated CD73 +, CD105 + placental cells are additionally 0CT-4 +. 在更具体的实施方案中,所述分离的CD73+、CD105+胎盘细胞还为〇CT-4+、CD34-、CD38-和CD45-。 In a more specific embodiment, said isolated CD73 +, CD105 + placental cells are additionally 〇CT-4 +, CD34-, CD38- and CD45-. 在另外具体的实施方案中,所述分离的CD73+、CD105+ 胎盘细胞与非所述细胞的胎盘细胞分离。 In another specific embodiment, said isolated CD73 +, CD105 + placental cells and non-separation of the cells. 在另外具体的实施方案中,所述分离的CD73 +、 CD105+胎盘细胞与不显示运些特性的胎盘细胞分离。 In another specific embodiment, said isolated CD73 +, CD105 + cells and placental transport properties of these isolated placental cells do not display.

[0111] 在另一个实施方案中,可用于此处所述治疗方法的细胞群为包含例如富集CD73+、 CD105+的分离的胎盘细胞,且当所述群在允许形成胚样体的条件下培养时,有助于在包括所述细胞的分离的胎盘细胞群中形成一个或多个胚样体的细胞群。 [0111] In another embodiment, a cell population useful for the methods of treatment described herein include, for example enriched in CD73 +, CD105 + isolated placental cells when said population and culture conditions that allow formation of embryoid-like bodies , the population of cells contribute to the formation of one or more embryoid-like bodies in cells comprising the isolated placental cell population in. 在不同的实施方案中, 所述细胞群中至少约10 %、至少约20 %、至少约30 %、至少约40 %、至少约50 %或至少约60%的细胞为分离的CD73+、CD105+胎盘细胞。 In various embodiments, the population of cells at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, or at least about 60% of cells are isolated CD73 +, CD105 + placental cell. 在另一个实施方案中,所述细胞群中至少约70%的细胞为所述分离的CD73+、CD105+胎盘细胞。 In another embodiment, the cell population of at least about 70% of cells are said isolated CD73 +, CD105 + placental cells. 在另一个实施方案中,所述细胞群中至少约90%、95%或99%的细胞为所述分离的〔073\〔0105+胎盘细胞。 In another embodiment, the population of the cells at least about 90%, 95% or 99% of the cells of the isolated [073 \ [0105+ placental cells. 在上述群的具体的实施方案中,所述分离的CD73+、CD105+胎盘细胞还为CD34-、CD38-或CD45-。 In a specific embodiment of the above populations, said isolated CD73 +, CD105 + placental cells are additionally CD34-, CD38- or CD45-. 在另外具体的实施方案中,所述分离的CD73+、CD105+胎盘细胞还为CD34-、CD38-和CD45-。 In another specific embodiment, said isolated CD73 +, CD105 + placental cells are additionally CD34-, CD38- and CD45-. 在另外具体的实施方案中, 所述分离的CD73+、CD105+胎盘细胞还为0CT-4+。 In another specific embodiment, said isolated CD73 +, CD105 + placental cells are additionally 0CT-4 +. 在另外具体的实施方案中,所述分离的CD73 +、CD105+胎盘细胞还为CD200+。 In another specific embodiment, said isolated CD73 +, CD105 + placental cells are additionally CD200 +. 在更具体的实施方案中,所述分离的CD73+、CD105+胎盘细胞还为〔034-、〔038-、〔045-、0(:1'-4+和〔0200+。在另外具体的实施方案中,所述细胞群与非所述分离的CD73+、CD105+胎盘细胞的胎盘细胞分离。在另外具体的实施方案中,所述细胞群与不显示运些标记的胎盘细胞分离。 In a more specific embodiment, said isolated CD73 +, CD105 + placental cells are additionally [034--, [038--, [045, 0 (: [1'-4 + and 0200+ another specific embodiment. , the cell population of the non-separated CD73 +, CD105 + placental cells is isolated. in another specific embodiment, said population of cells that do not display these markers transport placental cells are isolated.

[0112] 在另一个实施方案中,可用于此处所述治疗方法的分离的胎盘细胞为0CT-4+,且当在允许形成胚样体的条件下培养时,有助于在包括所述细胞的分离的胎盘细胞群中形成一个或多个胚样体。 When [0112] In another embodiment, be used to separate the therapeutic methods described herein placental cells 0CT-4 +, and when cultured under conditions that allow formation of embryoid-like bodies, including the help one or more embryoid-like bodies isolated placental cell population is formed. 在一个具体的实施方案中,所述分离的0CT-4+胎盘细胞还为CD73+和CD105+。 In a specific embodiment, the isolated 0CT-4 + placental cells are additionally CD73 + and CD105 +. 在另外具体的实施方案中,所述分离的0CT-4+胎盘细胞还为CD34-、CD38-或CD45-。 In another specific embodiment, said isolated placental 0CT-4 + cells were also CD34-, CD38- or CD45-. 在另外具体的实施方案中,所述分离的0CT-4+胎盘细胞还为CD200+。 In another specific embodiment, said isolated 0CT-4 + placental cells are additionally CD200 +. 在更具体的实施方案中, 所述分离的0CT-4+胎盘细胞还为CD73\CD105\CD200+、CD34-、CD38-和CD45-。 In a more specific embodiment, said isolated 0CT-4 + placental cells are additionally CD73 \ CD105 \ CD200 +, CD34-, CD38- and CD45-. 在另外具体的实施方案中,所述分离的0CT-4+胎盘细胞与非0CT-4+胎盘细胞的胎盘细胞分离。 In another specific embodiment, said isolated placental 0CT-4 + cells and non-placental 0CT-4 + cells are isolated placental cells. 在另外具体的实施方案中,所述分离的0CT-4+胎盘细胞与不显示运些特性的胎盘细胞分离。 In another specific embodiment, said isolated placental cells 0CT-4 + placental cells that do not display these characteristics of the separation operation.

[0113] 在另一个实施方案中,可用于此处所述治疗方法的细胞群为包含例如富集0CT-4+ 的分离的胎盘细胞,且当所述群在允许形成胚样体的条件下培养时,有助于在包括所述细胞的分离的胎盘细胞群中形成一个或多个胚样体的细胞群。 [0113] In another embodiment, cell populations described herein may be used for treatment of isolated placental 0CT-4 + cells including, for example enrichment, and the group under conditions that allow formation of embryoid-like bodies when incubation, a cell population contribute to the formation of one or more embryoid-like bodies in cells comprising the isolated placental cell population in. 在不同的实施方案中,所述细胞群中至少约10%、至少约20%、至少约30%、至少约40%、至少约50%或至少约60%的细胞为分离的0CT-4+胎盘细胞。 In various embodiments, the population of cells at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, or at least about 60% of the cells 0CT-4 + isolated placental cells. 在另一个实施方案中,所述细胞群中至少约70%的细胞为所述分离的0CT-4+胎盘细胞。 In another embodiment, the cell population of at least about 70% of cells are said isolated placental 0CT-4 + cells. 在另一个实施方案中,所述细胞群中至少约80%、90%、95%或99%的细胞为所述分离的0CT-4+胎盘细胞。 In another embodiment, the cell population of at least about 80%, 90%, 95%, or 99% of cells are said isolated placental 0CT-4 + cells. 在上述群的具体实施方案中,所述分离的0CT-4+ 胎盘细胞还为CD34-、CD38-或CD45-。 In a specific embodiment of the above populations, said isolated placental 0CT-4 + cells were also CD34-, CD38- or CD45-. 在另外具体的实施方案中,所述分离的0CT-4+胎盘细胞还为CD34-、CD38-和CD45-。 In another specific embodiment, said isolated placental 0CT-4 + cells were also CD34-, CD38- and CD45-. 在另外具体的实施方案中,所述分离的0CT-4+胎盘细胞还为CD73+ 和CD105+。 In further particular embodiments, the isolated 0CT-4 + placental cells are additionally CD73 + and CD105 +. 在另外具体的实施方案中,所述分离的0CT-4+胎盘细胞还为CD200+。 In another specific embodiment, said isolated 0CT-4 + placental cells are additionally CD200 +. 在更具体的实施方案中,所述分离的0CT-4+胎盘细胞还为〔073+、〔0105+、〔0200+、〔034-、〔038-和〔045-。 In a more specific embodiment, said isolated placental cells 0CT-4 + + also [073, [0105 + 0200 + [, [034--, and [038- [045. 在另外具体的实施方案中,所述细胞群与不是所述细胞的胎盘细胞分离。 In another specific embodiment, said population of cells that are not said cells are isolated placental cells. 在另外具体的实施方案中,所述细胞群与不显示运些标记的胎盘细胞分离。 In another specific embodiment, said population of cells that do not display these markers transport placental cells are isolated.

[0114] 在另一个实施方案中,可用于此处所述治疗方法的分离的胎盘细胞为化AA,B, C-、CD45-、CD133-和CD34-的分离的胎盘细胞。 [0114] In another embodiment, described herein can be used to separate the methods of treatment of isolated placental cells AA, B, C-, CD45-, CD133- and CD34- placental cells. 在另一个实施方案中,可用于脑或CNS中或周围血流破坏治疗的细胞群为包含分离的胎盘细胞的细胞群,其中所述分离的细胞群中至少约70%、至少约80%、至少约90%、至少约95%或至少约99%的细胞为HLA-A,B,C-、CD45-、 CD133-和CD34-的分离的胎盘细胞。 In another embodiment, the brain or CNS can be used for disruption of blood flow in or around the treated cell population is a population of cells comprising isolated placental cells, wherein said isolated population of cells, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% of cells are isolated HLA-a, B, C-, CD45-, CD133- and CD34- placental cells. 在一个具体的实施方案中,所述分离的胎盘细胞或分离的胎盘细胞群与非化4-4,8,(7^045-八0133-和〔034-胎盘细胞的胎盘细胞分离。在另外具体的实施方案中,所述分离的胎盘细胞为非母体来源的。在另外具体的实施方案中,所述分离的胎盘细胞群基本上无母体的组分;例如所述分离的胎盘细胞群中至少约40%、45%、 50%、55%、60%、65%、70%、75%、80%、85%、90%、95%、98% 或99% 的细胞为非母体来源的。 In a specific embodiment, said isolated placental cells or population of isolated placental cells of the non-4-4,8, (7 ^ 045- and 0133- eight isolated placental cells 034- [placental cells. In another specific embodiment, said isolated placental cells are non-maternal in origin in another specific embodiment, said isolated placental cells are substantially free of maternal components; for example the isolated placental cell population at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of the cells are non-maternal in origin .

[0115] 在另一个实施方案中,可用于此处所述治疗方法的分离的胎盘细胞为分离的CD10 +、CD13+、CD33\CD45-、CD11厂和CD133-胎盘细胞。 [0115] In another embodiment, it is used to separate the therapeutic methods described herein are isolated placental cells CD10 +, CD13 +, CD33 \ CD45-, CD11 plants and CD133- placental cells. 在另一个实施方案中,可用于脑或CNS中或周围血流破坏治疗的细胞群为包含分离的胎盘细胞的细胞群,其中所述分离的细胞群中至少约70%、至少约80%、至少约90%、至少约95%或至少约99%的细胞为CD10\CD13+、CD33 +、CD45-、CD117-和CD133-的分离的胎盘细胞。 In another embodiment, the brain or CNS can be used for disruption of blood flow in or around the treated cell population is a population of cells comprising isolated placental cells, wherein said isolated population of cells, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% of the cells CD10 \ CD13 +, the isolated CD33 +, CD45-, CD117- and CD133- placental cells. 在一个具体的实施方案中,所述分离的胎盘细胞或分离的胎盘细胞群与非所述分离的胎盘细胞的胎盘细胞分离。 In a specific embodiment, said isolated placental cells or isolated placental cells and placental cell population of the isolated placental cells non-isolated. 在另外具体的实施方案中,所述分离的CD10+、CD13\CD33\CD45-、CD11厂和CD133-胎盘细胞为非母体来源的,即具有胎儿基因型。 In another specific embodiment, said isolated CD10 +, CD13 \ CD33 \ CD45-, CD11 plants and CD133- cells are non-maternal placental origin, i.e., have the fetal genotype. 在另外具体的实施方案中,所述分离的胎盘细胞群中至少约40%、45%、 50%、55%、60%、65%、70%、75%、80%、85%、90%、95%、98% 或99% 的所述细胞为非母体来源的。 In another specific embodiment, said isolated placental cell population by at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% , 95%, 98% or 99% of the cells are non-maternal in origin. 在另外具体的实施方案中,所述分离的胎盘细胞或分离的胎盘细胞群与不显示运些特性的胎盘细胞分离。 In another specific embodiment, said isolated placental cells or population of isolated placental cells are placental cells that do not display these characteristics separation operation.

[0116] 在另一个实施方案中,可用于此处所述治疗方法的分离的胎盘细胞为分离的CDl〇-、CD33-、CD44+、CD45-和CD11厂胎盘细胞。 [0116] In another embodiment, it is used to separate the therapeutic methods described herein are isolated placental cells CDl〇-, CD33-, CD44 +, CD45- CD11, and placental cells factory. 在另一个实施方案中,可用于脑或CNS中或周围血流破坏治疗的细胞群为包含例如富集的分离的胎盘细胞的细胞群,其中所述细胞群中至少约70 %、至少约80 %、至少约90 %、至少约95 %或至少约99 %的细胞为分离的CD1(T、 CD33-、CD44+、CD45-和CD11厂胎盘细胞。在一个具体的实施方案中,所述分离的胎盘细胞或分离的胎盘细胞群与非所述细胞的胎盘细胞分离。在另外具体的实施方案中,所述分离的胎盘细胞为非母体来源的。在另外具体的实施方案中,所述细胞群中至少约40%、45 %、 50%、55%、60%、65%、70%、75%、80%、85%、90%、95%、98% 或99% 的所述细胞为非母体来源的。在另外具体的实施方案中,所述分离的胎盘细胞或分离的胎盘细胞群与不显示运些标记的胎盘细胞分离。 In another embodiment, it can be used in or around the brain or CNS cell population comprising the treatment of disruption of blood flow placental cells isolated e.g. enriched, wherein said cell population is at least about 70%, at least about 80 %, at least about 90%, at least about 95%, or at least about 99% of the cells isolated CD1 (T, CD33-, CD44 +, CD45- and CD11 plant placental cells. in a specific embodiment, the isolated placental cells are isolated placental cells or population of isolated placental cells are not said cells. in another specific embodiment, said isolated placental cells are non-maternal in origin. in another specific embodiment, said population of cells at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of the cells are non- maternal in origin. in another specific embodiment, said isolated placental cells or population of isolated placental cells that do not display these markers transport placental cells are isolated.

[0117] 在另一个实施方案中,可用于此处所述治疗方法的分离的胎盘细胞为分离的CD10-、CD13-、CD33-、CD45-和CD11厂胎盘细胞。 Isolated placental cells [0117] In another embodiment, the methods of treatment described herein may be used for separation of CD10-, CD13-, CD33-, CD45- and CD11 plant placental cells. 在另一个实施方案中,可用于脑或CNS中或周围血流破坏治疗的细胞群为包含例如富集的分离的〔010-人013-^033-、〔045-和〔011厂胎盘细胞的细胞群,其中所述细胞群中至少约70%、至少约80%、至少约70%、至少约95%或至少约99%的细胞为〔010-^013-、〔033-、〔045-和〔011厂胎盘细胞。 In another embodiment, the brain or CNS can be used for disruption of blood flow in or around the treated cell population is enriched, for example, comprise an isolated human [010 ^ 013 033--, [045 and [011 plants placental cells population of cells, wherein said population of cells of at least about 70%, at least about 80%, at least about 70%, at least about 95%, or at least about 99% of the cells [010 ^ 013--, [033--, [045- [011 plants and placental cells. 在一个具体的实施方案中, 所述分离的胎盘细胞或分离的胎盘细胞群与非所述细胞的胎盘细胞分离。 In a specific embodiment, said isolated placental cells or population of isolated placental cells isolated placental cells are not said cells. 在另外具体的实施方案中,所述分离的胎盘细胞为非母体来源的。 In another specific embodiment, said isolated placental cells are non-maternal in origin. 在另外具体的实施方案中,所述细胞群中至少约40%、45%、50%、55%、60%、65%、70%、75%、80%、85%、90%、95%、98% 或99% 的所述细胞为非母体来源的。 In another specific embodiment, said population of cells of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% , 98% or 99% of the cells are non-maternal in origin. 在另外具体的实施方案中,所述分离的胎盘细胞或分离的胎盘细胞群与不显示运些特性的胎盘细胞分离。 In another specific embodiment, said isolated placental cells or population of isolated placental cells are placental cells that do not display these characteristics separation operation.

[0118] 在另一个实施方案中,可用于此处所述治疗方法的分离的胎盘细胞为化AA,B,C +、CD45-、CD34-和CDI33-,并且还为〔010\〔013\〔038+、〔044\〔090+、〔0105\〔0200+和/或HLA-G%和/或CD117阴性。 [0118] In another embodiment, it is used to separate the therapeutic methods described herein placental cells of AA, B, C +, CD45-, CD34- and CDI33-, and further to 010 [\ [013 \ [038 + 044 [\ + [090, 0105 [\ [0200+ and / or HLA-G% and / or CD117 negative. 在另一个实施方案中,可用于脑或CNS中或周围血流破坏治疗的细胞群为包含分离的胎盘细胞的细胞群,其中所述细胞群中至少约20%、25%、30%、35%、 40%、45%、50%、55%、60%、65%、70%、75%、80%、85%、90%、95%、98% 或约99% 的细胞为分离的胎盘细胞,其为化4-4,8,(:-、〔045-、〔0:34-、〔0133-,而且还为〔010、〔013、〔038、 〔044、〔090、〔0105、〔0200和/或化4-6阳性,和/或〔0117阴性。在一个具体的实施方案中,所述分离的胎盘细胞或分离的胎盘细胞群与非所述细胞的胎盘细胞分离。在另外具体的实施方案中,所述分离的胎盘细胞为非母体来源的。在另外具体的实施方案中,所述细胞群中至少约40%、45%、50%、55%、60%、65%、70%、75%、80%、85%、90%、95%、98% 或99% 的所述细胞为非母体来源的。在另外具体的实施方案中,所述分离的胎盘细胞或分离的胎盘细胞群与不显示 In another embodiment, the brain or CNS can be used in the population of cells or the treatment of disruption of blood flow around the population of cells comprising isolated placental cells, wherein said population of cells of at least about 20%, 25%, 30%, 35 %, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or about 99% of the cells isolated placental cell, which is of 4-4,8, (: - [045--, [0:34 -, [0133-, but also as [010, [013, [038, [044, [090, [0105, and or [0200 4-6 of positive and / or negative [0117] in a specific embodiment, said isolated placental cells or isolated placental cells are isolated placental cell population and the non-cell /. in another specific embodiment, said isolated placental cells are non-maternal in origin. in another specific embodiment, said population of cells of at least about 40%, 45%, 50%, 55%, 60%, 65% , 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of the cells are non-maternal in origin. in another specific embodiment, said isolated placental cells or isolated placental cells that do not display 运些标记的胎盘细胞分离。 Transport of these marks are isolated placental cells.

[0119] 在另一个实施方案中,可用于此处所述治疗方法的分离的胎盘细胞为,例如通过抗体结合测定的CD200+和CD10+分离的胎盘细胞,W及例如通过抗体结合和RT-PCR测定的CD117^分离的胎盘细胞。 Isolated placental cells [0119] In another embodiment, the methods of treatment described herein may be used for, CD200 + and CD10 + cells isolated placental, W, and by antibody binding and RT-PCR assays such as, for example, by antibody binding assay the isolated placental cells are CD117 ^. 在另一个实施方案中,可用于治疗脑或CNS中或周围血流破坏的所述分离的胎盘细胞为分离的胎盘细胞,例如〔010+、〔029-、〔054+、〔0200\化4-6\化4类1-和球蛋白1 勺胎盘干细胞或胎盘多能细胞。 The isolated placental cells In another embodiment, the treatment may be used in or around the brain or CNS disruption of blood flow is isolated placental cells, e.g. 010 [+ [029--, 054 [+, 0200 [\ 4 of -6 \ are 1- and 4 of immunoglobulin spoon placental stem cells or placental multipotent cells. 在另一个实施方案中,可用于治疗脑或CNS中或周围血流破坏的分离的胎盘细胞为至少一种细胞标记的表达比间充质干细胞(例如骨髓来源的间充质干细胞)至少高两倍的胎盘细胞。 In another embodiment, useful for the treatment or around the brain or CNS are isolated placental disruption of blood flow is at least one cell marker expression ratio between mesenchymal stem cells (e.g., bone marrow-derived mesenchymal stem cells) is at least two fold placental cells. 在另外具体的实施方案中,所述分离的胎盘细胞为非母体来源的。 In another specific embodiment, said isolated placental cells are non-maternal in origin. 在另外具体的实施方案中,所述细胞群中至少约40%、45%、50%、 55%、60%、65%、70%、75%、80%、85%、90%、95%、98% 或99% 的所述细胞为非母体来源的。 In another specific embodiment, said population of cells of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% , 98% or 99% of the cells are non-maternal in origin.

[0120] 在另一个实施方案中,可用于此处所述治疗方法的分离的胎盘细胞为分离的胎盘细胞,例如胎盘干细胞或胎盘多能细胞,其为CD10\CD29\CD44\CD45-、CD54/ICAM\ 00626-、〔062[、〔062口-、〔080-、〔086-、〔0103-、〔0104-、〔0105+、〔0106八〔41\〔0144八6-巧粘蛋白i™、CD184/CXCR4-、02-微球蛋白肥-ir、HLA-Gi™和/ 或PDL!i™ 中的一种或多种。 [0120] In another embodiment, be used to separate the therapeutic methods described herein placental cells are isolated placental cells, e.g., placental stem cells or placental multipotent cells, that is CD10 \ CD29 \ CD44 \ CD45-, CD54 / ICAM \ 00626 -, [062 [, [062 - [080--, [086--, [0103--, [0104--, [0105 + 0106 eight [41 [\ 6- [0144 Qiao eight mucin i ™, CD184 / CXCR4-, 02- microglobulin fertilizer -ir, HLA-Gi ™ and / or the PDL! one or more of i ™. 在具体的实施方案中,所述分离的胎盘细胞至少为CD29+和CD54+。 In a specific embodiment, the isolated placental cells are at least CD29 + and CD54 +. 在另外具体的实施方案中,所述分离的胎盘细胞至少为CD44+和CD106+。 In another specific embodiment, the isolated placental cells are at least CD44 + and CD106 +. 在另外具体的实施方案中,所述分离的胎盘细胞至少为CD29+。 In another specific embodiment, the isolated placental cells are at least CD29 +.

[0121] 在另一个实施方案中,可用于此处所述治疗方法的细胞群包含分离的胎盘细胞, 并且所述细胞群中至少50 %、60 %、70 %、80 %、90 %、95 %、98 %或99 %的细胞为分离的胎盘细胞,其为CD10+、CD29-、CD44+、CD45-、CD54/ICANT、CD62-E-、CD62-L-、CD62-p-、CD8〇-、 〔〇86-、〔〇1〇3-、〔〇1〇4-、〔〇1〇5+、〔〇1〇6八〔41+、〇〇144八6-巧粘蛋白1。 [0121] In another embodiment, placental cell population can be used in the methods of treatment herein comprises an isolated, the cell population and at least 50%, 60%, 70%, 80%, 90%, 95 %, 98% or 99% of cells are isolated placental cells that are CD10 +, CD29-, CD44 +, CD45-, CD54 / iCANT, CD62-E-, CD62-L-, CD62-p-, CD8〇-, [〇86 -, [〇1〇3 -, [〇1〇4 -, [〇1〇5 + 41 + [[〇1〇6 eight, eight 6- 〇〇144 Qiao mucin 1. \〔〇184/〔乂〇?4-、02-微球蛋白1\化4-11\化4-1[、化4-61"和/或口0心"中的一种或多种。 One kind \ [〇184 / [square qe? 4-, 02- microglobulin 1 \ of 4-11 \ of 4-1 [, of 4-61 "and / or mouth Heart 0 " in or more. 在更具体的实施方案中, 所述细胞群中至少50%、60%、70%、80%、90%、95%、98%或99% 的细胞为CD10+、CD29-、 CD44+、CD45-、CD54/ICANT、CD62-E-、CD62-L-、CD62-P-、CD80-、CD8 扩、CD103-、CD104-、CD105+、 CD106/VCAM+、CD144/VE-巧粘蛋白i™、CD184/CXCR4-、02-微球蛋白i™、HLA-li™、HLA-n-、 HLA-gI™ 和PDLli™。 In a more specific embodiment, said cell population is at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% of the cells CD10 +, CD29-, CD44 +, CD45-, CD54 / iCANT, CD62-E-, CD62-L-, CD62-P-, CD80-, CD8 expansion, CD103-, CD104-, CD105 +, CD106 / VCAM +, CD144 / VE- Qiao mucin i ™, CD184 / CXCR4 -, 02- microglobulin i ™, HLA-li ™, HLA-n-, HLA-gI ™ and PDLli ™.

[0122] 在另一个实施方案中,可用于治疗脑或CNS中或周围血流破坏的所述分离的胎盘细胞为分离的胎盘细胞,其为CD10\CD29\CD34-、CD38-、CD44\CD45-、CD54\CD90\SH2+、 甜3+、SH4+、SSEA3-、SSEA4-、0CT-4+、和ABC-P+中的一种或多种或全部,其中ABC-p为胎盘特异性ABC转运蛋白(亦称乳腺癌耐受性蛋白(BCRP)和米托蔥酿耐受性蛋白(MXR)),其中所述分离的胎盘细胞通过灌注已经排干厮带并灌注W除去残余血液的哺乳动物例如人的胎盘的获得。 [0122] In another embodiment, useful for the treatment of brain or CNS or the disruption of blood flow around the isolated placental cells are isolated placental cells that are CD10 \ CD29 \ CD34-, CD38-, CD44 \ CD45 -, CD54 \ CD90 \ SH2 +, sweet 3 +, SH4 +, SSEA3-, SSEA4-, one kind 0CT-4 +, and ABC-P + one or more or all, where ABC-p is a placenta-specific ABC transporter protein (also known as breast cancer resistance protein (of BCRP) and onions stuffed mitoxantrone resistance protein (MXR)), wherein said isolated placental cells have been drained and perfused with servant mammal W remove residual blood by perfusion e.g. human placenta obtained.

[0123] 基因谱证实分离的胎盘细胞和分离的胎盘细胞群可与其他细胞,例如间充质干细胞,例如骨髓来源的间充质干细胞区分。 [0123] spectrum confirmed the gene isolated placental cells and placental cell population can be isolated from other cells, such as mesenchymal stem cells, e.g. bone marrow-derived mesenchymal stem cells differentiate. 此处所述的分离的胎盘细胞可在一种或多种基因表达的基础上与例如间充质干细胞区分,与骨髓来源的间充质干细胞相比,所述基因的表达显著高于分离的胎盘细胞或某些分离的厮带干细胞。 Herein may be isolated placental cells, as compared to mesenchymal stem cells and bone marrow-derived mesenchymal stem cells, for example, distinguished on the basis of one or more genes on the gene expression was significantly higher than that isolated isolated placental cells or certain stem cells with a servant. 特别地,可用于此处提供的治疗方法的分离的胎盘细胞可在一种或多种基因表达的基础上与间充质干细胞区分,当所述细胞在同等条件下培养时,分离的胎盘细胞中所述基因的表达比等量骨髓来源的间充质干细胞明显高(即至少高2倍),其中所述一种或多种基因为ACTG2、ADARB1、AMIG02、ARTS-I、 B4GALT6、BCHE、Cllorf9、CD200、C0L4Al、C0L4A2、CPA4、DMD、DSC3、DSG2、EL(WL2、F2I?Ll、 FLJ10781、GATA6、GPRl26、GPRC5B、HLA-G、ICAMl、IER3、IGFBP7、ILIA、IL6、ILI8、KRT18、 KRT8、LIPG、LRAP、MATN2、MEST、NFE2L3、NUAK1、PCDH7、PDLIM3、PKP2、RTN1、SERPINB9、 ST3GAL6、ST6GALNAC5、SLC12A8、TCF21、TGFB2、VTN、ZC3H12A 或上述任何基因的组合。参见例如美国专利申请公开号2007/0275362,其公开内容在此全文引入作为参考。在更具体的实施方案中,所述分离的胎盘细胞在包含DMEM-LG(例如来自Gibco);2%胎牛血清(例如来自Hyc In particular isolated placental cells, useful in the treatment methods provided herein may be based on one or more genes expressed on the mesenchymal stem cells differentiate, when the cells were cultured under the same conditions, the isolated placental cells expression of the gene ratio between an equivalent amount of bone marrow-derived mesenchymal stem cells was significantly higher (i.e., at least 2 times higher), wherein said one or more genes are ACTG2, ADARB1, AMIG02, ARTS-I, B4GALT6, BCHE, Cllorf9, CD200, C0L4Al, C0L4A2, CPA4, DMD, DSC3, DSG2, EL (WL2, F2I? Ll, FLJ10781, GATA6, GPRl26, GPRC5B, HLA-G, ICAMl, IER3, IGFBP7, ILIA, IL6, ILI8, KRT18, KRT8, LIPG, LRAP, MATN2, MEST, NFE2L3, NUAK1, PCDH7, PDLIM3, PKP2, RTN1, SERPINB9, ST3GAL6, ST6GALNAC5, SLC12A8, TCF21, TGFB2, VTN, ZC3H12A or any combination of these genes. see, e.g. U.S. Patent application Publication number 2007/0275362, the disclosure of which is hereby incorporated by reference in a more specific embodiment, said isolated placental cells containing DMEM-LG (e.g., from Gibco);. 2% fetal calf serum (e.g. from Hyc lone Labs); 1 X膜岛素-转铁蛋白-砸(ITS); 1 X亚油酸-牛血清白蛋白(LA-BSA); 10-9M 地塞米松(例如来自Sigma); 10-4Μ抗坏血酸2-憐酸盐(例如来自Sigma);表皮生长因子long/ mL(例如来自R&D SySterns);和血小板来源的生长因子(PDGF-BB) 1 Ong/mL(例如来自R&D Systems)的培养基中培养约3至约35个群倍增时表达所述一种或多种基因。在一个具体的实施方案中,所述分离的胎盘细胞-特异的或分离的厮带细胞-特异的基因为CD200。 lone Labs); 1 X film Insulin - Transferrin - hit (ITS); 1 X linoleic acid - bovine serum albumin (LA-BSA); 10-9M dexamethasone (e.g., from Sigma); 10-4Μ ascorbic acid 2-pity salt (e.g. from Sigma); epidermal growth factor long / mL (e.g., from R & D SySterns); and platelet-derived growth factor (PDGF-BB) 1 Ong / mL (e.g., from R & D Systems) culture media expression cultured for about 3 to about 35 population doublings during the one or more genes in a particular embodiment, the isolated placental cells - or isolated cells with specific servant - for the specific gene of CD200.

[0124] 运些基因的特定序列可在GenBank中找到,登录号为2008年3月的應_001615 (ACTG2)、BC065545(ADARB1)、(應J81847(AMIG02)、AY358590(ARTS-I)、BC074884 (B4GALT6)、BC008396(BCHE)、BC020196(ClIorf9)、BC031103(CD200)、NM_001845(C0L4A1)、 MM_001846(C0L4A2)、BC052289(CP A4)、BC094758(DMD)、AF293359(DSC3)、NM_001943 (DSG2)、AF33824UEL(WL2)、AY336105(F2I?L1)、NMJ) 18215 巧LJ 10781 )、AY416799(GATA6)、 BC075798(GPR1 26)、NM_016235(GPRC5B)、AF:M0038aCAMl)、BC000844(IER3)、BC066339 (IGFBP7)、BC013142(ILIA)、BT019749(IL6)、BC007461(IL1 8)、(BC072017)KRT18、 BC075839化RTS)、BC060825(LIPG)、BC06524(KLRAP)、BC0 10444(MATN2)、BC01 1908 (MEST)、BC068455(NFE2L3)、醒JH4840(NUAK1)、AB006755(PCDH7)、NM_014476(PDLIM3)、 BC126199(PKP-2)、BC090862(RTN1)、BC002538(SERPINB9)、BC023312(ST3GAL6)、BC001201 (ST6GALNAC5)、BC126160或BC065328(SLC12A8)、BC025697(TCF21)、BC096235(TGFB2)、 BC005046(VTN)和BC005001(ZC3H12A)。 [0124] transport specific sequence these genes can be found in GenBank accession number in March 2008 should _001615 (ACTG2), BC065545 (ADARB1), (should J81847 (AMIG02), AY358590 (ARTS-I), BC074884 (B4GALT6), BC008396 (BCHE), BC020196 (ClIorf9), BC031103 (CD200), NM_001845 (C0L4A1), MM_001846 (C0L4A2), BC052289 (CP A4), BC094758 (DMD), AF293359 (DSC3), NM_001943 (DSG2), AF33824UEL (WL2), AY336105 (? F2I L1), NMJ) 18215 Qiao LJ 10781), AY416799 (GATA6), BC075798 (GPR1 26), NM_016235 (GPRC5B), AF: M0038aCAMl), BC000844 (IER3), BC066339 (IGFBP7) , BC013142 (ILIA), BT019749 (IL6), BC007461 (IL1 8), (BC072017) KRT18, BC075839 of RTS), BC060825 (LIPG), BC06524 (KLRAP), BC0 10444 (MATN2), BC01 1908 (MEST), BC068455 (NFE2L3), wake JH4840 (NUAK1), AB006755 (PCDH7), NM_014476 (PDLIM3), BC126199 (PKP-2), BC090862 (RTN1), BC002538 (SERPINB9), BC023312 (ST3GAL6), BC001201 (ST6GALNAC5), BC126160 or BC065328 (SLC12A8), BC025697 (TCF21), BC096235 (TGFB2), BC005046 (VTN) and BC005001 (ZC3H12A).

[0125] 在更具体的实施方案中,当在同等条件下培养时,所述分离的胎盘细胞W比等量骨髓来源的间充质干细胞W可检测的更高水平表达ACTG2、ADARB1、ARTS-I、B4GALT6、BC肥、 ClIorf9、CD200、C0L4Al、C0L4A2、CPA4、DMD、DSC3、DSG2、EL(WL2、F2I?Ll、njl0781、GATA6、 GPR126、GPRC5B、ICAM1、IER3、IGFBP7、ILIA、IL6、IL18、KRT18、KRT8、LIPG、LRAP、MATN2、 MEST、N阳化3、NUAK1、PC畑7、PDLIM3、PKP2、RTNl、沈RPINB9、ST3GAL6、ST6GALNAC5、SLC 12A8、 TCF21、TGFB2、VTN 和ZC3H12A 中的每一种。 [0125] In a more specific embodiment, when cultured under the same conditions, the isolated placental cells than W between the same amount of bone marrow-derived mesenchymal stem cells W detectably higher expression levels ACTG2, ADARB1, ARTS- I, B4GALT6, BC fat, ClIorf9, CD200, C0L4Al, C0L4A2, CPA4, DMD, DSC3, DSG2, EL (WL2, F2I? Ll, njl0781, GATA6, GPR126, GPRC5B, ICAM1, IER3, IGFBP7, ILIA, IL6, IL18 , KRT18, KRT8, LIPG, LRAP, MATN2, MEST, N yang 3, NUAK1, PC Hata 7, PDLIM3, PKP2, RTNl, Shen RPINB9, ST3GAL6, ST6GALNAC5, SLC 12A8, TCF21, TGFB2, VTN, and ZC3H12A each one kind.

[0126] 上述参照基因的表达可通过标准技术评估。 [0126] expression of the reference genes can be assessed by standard techniques. 例如,可通过常规方法选择和构建基于基因序列的探针。 For example, the probe can be selected and constructed based on the gene sequence by a conventional method. 基因表达可例如在包括一种或多种所述基因的探针的微阵列上评估,例如Affyme比ixCBNEC拭巧⑥Human Genome U133A 2.0阵列或Af玲metrijtGENEC打IP⑥ Human Genome U133Plus 2.0(Santa Clara,California)。 Gene expression may be assessed, for example, on a probe comprising one or more genes in the microarray, e.g. Affyme clever than ixCBNEC swab or ⑥Human Genome U133A 2.0 array Af Ling metrijtGENEC play IP⑥ Human Genome U133Plus 2.0 (Santa Clara, California) . 由于所述修正序列的特异性探针可利用熟知的标准技术很容易地得到,因此即使具体GenBank登录号的序列被修正也可W评估运些基因的表达。 Since the specific probe sequence may be corrected using well known standard techniques readily obtained, even if the specific sequence of GenBank Accession No. W may be corrected evaluation operation expression of these genes.

[0127] 运些基因的表达水平可用于证实分离的胎盘细胞群的同一性,鉴定至少包含多个分离的胎盘细胞的细胞群等等。 Identity to the [0127] operation of these gene expression levels may be used to confirm the isolated placental cells, comprising identifying at least a plurality of isolated placental cells, and the like. 同一性已经被证实的分离的胎盘细胞群,可W是克隆的,例如扩增自单个分离的胎盘细胞的分离胎盘细胞群,或混合的干细胞群,例如包含扩增自多个分离的胎盘细胞的分离的胎盘细胞的细胞群,或包含此处所述的分离的胎盘细胞和至少一种其他类型细胞的细胞群。 Has confirmed the identity of the isolated placental cells, it can be cloned W is, for example, amplified from a single isolated placental cell populations of isolated placental cells, or a mixed population of stem cells, for example, comprising a plurality of amplified from isolated placental cells isolated placental cells or isolated placental cells comprising at least one cell population of other cell types described herein.

[0128] 运些基因的表达水平可用于选择分离的胎盘细胞群。 [0,128] op some level of gene expression can be used to select populations of isolated placental cells. 例如,如果一种或多种上列基因在来自所述细胞群的样品中的表达明显高于等量间充质干细胞群的样品,则可W选择该细胞群,如克隆-扩增的细胞。 For example, expression of a gene in a sample from the cell population of the above, if one or more of the same amount was significantly higher than among mesenchymal stem cells of the sample, W may select the population of cells, clonal - expanded cells . 所述选择可W来自多个分离的胎盘细胞群、来自多个细胞群,其同一性未知等。 W may be selected from a plurality of said isolated placental cell populations, from a plurality of cell populations, which identity is unknown and the like.

[0129] 可在比较一种或多种所述基因的表达水平与例如在间充质干细胞对照中所述一种或多种基因的表达水平,例如在等量骨髓来源的间充质干细胞中所述一种或多种基因的表达水平的基础上选择分离的胎盘细胞。 [0129] In comparison may be one or more of the expression level of the gene for example in the mesenchymal stem cell control in the expression levels of one or more genes, for example, between equal amounts of bone marrow-derived mesenchymal stem cells the selective separation of the one or more gene expression levels based on placental cells. 在一个实施方案中,包含等量间充质干细胞的样品中所述一种或多种基因的表达水平用作对照。 In one embodiment, it contains equal amounts between mesenchymal stem cells in the sample of the one or more expression level of a gene used as a control. 在另一个实施方案中,对于在一定条件下检测的分离的胎盘细胞,其对照为代表在所述条件下间充质干细胞中所述一种或多种基因表达水平的数值。 In another embodiment, for isolated placental cells tested under certain conditions, the control of one or more values representing the expression levels of genes between the condition of the mesenchymal stem cells.

[0130] 此处所述的分离的胎盘细胞在原代培养或培养基中增殖期间呈现上述特性(例如细胞表面标记和/或基因表达谱的组合),所述培养基包含,例如DMEM-LG(Gibco);2%胎牛血清(例如来自Hyclone Labs.); 1 X膜岛素-转铁蛋白-砸(ITS); 1 X亚油酸-牛血清白蛋白(LA-BSA); 10-9M地塞米松(Sigma); 10-4M抗坏血酸2-憐酸盐(Sigma);表皮生长因子lOng/mL (R&D Systems);和血小板来源的生长因子(PDGF-BB)10ng/mL(R&D Systems)和100U青霉素/1000U链霉素。 During the presentation [0,130] described herein isolated placental cells proliferate in primary culture medium or the above characteristics (e.g., cell surface markers or combinations and / gene expression profile) medium comprising, for example, DMEM-LG ( Gibco); 2% fetal calf serum (e.g., from Hyclone Labs); 1 X film Insulin - transferrin - hit (ITS);. 1 X linoleic acid - bovine serum albumin (LA-BSA); 10-9M dexamethasone (Sigma); 10-4M ascorbic acid 2-pity acid (Sigma); epidermal growth factor lOng / mL (R & D Systems); and platelet-derived growth factor (PDGF-BB) 10ng / mL (R & D Systems) and 100U penicillin / 1000U streptomycin.

[0131] 在所述分离的胎盘细胞或包含所述分离的胎盘细胞的细胞群另外具体的实施方案中,所述细胞或群已扩增例如传代至少约或不超过1、2、3、4、5、6、7、8、9、10、11、12、13、 14、15、16、17、18、19或20次,或增殖至少约或不超过1、2、3、4、5、6、7、8、9、10、12、14、16、18、 20、22、24、26、28、30、32、34、36、38或40次群倍增。 [0131] In the isolated placental cells or isolated placental cells comprising said cell population another specific embodiment, the cell population of amplified or passaged for example at least or no more than about 3, 4 ,, 12, 13, 14,15,16,17,18,19, or 20, or at least or no more than about proliferation 1,2,3,4,5 , 6,7,8,9,10,12,14,16,18, 20,22,24,26,28,30,32,34,36,38 or 40 population doublings. 在此处公开的分离的胎盘细胞或包含分离的胎盘细胞的细胞群另外具体的实施方案中,所述分离的胎盘细胞为胎儿来源的(即具有胎儿基因型)。 Disclosed herein are isolated placental cells or cell populations of isolated placental cells comprising another specific embodiment, said isolated placental cells are fetal in origin (i.e., have the fetal genotype).

[0132] 在分离的胎盘细胞的某些实施方案中,所述分离的胎盘细胞在生长培养基(即配制W促进增殖的培养基)中的培养期间,例如在生长培养基中的增殖期间通常不分化。 During the culture period (i.e., medium formulated to promote proliferation W) [0132] In certain embodiments of isolated placental cells, said isolated placental cells in growth medium, for example propagated in growth medium usually undifferentiated. 在另外具体的实施方案中,所述分离的胎盘细胞不需要滋养层W增殖。 In another specific embodiment, said isolated placental cells do not require trophoblast proliferation W layer. 在另外具体的实施方案中,所述分离的胎盘细胞仅由于没有饲养细胞层而在培养时不分化。 In another specific embodiment, said isolated placental cells only and the absence of a feeder cell layer without differentiation in culture.

[0133] 在另一个实施方案中,可用于脑或CNS中或周围血流破坏治疗的细胞为分离的胎盘细胞,如通过醒脱氨酶活力测定法评估的,其中多个所述分离的胎盘细胞为醒脱氨酶(八0)^阳性。 [0133] In another embodiment, the brain or CNS can be used in the disruption of blood flow surrounding the cells or treatment of isolated placental cells, as determined by wake deaminase activity assay evaluation, wherein a plurality of said isolated placental cells wake deaminase (80) ^ positive. 所述测定法为本领域已知(参见例如8〇311曰11和863,81〇^16111.1,173,787, (1978))。 The assay known in the art (see, e.g. 11 and 86 8〇311 said ^ 16111.1,173,787 3,81〇, (1978)). 在一个具体的实施方案中,所述ALD扣则定法利用ALDEFLU(m|(Aldagen, Inc.,Ashland,Oregon)作为醒脱氨酶活力标记。在一个具体的实施方案中,所述细胞群中的所述多个在约3%和约25%细胞之间。在另一个实施方案中,此处提供了分离的厮带细胞群,例如多能的分离厮带细胞,如通过利用ALD巨FLUO防作为醒脱氨酶活力指标的醒脱氨酶活力测定法评估的,其中多个所述分离的厮带细胞为醒脱氨酶阳性。在一个具体的实施方案中,所述细胞群中的所述多个在约3%和约25%细胞之间。在另一个实施方案中, 所述分离的胎盘细胞或分离的厮带细胞群显示比具有大致相等量和同样的条件下培养的骨髓来源的间充质干细胞的ALDH活力高至少Ξ倍或至少五倍。 In one particular embodiment, the fastening titration using the ALD ALDEFLU (m | (Aldagen, Inc., Ashland, Oregon) as a marker wake deaminase activity In a specific embodiment, said population of cells. said plurality is between about 3% and about 25% of the cells. in another embodiment, provided herein is an isolated population of cells with a servant, such as multi-band energy servant isolated cells, such as by ALD using giant FLUO anti- wake wake as deaminase activity index deaminase activity assay evaluation, wherein said plurality of isolated cells servant with ammonia awake decarboxylase positive. in a specific embodiment, the cell population It said plurality is between about 3% and about 25% of the cells. in another embodiment, the isolated placental cells or isolated cell populations display servant with bone marrow cultures having substantially equal ratio condition and the same quantity of - derived mesenchymal stem cells higher ALDH activity at least five-fold, or at least Ξ.

[0134] 在包含此处所述分离的胎盘细胞的任何细胞群的某些实施方案中,所述细胞群中的胎盘细胞基本上没有具有母体基因型的细胞;例如所述群中至少40 %、45 %、50 %、55 %、 60%、65%、70%、75%、80%、85%、90%、95%、98%或99%的胎盘细胞具有胎儿基因型。 [0134] In certain embodiments herein, comprising the isolated placental cells of any cell population, the cell population of placental cells is substantially free of cells having a maternal genotype; e.g. at least 40% of the population , 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of the placental cells have a fetal genotype. 在包含此处所述分离的胎盘细胞的任何细胞群的某些其他实施方案中,包含胎盘细胞的所述细胞群基本上没有具有母体基因型的细胞;例如所述群包括至少40%、45 %、50 %、55 %、 60%、65%、70%、75%、80%、85%、90%、95%、98% 或99% 的细胞具有胎儿基因型。 In certain other embodiments herein comprise the isolated placental cells of any cell population, said cell population comprises placental cells substantially free of cells having a maternal genotype; for example, the group comprising at least 40%, 45 %, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of cells having the fetal genotype.

[0135] 在任何上述分离的胎盘细胞或分离的胎盘细胞细胞群的具体实施方案中,所述细胞的核型或所述群中至少约95%或约99%的细胞核型是正常的。 [0135] In a specific embodiment of any of the above-described isolated placental cells or cell populations of isolated placental cells, the karyotype of the cells in the group or at least about 95%, or about 99% of normal karyotype. 在任何上述胎盘细胞或细胞群另外具体的实施方案中,所述细胞或所述细胞群中的细胞为非母体来源的。 In any of the above placental cells or cell populations another specific embodiment, said population of cells or the cells of the cells are non-maternal in origin.

[0136] 带有任何上述标记组合的分离的胎盘细胞或分离的胎盘细胞群可W任何比例组合。 [0136] The isolated placental cells or isolated placental cells labeled with a combination of any of the above W may be combined in any proportion. 任何两种或多种上述分离的胎盘细胞群可组合形成分离的胎盘细胞群。 Any two or more of the above isolated placental cell populations can be combined to form an isolated placental cell population. 例如,分离的胎盘细胞群可包含通过如上所述的一种标记组合定义的第一种分离的胎盘细胞群,和通过如上所述的另一种标记组合定义的第二种分离的胎盘细胞群,其中所述第一种细胞群和第二种细胞群W约1:99、2:98、3:97、4:96、5:95、10:90、20:80、30:70、40:60、50:50、60:40、70: 30、80:20、90:10、95:5、96:4、97:3、98:2或约99:1的比例组合。 For example, isolated placental cells can comprise a first population of isolated placental cell populations by means of a marking composition defined as described above, and separated by another marking defined by a combination of a second population of placental cells described above, wherein said first cell population and a second population of cells of about 1 W: 99,2: 98,3: 97,4: 96,5: 95,10: 90,20: 80, 30: 70, 40 : 60,50: 50,60: 40,70: 30,80: 20,90: 10, 95: 5,96: 4,97: 3, 98: 2, or about 99: combination 1 ratio. ^类似的方式,可获得任何Ξ种、四种、五种或W上上述分离的胎盘细胞或分离的胎盘细胞群组合。 ^ A similar manner, any available Ξ species, four, five or W above-described isolated placental cells or population of isolated placental cells in combination.

[0137] 可用于脑或CNS中或周围血流破坏治疗的分离的胎盘细胞可例如通过用或不用酶促消化(参见5.5.3章节)或灌流(参见5.5.4章节)破坏胎盘组织而获得。 [0137] may be used in or around the brain or CNS treatment of disruption of blood flow isolated placental cells can be obtained, for example by treatment with or without enzymatic digestion (see Section 5.5.3) or perfusion (see Section 5.5.4) placental tissue damage . 例如,分离的胎盘细胞群可W通过W下方法制备,该方法包括包括灌流已排干厮带血且已被灌流W除去残余血液的哺乳动物胎盘;用灌流液灌流所述胎盘;并且收集所述灌流液,其中灌流后所述灌流液包括包含分离的胎盘细胞的胎盘细胞群;并且从所述细胞群分离多个所述分离的胎盘细胞。 For example, isolated placental cells can be prepared by a W at W, the method comprising comprising servant blood perfusion is drained and perfused W has been removed residual blood mammalian placenta; perfused with the placental perfusate; and collecting the said perfusion fluid, wherein after said perfusate comprises placental perfusate cells comprises isolated placental cells; and separating a plurality of said isolated placental cells from said population of cells. 在一个具体的实施方案中,所述灌流液通过厮静脉和厮动脉并且在其从所述胎盘缓慢流出后收集。 In a specific embodiment, said perfusate servant arteries and veins by a servant and collected after it flows slowly from the placenta. 在另外具体的实施方案中,所述灌流液通过厮静脉并且从厮动脉收集,或通过厮动脉并且从厮静脉收集。 In another specific embodiment, said perfusate collected by servant servant artery and vein were collected, or by a servant from the artery and vein from the servant.

[0138] 在不同的实施方案中,含于胎盘灌流获得的细胞群内的所述分离的胎盘细胞为所述胎盘细胞群中的至少50%、60%、70%、80%、90%、95%、99%或至少99.5%。 [0138] In various embodiments, the population of cells contained within placental perfusate to obtain isolated placental cells are at least 50% of said population of placental cells, 60%, 70%, 80%, 90%, 95%, 99%, or at least 99.5%. 在另外具体的实施方案中,通过灌流收集的所述分离的胎盘细胞包含胎儿细胞和母体细胞。 In another specific embodiment, the perfusion collected by the isolated placental cells comprise fetal cells and maternal cells. 在另外具体的实施方案中,通过灌流收集的所述分离的胎盘细胞为至少50 %、60 %、70 %、80 %、 90%、95%、99%或至少99.5%的胎儿细胞。 In another specific embodiment, the perfusion collected by the isolated placental cells are at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or at least 99.5% fetal cells.

[0139] 在另外具体的实施方案中,此处提供了包含如此处所述通过灌流收集的分离的胎盘细胞群的组合物,其中所述组合物包含至少一部分用于收集所述分离的胎盘细胞的灌流液。 [0139] In another specific embodiment, provided herein comprising a composition as described herein is an isolated population of placental cells collected by perfusion, wherein said composition comprises placental cells, for collecting at least a portion of the separated the perfusion liquid.

[0140] 此处所述的分离胎盘细胞的分离群可通过W下方法制备:组织-分裂酶消化胎盘组织w获得包含所述细胞的胎盘细胞群,并且从其余胎盘细胞分离或基本上分离多个所述胎盘细胞。 Isolated placental cells isolates [0140] described herein can be prepared by the process of the W: Organization - split w enzymatic digestion of placental tissue to obtain a population of cells comprising said placental cells, and placental cells are isolated from the rest or substantially isolated polynucleotide the placental cells. 可消化整个或胎盘任一部分W获得此处所述的分离的胎盘细胞。 Digestible whole or any part of the placenta W to obtain isolated placental cells described herein. 在具体的实施方案中,例如所述胎盘组织可W是整个胎盘、羊膜、绒毛膜、羊膜和绒毛膜的组合或任何上述的组合。 In a specific embodiment, the placental tissue, for example, a combination may be W is a whole placenta, amnion, chorion, amnion and chorion, or any combination thereof. 在其他具体的实施方案中,所述组织-分裂酶为膜蛋白酶或胶原酶。 In other specific embodiments, the tissue - splitting enzyme is collagenase or protease film. 在不同的实施方案中,包含在消化胎盘而获得的细胞群内的所述分离的胎盘细胞为所述胎盘细胞群的至少50%、60%、70%、80%、90%、95%、99%或至少99.5%。 The isolated placental cells In various embodiments, the population of cells contained in the digestion of placental obtained for the population of placental cells are at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or at least 99.5%.

[0141] 如上所述胎盘细胞的分离群和分离的胎盘细胞群通常可包含大约至少或不超过1 X1〇5、5X1〇5、1X1〇6、5X1〇6、1Xx1〇7、5X1〇7、1X1〇8、5X1〇8、1X1〇9、5X1〇9、1X1〇i〇、5X i〇w、ixi〇ii或更多所述分离的胎盘细胞。 [0141] As described above isolated placental cell population of isolated placental cells, and may generally comprise about at least or no more than 1 X1〇5,5X1〇5,1X1〇6,5X1〇6,1Xx1〇7,5X1〇7, 1X1〇8,5X1〇8,1X1〇9,5X1〇9,1X1〇i〇, 5X i〇w, ixi〇ii or more of the isolated placental cells. 可用于此处所述治疗方法的分离的胎盘细胞群包含至少50%、55%、60%、65%、70%、75%、80%、85%、90%、95%98% 或99% 如通过例如台吩蓝排除测定的存活的分离的胎盘细胞。 The method described herein may be used for the treatment of isolated placental cells comprising at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% e.g., by blue exclusion table thiophene viable isolated placental cells measured.

[0142] 5.4.3培养生长 [0142] 5.4.3 culture growth

[0143] 对于任何哺乳动物细胞,5.4.2节所述的分离的胎盘细胞的生长在某种程度上取决于选择用于生长的具体培养基。 [0143] For any mammalian cells, grown in Section 5.4.2, said isolated placental cells depend somewhat on the particular medium selected for growth. 在最佳条件下,所述分离的胎盘细胞通常在约1天中数量倍增。 Under optimum conditions, the isolated placental cells typically doubling in the number of about 1 day. 在培养期间,此处所述分离的胎盘细胞粘附于培养底物,例如组织培养容器的表面(例如组织培养平皿塑料制品、纤连蛋白-涂布的塑料制品等等)并且形成单细胞层。 During culture, the isolated placental cells described herein adhere to the culture substrate, e.g., tissue culture container surface (e.g., tissue culture dish plastic products, fibronectin - coated plastic products, etc.) and form a single cell layer .

[0144] 当包含此处所述分离的胎盘细胞的胎盘细胞群在合适的条件下培养时,可形成胚样体,即在贴壁细胞层顶上的细胞培养物的立体团。 [0144] When included herein, the isolated placental cells, placental cells cultured under appropriate conditions, form embryoid-like bodies, i.e. groups in adherent cell culture perspective top cell layer. 胚样体内的细胞表达与早期干细胞有关的标记,例如0CT-4、化nog、SSEA3和SSEA4。 Cells expressing markers associated with early embryo stem cell-like in vivo, e.g. 0CT-4, of nog, SSEA3 and SSEA4. 胚样体内的细胞通常不像此处所述的分离的胎盘细胞那样粘附培养底物,但在培养期间仍然附着于贴壁细胞。 Embryoid-like cells in vivo usually not described herein as adherent isolated placental cells culture substrate, but remains attached to the adherent cells during culture. 胚样体细胞依赖于分离的贴壁胎盘细胞而存活,因为在不存在分离的贴壁胎盘细胞时不形成胚样体。 Embryoid-like body cells are dependent on the adherent isolated placental cells survive, because they do not form embryoid-like bodies when separating the absence of adherent placental cells. 分离的贴壁胎盘细胞因此促进包含所述分离的贴壁胎盘细胞的胎盘细胞群中一种或多种胚样体的生长。 The isolated adherent placental cells thus facilitate the growth of said population of placental cells comprising the isolated placental adherent cells of one or more embryoid-like bodies. 不希望受理论限制,认为与胚胎干细胞在滋养层细胞上生长一样,胚样体细胞在分离的贴壁胎盘细胞上生长。 Without wishing to bound by theory, that as the cells grow on the trophoblast cells, embryonic-like cells grown on the adherent isolated placental cells and embryonic stem cells.

[0145] 5.5获得分离的胎盘细胞的方法 [0145] 5.5 A method to obtain isolated placental cells

[0146] 5.5.1干细胞收集组合物 [0146] 5.5.1 stem cell collection composition

[0147] 此处进一步提供收集和分离胎盘细胞的方法,例如分离W上5.4.2节所述的分离的胎盘细胞的方法。 [0147] Further provided herein and methods for the collection of isolated placental cells, for example, Section 5.4.2, the method of separating the W isolated placental cells. 通常,所述细胞利用生理学可接受的溶液,例如细胞收集组合物,从哺乳动物胎盘获得。 Typically, the cells using a physiologically acceptable solution, such as a cell collection composition, is obtained from a mammalian placenta. 示例性细胞收集组合物在相关美国专利申请公开号2007/0190042中详细描述,名为('Improved Medium for Collecting PI曰cent曰1 Stem Cells 曰nd Preserving 化gans",其公开内容在此全文引入作为参考。 Exemplary cell collection composition in the related U.S. Patent Application Publication No. 2007/0190042 is described in detail, the name ( 'Improved Medium for Collecting PI said cent said 1 Stem Cells nd Preserving of said gans ", the disclosure of which is herein incorporated by reference.

[0148] 所述细胞收集组合物可包含适于例如此处所述分离的胎盘细胞的细胞收集和/或培养的任何生理学可接受的溶液,例如盐溶液(例如憐酸缓冲盐溶液、Kreb ' S溶液、改进的Kreb's溶液、Eagle's溶液、0.9%NaCl等等)、培养基(例如DMEM、H.DMEM等等)等。 [0148] The cell collection composition can comprise a cell adapted to collect the isolated placental cells described herein and / or any physiologically acceptable solution, such as culture, for example, a saline solution (e.g., buffered saline acid pity, Kreb ' S solution, modified Kreb's solution, Eagle's solution, 0.9% NaCl, etc.), medium (e.g. DMEM, H.DMEM etc.) and the like.

[0149] 所述细胞收集组合物可包含有助于保存分离的胎盘细胞的一种或多种组分,即在细胞收集至培养期间防止所述分离的胎盘细胞死亡或延缓所述分离的胎盘细胞死亡、减少细胞群中分离的胎盘细胞的死亡数等等。 [0149] The cell collection composition can comprise isolated placental cells help to preserve one or more components, i.e., during the cultivation to collect the isolated placental cells to prevent or delay the death of the isolated placental cells in cell death, and so reduce the number of deaths in a population of cells isolated placental cells. 所述组分可W是例如调亡抑制剂(例如级联抑制剂或JNK抑制剂);血管扩张剂(例如硫酸儀、抗高血压药物、屯、房利钢肤(ANP )、促肾上腺皮质激素、促肾上腺皮质激素-释放因子、硝普钢盐、阱苯化嗦、腺巧Ξ憐酸盐、腺巧、消炎痛或硫酸儀、憐酸二醋酶抑制剂等等);坏死抑制剂(例如2-QH-吗隙-3毒)-3讀胺基-马来酷亚胺、邮咯烧二硫代氨基甲酸盐或氯硝西泮);TNF-a抑制剂和/或携氧全氣化碳(例如全氣漠辛烧、全氣漠癸烧等等)。 The W component may be, for example inhibitors of apoptosis (e.g. cascade inhibitor or JNK inhibitor); vasodilators (e.g. sulfuric instrument, antihypertensives, village, Fannie steel skin (of ANP), adrenocorticotropin hormone, adrenocorticotropic hormone - releasing factor, nitroprusside salts of steel, well winded of benzene, Qiao Ξ pity salt gland, glandular Qiao, indomethacin or sulfuric instrument, pity acid esterase inhibitors, etc.); inhibitors of necrosis (e.g. 2-QH- it toxic gap -3) -3 read amine - maleic imide cool, slightly burning Post dithio carbamate or clonazepam); TNF-a inhibitor and / or carry full carbon oxygen gas (e.g. desert oct full gas burning, gas full burn like desert dec).

[0150] 所述细胞收集组合物可包含一种或多种组织降解酶,例如金属蛋白酶、丝氨酸蛋白酶、中性蛋白酶、核糖核酸酶或脱氧核糖核酸酶等等。 [0150] The cell collection composition can comprise one or more tissue-degrading enzymes, such as metalloproteases, serine proteases, a neutral protease, DNase or RNase like. 所述酶包括,但不限于胶原酶(例如胶原酶Ι、Π 、ΙΠ 或IV、来自溶组织梭状芽抱杆菌的胶原酶等等);分散酶、嗜热菌蛋白酶、弹性蛋白酶、膜蛋白酶、释放酶、透明质酸酶等等。 The enzymes include, but are not limited to, collagenases (e.g., collagenase Ι, Π, ΙΠ or IV, from Clostridium histolyticum collagenase Bacillus spores, etc.); dispase, thermolysin, elastase, protease film , the release of the enzyme, hyaluronidase, and so on.

[0151] 所述细胞收集组合物可包含杀菌或抑菌有效量的抗生素。 [0151] The cell collection composition can comprise an effective amount of a bactericidal or bacteriostatic antibiotic. 在某些非限制性实施方案中,所述抗生素为大环内醋(例如,托普霉素)、头抱菌素(例如,头抱氨节、头抱拉晚、头抱巧辛、头抱丙締、头抱克洛、头抱克朽或头抱径氨节)、克拉霉素、红霉素、青霉素(例如,青霉素V)或哇诺酬类(例如,氧氣沙星、环丙沙星、诺氣沙星)、四环素、链霉素等等。 In certain non-limiting embodiments, the antibiotic is a macrolide vinegar (e.g., tobramycin), cephalosporins (e.g., cephalosporin ammonia section, cephalosporin pull late, octyl cephalosporin Qiao, head hold propan association, Crowe cephalosporin, cephalosporin or cephalosporin rot g ammonia diameter section), clarithromycin, erythromycin, penicillin (e.g., penicillin V) or wow Connaught pay (e.g., oxygen, gatifloxacin, cyclopropyloxy gatifloxacin, Novo gas sand star), tetracycline, streptomycin and so on. 在具体的实施方案中,所述抗生素对革兰氏(+)和/或革兰氏(-)细菌有效,例如铜绿假单胞菌、金黄色葡萄球菌等等。 In a specific embodiment, the antibiotic against Gram (+) and / or Gram - bacteria effectively, e.g. Pseudomonas aeruginosa, Staphylococcus aureus, etc. (). 在一个实施方案中,所述抗生素为庆大霉素,例如培养基中约0.005%至约0.01%(w/v)〇 In one embodiment, the antibiotic is gentamicin, medium e.g. from about 0.005% to about 0.01% (w / v) square

[0152] 所述细胞收集组合物还可W包含一种或多种下列化合物:腺巧(约ImM至约50mM); 右旋葡萄糖(约20mM至约lOOmM);儀离子(约ImM至约50mM);分子量大于20,000道尔顿的大分子,在一个实施方案中,W足W维持内皮完整性和细胞存活力的量存在(例如W约25g/l 至约lOOg/1或约40g/l至约60g/l存在的合成或天然存在的胶质、多糖如葡聚糖或聚乙二醇);抗氧化剂(例如W约25μΜ至约100μΜ存在的下径基大茵香酸、二下基径基甲苯、谷脫甘肤、维生素C或维生素Ε);防止巧进入细胞的制剂(例如W约2μΜ至约25μΜ存在的异搏定);硝酸甘油(例如约〇.〇5g/L至约0.2g/L);抗凝血剂,在一个实施方案中,W足W防止残余血液凝固的量存在(例如W约1000单位/1至约100,000单位/1的浓度存在的肝素或水赔素);或含有化合物(例如W约Ι.ΟμΜ至约如Μ存在的氨氯化脉、乙基异丙基氨氯化脉、环己烧氨氯化脉、二甲 [0152] The cell collection composition may further comprise one or more W following compounds: Qiao gland (about 50mM to about ImM); dextrose (from about 20mM to about lOOmm); ion meter (from about ImM to about 50mM ); the amount of molecular weight greater than 20,000 daltons macromolecules, in one embodiment, W W sufficient to maintain endothelial integrity and cellular viability in the presence of (W, for example, from about 25g / l to about lOOg / 1, or about 40g / l to about 60g / l in the presence of synthetic or naturally occurring colloid, a polysaccharide such as dextran or polyethylene glycol); antioxidants (e.g., from about W to about 100μΜ 25μΜ presence in large diameter fennel acid group, di lower base diameter toluene, Gan valley off the skin, vitamin C or vitamin Epsilon); Qiao formulation to prevent entry into the cell (e.g., from about W to about 25μΜ 2μΜ present verapamil); nitroglycerin (e.g., about 〇.〇5g / L to about 0.2 g / L); anticoagulants, in one embodiment, W is W a sufficient amount to prevent the presence of residual blood coagulation (e.g., W present at a concentration of about 1000 units / 1 to about 100,000 units / 1 heparin or lose water hormone) ; or a compound containing (e.g., from about W to about Ι.ΟμΜ presence of ammonia as Μ pulse chloride, ethyl chloride, isopropyl ammonium veins, cyclohexyl burn pulse ammonium chloride, dimethyl 氨氯化脉或异下基氨氯化脉)的氨氯化脉。 Ammonium chloride or isobutyl pulse at pulse ammonium chloride group) pulse ammonia chloride.

[0153] 5.5.2胎盘的收集和处理 [0153] 5.5.2 Placental collection and processing of

[0154] 通常,在出生排出后立即回收人胎盘。 [0154] Generally, the discharge is recovered shortly after birth in the human placenta. 在优选的实施方案中,在告知患者同意并采集与胎盘相关的完整医疗史后,从患者处回收胎盘。 In a preferred embodiment, the patient informed consent and after a complete medical history of the collection associated with the placenta, the placenta is recovered from the patient. 优选的,在分娩后继续记录医疗史。 Preferably, after childbirth continue to record medical history. 此类医疗史可用于调整胎盘或从其收获的干细胞的收获后的应用。 Such medical history can be used to adjust the application from the placenta or after harvest harvested stem cells. 例如,根据医疗史,人胎盘干细胞可用于与胎盘相关的婴儿、或用于所述婴儿的父母、兄弟姐妹或其他亲戚的个人化药物。 For example, according to medical history, human placental stem cells can be used in connection with the placenta of the baby, or personalized medicine for the baby's parents, siblings or other relatives.

[0155] 在回收胎盘干细胞之前,优选去除厮带血和胎盘血。 [0155] Prior to recovery of placental stem cells, preferably blood and placental blood are removed servant. 在某些实施方案中,在分娩后回收胎盘中的厮带血。 In certain embodiments, after delivery of the placenta blood recovery servant. 所述胎盘可W采用常规的厮带血回收方法。 The placenta can servant W using conventional blood recovery process. 一般使用针头或插管,在重力帮助下,将胎盘放血(参见例如:Anderson,美国专利号5,372,581;化ssel等人,美国专利号5,415,665)。 Typically using a needle or cannula, under gravity assistance, exsanguinated placenta (see, e.g.: Anderson, U.S. Patent No. 5,372,581; of ssel et al., U.S. Patent No. 5,415,665). 所述针头或插管通常置于厮静脉内,并且可^轻轻地按揉胎盘帮助从胎盘中排出厮带血。 The needle or cannula is usually placed in the servant vein and the placenta can help ^ gently rubbing discharged servant blood from the placenta. 可W商业方式来实施此类厮带血回收,例如LifeBank USA,Cedar Knolls, NJ。 W may be a commercial manner, such embodiments servant blood recovery, e.g. LifeBank USA, Cedar Knolls, NJ. 优选地,对所述胎盘通过重力来放血而不进行其他操作,从而使厮带血回收过程中的组织破坏最小化。 Preferably, the placental blood by gravity to operate without other so that the servant blood recovery process to minimize tissue disruption.

[0156] 典型地,胎盘从分娩室或初生室转移至另一地点,例如实验室,用于厮带血回收和干细胞收集,例如,通过灌流或组织解离。 [0156] Typically, placental transfer from the primary chamber to the delivery chamber or another location, such as a laboratory, for recovery and servant blood stem cells collected, e.g., by perfusion or tissue dissociation. 所述胎盘优选在无菌的、保溫的转移装置(维持胎盘溫度在20-28Γ)中转移,例如,将厮带近端夹紧的胎盘放置在无菌、夹拉链封闭的塑料袋中,然后将其放置在保溫容器内。 The placenta is preferably (maintaining the temperature of the placenta in 20-28Γ) are transferred, the transfer device holding sterile, e.g., the proximal end of the clamping band servant placenta is placed in a sterile, closed plastic bag fastener clip, and then placing it in a heat-retaining container. 在另一个实施方案中,所述胎盘在实质上如未决美国专利申请号7,147,626中描述的厮带血收集试剂盒中转移,其公开内容在此引入作为参考。 In another embodiment, the placental transfer substantially as in copending U.S. Patent Application No. 7,147,626 described servant blood collection kit, the disclosure of which is hereby incorporated by reference. 优选地,在分娩后4至24小时将胎盘递送至实验室。 Preferably, 4 to 24 hours after birth the placenta is delivered to the laboratory. 在某些实施方案中,在厮带血回收前,夹紧厮带近端,优选在插入胎盘的4-5cm(厘米)内。 In certain embodiments, the blood servant before recovery, the clamping servant with a proximal end, preferably in the placenta is inserted into 4-5cm (cm). 在其他实施方案中,在厮带血回收后但是在胎盘的进一步处理前夹紧近端厮带。 In other embodiments, but recovered after the servant blood prior to further processing of the placenta in the proximal end of the clamping band servant.

[0157] 在收集干细胞前,可W将胎盘储存在无菌条件下,并储存在室溫或者5至25Γ (摄氏度)的溫度下。 [0157] stem cells prior to collection, the placenta may be W stored under sterile conditions and stored at room temperature or at a temperature of 5 to 25Γ (degrees Celsius). 所述胎盘可W在灌流胎盘W去除任何残留的厮带血前储存4至24小时,甚至48小时或更长时间。 The placenta may be W remove any residual blood in the perfusion of the placenta servant W stored from 4 to 24 hours before, 48 hours or even longer. 在一个实施方案中,在排出厮带血后约0小时至约2小时内收集所述胎盘。 In one embodiment, at from about 0 hours to collect the discharged servant placental blood within about 2 hours. 所述胎盘优选在5至25C下储存在抗凝剂溶液中。 The placenta is preferably at 5 to 25 C is stored in an anticoagulant solution. 合适的抗凝剂溶液是本领域公知的。 Suitable anticoagulant solutions are well known in the art. 例如,可W使用肝素或华法令钢(warfarin sodium)溶液。 For example, heparin or warfarin W steel (warfarin sodium) solution. 在优选的实施方案中,所述抗凝剂溶液含有肝素溶液(例如,在1:1000溶液中1 %w/w)。 In a preferred embodiment, the anticoagulant solution comprises a solution of heparin (e.g., in 1: 1000 solution of 1% w / w). 在收集胎盘干细胞前,放血的胎盘优选储存不超过36小时。 Before collecting placental stem cells, exsanguinated placenta is preferably stored does not exceed 36 hours.

[0158] 一旦按照上述一般性方法收集和制备,哺乳动物胎盘或其部分可W按照本领域已知的任何方法处理,例如,可W被灌流或解离,如用一种或多种组织解离酶来解离,W获得干细胞。 [0158] Once collected and prepared according to the general methods described above, W may be a mammalian placenta or a part thereof according to any method known in the art process, for example, W can be dissociated or perfusion, such as with one or more tissue Solutions dissociated from the enzyme, W obtain stem cells.

[0159] 5.5.3胎盘组织的物理解离和酶促消化 [0159] 5.5.3 Placental tissue was enzymatically digested and understood from

[0160] 在一个实施方案中,通过物理解离部分或全部器官,从哺乳动物胎盘中收集干细胞。 [0160] In one embodiment, the composition is understood from some or all organs collected from a mammalian placenta stem cells. 例如,可W将胎盘或其部分压碎(crush)、剪碎(shear)、绞碎(mince)、切块(dice)、切细(chop)、浸软(macerate)等。 For example, W can be crushed placenta or portion thereof (Crush), cut into pieces (Shear), minced (mince), cut (DICE), milled (CHOP), maceration (Macerata) and the like. 随后可培养所述组织W获得分离的胎盘细胞群。 The tissue culture may then be obtained W isolated placental cell population. 通常,所述胎盘组织利用例如培养基、盐溶液或干细胞收集组合物解离(参见5.5.1节及W下)。 Typically, the placental tissue dissociation media using, for example, saline solution or stem cell collection composition (see Section 5.5.1 and the W).

[0161] 在物理解离和/或酶促消化和干细胞回收前,所述胎盘可W被分割成多个部分。 [0161] In understanding thereof from the front and / or enzymatic digestion and stem cell recovery, the placenta may be W is divided into a plurality of portions. 可W从羊膜、绒毛膜、厮带、胎盘绒毛叶的全部或部分或其任何组合,包括从整个胎盘获得胎盘干细胞。 It can be W, chorion, servant with all or placental cotyledons, or any combination of parts, including obtaining from the entire placenta placental stem cells from the amniotic membrane. 优选地,分离的胎盘细胞获自包含羊膜和绒毛膜的胎盘组织。 Preferably, the isolated placental cells are obtained from placental tissue comprising amnion and chorion. 一般的,分离的胎盘细胞可W通过将胎盘组织解离为小块来获得,例如胎盘组织块的体积是约1、2、3、4、5、6、 7、8、9、10、20、30、40、50、60、70、80、90、100、200、300、400、500、600、700、800、900 或约1000 立方毫米。 Typically, isolated placental cells can be obtained by a small W is the dissociation placenta, placental tissue e.g. mass volume is from about 1,2,3,4,5,6, 7,8,9,10,20 , 30,40,50,60,70,80,90,100,200,300,400,500,600,700,800,900 or from about 1000 mm3. 任何物理解离的方法均可W使用,只要通过如台吩蓝排除测定的,所述解离方法能使所述器官中多数,更优选的大多数,更优选的至少60%、70%、80%、90%、95%、98%或99%的细胞存活。 The method of any isolated composition can be appreciated that the use of W, as long as the station through thiophene blue exclusion assay, said dissociating method enables most of the organ, and more preferably most, more preferably at least 60%, 70%, 80%, 90%, 95%, 98% or 99% of cell survival.

[0162] 所述分离的贴壁胎盘细胞通常可在排出后约Ξ天内,但优选在排出后约8小时和约18小时内的任何时候从胎盘或其部分收集。 [0162] The isolated adherent placental cells typically about Ξ days after the discharge, but any time after the discharge is preferably within about 8 hours and about 18 hours of collection from the placenta or a portion thereof.

[0163] 在一个具体的实施方案中,所述解离的组织在适于分离的胎盘细胞增殖的组织培养基中培养(参见例如W下5.6节,描述胎盘干细胞的培养)。 [0163] In one particular embodiment, the dissociated tissue culture (see, e.g. under Section W 5.6, described the culture of placental stem cells) in tissue culture medium suitable for the proliferation of isolated placental cells.

[0164] 在另外具体的实施方案中,分离的胎盘细胞通过胎盘组织的物理解离收集,其中所述物理解离包括酶促消化,其可通过利用一种或多种组织消化酶来完成。 [0,164] In another specific embodiment, isolated placental cells by placental tissue was collected from the understanding, wherein said composition comprises from enzymatic digestion appreciated that by using one or more of tissue digestion enzymes to complete. 胎盘或其部分还可W被物理的解离,并用一种或多种酶消化,然后将获得的材料浸入细胞收集组合物或与细胞收集组合物混合。 Placenta or a portion thereof may be isolated physically W solution, and digested with one or more enzymes, the obtained material is then immersed in the cell collection composition, or mixed with a cell collection composition.

[0165] 优选的细胞收集组合物包含一种或多种组织解离酶。 [0165] A preferred cell collection composition comprises one or more tissue dissociation enzyme. 可用于裂解胎盘组织的酶包括木瓜蛋白酶、脱氧核糖核酸酶、丝氨酸蛋白酶如膜蛋白酶、糜蛋白酶、胶原酶、分散酶或弹性蛋白酶。 Enzymatic disruption of placental tissue may be used include papain, deoxyribonucleases, serine proteases such as films, chymotrypsin, collagenase, dispase, or elastase. 丝氨酸蛋白酶可被血清中的α2微球蛋白所抑制,因此用于消化的介质通常是无血清的。 Serine proteases can be suppressed α2 microglobulin in serum and therefore the medium used for digestion is usually serum-free. 在酶促消化过程中通常使用抓ΤΑ和DNA酶来提高细胞回收的效率。 In the commonly used enzymatic digestion enzymes and DNA grasping ΤΑ to improve the efficiency of cell recovery. 消化物优选被稀释从而避免细胞陷入粘稠的消化物中。 Digestion is preferably diluted into the cell so as to avoid viscous digest.

[0166] 可使用任何组织酶切酶的组合。 [0166] may be used in combination with any tissue digestion enzyme. 膜蛋白酶的酶切消化典型浓度包括0.1%至约2% 膜蛋白酶,例如约0.25 %膜蛋白酶。 Typical concentrations of protease digestion membrane comprises from about 0.1% to 2% of a protease film, for example about 0.25% protease film. 可W组合使用蛋白酶,即在同一消化反应中使用两种或多种蛋白酶,或者可W按顺序相继使用两种或多种蛋白酶,从而释放胎盘细胞,例如胎盘干细胞和胎盘多能细胞。 W protease may be used in combination, i.e., the reaction of two or more proteases, or W may be used sequentially in the order of two or more proteases in the same digestion, thereby releasing the placental cells, e.g., placental stem cells and placental multipotent cells. 例如,在一个实施方案中,胎盘或其部分首先用合适量的I型胶原酶按约1至约2mg/ml消化例如30分钟,然后用0.25%的膜蛋白酶例如在37C消化10分钟。 For example, in one embodiment, a placenta or portion thereof first with an appropriate amount of collagenase I by about 1 to about 2mg / ml digestion example, 30 minutes, and then digested at e.g. 37 C 10 min 0.25% protease film. 丝氨酸蛋白酶优选在使用其它酶后再连续使用。 Serine protease is preferably used continuously after the other enzymes.

[0167] 在另一个实施方案中,在从干细胞收集组合物中分离干细胞之前,向含有干细胞的干细胞收集组合物,或者向在其中解离和/或消化组织的溶液中添加馨合剂来进一步解离组织,所述馨合剂例如乙二醇双(2-氨基乙酸)-Ν,Ν,Ν'Ν'-四乙酸化GTA)或乙二胺四乙酸化DTA)。 [0167] In another embodiment, prior to the separation of stem cells from the stem cell collection composition, or added Xin mixture to where the dissociation and / or digested tissue solution to the stem cell collection composition comprising stem cells further Solutions from the tissue, said hing agent such as ethylene glycol bis (2-acetate) -Ν, Ν, Ν'Ν'- tetraethylammonium acidified GTA) or ethylenediaminetetraacetic acidified DTA).

[0168] 酶切消化后,消化物例如用培养基洗涂Ξ次,并且将洗涂的细胞接种入培养瓶中。 After [0168] digestion, digests e.g. Ξ times with wash-coated medium, and the cells were seeded into washcoated culture flasks. 随后通过差异粘附分离细胞,并且鉴定其存活力、细胞表面标记、分化等等。 Then the cells isolated by differential adherence, and identifying its viability, cell surface markers, differentiation and the like.

[0169] 可W理解,当整个胎盘或胎盘的一部分同时含有胎儿和母体细胞(例如,胎盘的一部分包含绒毛膜或绒毛叶)时,分离的胎盘干细胞将包含来源于胎儿和母体源的胎盘细胞的混合物。 [0169] W can be appreciated, when the part of the placenta or placental contain both fetal and maternal cells (e.g., a portion of the placenta comprises the chorion or cotyledons), the isolated placental stem cells comprise placental cells derived from both fetal and maternal sources mixture. 当胎盘的部分不含有或只含有可忽略量的母体细胞(例如,羊膜)时,从其中分离的胎盘细胞将几乎只含有胎儿的胚胎细胞(即具有胎儿基因型的胎盘细胞)。 When the portion of the placenta contains no or only a negligible amount of the parent cells (e.g., amnion), from which the isolated placental cells almost exclusively of fetal embryonic cells comprising (i.e. placental cells having the fetal genotype).

[0170] 胎盘细胞,例如W上5.4.2节所述的胎盘细胞可通过差异膜蛋白酶消化(参见W下5.5.5节)从解离的胎盘组织分离,随后在新鲜增殖培养基中在一种或多种新的培养容器中培养,任选随后进行第二次差异膜蛋白酶消化步骤。 [0170] Placental cells, e.g., Section 5.4.2, the placental cells from the W film can be obtained by differences in protease digestion (see Section 5.5.5 under W) dissociated from placental tissue separation, followed by a fresh proliferation medium one or more new culture containers in culture, optionally followed by a second difference in film protease digestion step.

[0171] 5.5.4胎盘灌流 [0171] 5.5.4 Placental Perfusion

[0172] 胎盘细胞,例如W上5.4.2节所述的胎盘细胞还可W通过灌流哺乳动物胎盘而获得。 [0172] Placental cells, e.g., the W section 5.4.2 W placental cells can also be obtained by perfusion of a mammalian placenta. 灌流哺乳动物胎盘W获得胎盘细胞的方法公开在例如化riri,美国专利号7,045,148和7,255,729中,和美国专利申请公开号2007/0275362和2007/0190042中,每篇的公开内容在此整体引入作为参考。 Method W mammalian placenta to obtain placental perfusate cells are disclosed, for example, of riri, U.S. Patent Nos. 7,045,148 and 7,255,729, and U.S. Patent Application Publication No. 2007/0275362 and 2007/0190042, the disclosures of each incorporated by reference in entirety.

[0173] 可W利用例如细胞收集组合物作为灌流液,通过灌流例如胎盘脉管系统来收集胎盘细胞。 [0173] W may be, for example, using a cell collection composition as a perfusion fluid, placental cells collected by perfusion, for example, the placental vasculature. 在一个实施方案中,通过使灌流液流经厮动脉和/或厮静脉来灌流哺乳动物胎盘。 In one embodiment, to flow through perfusion by perfusion servant arteries and / or veins servant mammalian placenta. 可W利用如重力流入胎盘来实现灌流液在胎盘中的流动。 The gravity into the placental perfusate flow is achieved in the placenta W can be used. 优选地,利用累,例如蠕动累,来迫使灌流液流经胎盘。 Preferably, use tired, such as a peristaltic tired, to force the perfusion fluid flow through the placenta. 例如,可W用套管,如TEFLON愈或塑料套管对厮静脉进行插管,所述套管与无菌的连接装置,如无菌管道相连。 For example, W can be a cannula, such as a plastic sleeve of TEFLON or more servant vein cannulation, the cannula with a sterile connection apparatus, such as sterile piping connected. 无菌的连接装置与灌流歧管相连。 The sterile connecting device is connected to the perfusion manifold.

[0174] 在准备灌流中,胎盘优选按厮动脉和厮静脉位于胎盘最高点的方式来定位(如,悬挂)。 [0174] In preparation perfusion, the placenta is preferably press servant artery and vein in the servant highest point of the placenta manner oriented (e.g., suspended). 可W通过使灌流液在胎盘脉管系统或在胎盘脉管系统和相邻组织中的流通来灌流胎盘。 W may be in the placenta by perfusion fluid flows through the placental vasculature or adjacent vasculature and tissue perfusion to the placenta. 所述胎盘还可W通过使灌流液流经厮静脉并从厮动脉收集,或者流经厮动脉并从厮静脉收集而灌流。 The placenta can also be perfused from the servant W and collected by venous perfusion flow through the artery servant collected from vein servant, or flowing through arteries and servant.

[0175] 在一个实施方案中,厮动脉和厮静脉同时连接移液器,所述移液器通过可变的连接管与灌流液的储器相连。 [0175] In one embodiment, the servant arterial and venous servant simultaneously connected pipette, the pipette is connected to the reservoir by a variable connection pipe perfusate. 所述灌流液流入厮静脉和动脉。 The perfusate inflow servant veins and arteries. 所述灌流液渗出和/或流经血管壁进入胎盘的周围组织,并从在孕期附着于母体子宫上的胎盘表面合适的开放脉管中收集。 The perfusion fluid leakage and / or flow through the vessel walls into the surrounding tissue of the placenta, and is collected from the placenta during pregnancy is attached to the surface of the uterus of the mother suitable open vessel. 所述灌流液还可w通过厮带开口导入,并允许从与母体子宫壁接触的胎盘壁中的开口流出或渗出。 The perfusion fluid may also be introduced through the opening w servant band, and allow to flow or percolate from the wall of the placenta in contact with the maternal uterine wall opening. 通过可称为"盘式"法(pan method)的方法收集的胎盘细胞通常为胎儿和母体细胞的混合物。 Placental cells may be referred to by the method "disc" method (pan method) is generally collected in a mixture of fetal and maternal cells.

[0176] 在另一个实施方案中,所述灌注溶液通过厮静脉并且从厮动脉收集,或通过厮动脉并且从厮静脉收集。 [0176] In another embodiment, the perfusion solution through the vein and collected from the servant servant artery, or by a servant artery and collected from the vein servant. 通过可称为"闭合回路"法的方法收集的胎盘细胞通常几乎只有胎儿细胞。 Placental cells may be referred to by the method "closed loop" method usually collected almost exclusively fetal cells.

[0177] 可W理解,利用盘式法灌流获得的是胎儿和母体细胞的混合物,即通过该方法,灌流液在其从胎盘的母体侧渗出后被收集。 [0177] W can be appreciated that perfusion using the pan method is obtained a mixture of fetal and maternal cells, i.e., by this method, the perfusate was collected on being exuded from the maternal side of the placenta. 结果,通过该方法收集的细胞包含混合的胎儿和母体来源的胎盘细胞,例如胎盘干细胞或胎盘多能细胞群。 As a result, the cells collected by this method comprise placental cells derived from both fetal and maternal mixed, e.g., placental stem cells or placental multipotent cells. 相反,仅通过紧密回路方法中的胎盘脉管系统灌流,因为灌流液流经一根或两根胎盘血管,并仅通过剩余的血管收集,将导致胎盘细胞群的收集物几乎都是胎儿来源的。 In contrast, only by close loop method placental vasculature perfusion because perfusion flow through one or two placental vessels and is only collected through the remaining vessel, will result in collection of placental cells are fetal in origin almost .

[0178] 在一个实施方案中,可如下进行所述紧密回路灌流方法。 [0178] In one embodiment, it can be carried out by the close loop perfusion method. 在分娩后约48小时内获得产后胎盘。 Post-partum placenta is obtained within about 48 hours after birth. 夹紧厮带并且在夹子上割断。 Servant clamping band cut to the clip and. 厮带可丢弃或处理回收例如厮带干细胞和/或处理所述厮带膜用于生产生物材料。 Servant may be discarded or treated with e.g. servant recovered with stem cells and / or process for the production of films with the servant biomaterial. 灌流期间可保留羊膜,或可例如利用手指直接剖开而从绒毛膜分离。 Amniotic membrane can be retained during perfusion, or can be taken directly, for example with a finger separated from the chorion. 如果所述羊膜在灌流之前从绒毛膜分离,其可例如丢弃或通过酶促消化处理W获得干细胞,或生制备例如羊膜生物材料,如美国申请公开号2004/0048796中所述的生物材料,其公开内容在此全文引入作为参考。 If the separation of amnion from chorion, which may be obtained, for example, discarding or amniotic stem cells, for example, biological material, as described in US application 2004/0048796 said biomaterial by enzymatic digestion Publication No. W, raw or prepared prior to perfusion, it the disclosure of which is hereby incorporated by reference. 在例如利用无菌纱布清理胎盘全部可见的血块和残余血液后,如通过部分切割厮带膜W暴露厮带横截面而暴露厮带血管。 After cleaning the placenta, for example, using sterile gauze all visible blood clots and residual blood, such as a blood vessel is exposed by partially dicing tape servant servant W film strip exposed cross-section with a servant. 辨别血管,并通过例如推进经过每一血管切割端的闭合弹黃打开血管。 Identify blood vessels, and blood vessels opened by, for example through the cut end of each vessel closing spring propulsion. 装置,例如连接至灌流装置或蠕动累的塑料管材,随后被插入每一胎盘动脉中。 It means, for example to perfusion device or peristaltic tired plastic pipe, then inserted into each of the placental arteries. 累可W是适于所述目的的任何累,例如蠕动累。 W is tired can be adapted to any of the tired purposes, such as a peristaltic tired. 连接至无菌收集胆存器的塑料管状物,例如250mL收集袋的血袋,随后被插入胎盘静脉中。 Bile collector connected to a sterile plastic tube register, e.g. 250mL blood bag collection bag, is then inserted into the placental vein. 或者,连接至累的管状物被插入胎盘静脉中,并且连接至收集胆存器的管被插入一条或两条胎盘动脉中。 Alternatively, the tired connected to the tubular is inserted into the placental vein, and is connected to the collector of the register bladder tube is inserted in one or both of the placental arteries. 胎盘随后用大量灌流液灌流,例如约750mL灌流液。 Placental perfusate perfusion followed by a large number, e.g. about 750mL perfusate. 随后例如通过离屯、收集灌流液中的细胞。 Then, for example, by ex Tun collected perfusate cells. 在某些实施方案中,所述胎盘用灌流液灌流,例如100-300mL灌流液,W 在灌流收集胎盘细胞(如胎盘干细胞和/或胎盘多能细胞)之前除去残余血液。 In certain embodiments, the placental perfusion with perfusate, perfusate 100-300mL e.g., W collect placental cells (e.g., placental stem cells and / or placental multipotent cells) to remove residual blood prior to perfusion. 在另一个实施方案中,所述胎盘在灌流收集胎盘细胞之前不用灌流液灌流W除去残余血液。 In another embodiment, said placental perfusate without perfusion prior to perfusion to collect placental cells W remove residual blood.

[0179] 在一个实施方案中,在灌流过程中夹紧厮带近端,更优选,在厮带插入胎盘的4- 5cm(厘米)内夹紧。 [0179] In one embodiment, the perfusion process of the clamping band servant proximal end, more preferably, with the plug-in servant placenta 4- 5cm (cm) clamp.

[0180] 在放血过程中从哺乳动物胎盘首先收集的灌流液一般都被厮带血和/或胎盘血残留的血红细胞着色。 [0180] In the blood from a mammalian placenta during the first collecting perfusate are generally servant blood and / or placental blood colored red blood cells remaining. 随着灌流继续和残留的厮带血细胞从胎盘中洗出,灌流液颜色变得越来越浅。 With continued perfusion and residual blood cells are washed out of the servant placenta perfusate color becomes more shallow. 一般30至lOOmL(毫升)灌流液足W初步将胎盘放血,但根据观察的结果可W使用或多或少的灌流液。 Usually 30 to lOOmL (mL) Initial W perfusate foot placenta blood, but may be the result of observation using perfusate W or less.

[0181] 用于收集胎盘干细胞的灌流液体积可W根据收集的干细胞数量、胎盘大小、对单个胎盘进行的收集次数等变化。 Volume of perfusate [0181] used to collect placental stem cells may be W changes the number of stem cells collected, the size of the placenta, the placenta individual collection times and the like in accordance with. 在不同的实施方案中,灌流液的体积可W选自50mL至5000mL、50 血至4000mL、50 血至3000mL、lOOmL 至2000mL、250血至2000血、500血至2000mL、或750mL至2000mL。 In various embodiments, the volume of perfusion liquid may be W is selected from 50mL to 5000mL, 50 to 4000 mL blood, blood 50 to 3000mL, lOOmL to 2000mL, 250 to 2000 blood serum, blood 500 to 2000mL, or 750mL to 2000mL. 一般的,在放血后用700-800mL灌流液来灌流胎盘。 Generally, after exsanguination perfusion with 700-800mL to placental perfusate.

[0182] 胎盘可W在数小时或数天的过程中进行多次灌流。 [0182] placenta can be perfused a plurality of times during W hours or days of. 当胎盘进行多次灌流时,可W 在容器或其它合适的器皿中在无菌条件下维持或培养,并用细胞收集组合物或标准灌流液(例如,普通的盐溶液如憐酸盐缓冲液("PBS"))灌流,其中含有或不含抗凝剂(如,肝素、华法令钢、香豆素、双香豆素),和/或含有或不含抗微生物剂(如,β-琉基乙醇(O.lmM);抗生素如链霉素(如40-100μg/ml)、青霉素(如40U/ml)、两性霉素B(如0.化g/ml))。 When multiple perfused placenta, W can be maintained in a container or other suitable vessel under sterile conditions or the culture, and collecting the composition with a cell or a standard perfusion solution (e.g., common salt solution such as pity formate buffer ( "PBS")) perfusion, or containing no anticoagulant (e.g., heparin, warfarin steel, coumarin, dicumarol), and / or with or without an antimicrobial agent (e.g., [beta] sulfur yl ethanol (O.lmM); antibiotics such as streptomycin (e.g., 40-100μg / ml), penicillin (e.g., 40U / ml), amphotericin B (eg of 0.5 g / ml)). 在一个实施方案中,将分离的胎盘维持或培养一段时间而没有收集灌流液,从而所述胎盘在灌流和收集灌流液前,被维持或培养1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、 22、23或24个小时,或者2或3或更多天。 In one embodiment, the isolated placenta is maintained or cultured for a period of time without collecting the perfusate, such that the placenta prior to perfusion and collection of perfusate is maintained or cultured 1,2,3,4,5,6,7 , 8,9,10,11,12,13,14,15,16,17,18,19,20,21, 22, 23 or 24 hours, or 2 or 3 or more days. 灌流的胎盘可W被维持用于再进行一次或多次灌流,例如,再维持1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24 或更多个小时,并用如700-800mL灌流液再灌流第二次。 W perfused placenta can be maintained for further one or more perfusion, e.g., maintained for 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15 , 16,17,18,19,20,21,22,23,24 or more hours, and treated with reperfusion as 700-800mL perfusate second. 所述胎盘可W灌流1、2、3、4、5或更多次,例如每1、2、3、4、5或6小时一次。 The placenta can be perfused 1, 2, W or more times, for example every 5, or 6 hours. 在优选的实施方案中,重复灌流胎盘和收集灌流液(如细胞收集组合物),直至回收的有核细胞数低于100细胞/ml。 In a preferred embodiment, perfusion of the placenta and collection was repeated perfusate (e.g., cell collection composition) until the recovered nucleated cell count below 100 cells / ml. 对不同时间点的灌流液可W分别进一步处理,W回收时间依赖性的细胞群,如干细胞。 Different time points of the perfusate may be further processed separately W, W cell population recovered time-dependent, such as stem cells. 不同时间点的灌流液也可W被混合。 Perfusate different time points W may also be mixed. 在优选的实施方案中,在放血后约8小时和约18个小时之间一次或多次收集胎盘细胞。 In a preferred embodiment, one or more placental cells are collected between about 18 hours and about 8 hours after phlebotomy.

[0183] 优选的,灌流导致获得的胎盘干细胞数量显著多于从未用所述溶液灌流或者未经其它处理(例如,通过组织解离如酶促消化)的哺乳动物胎盘中可获得的干细胞数量。 [0183] Preferably, the number of placental stem cells perfused cause significantly higher than obtained with the solution never without perfusion or other processing (e.g., by enzymatic digestion of tissue, such as the dissociation) is available in a mammalian placenta stem cell number . 在本文的上下文中,"显著多于"是指至少多10%。 In this context, "significantly more" means at least 10% more. 灌流产生的胎盘干细胞显著多于例如,从培养胎盘或一部分胎盘的培养基中可分离的胎盘细胞数量。 Perfusion placental stem cells produced significantly more than for example, the number of placental cells may be isolated from the culture medium of the placenta or a portion of the placenta.

[0184] 通过用包含一种或多种蛋白酶或其他组织解离酶的溶液灌流,可W从胎盘中分离胎盘细胞。 [0184] by treatment with proteases comprising one or more perfusion solution or other tissue dissociation enzyme, W may be isolated placental cells from the placenta. 在具体的实施方案中,将胎盘或其一部分(例如,羊膜、羊膜和绒毛膜、胎盘小叶或绒毛小叶、厮带或任何上述的组合)置于25^37C,并在200mL培养基中用一种或多种组织解离酶解育30分钟。 In a specific embodiment, a placenta or portion thereof (e.g., amniotic membrane, amnion and chorion, placental lobule or villi leaflets, servant or any combination of the above tape) was placed 25 ^ 37 C, and 200mL medium enzymatic dissociation incubated for 30 minutes with one or more tissue. 收集灌流液中的细胞,降低溫度至4C,并用包含5mM抓TA、2mM二硫苏糖醇和琉基乙醇的冷却抑制剂混合物洗涂。 Collecting perfusate cells, lowering the temperature to 4 C, and treated with 5mM comprising grasping the TA, the mixture was cooled inhibitor 2mM dithiothreitol and thiol ethanol washcoat. 数分钟后,用冷却的(如4C)干细胞收集组合物洗涂所述胎盘细胞。 After a few minutes, with cooling (e.g., 4 C) stem cell collection composition of the wash-coated placental cells.

[0185] 5.5.5胎盘细胞的分离、分选和表征 [0185] 5.5.5 isolated placental cells, sorting and characterization

[0186] 分离的胎盘细胞,例如W上5.4.2节所述的细胞,不论是否由灌流或物理解离(如通过酶促消化)获得的,可W通过聚薦糖(Ficoll)梯度离屯、从其它细胞中初步纯化(即分离)。 [0186] The isolated placental cells, e.g. cells on section 5.4.2 of the W, whether or understood from perfusion was isolated (e.g., by enzymatic digestion) obtained by polymerizing W may be recommended sugar (Ficoll) gradient centrifugation Tun preliminary purified from other cells (i.e., separated). 此类离屯、可W遵循任何标准离屯、方法的速度等。 Such from Tun, W may follow any standard off velocity Tun, methods and the like. 例如,在一个实施方案中,从胎盘收集的细胞是通过在5000Xg室溫离屯、15分钟从灌流液中回收的,其将细胞与例如污染的残渣和血小板分离开。 For example, in one embodiment, cells from placenta are collected by Tun from room temperature at 5000Xg for 15 minutes recovered from perfusate, which cells such as pollution and the residue was separated from platelets. 在另一个实施方案中,胎盘灌流液被浓缩至约200mL,轻轻地铺在Ficoll 上,在22CW约llOOXg离屯、20分钟,收集低密度的细胞中间层用于进一步处理。 In another embodiment, placental perfusate is concentrated to about 200 mL, gently ground floor on Ficoll, at 22 CW from about llOOXg Tun 20 minutes, the intermediate layer of low density cells were collected for further processing.

[0187] 细胞沉淀可重悬于新鲜的干细胞收集组合物中,或适合维持干细胞的培养基中, 例如含有2U/ml肝素和2mM抓TA的IMDM无血清培养基(GibcoBRUNY)。 [0187] Cell pellets can be resuspended in fresh stem cell collection composition, or a medium suitable for maintaining the stem cells, for example, containing 2U / ml heparin and 2mM TA grip of IMDM serum-free medium (GibcoBRUNY). 可W利用例如LYMPHOP民邸⑥(Nycomed Pharma,Oslo,挪威),按照生产商的推荐方法来分离总单核细胞部分。 W may be using, for example LYMPHOP China Di ⑥ (Nycomed Pharma, Oslo, Norway) according to the method recommended by the manufacturer to isolate total mononuclear cell fraction.

[0188] 通过灌流或消化获得的胎盘细胞,例如,可W采用,如含有0.2 %抓TA(Sigma, St.Louis MO)的0.05%胃蛋白酶溶液,通过差异膜酶消化来进一步或初步地分离。 [0188] Placental cells obtained by perfusion or digestion, e.g., W can be employed, such as those containing 0.2% grip TA (Sigma, St.Louis MO) 0.05% pepsin solution, or to preliminarily separated by the difference in film further enzymatic digestion . 由于分离的胎盘细胞一般在约5分钟内从塑料表面脱离,而其它附着的群一般需要解育超过20-30 分钟才从塑料表面脱离,因此差异膜酶消化是可能的。 Since the isolated placental cells detached from plastic surfaces generally in about 5 minutes, while the other attachment group typically require more than 20-30 minutes incubation solution was removed from the plastic surface, so the difference in membrane enzyme digestion possible. 在膜酶消化和利用例如膜蛋白酶中和溶液(TNS,Cambrex)的膜蛋白酶中和后,可W收获脱离的胎盘细胞。 Placental cells, e.g. digest and use of the protease and the membrane solution (TNS, Cambrex) and membrane protease, W may be harvested from the membrane enzyme. 在分离附着细胞的一个实施方案中,等份的细胞,如5^0 X 106个细胞被放置在每个T-75瓶中,优选纤连蛋白包被的T75瓶中。 In one embodiment, attached cells are isolated, aliquots of cells, such as 5 ^ 0 X 106 cells were placed in each T75 flasks, preferably fibronectin-coated T75 flasks. 在运样的实施方案中,所述细胞可W用商购的间充质干细胞生长培养基(MSCGMKCambrex)培养,并且置于组织培养箱(37C,5%C02)中。 In an embodiment of the sample transport, the cells can be mesenchymal stem cells with W between commercially available growth medium (MSCGMKCambrex) culture and placed in a tissue culture incubator (37 C, 5% C02) in the. 在10-15天后,通过用PBS 洗涂从瓶中去除非附着细胞。 In 10-15 days, and washed with PBS to remove non-coated adherent cells from the flask. 然后用MSCGM替代PBS。 And replace with PBS MSCGM. 优选每天检查培养瓶中不同附着细胞类型的存在,并且特别的鉴别和扩增成纤维样细胞簇。 Preferably examined daily for the presence of the culture flasks of different types of cell adhesion, and particularly the identification and amplification of fibroblast-like cell clusters.

[0189] 从哺乳动物胎盘中收集的细胞数量和类型可W被监测:例如通过利用标准的细胞检测技术,如流式细胞仪、细胞分选、免疫细胞化学(例如,用组织特异性或细胞标志特异性抗体染色)、巧光活化细胞分选(FACS)、磁性活化细胞分选(MACS)来测量细胞的表面标记和形态学;通过利用光学或共聚焦显微镜来检测细胞形态学;和/或利用本领域公知的技术, 例如PCR和基因表达谱来检测基因表达的改变。 [0189] The number and type of cells collected from a mammalian placenta can be monitored W: for example, using standard cell detection techniques such as flow cytometry, cell sorting, immunocytochemistry (e.g., with tissue specific or cell flag-specific antibody staining), Qiao light activated cell sorting (FACS), magnetic activated cell sorting (the MACS) measuring surface markers and morphology of cells; cell morphology detected by using light or confocal microscopy; and / or using techniques well known in the art, such as PCR and gene expression profiling to detect changes in gene expression. 运些技术也可用于鉴别对于一种或多种特定标记呈阳性的细胞。 These techniques may also be transported for identifying one or more specific marker for positive cells. 例如,利用CD34的抗体,利用上述技术,可W确定细胞是否含有可检测量的CD34;如果是,则细胞是CD34+。 For example, using antibodies to CD34, using the techniques described above, W may be determined if a cell contains a detectable amount of CD34; if so, the cell is CD34 +. 同时,如果细胞产生足够的能被RT-PCR所检测的0CT- 4RNA,或者显著多于成体细胞的0CT-4RNA,则该细胞是0CT-4+。 Meanwhile, if the cell produces 0CT- 4RNA, or significantly more than adult cells 0CT-4RNA be sufficiently detected by RT-PCR, the cells are 0CT-4 +. 细胞表面标记(例如CD标记如CD34)的抗体,和干细胞特异性基因例如0CT-4的序列,也是本领域公知的。 Antibodies to cell surface markers (e.g., CD markers such as of CD34), the stem cell-specific genes, and for example, the sequence 0CT-4, also known in the art.

[0190] 胎盘细胞,特别是已经经过Ficoll分离、差别附着,或两者的结合所分离的细胞可W利用巧光活化细胞分选仪(FACS)来分选。 [0190] Placental cells, particularly has been subjected to Ficoll separation, differential adherence, or a combination of both isolated cells may be W clever use of light-activated cell sorter (FACS) sorting. 巧光活化细胞分选(FACS)是基于颗粒的巧光性质来分离颗粒(包括细胞)的普遍已知的方法化amarch,1987,Methods Enzymol,151:150- 165)。 Qiao light activated cell sorting (FACS) is used to separate particles based on the optical properties of the particles Qiao (including cells) generally known methods of amarch, 1987, Methods Enzymol, 151: 150- 165). 激光激发单个颗粒中的巧光部分产生微小的电荷,从而允许从混合物中电磁分离正电颗粒和负电颗粒。 Qiao laser excitation light portion of the individual particles produce small charge, thus allowing electromagnetic separation of the positively charged particles and negatively charged particles from the mixture. 在一个实施方案中,用不同的巧光标签来标记细胞表面标记特异性抗体或配体。 In one embodiment, light with a different label to mark clever cell surface marker-specific antibodies or ligands. 细胞经过细胞分选仪处理,从而可W基于其与所用抗体的结合能力来分离细胞。 Cells after cell sorter processing, whereby W can be isolated based on their ability to bind to the antibody-producing cells used. FACS分选的颗粒可W直接注入96-孔或38C孔板的单个孔中,从而便于分离和克隆。 FACS sorted particles may be directly injected into the individual wells W or 96-well plates 38C, thereby to facilitate separation and cloning.

[0191] 在一个分选技术方案中,来源于胎盘的干细胞基于标记CD34、CD38、CD44、CD45、 CD73、CD105、0CT-4和/或HLA-G的表达来分选。 [0191] In one aspect sorting, placenta derived stem cells based on expression of markers CD34, CD38, CD44, CD45, CD73, CD105,0CT-4 and / or HLA-G to sorting. 运可W结合基于细胞在培养中的附着性质来选择细胞的步骤来实现。 W may be transported in conjunction with the step of selecting cells based on adhesion properties of cells in culture is achieved. 例如,可W在基于标记表达的分选之前或之后进行组织培养塑料制品附着选择。 For example, W can be attached to tissue culture plastic sorting selected before or after the marker expression based. 例如,在一个实施方案中,首先基于CD34的表达来分选细胞;保留CD34-的细胞,并将CD200+HLA-G+的细胞与所有其它CD%-细胞分离。 For example, in one embodiment, based on CD34 expression is first sorting cells; retention CD34- cells, and CD200 + HLA-G + cells and all other CD% - cell separation. 在另一个实施方案中,基于标记CD200和/或HLA-G的表达来分选来源于胎盘的细胞;例如,表现出任一运些标记的细胞被分离W供进一步使用。 In another embodiment, based on the expression of markers CD200 and / or HLA-G of sorting cells derived from placenta; for example, some of the performance as a transport W labeled cells were isolated for further use. 在具体实施方案中,表达例如CD200和/或HLA-G的细胞可W进一步被分选,所述分选可W基于CD73和/或CD105的表达,或基于体SH2、SH3或甜4识别的表位,或基于CD34XD38或CD45的表达的缺失。 In a particular embodiment, for example, CD200 expression and / or HLA-G W cells may be sorted further, the sorting may be based on W express CD73 and / or CD105 or based on the volume SH2, SH3 or sweet identified 4 epitope, or based on the absence of CD45 expression or CD34XD38. 例如,在一个实施方案中,胎盘细胞通过CD200、化AG、 〔073、〔0105、〔034、〔038和〔045的表达或其缺失来分选,并且将〔0200+、化4-6+、〔073+、〔0105 +、CD34-、CD38-和CD45-的细胞与其它胎盘细胞分离,供进一步使用。 For example, in one embodiment, placental cells via CD200, of AG, [073, [0105, [034, 045 [038 and [expression or lack thereof to sorting, and the [0200+, of 4-6 + , 073 [+, [0105 +, CD34-, CD38- and CD45- cells isolated from other placental cells for further use.

[0192] 在任何上述分选胎盘细胞的实施方案的具体实施方案中,分选后保留的细胞群中至少50 %、60 %、70 %、80 %、90 %或95 %的细胞为所述分离的胎盘细胞。 [0192] In a specific embodiment of any of the above embodiments of sorted placental cells in a cell population after sorting retain at least 50%, 60%, 70%, 80%, 90% or 95% of the cells of the isolated placental cells. 可通过W上5.4.2 节所述的一种或多种任何标记分选胎盘细胞。 Any marker can be sorted by means of a placental cells described in Section 5.4.2 of the W or more.

[0193] 在一个具体的实施方案中,从其他胎盘细胞中分选(即分离)(1)附着于组织培养塑料制品,和(2)CD10+、CD34-和CD105+的胎盘细胞。 [0193] In one specific embodiment, the placental cells from other sorted from (i.e., isolated) (1) adherent to tissue culture plastic, and (2) CD10 +, CD34-, and CD105 + placental cells. 在另外具体的实施方案中,从其他胎盘细胞分选(即分离)(1)附着于组织培养塑料制品,和(2)CD10+、CD34-、CD105+和CD200+的胎盘细胞。 In another specific embodiment, the placental cells from other sorting (i.e., separation) (1) adherent to tissue culture plastic, and (2) CD10 +, CD34-, CD105 + and CD200 + placental cells. 在另外具体的实施方案中,从其他胎盘细胞分选(即分离)(1)附着于组织培养塑料制品,和(2)〔010+、〔034-、〔045-、〔090+、〔0105+和〔0200+的胎盘细胞。 In another specific embodiment, from other placental cell sorting (i.e., separation) (1) adherent to tissue culture plastic, and (2) [010, [034--, [045--, [090 +, [0105 [0200+ + and placental cells.

[0194]对于抗体-介导的胎盘细胞(例如胎盘干细胞或胎盘多能细胞)的检测和分选,可与适于检测和分选细胞的任何巧光团或其他标记物(例如巧光-活化细胞分选)结合,使用对特定标记特异性的任何抗体。 [0194] For antibody - mediated placental cells (e.g., placental stem cells or placental multipotent cells) detection and sorting, and may be adapted to detect any He Qiaoguang sorted cells or other marker group (e.g. Qiao light - activated cell sorting) binding any antibody specific for a particular marker. 特异性标记的抗体/巧光组合包括,但不限于与巧光素异硫氯酸醋(FITC)偶联的HLA-G(获自Serotec,Raleigh, Nodh Carol ina)、CD10 (获自BD Immunocytometry Systems , San Jose,Cal if ornia)、CD44(获自BD Biosciences Pharmingen,San Jose,California)和CD 105(获自R&D Systems Inc . ,Minneapolis , 11111163〇1曰)单克隆抗体;与藻红蛋白"6)偶联的〔044、〔0200、〔0117和〔0 13(80 Biosciences Pharmingen)单克隆抗体;与别藻蓝蛋白(APC)偶联的链亲和素和CD38单克隆抗体(BD Biosciences Pharmingen);和生物素化的CD90(BD Biosciences Pharmingen)。 可用的其他抗体包括,但不限于,CD133-APC(Milten}d)、邸R-Biotin(CD309,Abeam)、细胞角蛋白K-Fi tc (Sigma或Dako)、HLA ABC-Fi tc (抓)、HLA DR,DQ,DP-PE (抓)、β-2-微球蛋白-PE (抓)、CD80-PE(抓)和CD86-APC(抓)。 Antibody / clever combination of light specific markers include, but are not limited to, an optical element Qiao acid isothiocyanato vinegar (FITC) conjugated HLA-G (available from Serotec, Raleigh, Nodh Carol ina), CD10 (available from BD Immunocytometry Systems, San Jose, Cal if ornia), CD44 (available from BD Biosciences Pharmingen, San Jose, California), and CD 105 (available from R & D Systems Inc, Minneapolis, said 11111163〇1) a monoclonal antibody;. phycoerythrin " 6) coupled [044, [0200, [0117 and [0 13 (80 Biosciences Pharmingen) monoclonal antibody; with allophycocyanin (APC) conjugated streptavidin and monoclonal antibodies CD38 (BD Biosciences Pharmingen ); and a biotinylated CD90 (BD Biosciences Pharmingen) other antibodies can be used include, but are not limited to, CD133-APC (Milten} d), Di R-biotin (CD309, Abeam), cytokeratin K-Fi tc. (Sigma or Dako), HLA ABC-Fi tc (grasping), HLA DR, DQ, DP-PE (grasping), β-2- microglobulin -PE (grasping), CD80-PE (grip) and CD86-APC (Grab).

[01巧]其他可用的抗体/标记组合包括,但不限于CD45TerCP(巧叶绿素蛋白);CD44TE; CD19-PE;CD10-F(巧光素);HLA-GF和厂氨基-放线菌素-D(7-AAD) ;HLA-ABC-F等等。 [Qiao 01] Other useful antibody / marker combination including, but not limited to CD45TerCP (Qiao chlorophyll protein); CD44TE; CD19-PE; CD10-F (Qiao optical element); HLA-GF and plant amino - actinomycin - D (7-AAD); HLA-ABC-F and the like.

[0196] 此处提供的分离的胎盘细胞可利用例如与藻红蛋白-切5(PE切5)偶联的链亲和素和与生物素偶联的CD117或CD133单克隆抗体测定CD117或CD133;然而利用本系统,由于背景相对高,所述细胞似乎为CD117或CD133阳性。 [0196] The isolated placental cells provided herein can utilize, for example, phycoerythrin - conjugated streptavidin and biotin conjugated monoclonal antibody assay CD133 CD117 or CD117 or CD133 cut 5 (the PE cut 5) ; however, using the present system, since the background is relatively high, the cell appears to be CD117-positive or CD133.

[0197] 所述分离的胎盘细胞可用单个标记的抗体标记并且检测和分选。 [0197] The isolated placental cells can be labeled antibody and the labeled single detection and sorting. 胎盘细胞还可W 同时用多个不同标记的抗体标记。 W placental cells can also be simultaneously labeled with a plurality of differently labeled antibodies.

[0198] 在另一个实施方案中,可W使用磁珠来分离细胞。 [0198] In another embodiment, W can be isolated using magnetic beads to the cells. 可W利用磁性活化细胞分选(MACS)技术分选细胞,所述技术是基于颗粒结合磁珠(0.5-100μηι直径)的能力来分选颗粒的方法。 W may be using a magnetic activated cell sorting (the MACS) cell sorting techniques, the technology is based on the ability to bind magnetic beads (0.5-100μηι diameter) to points a method selected from the particles. 对磁微珠可W进行多种有效的修饰,包括共价添加特异性识别特定细胞表面分子或半抗原的抗体。 Magnetic beads can be effectively W various modifications, including covalent addition of specifically recognizes a particular cell surface molecule or hapten. 所述磁珠随后与细胞混合,从而结合。 The cells were then mixed with the beads, thereby binding. 然后将细胞通过磁场,W分离出具有特定细胞表面标记的细胞。 The cells were then isolated from cells having a specific cell surface marker by the magnetic field, W. 在一个实施方案中,可随后分离运些细胞,并与偶联了其它细胞表面标记抗体的磁珠再混合。 In one embodiment, these cells can then isolated operation, the labeled antibody and magnetic beads conjugated with other cell surface remix. 所述细胞再次通过磁场,分离结合了两种抗体的细胞。 The cells were again through a magnetic field, separation of bound cells both antibodies. 然后可W将此类细胞稀释放入不同的培养皿中,例如微滴培养皿中,用于克隆分离。 Such cells may then be diluted W into different petri dish droplets e.g., for clonal isolation.

[0199] 分离的胎盘细胞还可W基于细胞形态学和生长特征来表征和/或分选。 [0199] The isolated placental cells can also W based on cell morphology and growth characteristics characterized and / or sorted. 例如,分离的胎盘细胞可W表征为在培养中具有成纤维细胞样表型,和/或基于成纤维细胞样表型来选择。 For example, isolated placental cells can be characterized as having a W fibroblast-like phenotype, and / or into a cell-like phenotype-based fibers selected in culture. 所述分离的胎盘细胞还可W表征为具有形成胚样体的能力,和/或基于其形成胚样体的能力来选择。 The isolated placental cells can also be characterized as having the ability to W embryoid-like bodies, and / or based on its ability to form embryoid-like bodies are formed is selected. 在一个实施方案中,例如,将形状为成纤维细胞样,表达CD73和CD105,并在培养中产生一个或多个胚样体的胎盘细胞与其它胎盘细胞分离。 In one embodiment, for example, a fibroblast-like shape, express CD73 and CD105, and produce isolated placental cells from other placental cells one or more embryoid-like bodies in culture. 在另一个实施方案中,将培养中产生一个或多个胚样体的0CT-4+胎盘细胞与其它胎盘细胞分离。 In another embodiment, the generated 0CT-4 + placental cells are isolated from other placental cells one or more embryoid-like bodies in culture.

[0200] 在另一个实施方案中,分离的胎盘细胞可W通过集落形成单位试验来鉴别和表征。 [0200] In another embodiment, the isolated placental cells are identified by colony forming units of test and characterize W. 集落形成单位试验是本领域公知的,例如MESEN CULT™培养基(stem Cell Technologies,Inc.,Vancouver British Columbia)。 Colony forming unit assays are known in the art, e.g. MESEN CULT ™ medium (stem Cell Technologies, Inc., Vancouver British Columbia).

[0201] 利用本领域已知的标准技术,例如台吩蓝排除试验、醋酸巧光素摄取试验、舰化丙锭摄取试验(评估活力);和胸腺喀晚核巧摄取试验、MTT细胞增殖试验(评估增殖)来分析分离的胎盘细胞的活力、增殖潜力和寿命。 [0201] using standard techniques known in the art, e.g. thiophene station blue exclusion test, Qiao phototropins acid uptake assay, propidium ship uptake assay (assessed viability); Qiao and thymus Ka late nuclear uptake assay, the MTT cell proliferation assay (evaluation proliferation) to analyze the viability of isolated placental cells, proliferation potential, and longevity. 寿命可W通过本领域公知的方法确定,例如通过延长培养中群倍增的最大数来确定。 W life can be determined by methods known in the art, for example, be determined by the maximum number of population doubling in an extended culture.

[0202] 利用本领域已知的其它技术,也可W将分离的胎盘细胞,例如W上5.4.2节所述的分离的胎盘细胞,和其它胎盘细胞分开,例如:选择性生长期望的细胞(阳性分选)、选择性破坏不需要细胞(阴性选择)、基于混合群与例如大豆凝聚素的差异细胞可凝集性的分离、 冻融步骤、过滤、常规离屯、和区带离屯、、离屯、冲洗(逆流离屯、)、单位重力分离、逆流分布、电泳等等。 [0202] using other techniques known in the art, W may be isolated placental cells, e.g., isolated placental cells described in Section 5.4.2 of the W, and separated from other placental cells, for example: selective growth of desired cells (positive separation), does not require selective destruction of cells (negative selection), for example, based on the mixed population of soybean agglutinin differential cell agglutinability separation of freeze-thaw step, filtration, from the conventional Tun, zonal and village, , from the village, flush (counter-current from the Tun,), unit gravity separation, countercurrent distribution, electrophoresis and the like.

[0203] 5.6分离的胎盘细胞的培养 [0203] The isolated placental cells cultured in 5.6

[0204] 5.6.1 培养基 [0204] 5.6.1 Medium

[0205] 分离的胎盘细胞、或分离的胎盘细胞群、或从其中生长出胎盘干细胞的细胞或胎盘组织可被用于起始或接种细胞培养物。 [0205] The isolated placental cells or population of isolated placental cells, or cells grown from which placental stem cells or placental tissue may be used to initiate, or seed, cell cultures. 细胞一般转移到无菌的组织培养容器内,所述容器使用或未使用胞外基质或配体包被,例如层粘连蛋白、胶原(如天然的或变性的)、明胶、 纤连蛋白、鸟氨酸、玻连蛋白和胞外膜蛋白(例如:MATRIGEL瑕(BD Discovery Labware,Bedford,Mass.))。 Cells are typically transferred to a sterile tissue culture container, the container or without the use of the extracellular matrix or ligands coating, for example laminin, collagen (e.g., native or denatured), gelatin, fibronectin, ornithine acid, vitronectin, and extracellular membrane protein (e.g.: MATRIGEL flaw (BD Discovery Labware, Bedford, Mass).).

[0206] 可W在本领域认为适合细胞,例如适合干细胞培养的任何培养基和任何条件下, 培养分离的胎盘细胞。 [0206] W may be present in the art that suitable cells, for example, any dry media and any cell culture conditions, culturing the isolated placental cells. 优选的,培养基包含血清。 Preferably, the culture medium comprises serum. 分离的胎盘细胞可W培养在例如:DMEM-LG (Du化ecco改良的必需培养基,低糖)/MCDB201(鸡成纤维细胞基础培养基),其含有ITS(膜岛素-转铁蛋白-砸)、LA+BSA(亚油酸-牛血清白蛋白)、右旋糖、k抗坏血酸、PDGF、EGF、IGF- 1,和青霉素/链霉素;含有10%胎牛血清(FBS)的DMEM-HG(高糖);含有15%FBS的DMEM-HG; 含有10%FBS、10%马血清和氨化可的松的IMDM(Iscove改良的Du化ecco培养基);含有10% FBS、EGF和肝素的Ml99;含有10%FBS、GLUTAMAXTm和庆大霉素的α-ΜΕΜ(最低必需培养基);含有10%FBS、化UTAMAX™和庆大霉素等的DMEM。 Isolated placental cells can be cultured in, for example, W: DMEM-LG (Du of ecco modified essential medium, low glucose) / MCDB201 (chicken fibroblast basal medium) containing ITS (Insulin membrane - transferrin - smashing ), LA + BSA (linoleic acid - bovine serum albumin), dextrose, k ascorbic acid, PDGF, EGF, IGF- 1, and penicillin / streptomycin; DMEM-medium containing 10% (FBS) of HG (high glucose); containing 15% FBS in DMEM-HG; containing 10% FBS, 10% horse serum and ammoniated cortisone IMDM (Iscove modified ecco Du of medium); containing 10% FBS, EGF, and heparin Ml99; comprising 10% FBS, GLUTAMAXTm and gentamicin α-ΜΕΜ (Minimal essential medium); containing DMEM 10% FBS, gentamicin of UTAMAX ™ and the like.

[0207] 可用于培养胎盘细胞的其它培养基包括DMEM(高糖或低糖)、Eagle基础培养基、 Ham的F10培养基(F10)、Ham的F12培养基(F12)、Iscove的改良Du化ecco培养基、间充质干细胞生长培养基(MSCGM)、Liebovitz的レ15培养基、MCDB、DMIEM/F12、RPMI 1640、改良的DMEM (Gibco)、DMEM/MCDB201(Sigma)和CE化-GR0 FREE。 Other medium [0207] may be used to culture placental cells include DMEM (high glucose or sugar), Eagle basal medium, Ham's F10 medium (F10), Ham's F12 medium (F12), Iscove's Modified Du of ecco medium, mesenchymal Stem cell growth medium (MSCGM), Liebovitz's Rayon 15 medium, MCDB, DMIEM / F12, RPMI 1640, modified DMEM (Gibco), DMEM / MCDB201 (Sigma) and CE of -GR0 FREE.

[0208] 培养基中可W添加一种或多种组分,包括例如:血清(如:胎牛血清(FBS),优选约2-15%(乂八);马血清化5);人血清化5));护琉基乙醇(816),优选约0.001%(乂八);一种或多种生长因子,例如,血小板衍生的生长因子(PDGF)、表皮生长因子化GF)、基础成纤维细胞生长因子(bFGF)、膜岛素-样生长因子-l(IGF-l)、白血病抑制因子化IF)、血管内皮生长因子(VEGF)、和促红细胞生成素巧P0);氨基酸,包括心鄉氨酸;和用于控制微生物污染的一种或多种抗生素和/或抗真菌剂,例如青霉素G、硫酸链霉素、两性霉素B、庆大霉素和制霉菌素,单独或组合使用。 [0208] W medium may be added one or more components including, for example: serum (eg: fetal bovine serum (FBS), preferably about 2-15% (qe eight); horse serum for 5); human serum of 5)); sulfur protecting group ethanol (816), preferably from about 0.001% (qe eight); one or more growth factors, e.g., platelet-derived growth factor (PDGF), epidermal growth factor of GF), into base fibroblast growth factor (bFGF), membrane Insulin - like growth factor -l (IGF-l), leukemia inhibitory factor of the IF), vascular endothelial growth factor (VEGF), and erythropoietin Qiao P0); amino acids, comprising heart rural acid; and one or more antibiotics for the control of microbial contamination and / or antifungal agents, such as penicillin G, streptomycin sulfate, amphotericin B, gentamicin, and nystatin, either alone or in combination.

[0209] 所述分离的胎盘细胞可在标准组织培养条件下培养,例如在组织培养皿或多孔平皿中培养。 [0209] The isolated placental cells can be cultured tissue culture conditions, such as in a tissue culture dish or in a standard porous plate. 还可W利用悬滴法培养所述分离的胎盘细胞。 W may also be cultured using a hanging drop method of the isolated placental cells. 该方法中,分离的胎盘细胞W约5mL培养基中每毫升1 X 104个细胞悬浮,并且将一滴或多滴培养基置于组织培养容器,例如lOOmL陪氏培养皿的盖子内。 In this method, isolated placental cells in about 5mL medium W 1 X 104 per ml cells were suspended, and one or more drops will be placed in a tissue culture medium container, for example the lid of Petri dish lOOmL. 所述滴可例如单滴落下或例如自多通道移液管多滴落下。 The droplets may, for example, for example, from a multichannel pipette multiple single drip dripping or lower. 小屯、 反转盖子并且置于平皿底部之上,其含有大量液体,例如足W维持平皿空气中含水量的无菌PBS,然后培养干细胞。 Xiaotun, inverted and placed on top of the bottom cover plate, which contains a large amount of liquid, for example, sterile PBS sufficient to maintain the W dish water content in the air, and then cultured stem cells.

[0210] 在一个实施方案中,分离的胎盘细胞在用于维持分离的胎盘细胞中未分化表型的化合物存在下培养。 [0210] In one embodiment, the compound isolated placental cells in placental cells for maintaining the isolated undifferentiated phenotype in the presence of culture. 在一个具体的实施方案中,所述化合物为取代的3,r二氨化晚醇[4,5^ d]喀晚。 In a particular embodiment, the compound is a substituted 3, r two nights amide alcohol [4,5 ^ d] Ka night. 在更具体的实施方案中,所述化合物具有下列化学结构: In a more specific embodiment, the compound has the following chemical structure:

[0211] [0211]

Figure CN105796602AD00401

[0212]化合物可例如约ΙμΜ至约ΙΟμΜ的浓度与分离的胎盘细胞或分离的胎盘细胞群接触。 [0212] compound may be, for example, from about to about ΙΟμΜ ΙμΜ the concentration of isolated placental cells or population of isolated placental cells contacted.

[0213] 5.6.2胎盘细胞的扩增和增殖 [0213] 5.6.2 Expansion and Proliferation of Placental Cells

[0214] 分离的胎盘细胞或分离的胎盘细胞(例如,与至少50%的在体内通常与干细胞或干细胞群相伴的胎盘细胞分离开的胎盘细胞或胎盘细胞群)群一旦分离,就可W在体外增殖和扩增所述细胞或细胞群。 [0214] The isolated placental cells or isolated placental cells (e.g., at least 50% of the in vivo generally stem cells or stem cell population accompanied placental cells separated from placental cells or placental cells) group once isolated, W is and expanding the cell proliferation or cell population. 例如,可W在组织培养容器(例如皿、瓶、多孔板等)中培养分离的胎盘细胞群,培养时间足W使细胞增殖至70-90%汇合度,即,直到细胞及其后代占据组织培养容器70-90%的培养表面区域。 For example, W can be an isolated population of placental cells cultured in tissue culture containers (e.g., dishes, bottles, multiwell plates, etc.), the incubation time sufficient to cell proliferation W 70-90% confluence, i.e., until the cells and their progeny occupy tissue 70-90% of the culture the culture vessel surface area.

[0215] 分离的胎盘细胞W可W允许细胞生长的密度接种在培养容器内。 [0215] The isolated placental cells were seeded at a density W to W allow cell growth in the culture vessel. 例如,细胞可WW低密度(例如约1,000至约5,000细胞/cm2)至高密度(例如约50,000或更多细胞/cm2)接种。 For example, cell density may be WW (e.g., from about 1,000 to about 5,000 cells / cm2) to high density (e.g., about 50,000 or more cells / cm2) were seeded. 在优选的实施方案中,在约0%至约5%体积C〇2的空气中培养细胞。 In a preferred embodiment, the cells are cultured in air at from about 0% to about 5% by volume of C〇2. 在一些优选的实施方案中,在约2%至约25%体积化的空气中培养细胞,优选在约5%至约20%体积化的空气中培养。 In some preferred embodiments, the cells are cultured at about 2% to about 25% by volume of air, preferably from about 5% to about 20% by volume of air in the culture. 细胞优选在约25C至约40C,优选37C培养。 Cells preferably at about 25 C to about 40 C, preferably 37 C culture. 细胞优选在培养箱中培养。 The cells are preferably cultured in an incubator. 培养基可W 是静态的或揽动的,例如,利用生物反应器。 W medium can be static or dynamic embrace, for example, using a bioreactor. 胎盘细胞,例如胎盘干细胞或胎盘多能细胞优选生长在低氧化压力下(例如,添加谷脫甘肤、抗坏血酸、过氧化氨酶、生育酪、N-乙酷半脫氨酸等)。 Placental cells, e.g., placental stem cells or placental multipotent cells, preferably are grown under low oxidative stress (e.g., Gan added valley off the skin, ascorbic acid peroxide, ammonia lyase, fertility casein, N- acetyl Cysteine cool the like).

[0216] -旦获得小于100%,例如70%-90%汇合度,细胞就可W传代。 [0216] - less than 100% Once obtained, for example, 70% to 90% confluence, the cells can be passaged W. 例如,所述细胞可W利用本领域公知的技术进行酶促处理,例如膜蛋白酶消化,从而将其与组织培养表面分离。 For example, the cells may W using art-known techniques enzymatic treatment, such as a membrane protease digestion, so as to be separated from the tissue culture surface. 移液除去细胞和计数细胞后,约1〇,〇〇〇-1〇〇,〇〇〇细胞/cm2被传代到含有新鲜培养基的新培养容器内。 After pipetting to remove cells and the cells were counted, about 1〇, 〇〇〇-1〇〇, 〇〇〇 cells / cm2 are passaged into fresh medium containing the new culture container. 一般的,新培养基与从中去除分离的胎盘细胞的培养基是相同类型的培养基。 Typically, the new medium with the medium removed therefrom isolated placental cells are the same type of medium. 分离的胎盘细胞可传代约、至少或不超过至少1、2、3、4、5、6、7、8、9、10、12、14、16、18或20次或更多次。 Isolated placental cells can be passaged about, at least or no more than 20, or at least 1,2,3,4,5,6,7,8,9,10,12,14,16,18 or more times.

[0217] 5.6.3分离的胎盘细胞群 [0217] The isolated placental cell populations 5.6.3

[0218] 此处还提供分离的胎盘细胞群,例如W上5.4.2节所述的分离的胎盘细胞,可用于治疗脑或CNS中或周围的血流破坏。 [0218] Also provided herein is a population of isolated placental cells, for example, isolated placental cells described in Section 5.4.2 of the W, can be used for the treatment of disruption of blood flow in or around the brain or CNS of. 分离的胎盘细胞群可W直接分离自一个或多个胎盘; 即,所述细胞群可W是包含分离的胎盘细胞的胎盘细胞群,其中所述分离的胎盘细胞获自或包含于灌流液,或者来源于或包含于解离的胎盘组织,例如胎盘组织消化物(即,通过酶促消化胎盘或其部分所获得的细胞收集物)。 The isolated placental cells can be isolated directly from a W or more placenta; i.e., W may be a population of cells comprising isolated placental cells are placental cells, wherein said isolated placental cells are obtained from or contained in perfusate, or from or contained in placental tissue dissociation, e.g. digested placental tissue (i.e., by enzymatic digestion of placental cell collection or a portion thereof is obtained). 还可W培养和扩增此处所述的分离的胎盘细胞来产生所述分离的胎盘细胞群。 W may also be cultured and expanded isolated placental cells described herein to produce the isolated placental cell population. 还可W培养和扩增包含分离的胎盘细胞的胎盘细胞群来产生胎盘细胞群。 W may also be cultured and expanded placental cells comprising the isolated placental cells to produce a population of placental cells.

[0219] 可用于此处提供的治疗方法的胎盘细胞群包含分离的胎盘细胞,例如如此处5.4.2节所述的分离的胎盘细胞。 [0219] useful in the treatment methods provided herein placental cells comprising isolated placental cells, e.g. as herein described in Section 5.4.2, isolated placental cells. 在不同的实施方案中,在胎盘细胞群中,至少10%、20%、 30%、40%、50%、60%、70%、80%、90%、95%或99%的细胞是分离的胎盘细胞。 In various embodiments, the population of placental cells, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of cells are isolated placental cells. 即,分离的胎盘细胞群可W包含例如多至1%、5%、10%、20%、30%、40%、50%、60%、70%、80%、 90%的非分离的胎盘细胞的细胞。 That is, the isolated placental cells can W comprises, for example, up to 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% non-isolated placental cells.

[0220] 可用于治疗脑或CNS中或周围血流破坏的分离的胎盘细胞群可例如通过选择来源于酶促消化或灌流的分离的胎盘细胞而制备,所述分离的胎盘细胞表达特定的标记和/或特定的培养或形态特征。 Placental Cells [0220] useful for the treatment of brain or CNS disruption of blood flow in or around the isolated placental cells can be prepared for example by selecting from enzymatic digestion or perfusion of isolated placental cells, the isolated expression of a particular marker and / or particular culture or morphological characteristics. 例如,在一个实施方案中,细胞群可W通过如下步骤制备:选择胎盘细胞,所述胎盘细胞(a)贴壁于底物,和(b)表达CD200和HLA-G;和从其它细胞中分离所述细胞W形成细胞群。 For example, in one embodiment, the cell population of W can be prepared by the steps of: selecting placental cells, placental cells, said (a) adherent to the substrate, and (b) express CD200 and HLA-G; and from the other cells W separating the cells form a cell population. 在另一个实施方案中,细胞群可W通过如下步骤产生:选择胎盘细胞, 其中所述胎盘细胞表达CD200和HLA-G;和从其它细胞中分离所述细胞W形成细胞群。 In another embodiment, W can be a cell population is produced by steps of: selecting placental cells, wherein said placental cells express CD200 and HLA-G; and isolating said cells from other cells W form a cell population. 在另一个实施方案中,细胞群可W通过如下步骤产生:选择胎盘细胞,所述胎盘细胞(a)贴壁于底物,和(b)表达CD73XD105和200200;和从其它细胞中分离所述细胞W形成细胞群。 In another embodiment, W can be a cell population is produced by steps of: selecting placental cells, placental cells, said (a) adherent to the substrate, and (b) expressing CD73XD105 and 200,200; and isolating said cells from other W cells form a cell population. 在另一个实施方案中,细胞群可W通过如下步骤产生:鉴定胎盘细胞,其中所述胎盘细胞表达CD73、CD 105和CD200;和从其它细胞中分离所述细胞W形成细胞群。 In another embodiment, a cell population may be W is generated by the steps: identifying placental cells, wherein said placental cells express CD73, CD 105 and of CD200; and isolating said cells from other cells W form a cell population. 在另一个实施方案中, 细胞群可W通过如下步骤产生:选择胎盘细胞,所述胎盘细胞(a)贴壁于底物,和(b)表达CD200和0CT-4;和从其它细胞中分离所述细胞W形成细胞群。 In another embodiment, W can be a cell population is produced by steps of: selecting placental cells, placental cells, said (a) adherent to the substrate, and (b) express CD200 and 0CT-4; and isolated from the other cells W the cells form a cell population. 在另一个实施方案中,细胞群可W通过如下步骤产生:选择胎盘细胞,其中所述胎盘细胞表达CD200和0CT-4;和从其它细胞中分离所述细胞W形成细胞群。 In another embodiment, W can be a cell population is produced by steps of: selecting placental cells, wherein said placental cells express CD200 and 0CT-4; and isolating said cells from other cells W form a cell population. 在另一个实施方案中,细胞群通过如下步骤产生:选择胎盘细胞,其中所述胎盘细胞(a)贴壁于底物,(b)表达CD73和CD105,和(C)当包含所述干细胞的胎盘细胞群在允许胚样体形成的条件下培养时,有助于在所述群中形成一个或多个胚样体;和从其它细胞中分离所述细胞W形成细胞群。 In another embodiment, a cell population is produced by the steps of: selecting placental cells, wherein said placental cells (a) adherent to a substrate, (b) express CD73 and CD105, and (C) when said stem cell comprising when the population of placental cells cultured under conditions that allow formation of embryoid-like bodies, contribute to the formation of one or more embryoid-like bodies in a population; and isolating said cells from other cells W form a cell population. 在另一个实施方案中,细胞群通过如下步骤产生:选择胎盘细胞,其中所述胎盘细胞表达CD73和CD105,和当包含所述干细胞的胎盘细胞群在允许胚样体形成的条件下培养时,有助于在所述群中形成一个或多个胚样体;和从其它细胞中分离所述细胞W形成细胞群。 In another embodiment, a cell population is produced by the following steps: selecting placental cells, wherein said placental cells express CD73 and CD105, and when said stem cells under conditions comprising a population of placental cells that allow formation of embryoid-like bodies in culture, contribute to the formation of one or more embryoid-like bodies in a population; and isolating said cells from other cells W form a cell population. 在另一个实施方案中,细胞群可W通过如下步骤产生:选择胎盘细胞,所述胎盘细胞(a)贴壁于底物,和(b)表达CD73、CD 105和HLA-G;和从其它细胞中分离所述细胞W形成细胞群。 In another embodiment, W can be a cell population is produced by steps of: selecting placental cells, placental cells, said (a) adherent to the substrate, and (b) the expression of CD73, CD 105, and HLA-G; and the other the cells were isolated W form a cell population. 在另一个实施方案中,细胞群通过如下步骤产生: 选择胎盘细胞,其中所述胎盘细胞表达CD73、CD105和HLA-G;和从其它细胞中分离所述细胞W形成细胞群。 In another embodiment, the cell population is produced by the steps: selecting placental cells, wherein said placental cells express CD73, CD105 and HLA-G; and isolating said cells from other cells W form a cell population. 在另一个实施方案中,生产细胞群的方法包括选择胎盘细胞,其中所述胎盘细胞(a)贴壁于底物;(b)表达0CT-4,和(C)当包含所述干细胞的胎盘细胞群在允许胚样体形成的条件下培养时,有助于在所述群中形成一个或多个胚样体;和从其它细胞中分离所述细胞W形成细胞群。 In another embodiment, a method of producing a cell population comprises selecting placental cells, wherein said placental cells (a) adherent to the substrate; (b) expressing 0CT-4, and (C) when said placental stem cells comprising when the cell population is cultured under conditions that allow formation of embryoid-like bodies, contribute to the formation of one or more embryoid-like bodies in a population; and isolating said cells from other cells W form a cell population. 在另一个实施方案中,细胞群通过如下步骤产生:选择胎盘细胞,其中所述胎盘细胞表达0CT-4,和当包含所述干细胞的胎盘细胞群在允许胚样体形成的条件下培养时,有助于在所述群中形成一个或多个胚样体;和从其它细胞中分离所述细胞W形成细胞群。 In another embodiment, a cell population is produced by the steps of: selecting placental cells, wherein said placental cells express 0CT-4, and when cultured under conditions include the stem cell population of placental cells that allow formation of embryoid-like bodies, contribute to the formation of one or more embryoid-like bodies in a population; and isolating said cells from other cells W form a cell population.

[0221] 在另一个实施方案中,细胞群通过如下步骤产生:选择胎盘细胞,所述胎盘细胞(a) 贴壁于底物,和(b)表达CD10和CD105,不表达CD34;和从其它细胞中分离所述细胞W形成细胞群。 [0221] In another embodiment, a cell population is produced by the steps of: selecting placental cells, placental cells, said (a) adherent to the substrate, and (b) the expression of CD10 and CD105, the expression of CD34 is not; and the other the cells were isolated W form a cell population. 在另一个实施方案中,细胞群通过如下步骤产生:选择胎盘细胞,其中所述胎盘细胞表达CD10和CD105,不表达CD34;和从其它细胞中分离所述细胞W形成细胞群。 In another embodiment, the cell population is produced by the steps: selecting placental cells, wherein said placental cells express CD10 and CD105, the expression of CD34 is not; and isolating said cells from other cells W form a cell population. 在另一个实施方案中,细胞群通过如下步骤产生:选择胎盘细胞,所述胎盘细胞(a)贴壁于底物,和(b) 表达CD10、CD105和CD200,不表达CD34;和从其它细胞中分离所述细胞W形成细胞群。 In another embodiment, a cell population is produced by the steps of: selecting placental cells, placental cells, said (a) adherent to the substrate, and (b) the expression of CD10, CD105 and CD200, do not express of CD34; and from the other cells W separating the cells form a cell population. 在另一个实施方案中,细胞群通过如下步骤产生:选择胎盘细胞,其中所述胎盘细胞表达CD10、CD105和CD200,不表达CD34;和从其它细胞中分离所述细胞W形成细胞群。 In another embodiment, the cell population is produced by the steps: selecting placental cells, wherein said placental cells express CD10, CD105 and CD200, do not express of CD34; and isolating said cells from other cells W form a cell population. 在另外更具体的实施方案中,细胞群通过如下步骤产生:选择胎盘细胞,所述胎盘细胞(a)贴壁于底物,和(b)表达CD 10、CD90、CD 105和CD200,不表达CD34和CD45;和从其它细胞中分离所述细胞W形成细胞群。 In another more specific embodiment, the cell population is produced by the steps of: selecting placental cells, placental cells, said (a) adherent to the substrate, and (b) the expression of CD 10, CD90, CD 105, and CD200, did not express CD34 and CD45; and isolating said cells from other cells form a cell population W. 在另外更具体的实施方案中,细胞群通过如下步骤产生:选择胎盘细胞, 其中所述胎盘细胞表达CD 10、CD90、CD 105和CD200,不表达CD34和CD45;和从其它细胞中分离所述细胞W形成细胞群。 In another more specific embodiment, the cell population is produced by steps of: selecting placental cells, wherein said placental cells express CD 10, CD90, CD 105, and CD200, CD34 and do not express CD45; and isolating said cells from other W cells form a cell population.

[0222] 所述细胞群或其组合可用于治疗脑或CNS中或周围血流破坏,例如血流破坏的症状或所述血流破坏引起的神经性缺损。 [0222] The population of cells may be useful in the treatment or combinations thereof in or around the brain or CNS disruption of blood flow, for example, or the disruption of blood flow caused by neurological deficit symptoms of disruption of blood flow.

[0223] 在任何上述实施方案中,所述分离的细胞群的选择可另外包括选择表达ABC-P( - 种胎盘特异性ABC转运蛋白;参见例如Allikmets等,CancerRes. 58(23): 5337-9( 1998))的胎盘细胞。 [0223] In any of the above embodiments, the isolated population of cells selected may additionally comprise selecting expression ABC-P (- species placenta-specific ABC transporter protein; see, e.g., Allikmets et, CancerRes 58 (23): 5337-. 9 (1998)) of the placental cells. 所述方法还可W包括选择呈现例如间充质干细胞特异性特征的至少一种细胞, 例如表达CD44,表达CD90,或表达前述的组合。 The method may further comprise selecting W, for example, at least one cell presenting mesenchymal stem cell-specific features, such as the expression of CD44, expression of CD90, or a combination of the foregoing expression.

[0224] 在上述实施方案中,底物可W是能实现细胞例如分离的胎盘细胞的培养和/或选择的任何表面。 [0224] In the above embodiments, the substrate W may be cultured cells can be achieved, for example, isolated placental cells, and / or any selected surface. 一般的,底物是塑料制品,例如组织培养皿或多孔板塑料。 Typically, the substrate is plastic, for example, tissue culture dish or multiwell plate plastic. 组织培养塑料制品可W用生物分子例如层粘连蛋白或纤粘连蛋白包被。 Tissue culture plastic can be biomolecules such as W with laminin or fibronectin-coated.

[0225] 可W通过细胞选择领域任何已知的方法来选择胎盘细胞群的细胞(例如分离的胎盘细胞)。 [0225] W may be selected cell population of placental cells (e.g., isolated placental cells) by any known method of selecting the field of cell. 例如,可W利用抗一种或多种细胞表面标记的抗体来选择细胞,例如,在流式细胞仪或FACS中。 For example, a W using antibodies against one or more cell surface markers selected cell, e.g., in a flow cytometer or FACS. 利用与磁珠连接的抗体可W实现选择。 Using antibodies with magnetic beads attached may choose W achieved. 特异性针对某些干细胞相关标记的抗体是本领域已知的。 Antibodies specific for certain stem cell-related markers are known in the art. 例如,0CT-4抗体(Abeam,Cambr idge,MA)、CD200抗体(Abeam)、HLA-G抗体(Abcam)、CD73抗体(BD Biosciences Pha;rmingen,San Diego,CA)、CD105抗体(Abeam; BioDesign International,Saco,ME)等。 For example, 0CT-4 antibody (Abeam, Cambr idge, MA), CD200 antibody (Abeam), HLA-G antibody (Abcam), CD73 antibody (BD Biosciences Pha; rmingen, San Diego, CA), CD105 antibody (Abeam; BioDesign International, Saco, ME) and so on. 其他标记的抗体也是可商购的,例如:可W从如StemCell Technologies或BioDesign International获得的CD:M、CD38和CD45抗体。 Other labeled antibodies are also commercially available, for example: CD W can be obtained from such StemCell Technologies or BioDesign International: M, CD38 and CD45 antibody.

[0226] 分离的胎盘细胞群可W包含不是干细胞的胎盘细胞,或者不是胎盘细胞的细胞。 [0226] The isolated placental cells can comprise W placental cells are not stem cells, placental cells or not.

[0227] 此处提供的分离的胎盘细胞群可W与一个或多个非干细胞或非胎盘细胞的群组合。 [0227] The isolated placental cells herein may be provided with one or more W groups combination of non-stem cells or non-placental cells. 例如,分离的胎盘细胞群可W组合血液(例如:胎盘血或厮带血)、血液来源的干细胞(例如:来源于胎盘血或厮带血的干细胞)、厮带干细胞、血液来源的有核细胞的群、骨髓来源的间充质干细胞、骨来源的干细胞群、原始骨髓、成人(成体)干细胞、包含在组织内的干细胞群、培养的干细胞、完全分化的细胞的群(例如:软骨细胞、成纤维细胞、羊膜细胞、成骨细胞、肌细胞、屯、肌细胞等)等。 For example, isolated placental cells can be W composition of blood (e.g.: placental blood or servant blood), blood-derived stem cells (for example: derived from placental blood or servant blood stem cells), servant with stem cells, blood-derived nucleated population of cells, bone marrow-derived mesenchymal stem cells, populations of stem cells of bone origin, original bone marrow, adult (somatic) stem cells, populations of stem cells included in the tissue, cultured stem cells, fully differentiated population of cells (for example: chondrocytes , fibroblasts, amniotic cells, osteoblasts, muscle cells, Tun, muscle cells, etc.) and the like. 在一个具体的实施方案中,可用于治疗脑或CNS中或周围血流破坏的细胞群包含分离的胎盘细胞和分离的厮带细胞。 In a specific embodiment, useful for the treatment of brain or CNS disruption of blood flow in or around the cell population comprising isolated placental cells, and cells isolated with servant. 比较每个群中的总有核细胞数量, 分离的胎盘细胞群内的细胞可W与大量另一种类型的细胞相组合,组合比例为约100,000, 000:1、50,000,000:1、20,000,000:1、10,000,000:1、5,000,000:1、2,000,000:1、1,000, 000:1、500,000:1、200,000:1、100,000:1、50,000:1、20,000:1、10,000:1、5,000:1、2,000: 1、1,000:1、500:1、200:1、100:1、50:1、20:1、10:1、5:1、2:1、1:1;1:2、1:5、1:10、1:100、1: 200、1:500、1:1,000、1:2,000、1:5,000、1:10,000、1:20,000、1:50,000、1:100,000、1:500, 000、1:1,000,000、1:2,000,000、1:5,000,000、1:10,000,000、1:20,000,000、1:50,000, 000,或约1:100,000,000。 Comparing the total number of nuclei in each cluster cells, cells within the population of isolated placental cells can be another type W with a large number of cells in combination, the combination ratio of about 100,000, 000: 1,50,000,000: 1,20,000,000: 1 , 10,000,000: 1,5,000,000: 1,2,000,000: 1, 1,000, 000: 1,500,000: 1,200,000: 1,100,000: 1,50,000: 1,20,000: 1,10,000 : 1,5,000: 1,2,000: 1, 1,000: 1,500: 1,200: 1,100: 1, 50: 1,20: 1,10: 1,5: 1,2: 1,1 : 1; 1: 2,1: 5,1: 10,1: 100, 1: 200, 1: 500, 1: 1,000,: 2,000,1: 5,000,: 10,000, 1: 20,000,1 : 50,000, 1: 100,000: 500, 000,1: 1,000,000,1: 2,000,000,1: 5,000,000,1: 10,000,000,1: 20,000,000,1: 50,000, 000, or about 1: 100,000 , 000. 分离的胎盘细胞群中的细胞也可W与多种细胞类型的大量细胞组合。 Isolated placental cell population can be a large number of cells in the cell in combination with a variety of cell types W.

[02%]在一个实施方案中,分离的胎盘细胞群与大量造血干细胞组合。 [02%] In one embodiment, the isolated placental cell population with a large number of hematopoietic stem cell composition. 此类造血干细胞可w是例如,包含在未处理的胎盘、厮带血或外周血中;在来源于胎盘血、厮带血或外周血的总有核细胞中;在来源于胎盘血、厮带血或外周血的分离的CD34+细胞群中;在未处理的骨髓中;在来源于骨髓的总有核细胞中;在来源于骨髓的分离的CD34+细胞群中;等等。 Such hematopoietic stem cells can be, for example w, contained in the unprocessed placental, servant blood or peripheral blood; derived from the placental blood, total nucleated cells in peripheral blood or blood servant; derived from the placental blood, servant blood or isolated CD34 + peripheral blood cell population; in unprocessed bone marrow; in total nucleated cells from the bone marrow; CD34 + cells in the isolated population of bone marrow derived in; and the like.

[0229] 5.7胎盘细胞库的制备 Placental cell bank was prepared in [0229] 5.7

[0230] 来自产后胎盘的分离的细胞,例如W上5.4.2节所述的分离的胎盘细胞可多种不同的方式培养W制备一组批次,例如其中每个批次为多个可单独施用的剂量。 [0230] The isolated cells from postpartum placentas, e.g. culturing the isolated section 5.4.2 on the W placental cells can be prepared in many different ways W batch set, for example, wherein each batch is a plurality of individually administered dose. 运样的批次可例如,获自来自胎盘灌流液的细胞或获自来自酶消化胎盘组织的细胞。 Sample transport batches can e.g., be obtained from cells from placental perfusate or from cells obtained from enzymatic digestion of placental tissue. 获自多个胎盘的胎盘细胞批次组可被安排在分离的胎盘细胞库中W例如长期保存。 Obtained from a plurality of lots of placental cells in placental group W may be arranged, for example, long-term storage in a library of isolated placental cells. 通常,塑料贴壁组织培养的胎盘细胞获自胎盘材料的原代培养W形成种子培养物,其在受控制的条件下扩增W形成大约相当于倍增量的细胞群。 Typically, tissue culture plastic-adherent placental cells are obtained from primary culture of placental material W form a seed culture, which is formed under amplification conditions W controlled cell population doubling roughly equivalent amounts. 批次优选源自单个胎盘组织,但可源自多个胎盘组织。 Batch preferably derived from a single placenta, but can be derived from a plurality of placental tissue.

[0231] 在一个实施方案中,如下获得胎盘细胞批次。 [0231] In one embodiment, placental cell lots is obtained as follows. 胎盘组织例如通过切碎,用合适的酶,例如膜蛋白酶或胶原酶(参见W上5.5.3节)消化而被首次解离。 Placenta example by chopping with a suitable enzyme such as a protease or membrane collagenase (see Section 5.5.3 on W) to be dissociated first digestion. 所述胎盘组织优选包括,例如来自单个胎盘的完整的羊膜、完整的绒毛膜或两者,但可仅包括羊膜或绒毛膜的一部分。 The placental tissue preferably comprises, for example, from a single placenta intact amnion and chorion, or both intact, but may include only a portion of the amnion or chorion. 培养消化组织例如约1-3周,优选约2周。 E.g. digestive tissue culture for about 1-3 weeks, preferably about 2 weeks. 除去非贴壁细胞后,例如通过膜蛋白酶消化收集形成的高密度克隆。 After removing non-adherent cells, for example, the film formed was collected by protease digestion of the high-density clone. 收集运些细胞并重悬浮于合适体积的培养基中,随后用于接种扩大培养。 These cells were collected and resuspended in transport volume of a suitable medium, then used to inoculate culture expansion. 扩大培养可W是单独的细胞培养物装置的任意组合,例如NUNC⑧的Cell 化ctory。 W may be a separate expansion for any combination of the cell culture apparatus, e.g. NUNC⑧ of the Cell ctory. 细胞可W细分至任何程度W便用例如1 X 1〇3、2 X 1〇3、3 X 103、4X 1〇3、5 X 1〇3、6 X 103、7X103、8X103、9X103、1X104、1X104、2X104、3X104、4X104、5X104、6X104、7X104、 8 X 104、9 X 1〇4或10 X 1〇4细胞/cm2接种扩大培养。 Cells can be subdivided to any degree W W X 1〇3,2. 1, for example they 1〇3,3 X X X 103,4X 1〇3,5 1〇3,6 X 103,7X103,8X103,9X103,1X104 , 1X104,2X104,3X104,4X104,5X104,6X104,7X104, 8 X 104,9 X 10 X 1〇4 or 1〇4 cells / cm2 seeded expansion culture. 优选的,约1 X 1〇3至约1 X 1〇4细胞/cm2用于接种每次扩大培养。 Preferably, about 1 X to about 1 X 1〇4 1〇3 cells / cm2 were used to inoculate each expansion culture. 扩大培养次数在数量上可多可少,取决于从其获得细胞的具体胎盘。 The number of expansion for the number may be more or less, depending on the specific placental cells are obtained therefrom.

[0232] 扩大培养物生长至培养的细胞密度达到某个值,例如约1 X 105个细胞/cm2。 [0232] expansion cell density of the culture was grown to reach a certain value, for example, about 1 X 105 cells / cm2. 如上所述此时可W收集并且冷藏细胞,或者传代进入新的扩大培养。 W described above can now be collected and frozen cells, or passaged into new expansion cultures. 使用之前,细胞可W传代例如2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20次。 Prior to use, cells may be passaged, for example, W 18, 19 or 20 times. 优选在扩大培养期间记录群倍增的累计数。 The cumulative number of population doublings is preferably recorded during expansion culture. 来自培养物的细胞可W扩增2、3、4、5、6、7、8、9、10、12、14、16、18、20、22、24、 26、28、30、32、34、36、38或40次倍增,或多达60次倍增。 Cells from culture may be amplified 2,3,4,5,6,7,8,9,10,12,14,16,18,20,22,24 W, 26,28,30,32,34 , 36, 38 or 40 doublings, or up to 60 doublings. 然而优选在将所述细胞群等分为单次剂量之前群倍增数为约15至约30次。 Preferably, however, in the cell population doublings before divided into a single dose group is from about 15 to about 30 times. 所述细胞可W在整个扩增过程中连续培养,或可W 在扩增期间的一个或多个时间点冷冻。 The cells can be continuously cultured in the W of the entire amplification process, or W may be one or more time points during the amplification frozen.

[0233] 用于单次剂量的细胞可W冷冻,例如冷藏便于W后的使用。 [0233] cell for a single dose of freeze-W, such as a refrigerator easy to use after W. 单次剂量可W包含,例如每毫升约一百万至约五千万个细胞,并且可W包含约总计约1〇6至约l〇w个细胞。 W single dose may contain, for example, about one million per milliliter to about five million cells, and may comprise from about a total of about W to about l〇w 1〇6 cells.

[0234] 因此在一个实施方案中,可W通过如下方法制备胎盘细胞库,其包括:扩增来自人分娩后胎盘的原代培养胎盘细胞,用于第一次大量的群倍增;冷藏所述胎盘细胞W形成主细胞库;扩增来自主细胞库的大量胎盘细胞用于第二次大量的群倍增;冷藏所述胎盘细胞W形成工作细胞库;扩增来自工作细胞库的大量胎盘细胞用于第Ξ次大量的群倍增;和W 单次剂量冷藏所述胎盘细胞,其中所述单次剂量共同组成胎盘细胞库。 [0234] Thus, in one embodiment, W can be prepared by methods placental cell bank, which comprises: delivery amplification from human placenta after primary culture placental cells, for the first time a large number of population doublings; the refrigerator placental cells are formed W master cell bank; amplifying a plurality of placental cells from master cell banks for a second group of a large number of doublings; W refrigerator said placental cells are formed the working cell bank; amplifying a plurality of placental cells from the working cell bank with a large number of times on the first Ξ population doublings; W and a single dose of the refrigerator placental cells, wherein said single dose of placental cell bank composed. 选择性地,来自所述第Ξ次大量群倍增的大量胎盘细胞可W扩增用于第四次大量群倍增并且W单次剂量量冷藏,其中所述单次剂量共同组成胎盘干细胞。 Alternatively, a large number of times from the first population doublings Ξ plurality of placental cells can be used to amplify the fourth W large population doublings and frozen W single dose amounts, wherein the single dose composed placental stem cells.

[0235] 在另外具体的实施方案中,所述原代培养胎盘细胞包含来自胎盘灌流液的胎盘细胞。 [0235] In another specific embodiment, said primary culture placental cells comprise placental cells from placental perfusate. 在另外具体的实施方案中,所述原代培养胎盘细胞包含来自消化的胎盘组织的胎盘细胞。 In another specific embodiment, said primary culture placental cells comprise placental cells from digested placental tissue. 在另外具体的实施方案中,所述原代培养胎盘细胞包含来自胎盘灌流液和消化的胎盘组织的胎盘细胞。 In another specific embodiment, said primary culture placental cells comprise placental cells from placental tissue and digested placental perfusate. 在另外具体的实施方案中,所述胎盘细胞原代培养中所有所述的胎盘细胞来自同一个胎盘。 In another specific embodiment, said placental cell primary culture placental cells from all of the same placenta. 在另外具体的实施方案中中,所述方法进一步包括从来自所述工作细胞库的大量胎盘细胞选择CD200+或HLA-G+胎盘细胞W形成单次剂量。 In another specific embodiment, the method further comprises selecting CD200 + or HLA-G + W placental cells are formed in a single dose from a plurality of placental cells from the working cell bank. 在另外具体的实施方案中,所述单次剂量包含约1〇4至约1〇5个胎盘细胞。 In another specific embodiment, said individual doses comprise from about to about 1〇5 1〇4 placental cells. 在另外具体的实施方案中,所述单次剂量包含约105至约106个胎盘细胞。 In another specific embodiment, said individual doses comprise from about 105 to about 106 placental cells. 在另外具体的实施方案中,所述单次剂量包含约10 6至约1〇7个胎盘细胞。 In another specific embodiment, said individual doses comprise from about 106 to about 1〇7 placental cells. 在另外具体的实施方案中,所述单次剂量包含约1〇7至约1〇8个胎盘细胞。 In another specific embodiment, said individual doses comprise from about to about 1〇8 1〇7 placental cells. 在另外具体的实施方案中,所述单次剂量包含约1〇8至约1〇9个胎盘细胞。 In another specific embodiment, said individual doses comprise from about to about 1〇9 1〇8 placental cells. 在另外具体的实施方案中,所述单次剂量包含约1 〇9至约10 W个胎盘细胞。 In another specific embodiment, said individual doses comprise from about 1 to about 10 W 〇9 placental cells.

[0236] 在一个优选的实施方案中,检测提供胎盘的供者(例如母体)是否存在至少一种病原体。 [0236] In a preferred embodiment, the assay provides placental donor (e.g., parent) whether at least one pathogen. 如果所述母体检测为所检测的病原体阳性,则丢弃来自该胎盘的全部批次。 Detecting if the parent is a pathogen positive was detected, the entire batch is discarded from the placenta. 所述检验可W在制备胎盘细胞批次期间的任何时候进行,例如扩增培养期间。 The test may be performed at any time during W prepared lots of placental cells, e.g. during expansion culture. 检测其存在的病原体可W包括,但不限于甲型肝炎、乙型肝炎、丙型肝炎、下型肝炎、戊型肝炎、人类免疫缺陷性病毒(I和II型)、巨细胞病毒、瘤疹病毒等等。 Detecting the presence of pathogens which W may include, but are not limited to, hepatitis A, hepatitis B, hepatitis C, hepatitis lower, hepatitis E, human immunodeficiency virus (I and II), cytomegalovirus, measles tumor virus and so on.

[0237] 5.8胎盘细胞的保存 [0237] Placental cells saved 5.8

[0238] 分离的胎盘细胞,例如W上5.4.2节所述分离的胎盘细胞可W保存,即置于允许长期储存的条件下,或置于抑制例如调亡或坏死所致的细胞死亡的条件下。 [0238] The isolated placental cells, for example, a W Section 5.4.2, the isolated placental cells can be saved W, i.e., placed under conditions allowing long term storage, or placed, for example, inhibiting apoptosis or necrosis induced cell death conditions.

[0239] 如相关美国申请公开号2007/0190042中所述,可W利用例如包含调亡抑制剂、坏死抑制剂和/或携氧全氣化碳的组合物保存胎盘细胞。 [0239] The related U.S. Application Publication No. 2007/0190042 said, may comprise, for example, using a W apoptosis inhibitor, necrosis inhibitor and / or an oxygen-carrying carbon full gas composition was stored placental cells. 在一个实施方案中,本发明提供了保存细胞群的方法,所述群可用于治疗脑或CNS中或周围的血流破坏,包括将所述细胞群与含有调亡抑制剂和携氧全氣化碳的细胞收集组合物相接触,与未接触调亡抑制剂的细胞群相比,其中所述调亡抑制剂存在的量和时间可足W降低或预防细胞群中的调亡。 In one embodiment, the present invention provides a method of preserving a population of cells, the group may be the treatment of disruption of blood flow to or around the brain or CNS, comprising a population of cells containing the full apoptosis inhibitor and oxygen-carrying gas the carbon was contacted with a cell collection composition, as compared with the population of cells not contacted with the inhibitor of apoptosis, wherein the apoptosis inhibitor may be present in a sufficient amount of time and W to reduce or prevent apoptosis in the cell population. 在具体的实施方案中,所述调亡抑制剂是级联蛋白抑制剂。 In a specific embodiment, said inhibitor of apoptosis cascade inhibitors. 在另外具体的实施方案中,所述调亡抑制剂是JNK抑制剂。 In another specific embodiment, said inhibitor of apoptosis is a JNK inhibitor. 在更具体的实施方案中,所述JNK抑制剂不调节所述细胞的分化或增殖。 In a more specific embodiment, said JNK inhibitor does not modulate differentiation or proliferation of said cells. 在另一个实施方案中,所述细胞收集组合物包含乳液中的所述调亡抑制剂和在另外相中的所述携氧全氣化碳。 In another embodiment, the cell collection composition comprises said inhibitor of apoptosis in the emulsion and in the further gas phase full oxygen-carrying carbon. 在另一个实施方案中,所述细胞收集组合物包含所述调亡抑制剂和在乳剂中的所述携氧全氣化碳。 In another embodiment, the cell collection composition comprises said inhibitor of apoptosis and said oxygen-carrying emulsion in full gas carbon. 在另一个实施方案中,所述细胞收集组合物还包含乳化剂,例如卵憐脂。 In another embodiment, the cell collection composition additionally comprises an emulsifier, e.g. pity egg lipid. 在另一个实施方案中,在接触细胞时,所述调亡抑制剂和所述全氣化碳处于约〇C和约25Γ之间。 In another embodiment, the cells upon contact, said apoptosis inhibitor and said carbon is between about full air square C and about 25Γ. 在另外更具体的实施方案中,在接触细胞时,所述调亡抑制剂和所述全氣化碳处于约2Γ和约10C之间,或约2Γ和约fTC之间。 In another more specific embodiment, when contacting the cells, the apoptosis inhibitor and the whole gasifying 2Γ between about and about 10 C, or between about 2Γ about fTC. 在另外更具体的实施方案中,所述接触是在转移所述细胞群的过程中进行的。 In another more specific embodiment, said contacting is performed during the transfer of the cell population. 在另外更具体的实施方案中,所述接触是在冷冻和融化所述细胞群的过程中进行的。 In another more specific embodiment, said contacting is carried out in the freezing and thawing of said population of cells in the process.

[0240] 胎盘细胞群可W例如通过W下方法保存,包括将所述细胞群与调亡抑制剂和器官防腐化合物相接触,与未接触调亡抑制剂的细胞群相比,其中所述调亡抑制剂存在的量和时间可足W降低或预防细胞群中的调亡。 [0240] W, for example, placental cells can be preserved by the method in W, comprising contacting the cell population with an inhibitor of apoptosis and an organ preservative compounds, as compared to a population of cells not contacted with the inhibitor of apoptosis, wherein said modulation and the amount of inhibitor present in dead time W may be sufficient to reduce or prevent apoptosis in the cell population. 在一个具体的实施方案中,所述器官防腐化合物是UW溶液(描述于美国专利号4,798,824中;其也被称为ViaSpan;还参见Southard等人, Transplantation 49(2) :251-257( 1990))或者是Stern等人的美国专利号5,552,267中描述的溶液。 In a specific embodiment, the organ preservation compound is UW solution (described in U.S. Patent No. 4,798,824; also known as ViaSpan; see also Southard et al, Transplantation 49 (2): 251- solution described in U.S. Patent No. 5,552,267 257 (1990)) or a Stern et al. 在另一个实施方案中,所述器官防腐化合物是径乙基淀粉、乳糖酸、棉子糖,或其组合。 In another embodiment, the diameter of the organ preservative compound is ethyl starch, lactobionic acid, raffinose, or combinations thereof. 在另一个实施方案中,所述细胞收集组合物还包含位于两相或位于乳液中的携氧全氣化碳。 In another embodiment, the cell collection composition further comprises a two-phase located at or in the full oxygen-carrying gas emulsion carbon.

[0241] 在本方法的另一实施方案中,胎盘细胞在灌流过程中与包含调亡抑制剂和携氧全氣化碳,器官防腐化合物,或其组合的细胞收集组合物相接触。 [0241] In another embodiment of the method, placental perfusate cells are contained during apoptosis inhibitor and oxygen-carrying carbon full gas, organ preservative compound, or a combination thereof contacting the cell collection composition. 在另一个实施方案中,在组织破坏(例如,酶促消化)过程中,所述细胞产生接触。 In another embodiment, the tissue damage (e.g., enzymatic digestion) during contacting of said cells. 在另一个实施方案中,在灌流收集后, 或在组织破坏(例如,酶促消化)后,胎盘细胞与所述细胞收集化合物相接触。 In another embodiment, the perfusion after collection or after tissue disruption (e.g., enzymatic digestion), the placental cells are contacted cell collection compound.

[0242] -般的,在胎盘细胞收集、富集和分离过程中,优选最小化或消除由于缺氧和机械压力导致的细胞应激。 [0242] - as in placental cell collection, enrichment and isolation process is preferably minimized or eliminated due to hypoxia and mechanical stress caused by cellular stress. 因此,在本方法的另一实施方案中,在收集、富集或分离过程中,细胞或细胞群在所述保存中暴露在低氧条件下少于6个小时,其中所述低氧条件是氧浓度低于正常的血氧浓度。 Accordingly, in a further embodiment of the method, the collection, enrichment or isolation, the cell or population of cells exposed to hypoxic conditions in less than six hours during said preservation medium, wherein the hypoxic conditions are oxygen concentration below the normal oxygen concentration. 在更具体的实施方案中,所述细胞群在所述保存中暴露在所述低氧条件下少于2个小时。 In a more specific embodiment, said population of cells exposed to hypoxic conditions than in the two hours in the storage. 在另外更具体的实施方案中,在收集、富集或分离过程中,所述细胞群暴露在所述低氧条件下少于1个小时、或少于30分钟、或不暴露于低氧条件下。 In another more specific embodiment, the collection, enrichment or separation process, the cell population exposed to less than 1 hour at the hypoxic conditions, or less than 30 minutes, or is not exposed to hypoxic conditions under. 在另外具体的实施方案中,在收集、富集或分离过程中,所述细胞群不暴露于剪切力下。 In another specific embodiment, collection, enrichment or separation process, the cell population is not exposed to shear forces.

[0243] 胎盘细胞可W冷藏,例如置于小容器(如安飯瓶)中的冷冻保存培养基中。 [0243] W placental cells can be refrigerated, placed e.g. cryopreservation medium small containers (e.g., bottles An rice) was. 合适的冷冻保存培养基包括但不限于如下培养基,其包括例如生长培养基或细胞冷冻培养基,例如可商购的细胞冷冻培养基,例如C2695、C2639或C6039(Sigma)。 Suitable cryopreservation medium includes, but is not limited to, culture medium including, for example, growth media or cell freezing medium, for example commercially available cell freezing medium, for example, C2695, C2639 or C6039 (Sigma). 冷冻保存培养基优选包含DMS0(二甲亚讽),其浓度为约2%至约15%(v/v),例如约10%(v/v)。 Cryopreservation medium preferably comprises DMSO (dimethylsulfoxide ridicule), at a concentration of from about 2% to about 15% (v / v), for example about 10% (v / v). 冷冻保存培养基可W 包含其它试剂,例如甲基纤维素和/或甘油。 W comprises cryopreservation medium may be other agents, such as methylcellulose and / or glycerol. 在冷冻保存过程中,胎盘细胞优选W约rc/分钟冷却。 In the cryopreservation process, the placental cells are preferably from about W rc / min cooling. 优选的冷冻保存溫度为约-80C至约-180C,优选约-125C至约-140C。 The preferred cryopreservation temperature is about -80 C to about -180 C, preferably from about -125 C to about -140 C. 在解冻使用前,冷冻保存的细胞可W转移到液氮中。 Thaw before use, the cells can be cryopreserved W transferred to liquid nitrogen. 在一些实施方案中,例如,一旦安飯瓶达到约-90 C,就将其转移至液氮储存区域。 In some embodiments, e.g., up to about once bottle security rice -90 C, they are transferred to a liquid nitrogen storage area. 还可W利用控制-速率的冷冻箱完成冷冻保存。 W may also be controlled using the - rate freezer to complete cryopreservation. 冷冻保存的细胞优选在溫度约25C至约40C,优选在溫度约37C下解冻。 Cryopreserved cells preferably at a temperature of about 25 C to about 40 C, preferably at a temperature of about thawed at 37 C.

[0244] 5.9包含分离的胎盘细胞的组合物 [0244] The isolated placental cells comprising 5.9 composition

[0245] 此处例如5.4.2节所述的胎盘细胞可W与用于治疗脑或CNS中或周围血流破坏的任何生理学可接受或医学可接受的化合物、组合物或装置组合。 [0245] Here, for example, Section 5.4.2, the placental cells may be useful in the treatment and W in the brain or CNS, composition or combination thereof or any physiologically acceptable compound or medically acceptable disruption of blood flow around the device. 可用于此处提供的治疗方法的组合物可包含任何一种或多种在此所述的胎盘细胞(参见W上5.4.2节)。 Useful in the treatment methods provided herein compositions may comprise any one or more of the placental cells described herein (see section on W 5.4.2). 在某些实施方案中,所述组合物为药学可接受的组合物,例如包含在药学可接受载体中的胎盘细胞的组合物。 In certain embodiments, the composition is a pharmaceutically acceptable composition, such as a composition comprising placental cells in a pharmaceutically acceptable carrier. 参见W下5.9.2节。 See the following section W 5.9.2.

[0246] 在某些实施方案中,包含所述分离的胎盘细胞的组合物还包含基质,例如脱细胞基质或合成的基质。 [0246] In certain embodiments, the isolated placental cells comprising said composition further comprises a matrix, for example an acellular matrix or synthetic matrix. 在更具体的实施方案中,所述基质为立体支架。 In a more specific embodiment, said matrix is a three-dimensional scaffold. 在另外更具体的实施方案中,所述基质包含胶原、明胶、层粘连蛋白、纤粘连蛋白、果胶、鸟氨酸或玻连蛋白。 In another more specific embodiment, said matrix comprises collagen, gelatin, laminin, fibronectin, pectin, ornithine, or vitronectin. 在另外更具体的实施方案中,所述基质为羊膜或羊膜来源的生物材料。 In another more specific embodiment, said matrix is amniotic membrane or amniotic membrane biomaterial derived. 在另外更具体的实施方案中,所述基质包含胞外膜蛋白。 In another more specific embodiment, said matrix comprises an extracellular membrane protein. 在另外更具体的实施方案中,所述基质包含合成的化合物。 In another more specific embodiment, the matrix comprises a synthetic compound. 在另外更具体的实施方案中,所述基质包含生物活性化合物。 In another more specific embodiment, said matrix comprises a bioactive compound. 在另外更具体的实施方案中, 所述生物活性化合物为生长因子、细胞因子、抗体或小于5,000道尔顿的有机分子。 In another more specific embodiment, said bioactive compound is a growth factor, cytokine, antibody, or organic molecule of less than 5,000 daltons.

[0247] 在另一个实施方案中,可用于此处提供的治疗方法的组合物包含通过任何上述胎盘细胞或任何上述胎盘细胞群条件化的培养基。 [0247] In another embodiment, the methods provided herein for treating a composition comprising culture medium by any of the above placental cells or cell populations of any of the foregoing placental conditioned.

[0248] 5.9.1冷冻保存的分离的胎盘细胞 [0248] 5.9.1 cryopreserved isolated placental cells

[0249] 可用于治疗脑或CNS中或周围血流破坏的所述分离的胎盘细胞群可W保存,例如冷冻保存供后期使用。 The [0249] useful for the treatment of brain or CNS disruption of blood flow in or around the isolated placental cell population can be saved W, for example, cryopreserved for later use. 冷冻保存细胞,例如干细胞的方法是本领域公知的。 Cryopreservation of cells, such as stem cells, the present methods are known in the art. 胎盘细胞群可W 制备成易于向个体施用的形式,例如包含在适合医学使用的容器内的分离的胎盘细胞群。 W population of placental cells can be administered to an individual to be easily prepared in the form of, for example, comprising an isolated population of placental cells in a suitable container for use in medicine. 此类容器可w是例如,注射器、无菌的塑料袋、瓶、罐,或其它可w方便配制胎盘细胞群的容器。 Such containers can be, for example, w, a syringe, sterile plastic bags, bottles, cans, or other containers can conveniently be formulated w placental cell population. 例如,所述容器可W是适合将液体静脉施用至接受者的血袋或其它塑料的、医学可接受的袋。 For example, the container may be adapted to the liquid W is administered intravenously to the recipient of the blood bag or other plastic, and medically acceptable bag. 所述容器优选为允许冷冻保存组合细胞群的容器。 The container is preferably a container to allow cell population cryopreservation composition.

[0250] 冷冻保存的分离的胎盘细胞群可W包括来源于单个供体,或多个供体的分离的胎盘细胞。 [0250] cryopreserved isolated placental cells can comprise W derived from a single donor, or a separate body for a plurality of placental cells. 所述分离的胎盘细胞群可W与目标接受者是完全的HLA匹配,或者部分的HLA-不匹配、或完全的HLA-不匹配。 The isolated placental cells can be completely W intended recipient of HLA-matched or partially of HLA- mismatched, completely or HLA- mismatched.

[0251] 因此,在一个实施方案中,分离的胎盘细胞可容器中包含塑料贴壁组织培养胎盘细胞群的组合物形式用于治疗脑或CNS中或周围血流破坏。 [0251] Thus, in one embodiment, the isolated placental cells can comprise a plastic container adherent placental cells in tissue culture in the form of a composition for treating in or around the brain or CNS disruption of blood flow. 在具体的实施方案中,所述分离的胎盘细胞冷冻保存。 In a specific embodiment, said isolated placental cells are cryopreserved. 在另外具体的实施方案中,所述容器是袋、瓶或罐。 In another specific embodiment, the container is a pouch, bottle or can. 在更具体的实施方案中,所述袋为无菌塑料袋。 In a more specific embodiment, said bag is a sterile plastic bag. 在更具体的实施方案中,所述袋适合、允许或有助于静脉施用所述分离的胎盘细胞群,例如通过静脉注射注入。 In a more specific embodiment, said bag is suitable, allows or facilitates intravenous administration of said isolated placental cells, e.g., by intravenous injection. 所述袋可W包括相互连接的多个内腔或隔室,其允许在施用前或施用过程中将分离的胎盘细胞与一种或多种其它溶液,例如药物进行混合。 The bag can comprise multiple lumens or W interconnected compartments, which allow separation in a prior or during application placental cells with one or more other solutions, e.g. by mixing the drug. 在另外具体的实施方案中,所述组合物包含有助于冷冻保存所述组合细胞群的一种或多种化合物。 In another specific embodiment, the composition comprises facilitate cryopreservation combination of one or more compounds of said cell population. 在另外具体的实施方案中,所述分离的胎盘细胞群包含在生理可接受的水溶液中。 In another specific embodiment, said isolated placental cell population contained in a physiologically acceptable aqueous solution. 在更具体的实施方案中,所述生理可接受的水溶液为0.9%化C1溶液。 In a more specific embodiment, said physiologically-acceptable aqueous solution of 0.9% of C1. 在另外具体的实施方案中,所述分离的胎盘细胞群包含与所述细胞群的接受者HLA匹配的胎盘细胞。 In another specific embodiment, said isolated population of placental cells comprising said cell population of the recipient HLA matching placental cells. 在另外具体的实施方案中,所述组合的细胞群包含与所述细胞群的接受者至少部分HLA不匹配的胎盘细胞。 In another specific embodiment, said population of cells comprising a combination of a recipient of said cell population is at least partially HLA-mismatched placental cells. 在另外具体的实施方案中,所述分离的胎盘细胞来源于多个供体。 In another specific embodiment, said isolated placental cells are derived from a plurality of donors.

[0252] 在某些实施方案中,容器中分离的胎盘细胞为分离的CD10+、CD34-、CD105+胎盘细胞,其中所述细胞已经被冷冻保存且包含在容器中。 [0252] In certain embodiments, the container in the isolated placental cells are isolated CD10 +, CD34-, CD105 + placental cells, wherein said cells have been cryopreserved and contained in a container. 在具体的实施方案中,所述CD10+、 CD3r、CD105+胎盘细胞还为CD200+。 In a specific embodiment, the CD10 +, CD3r, CD105 + placental cells are additionally CD200 +. 在更具体的实施方案中,所述CD10+、CD3r、CD105+、 CD200+胎盘细胞还为CD45-或CD90+。 In a more specific embodiment, the CD10 +, CD3r, CD105 +, CD200 + CD45- or placental cells are additionally CD90 +. 在更具体的实施方案中,所述CD10+、CD:34-、CD105+、 CD200+胎盘细胞还为CD45-和CD90+。 In a more specific embodiment, the CD10 +, CD: 34-, CD105 +, CD200 + and CD45- placental cells are also CD90 +. 在另外具体的实施方案中,所述CD34-、CD10+、CD105+胎盘细胞还为W 下的一种或多种:CD13\CD29+、CD33+、CD38-、CD44+、CD45-、CD54+、CD62E-、 CD62L-、CD62P-、甜3+(CD73+)、甜4+(CD73+)、CD8〇-、CD86-、CD90+、W2+(CD105+)、CD106/VCAM+、 CDll厂、CD 144/VE-巧粘蛋白i™、CD184/CXCR4-、CD200+、CD133-、0CT-4\SSEA3-、SSEA4-、 ABC-P+、邸R- (VEGFR2-)、HLA-A,B,C\ HLA-DP、DQ、DR-、HLA-G+或程序性死亡-1 配体(PDL1) +或其任何组合。 In another specific embodiment, the CD34-, CD10 +, CD105 + placental cells are additionally one or more of the W: CD13 \ CD29 +, CD33 +, CD38-, CD44 +, CD45-, CD54 +, CD62E-, CD62L- , CD62P-, sweet 3+ (CD73 +), sweet 4+ (CD73 +), CD8〇-, CD86-, CD90 +, W2 + (CD105 +), CD106 / VCAM +, CDll plant, CD 144 / VE- Qiao mucin i ™, CD184 / CXCR4-, CD200 +, CD133-, 0CT-4 \ SSEA3-, SSEA4-, ABC-P +, Di R- (VEGFR2 -), HLA-A, B, C \ HLA-DP, DQ, DR-, HLA + -G +, or any combination or programmed death 1 ligand (PDL1). 在更具体的实施方案中,所述CD34-、CD10+、CD105+胎盘细胞还为CD13+、CD29\ CD33+、CD38-、CD44\CD45-、CD54/ICAM+、CD62E-、CD6 化-、CD62P-、S册+(CD73+)、SH4+(CD73+)、 0080-、〔086-、〔090\5肥+(〔0105+)、〔0106/〔41\〔011厂、〔0 144八6-巧粘蛋白1\〔0184/ CXCR4-、CD200+、CD 133-、0CT-4+、SSEA3-、SSEA4-、ABC-P+、邸扩(66尸尺2-)、化4-4,8,(:\化八- 0口、09、0扩、化4-6+或程序性死亡-1配体。化1) +。 In a more specific embodiment, the CD34-, CD10 +, CD105 + placental cells are additionally CD13 +, CD29 \ CD33 +, CD38-, CD44 \ CD45-, CD54 / ICAM +, CD62E-, CD6 of -, CD62P-, S Volume + (CD73 +), SH4 + (CD73 +), 0080 -, [086--, [090 \ 5 fertilizer + ([0105 +), [0106 / [41 \ [011 plants, [0144 eight 6- Qiao mucin 1 [deg.] \ [0184 / CXCR4-, CD200 +, CD 133-, 0CT-4 +, SSEA3-, SSEA4-, ABC-P +, Di expansion ( 66 feet dead 2-), of 4-4,8, (: \ of eIGHT - 0, 09,0 expansion, of 4-6 +, or programmed death of 1 ligand 1) +.

[0253] 在某些实施方案中,上述引用的分离的胎盘细胞为分离的CD200+、HLA-G+胎盘细胞,其中所述细胞已经被冷冻保存且包含在容器中。 [0253] In certain embodiments, the above-referenced isolated placental cells are isolated CD200 +, HLA-G + placental cells, wherein said cells have been cryopreserved and contained in a container. 在另一个实施方案中,所述分离的胎盘细胞为CD73\CD105\CD200+细胞,其已经被冷冻保存且包含在容器中。 In another embodiment, the isolated placental cells are CD73 \ CD105 \ CD200 + cells, which had been stored frozen and contained in a container. 在另一个实施方案中,所述分离的胎盘细胞为CD200+、0CT-4+干细胞,其已经被冷冻保存且包含在容器中。 In another embodiment, the isolated placental cells are CD200 +, 0CT-4 + stem cells which have been cryopreserved and contained in a container. 在另一个实施方案中,所述分离的胎盘细胞为CD73+、CD105+细胞,其已经被冷冻保存且包含在容器中,并且当所述分离的胎盘细胞与胎盘细胞群在允许所述形成胚样体的条件下培养时, 有助于形成一个或多个胚样体。 In another embodiment, the isolated placental cells are CD73 +, CD105 + cells, which had been stored frozen and contained in a container, and when the isolated placental cells and placental cells that allow the formation of embryoid-like bodies when cultured under the conditions, contribute to the formation of one or more embryoid-like bodies. 在另一个实施方案中,所述分离的胎盘细胞为CD73+、CD105 +、HLA-G+细胞,其已经被冷冻保存且包含在容器中。 In another embodiment, the isolated placental cells are CD73 +, CD105 +, HLA-G + cells, which had been stored frozen and contained in a container. 在另一个实施方案中,所述分离的胎盘细胞为0CT-4+细胞,其已经被冷冻保存且包含在容器中,并且当所述分离的胎盘细胞与胎盘细胞群在允许所述形成胚样体的条件下培养时,有助于形成一个或多个胚样体。 In another embodiment, the isolated placental cells are 0CT-4 + cells, which had been stored frozen and contained in a container, and when the isolated placental cells and placental cells that allow the formation of embryoid when cultured under conditions body, contribute to the formation of one or more embryoid-like bodies.

[0254] 在另外具体的实施方案中,上述引用的分离的胎盘细胞为胎盘干细胞或如通过流式细胞术检测到的CD34-、CD10+和CD105+胎盘多能细胞。 [0254] In another specific embodiment, the above-referenced isolated placental cells are placental stem cells or as determined by flow cytometry to CD34-, CD10 + and CD105 + placental multipotent cells. 在更具体的实施方案中,所述分离的CD34-、CD10+、CD105+胎盘细胞为胎盘干细胞。 In a more specific embodiment, the isolated CD34-, CD10 +, CD105 + placental cells are placental stem cells. 在另外更具体的实施方案中,所述分离的CD3r、CD10 +、CD105+胎盘细胞为多能胎盘细胞。 In another more specific embodiment, said isolated CD3r, CD10 +, CD105 + placental cells are multipotent placental cells. 在另外具体的实施方案中,所述分离的CD34-、CD10+、CD105+胎盘细胞具有分化为神经表型细胞、成骨表型细胞或成软骨表型细胞的潜能。 In another specific embodiment, the isolated CD34-, CD10 +, CD105 + placental cells differentiate into neural phenotype cells, osteoblast phenotype cells, or to the potential of the cartilage phenotype. 在更具体的实施方案中,所述分离的CD34-、CD10+、CD105+胎盘细胞还为CD200+。 In a more specific embodiment, the isolated CD34-, CD10 +, CD105 + placental cells are additionally CD200 +. 在另外更具体的实施方案中,如流式细胞计检测的,所述分离的CD34-、CD10+、CD105+胎盘细胞还为CD90+或CD45-。 In another more specific embodiment, as detected by flow cytometry, the isolated CD34-, CD10 +, CD105 + placental cells are additionally CD90 + or CD45-. 在另外更具体的实施方案中,如流式细胞计检测的,所述分离的CD34-、 CD10+、CD105+胎盘细胞还为CD90+或CD45-。 In another more specific embodiment, as detected by flow cytometry, the isolated CD34-, CD10 +, CD105 + placental cells are additionally CD90 + or CD45-. 在更具体的实施方案中,如流式细胞计检测的, 所述分离的〔034-、〔010\〔0105\〔0200+胎盘细胞还为〔090+或〔045-。 In a more specific embodiment, as detected by flow cytometry, the isolated [034--, [010 \ [0105 \ [0200+ 090+ placental cells are additionally or [[045. 在另外更具体的实施方案中,如流式细胞计检测的,所述分离的CD34-、CD10+、CD105\CD200+胎盘细胞还为CD90+ 和CD45-。 In another more specific embodiment, as detected by flow cytometry, the isolated CD34-, CD10 +, CD105 \ CD200 + placental cells are additionally CD90 + and CD45-. 在另外更具体的实施方案中,如流式细胞计检测的,所述CD:34-、CD10+、CD105、 CD200\CD90+、CD45-细胞还为CD80-和CD86-。 In another more specific embodiment, as detected by flow cytometry, the CD: 34-, CD10 +, CD105, CD200 \ CD90 +, CD45- and CD80- cells were also CD86-. 在另外更具体的实施方案中,所述CD34-、CD10 +、CD 105+细胞还为W 下的一种或多种:CD29+、CD38-、CD44+、CD54+、CD8〇-、CD86-、S册+或甜4+。 In another more specific embodiment, the CD34-, CD10 +, CD 105+ cells are additionally one or more of the W: CD29 +, CD38-, CD44 +, CD54 +, CD8〇-, CD86-, S Volume + 4+ or sweet. 在更具体的实施方案中,所述分离的CD34-、CD10+、CD105+胎盘细胞还为CD44+。 In a more specific embodiment, the isolated CD34-, CD10 +, CD105 + placental cells are additionally CD44 +. 在W上任何分离的CD34-、CD10+、CD105+胎盘细胞的具体的实施方案中,所述细胞还为W下的一种或多种:CD 11 厂、CD 13 3-、邸R- (VEGFR2-)、HLA-A、B、C+、HLA-DP、DQ、DR-和/或PDL Γ。 W in any of the isolated CD34-, CD10 +, CD105 specific embodiments + placental cells, the cells are additionally one or more of the W: CD 11 plants, CD 13 3-, Di R- (VEGFR2- ), HLA-A, B, C +, HLA-DP, DQ, DR- and / or PDL Γ.

[0255] 在任何上述冷冻保存的分离的胎盘细胞具体的实施方案中,所述容器为袋。 [0255] In any of the foregoing cryopreserved isolated placental cells specific embodiment, the container is a bag. 在不同的实施方案中,所述容器包含大约、至少、或不超过1 X 1〇6所述分离的胎盘细胞、5X 106所述分离的胎盘细胞、1 X 1〇7所述分离的胎盘细胞、5 X 107所述分离的胎盘细胞、1 X 108所述分离的胎盘细胞、5 X 108所述分离的胎盘细胞、1 X 109所述分离的胎盘细胞、5 X 109所述分离的胎盘细胞、1 X l〇w所述分离的胎盘细胞或5 X l〇w所述分离的胎盘细胞。 In various embodiments, said container comprises about, at least, or not more than the isolated placental cells 1〇6 X 1, the 5X 106 isolated placental cells, 1 X 1〇7 the isolated placental cells , 5 X 107 isolated placental cells, 1 X 108 the isolated placental cells, 5 X 108 of the isolated placental cells, said isolated placental 1 X 109 cells, 5 X 109 of the isolated placental cells , 1 X l〇w said isolated placental cells or 5 X l〇w said isolated placental cells. 在任何上述冷冻保存群的其他具体的实施方案中,所述分离的胎盘细胞已传代约、至少或不超过5次、不超过10次、不超过15次或不超过20次。 In any of the other specific embodiments cryopreserved populations, said isolated placental cells have been passaged about, at least, or no more than 5, no more than 10, no more than 15, or no more than 20 times. 在任何上述冷冻保存的分离的胎盘细胞另外具体的实施方案中,所述分离的胎盘细胞已在所述容器内扩增。 In any of the foregoing cryopreserved isolated placental cells another specific embodiment, said isolated placental cells amplified within the container.

[0巧6] 5.9.2药物组合物 [Qiao 0 6] the pharmaceutical composition 5.9.2

[0257] 分离的胎盘细胞群或包含分离的胎盘细胞的细胞群,可W配制成体内使用的药物组合物,例如用于此处提供的治疗方法中。 [0257] The isolated placental cells or population of isolated placental cells comprising a cell population, W may be formulated into pharmaceutical compositions for use in vivo, for example, provided herein are methods of treating. 此类药物组合物在制药可接受的载体(例如用于体内施用的盐溶液或其它生理学可接受的溶液)中包含分离的胎盘细胞群,或包含分离的胎盘细胞的细胞群。 Such pharmaceutical compositions in a pharmaceutically acceptable carrier (e.g., for in vivo administration of saline solution or other physiologically acceptable solution) comprising isolated placental cells, or populations of cells comprising isolated placental cells. 包含本发明分离的胎盘细胞的药物组合物可W包含本发明其他部分描述的任何分离的胎盘细胞群或分离的胎盘细胞或任何其组合。 Any population of isolated placental cells comprising the isolated placental cells according to the present invention, a pharmaceutical composition of the present invention may comprise other parts W described or isolated placental cells, or any combination thereof. 所述药物组合物可W包含胎儿的分离的胎盘细胞、母体的分离的胎盘细胞,或同时包括胎儿和母体的分离的胎盘细胞。 Placental cells, the pharmaceutical composition may comprise isolated W fetus, maternal isolated placental cells, including isolated or both fetal and maternal placental cells. 本发明的药物组合物还可W包含来源于单个个体或胎盘的分离的胎盘细胞,或来源于多个个体或胎盘的分离的胎盘细胞。 The pharmaceutical compositions of the present invention may further comprise W isolated placental cells derived from a single subject or placenta, or from individual or multiple isolated placental placental cells.

[0258] 本发明的药物组合物可W包含任何数量的分离的胎盘细胞。 [0258] The pharmaceutical compositions of the present invention may comprise separate any number W of placental cells. 例如,在不同的实施方案中,单个单位剂量的分离的胎盘细胞可W包含约、至少、或不超过1 X 1〇5、5 X 105、1 X 1〇6、5X1〇6、1X1〇7、5X1〇7、1X1〇8、5X1〇8、1X1〇9、5X1〇9、1X1〇w、5X1〇w、1X1〇ii 或更多个分离的胎盘细胞。 For example, In various embodiments, a single unit dose of isolated placental cells can comprise about W, at least, or no more than 1〇5,5 X 105,1 X 1 X 1〇6,5X1〇6,1X1〇7 , 5X1〇7,1X1〇8,5X1〇8,1X1〇9,5X1〇9,1X1〇w, 5X1〇w, 1X1〇ii or more isolated placental cells.

[0259] 本发明的药物组合物包含含有50%或更多活细胞的细胞群(即,群中至少50%的细胞是功能性的或活的)。 [0259] The pharmaceutical compositions of the present invention comprises a cell population containing 50% or more viable cells (i.e., at least 50% of the population of cells are functional or living). 优选的,群中至少60%的细胞是活的。 Preferably, at least 60% of the population are viable cells. 更优选的,药物组合物的群中至少70%、80%、90%、95%或99%的细胞是活的。 More preferably the group of the pharmaceutical composition of at least 70%, 80%, 90%, 95% or 99% of the cells are viable.

[0260] 本发明提供的药物组合物可W包含一种或多种例如有助于移植的化合物(例如抗T细胞受体、免疫抑制剂等等);稳定剂如白蛋白、葡聚糖40、明胶、径乙基淀粉、勃脉力等等。 [0260] The present invention provides a pharmaceutical composition may comprise one or more compounds W contribute to transplant, for example, (e.g., anti-T cell receptor, an immunosuppressive agent, etc.); stabilizers such as albumin, dextran 40 , gelatin, starch diameter ethyl, Plasmalyte like. [0261 ]在一个实施方案中,当配制为注射溶液时,所述药物组合物包含约1 %至1.5%的HSA和约2.5%的葡聚糖。 [0261] In one embodiment, when formulated as an injectable solution, the pharmaceutical composition comprises from about 1% to 1.5% HSA, and about 2.5% dextran. 在一个优选的实施方案中,所述药物组合物包含每毫升约5 X 1〇6 至约2X107个细胞,其中所述细胞包含于包括5%服A和10%葡聚糖,任选包含免疫抑制剂, 例如1 Omg/kg环胞素A的溶液中。 In a preferred embodiment, the pharmaceutical composition comprises from about 5 X 1〇6 per milliliter to about 2X107 cells, wherein said cells are contained comprises 5% A and 10% dextran clothes, optionally containing immune inhibitors, for example 1 Omg / kg cyclosporine a in the solution.

[0262] 在其他的实施方案中,所述药物组合物例如溶液,包含大量细胞,例如分离的胎盘,如胎盘干细胞或胎盘多能细胞,其中所述药物组合物包含每毫升约l.〇〇. 3 X106至约5.0 1.5 X 106个细胞。 [0262] In other embodiments, the pharmaceutical compositions such as solutions, comprising a plurality of cells, such as separation of the placenta, placental stem cells or such placental multipotent cells, wherein said pharmaceutical composition comprises from about l.〇 per milliliter square. 3 X106 to about 5.0 1.5 X 106 cells. 在其他的实施方案中,所述药物组合物包含每毫升约1.5 X 106至约3.75 X 106个细胞。 In other embodiments, the pharmaceutical composition comprises from about 1.5 X 106 per milliliter to about 3.75 X 106 cells. 在其他的实施方案中,所述药物组合物包含约1 X 106个细胞/mL至约50 X 1〇6个细胞/mL、约1 X 106个细胞/mL至约40 X 106个细胞/mL、约1 X 106个细胞/mL至约30 X 1〇6 个细胞/mL、约1 X 106个细胞/血至约20X 106个细胞/mL、约1 X 106个细胞/mL至约15 X 106个细胞/mL、约1 X 106个细胞/mL至约10 X 106个细胞/mL。 In other embodiments, the pharmaceutical composition comprises from about 1 X 106 cells / mL to about 50 X 1〇6 cells / mL, about 1 X 106 cells / mL to about 40 X 106 cells / mL , about 1 X 106 cells / mL to about 30 X 1〇6 cells / mL, about 1 X 106 cells / of blood to about 20X 106 cells / mL, about 1 X 106 cells / mL to about 15 X 106 cells / mL, about 1 X 106 cells / mL to about 10 X 106 cells / mL. 在某些实施方案中,所述药物组合物不包含可见的细胞块(即无大的细胞块)或基本上无所述可见的细胞块。 In certain embodiments, the pharmaceutical composition comprises no visible cell clumps (i.e., no big cell clumps), or substantially free of the visible cell clumps. 如此处所用的,"大的细胞块"指不放大即可见的细胞聚集体,例如肉眼可见的,并且泛指大于约150微米的细胞聚集体。 As used herein, "large-cell block" refers to see without magnification cell aggregates, for example, visible, refers to greater than about 150 microns and cell aggregates. 在一些实施方案中,所述药物组合物包含约2.5%、3.0%、3.5%、4.0%、4.5%、 5.0%、5.5%、6.0%、6.5%、7.0%、7.5%、8.0%、8.5%、9.0%、9.5%或10% 的葡聚糖,例如葡聚糖-40。 In some embodiments, the pharmaceutical composition comprises about 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5 %, 9.0%, 9.5% or 10% dextran, such as dextran -40. 在一个具体的实施方案中,所述组合物包含约7.5%至约9%葡聚糖-40。 In a specific embodiment, the composition comprises from about 7.5% to about 9% dextran-40. 在一个具体的实施方案中,所述组合物包含约5.5%葡聚糖-40。 In a particular embodiment, the composition comprises about 5.5% dextran-40. 在某些实施方案中,所述药物组合物包含约1%至约15%的人血清白蛋白化SA)。 In certain embodiments, the pharmaceutical composition comprises from about 1% to about 15% of human serum albumin SA). 在具体的实施方案中,所述药物组合物包含约1%、2%、3%4%、5%、6%、7%、8%、9%、10%、11%、12%、13%、14%或15%服4。 In specific embodiments, the pharmaceutical composition comprises from about 1%, 2%, 3% 4% 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13 %, 14% or 15% for 4. 在一个具体的实施方案中,所述细胞已经被冷冻保存且融解。 In a specific embodiment, said cells have been cryopreserved and thawed. 在另外具体的实施方案中,所述细胞已通过70μΜ至100μΜ过滤器过滤。 In another specific embodiment, the cells are passed to the 70μΜ 100μΜ filter. 在另外具体的实施方案中,所述组合物不包含可见的细胞块。 In another specific embodiment, the composition comprises no visible cell clumps. 在另外具体的实施方案中,所述组合物包含每1〇6个细胞少于约200个细胞块,其中所述细胞块仅在显微镜下可见,例如光学显微镜。 In another specific embodiment, the composition comprises cells per 1〇6 cell mass less than about 200, wherein the cell mass is visible only under a microscope, such as an optical microscope. 在另外具体的实施方案中,所述组合物包含每1〇6个细胞少于约150个细胞块,其中所述细胞块仅在显微镜下可见,例如光学显微镜。 In another specific embodiment, the composition comprises cells per 1〇6 cell mass less than about 150, wherein the cell mass is visible only under a microscope, such as an optical microscope. 在另外具体的实施方案中,所述组合物包含每1〇6个细胞少于约100个细胞块,其中所述细胞块仅在显微镜下可见,例如光学显微镜。 In another specific embodiment, the composition comprises cells per 1〇6 cell mass less than about 100, wherein the cell mass is visible only under a microscope, such as an optical microscope.

[0263] 在一个具体的实施方案中,所述药物组合物包含每毫升约1.00.3 X 106个细胞、 约5.5%葡聚糖-40(*八)、约10%服4(*八)和约5%0150(八)。 [0263] In one particular embodiment, the pharmaceutical composition comprises per milliliter to about 1.0 0.3 X 106 cells, about 5.5% dextran-40 (* h), for 4 to about 10% (* h) and about 5% 0150 ( eight).

[0264] 在其他的实施方案中,所述药物组合物包含大量细胞,例如在包含10%葡聚糖-40 的溶液中的大量分离的胎盘细胞,其中所述药物组合物包含每毫升约1.〇〇. 3 Χ106个细胞至每毫升约5.01.5Χ106个细胞,并且其中所述组合物不包含肉眼可见的细胞块(即不包含大的细胞块)。 [0264] In other embodiments, the pharmaceutical composition comprises a plurality of cells, for example, contains a large number of separate solution 10% dextran -40 in the placental cells, wherein said pharmaceutical composition comprises from about 1 per ml .〇 square. 3 Χ106 cells per milliliter to about 5.0 1.5Χ106 cells, and wherein said composition comprises no visible cell clumps (i.e., without large cell clumps). 在一些实施方案中,所述药物组合物包含每毫升约1.5 X ΙΟ6至每毫升约3.75Χ106个细胞。 In some embodiments, the pharmaceutical composition comprises from about 1.5 X ΙΟ6 per milliliter to about 3.75Χ106 cells per ml. 在一个具体的实施方案中,所述细胞已经被冷冻保存且融解。 In a specific embodiment, said cells have been cryopreserved and thawed. 在另外具体的实施方案中,所述细胞已通过70μΜ至100μΜ过滤器过滤。 In another specific embodiment, the cells are passed to the 70μΜ 100μΜ filter. 在另外具体的实施方案中,所述组合物包含每1〇6个细胞少于约200个微细胞块即仅放大可见的细胞块)。 In another specific embodiment, the composition comprises cells per 1〇6 less than about 200 micro cell clumps are visible only enlarged i.e. cell mass). 在另外具体的实施方案中,所述药物组合物包含每1〇6个细胞少于约150个微细胞块。 In another specific embodiment, the pharmaceutical composition comprises cells per 1〇6 less than about 150 micro cell clumps. 在另外具体的实施方案中,所述药物组合物包含每1〇6个细胞少于约100个微细胞块。 In another specific embodiment, the pharmaceutical composition comprises cells per 1〇6 less than about 100 micro cell clumps. 在另外具体的实施方案中,所述药物组合物包含少于15%、14%、13%、12%、11%、10%、9%、8%、7%、6%、5%、 4%、3%或2%的0150,或少于1%、0.9%、0.8%、0.7%、0.6%、0.5%、0.4%、0.3%、0.2% 或0.1%的0150。 In another specific embodiment, the pharmaceutical composition comprises less than 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4 %, 3% or 2% of 0150, or less than 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2% or 0.1% of 0150.

[0265] 此处进一步提供包含细胞的组合物,其中所述组合物通过本发明公开的方法制备。 [0265] Further provided herein is a composition comprising a cell, wherein the method of preparing the composition disclosed by the present invention. 例如在一个实施方案中,药物组合物包含细胞,其中所述药物组合物通过W下方法制备,其包括过滤包含胎盘细胞例如胎盘干细胞或胎盘多能细胞的溶液W形成过滤的含有细胞的溶液;例如在冷冻保存之前用第一种溶液稀释所述过滤的含有细胞的溶液至每毫升约1至50Χ106、1 至40Χ106、1至30Χ106、1 至20Χ106、1至15Χ106 或1 至1〇Χ1〇6 个细胞;和用第二种包含葡聚糖而不包含人血清白蛋白化SA)的溶液稀释得到的过滤的含有细胞的溶液W 形成所述组合物。 For example, in one embodiment, a pharmaceutical composition comprising a cell, wherein the preparation method of the pharmaceutical composition through the W, which comprises a filter comprising placental cells, e.g., placental stem cells or placental multipotent solution W cells formed cell-containing solution by filtration; for example, a solution containing the cells before cryopreservation solution was diluted with the first filter per milliliter to about 1 to 50Χ106,1 to 40Χ106,1 to 30Χ106,1 to 20Χ106,1 to 15Χ106 or 1 to 1〇Χ1〇6 cells; and the second composition comprising a solution comprising dextran but not comprising cells W filtered dilute solution of human serum albumin SA) obtained by forming. 在某些实施方案中,所述稀释不超过每毫升约15 X 106个细胞。 In certain embodiments, the dilution per milliliter of not more than about 15 X 106 cells. 在某些实施方案中,所述稀释不超过每毫升约1〇3 X 106个细胞。 In certain embodiments, the dilution is not more than about 1〇 per milliliter 3 X 106 cells. 在某些实施方案中,所述稀释不超过每毫升约7.5 X 106个细胞。 In certain embodiments, the dilution is not more than about 7.5 X 106 per ml cells. 在其他的某些实施方案中,如果过滤的含有细胞的溶液在稀释之前包含每毫升少于约15 X 106个细胞,则过滤为任选的。 In certain other embodiments, if the cell-containing solution is filtered prior to dilution contains per milliliter less than about 15 X 106 cells, the filter is optional. 在其他的某些实施方案中,如果过滤的含有细胞的溶液在稀释之前包含每毫升少于约10 3 X 106个细胞,则过滤为任选的。 In certain other embodiments, if the cell-containing solution is filtered prior to dilution contains per milliliter less than about 10 3 X 106 cells, the filter is optional. 在其他的某些实施方案中,如果过滤的含有细胞的溶液在稀释之前包含每毫升少于约7.5 X 106个细胞,则过滤为任选的。 In certain other embodiments, if the cell-containing solution is filtered prior to dilution contains per milliliter less than about 7.5 X 106 cells, the filter is optional.

[0266] 在一个具体的实施方案中,所述细胞在用第一种稀释溶液稀释和用第二种稀释溶液稀释之间冷冻保存。 [0266] In one particular embodiment, the cells are diluted with a first dilution solution and stored frozen between dilute with said second dilution solution. 在另外具体的实施方案中,第一种稀释溶液包含葡聚糖和HSA。 In another specific embodiment, said first dilution solution comprises dextran and HSA. 第一种稀释溶液或第二种稀释溶液中的葡聚糖可W是任何分子量的葡聚糖,例如具有约lOkD至约150kD分子量的葡聚糖。 The first dilution solution or second dilution solution can be dextran of any molecular weight dextran W is, for example, about 150kD molecular weight dextran to about lOkD. 在一些实施方案中,第一种稀释溶液或第二种溶液中的葡聚糖为约2.5%'3.0%'3.5%'4.0%'4.5%、5.0%5.5%'6.0%'6.5%'7.0%'7.5%'8.0%、 8.5%、9.0%、9.5%或10%葡聚糖。 In some embodiments, the first dilution solution or second solution is about 2.5% dextran '3.0%' 3.5% '4.0%' 4.5% 5.0% 5.5% '6.0%' 6.5% '7.0 % '7.5%' 8.0%, 8.5%, 9.0%, 9.5% or 10% dextran. 在另外具体的实施方案中,第一种稀释溶液或第二种稀释溶液中的葡聚糖为葡聚糖-40。 In another specific embodiment, said first dilution solution or second dilution solution is dextran is dextran-40. 在另外具体的实施方案中,第一种稀释溶液和第二种稀释溶液中的葡聚糖为葡聚糖-40。 In another specific embodiment, said first dilution solution and said second dilution solution is dextran is dextran-40. 在另外具体的实施方案中,第一种稀释溶液中的葡聚糖-40 为5.0%的葡聚糖-40。 In another specific embodiment, the dextran-40 in said first dilution solution is 5.0% dextran-40. 在另外具体的实施方案中,第一种稀释溶液中的葡聚糖-40为5.5% 的葡聚糖40。 In another specific embodiment, the dextran-40 in said first dilution solution is 5.5% dextran 40. 在另外具体的实施方案中,第二种稀释溶液中的葡聚糖-40为10%的葡聚糖- 40。 In another specific embodiment, said second dilution solution is dextran-40 10% dextran - 40. 在另外具体的实施方案中,包含HSA的溶液中的HSA为1至15%的HSA。 In another specific embodiment, the HSA solution comprising HSA is 1 to 15% HSA. 在另外具体的实施方案中,包含HSA的所述溶液中的HSA为约1%、2%、3%4%、5%、6%、7%、8%、9%、10%、 11 %、12%、13%、14%或15%的HSA。 In another specific embodiment, said HSA in solution comprising HSA is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11% , 12%, 13%, 14% or 15% HSA. 在另外具体的实施方案中,包含HSA的溶液中的HSA为10%的HSA。 In another specific embodiment, the solution comprises HSA HSA in 10% HSA. 在另外具体的实施方案中,第一种稀释溶液包含HSA。 In another specific embodiment, said first dilution solution comprises HSA. 在更具体的实施方案中, 第一种稀释溶液中的HSA为10%的HSA。 In a more specific embodiment, a first dilute solution of HSA was 10% HSA. 在另外具体的实施方案中,第一种稀释溶液包含防冻剂。 In another specific embodiment, said first dilution solution comprises a cryoprotectant. 在更具体的实施方案中,所述防冻剂为DMS0。 In a more specific embodiment, said cryoprotectant is DMSO. 在另外具体的实施方案中,第二种稀释溶液中的葡聚糖-40为约10 %的葡聚糖-40。 In another specific embodiment, the second dilution solution is dextran-40 is about 10% dextran-40. 在另外具体的实施方案中,包含细胞的组合物包含约7.5%至约9%的葡聚糖。 In another specific embodiment, a composition comprising cells comprises about 7.5% to about 9% dextran. 在另外具体的实施方案中,所述药物组合物包含每毫升约1.0 0.3 X 106个细胞至每毫升约5.0 1.5 X 106个细胞。 In another specific embodiment, the pharmaceutical composition comprises per milliliter to about 1.0 0.3 X 106 cells per milliliter to about 5.0 1.5 X 106 cells. 在另外具体的实施方案中,所述药物组合物包含每毫升约1.5 X 106个细胞至每毫升约3.75 X 106个细胞。 In another specific embodiment, the pharmaceutical composition comprises from about 1.5 X 106 per ml cells per milliliter to about 3.75 X 106 cells.

[0267] 在另一个实施方案中,所述药物组合物通过W下方法制备,其包括(a)冷冻保存之前过滤包含胎盘细胞,例如胎盘干细胞或胎盘多能细胞的含有细胞的溶液W产生过滤的含有细胞的溶液;(b)冷冻保存过滤的含有细胞的溶液中的细胞,其为每毫升约1至50 X 106、1 至40X 106、1至30X 106、1至20X 106、1至15X106或1至l〇Xl〇6个细胞;(C)解冻所述细胞;和(d)用葡聚糖-40溶液W约1:1至约l:ll(v/v)的比例稀释过滤的含有细胞的溶液。 [0267] In another embodiment, the pharmaceutical composition is prepared by the method of W, placental cells, comprising a filter which comprises (a) prior to cryopreservation, e.g., placental stem cells or placental multipotent cells containing W was produced was filtered the cell-containing solution; (b) cells cryopreserved filtered cell-containing solution, which is about 1 milliliter per 50 X 106,1 to 40X 106,1 to 30X 106,1 to 15X106 to 20X 106,1 or 1 to l〇Xl〇6 cells; (C) thawing the cells; and (d) using dextran-40 solution W from about 1: 1 to about L: ratio ll (v / v) dilution of the filtration cell containing solution. 在某些实施方案中,如果步骤(a)之前细胞数少于每毫升约103X106个细胞,则过滤为任选的。 In certain embodiments, if the number of cells prior to step (a) is less than about 10 3X106 per ml of cells, the filter is optional. 在更具体的实施方案中,步骤(b)中的细胞W每毫升约103X106个细胞冷冻保存。 In a more specific embodiment, the cell W in step (b) is from about 10 3X106 per ml cells were cryopreserved. 在更具体的实施方案中,步骤(b)中的细胞冷冻保存于包含约5%至约10%葡聚糖-40和HSA的溶液中。 In a more specific embodiment, the cells are frozen in step (b) is stored in a solution comprising about 5% to about 10% dextran-40 and the HSA. 在某些实施方案中,步骤(b)中的稀释为不超过每毫升约15X106个细胞。 In certain embodiments, the dilution step (b) is no more than about 15X106 cells per ml.

[0268] 在另一个实施方案中,所述药物组合物通过W下方法制备,其包括:(a)将胎盘细胞,例如胎盘干细胞或胎盘多能细胞悬浮于包含10%HSA的5.5%葡聚糖-40溶液中W形成含有细胞的溶液;(b)用70μΜ过滤器过滤所述含有细胞的溶液;(C)用包含5.5%葡聚糖-40、 10%HSA和5%DMS0的溶液稀释含有细胞的溶液至每毫升约1至50Χ106、1至40Χ106、1至30 X 106、1至20 X 106、1至15 X 106或1至10 X 106个细胞;(d)冷冻保存所述细胞;(e)解冻所述细胞;和(d)用10%葡聚糖-40W约1:1至约l:ll(v/v)的比例稀释所述含有细胞的溶液。 [0268] The pharmaceutical composition prepared by the method of W In another embodiment, comprising: (a) the placental cells, e.g. placental stem cells or placental multipotent cells were suspended in 5.5% dextran comprising of 10% HSA -40 W sugar solution is a solution containing the cells; (b) filters with 70μΜ solution containing the cells; (C) -40, 10% HSA and diluted with 5.5% dextran solution in 5% DMS0 containing to a solution containing cells per milliliter to about 1 to 50Χ106,1 40Χ106,1 to 30 X 106,1 to 20 X 106,1 to 1 to 15 X 106 or 10 X 106 cells; (d) storage of the frozen cells ; (e) thawing the cells; and (d) with 10% dextran -40W from about 1: 1 to about L: the ratio of ll (v / v) diluted solution containing the cells. 在某些实施方案中,步骤(C)中的稀释物不超过每毫升约15X106个细胞。 In certain embodiments, step (C) were diluted to no more than about 15X106 cells per ml. 在某些实施方案中, 步骤(C)中的稀释物不超过约103X106个细胞/mL。 In certain embodiments, step (C) is diluted to no more than about 10 3X106 cells / mL. 在某些实施方案中,步骤(C)中的稀释物不超过约7.5 X 106个细胞/mL。 In certain embodiments, step (C) were diluted to no more than about 7.5 X 106 cells / mL.

[0269] 在另一个实施方案中,包含细胞的所述组合物通过W下方法制备,其包括:(a)离屯、大量细胞W收集细胞;(b)将所述细胞重悬浮于5.5%葡聚糖-40中;(C)离屯、所述细胞W 收集细胞;(d)将所述细胞重悬浮于包含10%HSA的5.5%葡聚糖-40溶液中;(e)通过70μΜ过滤器过滤所述细胞;(f)将所述细胞稀释于5.5%葡聚糖-40、10%HSA和5%DMS0中至每毫升约1至50X 106、1至40X 106、1至30X 106、1至20X 106、1至15X106或1至l〇Xl〇6个细胞;(g) 冷冻保存所述细胞;化)解冻所述细胞;和(i)用10%葡聚糖-4〇Wl:l至1:11稀释所述细胞。 [0269] In another embodiment, the compositions comprising cells prepared by the method of W, comprising: (a) from the village, a large number of cells collected W cells; (b) the cells were resuspended in 5.5% in dextran-40; (C) from the village, the cells were collected by W cells; (d) the cells were resuspended in a solution containing 5.5% dextran-40 in 10% HSA; (e) by 70μΜ filters said cells; (f) diluting the cells in 5.5% dextran -40,10% HSA and 5% DMS0 in per milliliter to about 1 to 50X 106,1 to 30X 106 to 40X 106,1 , to 20X 106,1 to 1 or 1 to l〇Xl〇6 15X106 cells; (G) cryopreserving said cells; of) thawing the cells; and (i) with 10% dextran -4〇Wl : l to the cells was diluted 1:11. 在某些实施方案中,步骤(f)中的稀释物不超过每毫升约15X106个细胞。 In certain embodiments, step (f) is diluted to no more than about 15X106 cells per ml. 在某些实施方案中,步骤(f)中的稀释物不超过约103X106个细胞/mL。 In certain embodiments, step (f) dilution of no more than about 10 3X106 cells / mL. 在某些实施方案中,步骤(f)中的稀释物不超过约7.5 X 106个细胞/mL。 In certain embodiments, step (f) is diluted to no more than about 7.5 X 106 cells / mL. 在其他的某些实施方案中,如果细胞数少于每毫升约10 3 X 106个细胞,贝脚滤为任选的。 In certain other embodiments, if the number of cells per milliliter less than about 10 3 X 106 cells, filtration is optional foot shell.

[0270] 组合物,例如本发明包含分离的胎盘细胞的药物组合物可W包含此处所述的任何分离的胎盘细胞。 [0270] composition of the present invention comprises, for example, isolated placental cells W pharmaceutical composition may comprise any of the isolated placental cells described herein.

[0271] 可W使用其他可注射的制剂,其适于施用细胞产品。 [0271] W may be other injectable formulations, which are suitable for administration cell products.

[0272] 在一个实施方案中,药物组合物包含分离的胎盘细胞,即基本上或完全为非母体来源的,即具有胎儿基因型;例如至少约90%、95%、98%、99%或约100%为非母体来源的。 [0272] In one embodiment, the pharmaceutical composition comprises isolated placental cells, i.e., substantially or completely non-maternal in origin, i.e., have the fetal genotype; e.g., at least about 90%, 95%, 98%, 99%, or to about 100% are non-maternal origin. 例如在一个实施方案中,药物组合物包含分离的胎盘细胞群,其为CD200+和HLA-G+;CD105+ 和CD200+; CD200+和0CT-4+; CD73+、CD105+和HLA-G+; CD73+和CD105% 并且当所述胎盘细胞群在允许形成胚样体的条件下培养时,有助于在包含所述分离的胎盘细胞群的胎盘细胞群中形成一个或多个胚样体;或0CT-4+,并且当所述胎盘细胞群在允许形成胚样体的条件下培养时,有助于在包含所述分离的胎盘细胞群的胎盘细胞群中形成一个或多个胚样体;或上述组合,其中至少70%、80%、90%、95%或99%所述分离的胎盘细胞为非母体来源的。 For example, in one embodiment, the pharmaceutical composition comprises isolated placental cells, which are CD200 +, and HLA-G +; CD105 + and CD200 +; CD200 + and 0CT-4 +; CD73 +, CD105 +, and HLA-G +; CD73 + and CD105% and when when the population of placental cells that allow formation of embryoid-like bodies under conditions of culture, contributes to the formation of one or more embryoid-like bodies in a population of placental cells comprise said isolated placental cell population; or 0CT-4 +, and when culturing said population of placental cells under conditions that allow formation of embryoid-like bodies, contribute to the formation of one or more embryoid-like bodies in a population of placental cells comprising said isolated placental cell population; or a combination thereof, wherein at least 70%, 80%, 90%, 95%, or 99% of said isolated placental cells are non-maternal in origin. 在另一个实施方案中,药物组合物包含分离的胎盘细胞群,其为CD10+、CD105+和CD34-;CD10\ CD105+、CD200+和〔0:34-;〔010\〔0105+、〔0200\〔0:34-和〔090+或0045-中的至少一种;〔010\ 0090+、〔0105+、〔0200+、〔034-和〔045-;〔010\〔090\〔0105\〔0200\〔0:34-和〔045-;〔0200+和HLA-G+; CD73+、CD105+和CD200+; CD200+和0CT-4+; CD73+、CD105+和HLA-G+; CD73+和CD105% 并且当所述胎盘细胞群在允许所述形成胚样体的条件下培养时,有助于在包含所述分离的胎盘细胞的胎盘细胞群中形成一个或多个胚样体;0CT-4+,并且当所述胎盘细胞群在允许所述形成胚样体的条件下培养时,有助于在包含所述分离的胎盘细胞的胎盘细胞群中形成一个或多个胚样体;或W下的一种或多种:CD117-、CD 133-、KDR-、CD8〇-、CD86-、HLA-A、B、C\ HLA-DP、DQ、DR-和/或PDLr;或上述组合,其中至少70 %、80%、90%、95 %或99%所述分离的胎盘细胞 In another embodiment, the pharmaceutical composition comprises isolated placental cells, which are CD10 +, CD105 + and CD34-; CD10 \ CD105 +, CD200 +, and [0:34 -; [010 \ [0105 +, [0200 \ [0 : 34- [090+ and 0045- or at least one of; [010 \ 0090 + + [0105, + [0200, and [034- [045--; [010 \ 090 [\ [0105 \ [0200 \ [0: 34- and [045--; [0200+ and HLA-G +; CD73 +, CD105 + and CD200 +; CD200 + and 0CT-4 +; CD73 +, CD105 +, and HLA-G +; CD73 + and CD105% and when said population of placental cells when cultured under conditions that allow the formation of embryoid-like bodies, contribute to the formation of one or more embryoid-like bodies in a population of placental cells comprising the isolated placental cells; 0CT-4 +, and when the placental cells when the population is cultured under conditions that allow the formation of embryoid-like bodies, contribute to the formation of one or more embryoid-like bodies in a population of placental cells comprising the isolated placental cells; or in one or more of W: CD117-, CD 133-, KDR-, CD8〇-, CD86-, HLA-A, B, C \ HLA-DP, DQ, DR- and / or PDLr; or a combination thereof, wherein at least 70%, 80%, 90%, 95% or 99% of said isolated placental cells 为非母体来源的。 Non-maternal in origin. 在一个具体的实施方案中,药物组合物还包含不获自胎盘的干细胞。 In a specific embodiment, the pharmaceutical composition further comprises not stem cells obtained from placenta.

[0273 ]此处提供的组合物,例如药物组合物中分离的胎盘细胞可W包含源自单个供体或源自多个供体的胎盘细胞。 [0273] The composition provided herein, for example, the pharmaceutical composition may be isolated placental cells derived from a single donor W or comprise placental cells derived from a plurality of donors. 分离的胎盘细胞可W是与指定接受者完全HLA-相配的、或部分HLA-不匹配、或完全HLA-不匹配的。 Isolated placental cells can be completely W is HLA- matched intended recipient, or partially HLA- mismatched, completely or HLA- mismatched.

[0274] 5.9.3包含分离的胎盘细胞的基质 [0274] 5.9.3 isolated placental cells comprising a matrix

[0275] 本发明进一步提供包含基质、水凝胶、支架等的组合物,其中包含胎盘干细胞或分离的胎盘细胞群。 [0275] The present invention further provides a substrate comprising, hydrogels, scaffolds, and the like compositions, which comprise placental stem cells or isolated placental cells. 所述组合物可用于代替用于治疗脑或CNS中或周围血流破坏的液体悬浮液中的细胞,或在用于治疗脑或CNS中或周围血流破坏的液体悬浮液中的细胞之外使用。 The composition may be used for the treatment in place in or around the brain or CNS disruption of blood flow in the liquid suspension of cells, or for the treatment of brain or CNS disruption of blood flow in or around the liquid suspension of cells other than use.

[0276] 此处所述的分离的胎盘细胞可W接种在天然基质上,例如胎盘生物材料,如羊膜材料。 [0276] The isolated placental cells described herein may be seeded onto a natural matrix W, e.g., placental biomaterials, such as amniotic membrane material. 此类羊膜材料可W是例如:直接从哺乳动物胎盘切割的羊膜;固定的或热处理的羊膜、基本干燥(即,<20%H20)的羊膜、绒毛膜、基本干燥的绒毛膜、基本干燥的羊膜和绒毛膜等。 Such amniotic material W may be, for example: cutting placental amniotic directly from the mammal; fixed or heat-treated amniotic membrane, substantially dry (i.e., <20% H20) amnion, chorion, substantially dry chorionic membrane, substantially dry amnion and chorionic so on. 优选的可W接种分离的胎盘细胞的胎盘生物材料在化riri,美国申请公开号2004/ 0048796中描述,其公开内容在此全文引入作为参考。 Preferred placental biomaterials W isolated placental cells were seeded at riri of, U.S. Application Description 2004 / Publication No. 0,048,796, the disclosure of which is hereby incorporated by reference.

[0277] 此处所述的分离的胎盘细胞可W悬浮在适合例如注射的水凝胶溶液中。 [0277] The isolated placental cells described herein, W can be suspended in a hydrogel solution suitable, for example injectable. 适合此类组合物的水凝胶包括自组装的肤,例如RAD16。 Suitable such compositions include self-assembling hydrogel skin, e.g. RAD16. 在一个实施方案中,包含细胞的水凝胶溶液可W在例如模具中硬化,从而形成其中分散有细胞的、用于移植的基质。 In an embodiment, a hydrogel solution comprising the cells, for example, W may be cured in a mold, thereby forming cells dispersed therein, the matrix for transplantation. 也可W培养此类基质中的分离的胎盘细胞使得细胞在移植前有丝分裂扩增。 W may also be cultured isolated placental cells in such matrices such cells prior to transplantation mitotically expanded. 水凝胶是例如通过共价键、离子键或氨键交联的有机聚合物(天然的或合成的),从而产生Ξ维开放的晶格结构,将水分子陷于其中形成凝胶。 Hydrogels, for example, through a covalent bond, ionic bond or amino bond crosslinked organic polymer (natural or synthetic), thereby producing Ξ dimensional open-lattice structure, wherein the trapped water molecules to form a gel. 水凝胶形成材料包括多糖,例如褐藻胶及其盐,肤、聚麟嗦和离子交联的聚丙締酸脂,或嵌段聚合物,例如分别通过溫度或抑交联的聚环氧乙烧-聚丙二醇嵌段共聚物。 Hydrogel-forming materials include polysaccharides such as alginate and salts of polyacrylic acid lipid association, skin, and poly Lin winded ionic crosslinking, or block polymers, for example, respectively by the temperature or suppressing cross-linked polyethylene oxide burn - polypropylene glycol block copolymers. 在一些实施方案中,水凝胶或基质是可生物降解的。 In some embodiments, the hydrogel or matrix is biodegradable.

[0278] 在一些实施方案中,所述制剂包含原位可聚合凝胶(参见例如,美国专利申请公开号2002/0022676,其公开内容在此全文引入作为参考;Anseth等人,J. Control Release,78 (1-3) :199-209(2002) ;Wang等人,Biomaterials,24(22):3969-80(2003))。 [0278] In some embodiments, the formulation comprises an in situ polymerizable gel (see, e.g., U.S. Patent Application Publication No. 2002/0022676, the disclosure of which is hereby incorporated by reference;. Anseth et al., J Control Release , 78 (1-3): 199-209 (2002); Wang et al., Biomaterials, 24 (22): 3969-80 (2003)).

[0279] 在一些实施方案中,所述聚合物在水性溶液(例如水、缓冲盐溶液、或乙醇水溶液) 中至少是部分溶解的,所述聚合物具有带电的侧基,或其单价离子盐。 [0279] In some embodiments, the polymer in aqueous solutions (e.g., water, buffered salt solutions, or aqueous ethanol) are at least partially soluble, said polymer having a charged side groups, or a monovalent ionic salt thereof . 具有酸性侧基、可W 与阳离子反应的聚合物实例是聚(麟嗦)、聚(丙締酸)、聚(甲基丙締酸)、丙締酸和甲基丙締酸的共聚物、聚(醋酸乙締醋),w及横化聚合物,例如横酸化聚苯乙締。 Having acidic side groups, examples of the polymer can be reacted with cations W is a poly (Lin repetitious), poly (propyl association acid), poly (methyl propionic acid association), propionic acid and methacrylic association associating acid copolymer, poly (vinyl acetate association vinegar), w and cross-polymers, for example polystyrene cross acidified association. 通过丙締酸或甲基丙締酸与乙締酸单体或聚合物反应形成的具有酸性侧基的共聚物也可W使用。 Copolymer having acidic side groups formed by the association of propionic acid or methacrylic acid and acetic associated associative acid monomer or polymer may also be a reaction using W. 酸性基团的实例是簇酸基、横酸基、面代醇基(优选氣代)、酪0H基和酸性0H基。 Examples of acidic groups are the cluster acid group, an acid group horizontal, surface behalf alcohol (preferably gas generation), casein 0H 0H group and an acidic group.

[0280]此处所述的分离的胎盘细胞或其共培养物可W接种在立体框架或支架上,并植入体内。 [0280] The isolated placental cells described herein, or co-cultures thereof can be seeded onto the stereotaxic frame W or scaffold and implanted in vivo. 此类框架可W与刺激组织形成的任何一种或多种生长因子、细胞、药物或其它组分联合植入。 Such frameworks may be any of a W and stimulate tissue formation or more growth factors, cells, drugs or other components implanted in combination.

[0%1]可W使用的支架的实例包括非织拉(nonwoven mat)、多孔泡沫、或自组装肤。 [0% 1] Examples of stents include the use of W nonwoven pull (nonwoven mat), porous foams, or self assembling skin. 非织拉可W利用含有合成的可吸收的乙醇酸和乳酸共聚物(例如:PGA/PLA)的纤维形成(VICR化,Ethicon,Inc.,Somerville,NJ)。 W can be pulled by using nonwoven glycolic and lactic acids containing synthetic absorbable copolymer (e.g.: PGA / PLA) fibers formed (VICR of, Ethicon, Inc., Somerville, NJ). 由例如聚(e-己内醋)/聚(乙醇酸KPCL/PGA) 共聚物组成的泡沫也可作为支架使用,其通过例如冷冻干燥或低压冻干处理形成(参见例如美国专利号6,355,699)。 For example, poly (e- caprolactone inner) / poly (glycolic acid KPCL / PGA) copolymer foam may also be used as a stent, for example, by freeze-drying or lyophilization process is formed (see, e.g. U.S. Pat. No. 6,355,699).

[0282] 在另一个实施方案中,分离的胎盘细胞还可W接种在拉上,或与其接触,所述拉可W由生物可吸收材料如PGA、PLA、P化共聚物或渗合物或透明质酸制成的复丝组成。 [0282] In another embodiment, isolated placental cells can also be seeded pull W, or in contact with, the W can be pulled by a bioabsorbable material such as PGA, PLA, P or copolymer compound or infiltration multifilament made of hyaluronic acid composition.

[0283] 在另一个实施方案中,此处提供的分离的胎盘细胞可W接种在可W是复合结构的泡沫支架上。 [0283] In another embodiment, isolated placental cells provided herein may be seeded W W is a foam scaffold structure. 此类泡沫支架可W模制成有用的形状,例如需要修复、取代或强化的机体特定结构的一部分。 Such foam scaffolds can be part W molded into a useful shape, such as the need to repair, replace or enhance a particular body structure. 在一些实施方案中,在接种细胞前,用例如0.1M乙酸处理框架,再将框架解育在聚赖氨酸、PBS和/或胶原中,从而增加细胞附着。 In some embodiments, the cells prior to inoculation, with 0.1M acetic acid for example, the process frame, the frame then incubated in solution polylysine, PBS, and / or collagen, thereby increasing cell attachment. 可W修饰基质的外表面来改善细胞的附着或生长,W及组织的分化,例如通过血浆包被基质,或添加一种或多种蛋白质(例如:胶原、弹性纤维、网状纤维)、糖蛋白、葡萄糖胺聚糖(例如:硫酸肝素、4-硫酸软骨素、6-硫酸软骨素、硫酸皮肤素、硫酸角质素等)、细胞基质,和/或其它材料,例如但不限于明胶、褐藻胶、 琼脂、琼脂糖和植物胶等。 The outer surface of the substrate W may be modified to improve the attachment or growth of cells differentiated, W and tissue, such as by plasma coating the matrix, or addition of one or more proteins (e.g.: collagen, elastic fibers, reticular fibers), sugar proteins, glycosaminoglycans (e.g.: heparin sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratan sulfate, etc.), a cellular matrix, and / or other materials, such as but not limited to, gelatin, alginic gum, agar, agarose, and plant gum.

[0284] 在一些实施方案中,所述支架包括使其具有非血栓形成性的材料,或用所述材料处理。 [0284] In some embodiments, the stent comprises a material formed to have a non-thrombotic properties, or processing of the material. 运类处理和材料还可W促进和维持内皮生长、迁移和胞外基质沉积。 Transport and handling material W may also promote and sustain endothelial growth, migration, and extracellular matrix deposition. 运类材料和处理的实例包括但不限于天然材料例如基膜蛋白(如层粘连蛋白和IV型胶原)、合成材料例如EPTFE,和含娃嵌段聚氨醋脈(se卵ented polyurethaneurea silicones)例如PURSPANTM (The 化lymer Technology Group,Inc.,Berkeley,&lif.)。 Examples of transport and processing based material including but not limited to, natural materials such as basement membrane proteins (e.g., laminin, and type IV collagen), synthetic materials such as the EPTFE, and (ented polyurethaneurea silicones se eggs) containing vinegar pulse polyurethane block baby e.g. PURSPANTM (The technology lymer Technology Group, Inc., Berkeley, & lif.). 所述支架还可W包含抗血栓形成剂例如肝素;所述支架还可W在用分离的胎盘细胞接种之前进行处理W改变表面电荷(例如用血浆包被)。 W may further comprise the stent antithrombotic agent such as heparin; the stent may also be treated W W alter the surface charge (e.g., coating with plasma) prior to inoculation with the isolated placental cells.

[0285] 在一个实施方案中,所述分离的胎盘细胞W约0.5 X 106至约8 X 106个细胞/mL接种在合适的支架上或与其接触。 [0285] In one embodiment, the isolated placental cells W from about 0.5 X 106 to about 8 X 106 cells / mL were seeded on a suitable scaffold or in contact therewith.

[02化]5.10永生化的胎盘细胞系 [Of 02] 5.10 immortalized placental cell lines

[0287] 哺乳动物胎盘细胞通过用任何含有生长促进基因的合适的载体转染可W条件性永生化,即,编码在适宜条件下促进所转染的细胞生长的蛋白质的基因,从而所述生长促进蛋白质的产量和/或活性可通过外部因子调控。 [0287] Any mammalian placental cells by treatment with a suitable vector containing a growth-promoting gene transfection W conditionally immortalized, i.e., a gene encoding a protein promoting the transfected cells grown under suitable conditions such that the growth promote protein production and / or activity can be modulated by external factors. 在优选的实施方案中,所述生长促进性基因是癌基因,例如但不限于:v-myc、N-myc、c-myc、p53、SV40大T抗原、多瘤病毒大T抗原、Ela腺病毒或人乳头瘤病毒的E7蛋白。 In a preferred embodiment, said growth-promoting gene is an oncogene such as, but not limited to: v-myc, N-myc, c-myc, p53, SV40 large T antigen, polyoma large T antigen, adenovirus Ela virus or human papillomavirus E7 protein.

[0288] 生长促进性蛋白质的外部调控可W通过将生长促进性基因置于外部可调控启动子的控制之下来实现,例如其活性能够通过例如改变所转染的细胞的溫度或者与细胞接触的培养基的组成来控制的启动子,在一个实施方案中,可W采用四环素(tet)控制的基因表达系统(参见Gossen等,Proc .化tl .Acad. Sci . USA 89 : 5547-5551,1992;Hoshimaru等, Proc .Natl .Acad. Sci .USA 93:1518-1523,1996)。 [0288] External regulation of the growth-promoting protein can be grown by W-promoting gene under the control of an external promoter regulatable promoter down implemented, for example, the activity can be, for example, changing the temperature of the transfected cells or in contact with the cells through ... to control the composition of the medium of a promoter, in one embodiment, W can be controlled gene expression system using tetracycline (Tet) (see Gossen et al, Proc of tl .Acad Sci USA 89: 5547-5551,1992 .; Hoshimaru the like, Proc .Natl .Acad Sci .USA 93: 1518-1523,1996). 不存在tet时,此载体内tet控制的转录激活子(tTA)强有力地激活来自地的转录,其为与操纵子序列融合的来自人巨细胞病毒的最小启动子。 When the absence of tet, a tet controlled transcription activator within this vector (the tTA) strongly activates transcription from the ground, which is a minimal promoter from human cytomegalovirus fused to the operator sequence. tTA是大肠杆菌巧scherichiacoli)转座子-10来源的tet抗性操纵子的阻遏蛋白(tetR)和单纯瘤疹病毒VP16酸性结构域的融合蛋白。 tTA is a fusion protein in E. coli Qiao scherichiacoli) -10 transposon-derived tet resistance operon repressor protein (tetR) and Herpes simplex VP16 acidic tumor domains. 无毒低浓度的tet(例如0.01-1.0 yg/mU几乎完全消除tTA的转录激活作用。 Tet low concentration of non-toxic (e.g. 0.01-1.0 yg / mU of almost completely eliminating the tTA transcriptional activation.

[0289] 在一个实施方案中,载体还含有编码可选择性标记(例如赋予药物抗性的蛋白质) 的基因。 [0289] In one embodiment, the vector further contains a gene encoding a selectable marker (e.g., a protein that confers drug resistance) is. 细菌新霉素抗性基因(neoR)是一种运样的所述方法可W采用的标记。 Bacterial neomycin resistance gene (the neoR) is a method of labeling the sample transport W may be employed. 携带neoR的细胞可W通过本领域普通技术人员公知的方法来选择,例如向生长培养基中添加例如100- 200yg/mL的G418。 W carrying the neoR cells can be selected by those of ordinary skill in the art well-known methods, such as adding e.g. 100- 200yg / mL G418 to the growth medium.

[0290] 转染可W通过本领域普通技术人员公知的多种方法中的任何一种来实现,包括但不限于逆转录病毒感染。 [0290] Transfection can be achieved by any of a W number of methods known to those of ordinary skill in the art, including, but not limited to, retroviral infection. 通常,细胞培养物可W通过与从载体生产细胞系收集的条件培养基和含N2添加剂的DMEM/F12的混合物解育来转染。 Typically, a cell culture may be a mixture of W and by conditioned medium collected from the producer cell lines and vectors containing N2 supplements DMEM / F12 transfected sterile solution. 例如,如上文所述制备的胎盘细胞培养物可W在例如5天后,通过在体外在一体积的条件培养基和两体积的含N2添加剂的DMEM/ F12中解育大约20小时进行感染。 For example, placental cells prepared as described above in culture may be, for example, W 5 days, infected in vitro in DMEM conditioned medium and two volumes of a volume of additive-containing N2 / F12 incubated in solution for about 20 hours passed. 携带可选择标记的经转染的细胞可W随之如上文所述进行选择。 Carrying a selectable marker may be the transfected cells as described above W will be selected.

[0291] 在转染之后,培养物在允许增殖的表面上传代,例如允许至少30%的细胞在24小时的周期内倍增。 [0291] Following transfection, cultures were passaged to allow the proliferation of the surface, for example, allows at least 30% of the cells to double in a 24 hour period. 优选底物是聚鸟氨酸/层粘连蛋白底物(由包被聚鸟氨酸(1化g/mU和/或层粘连蛋白(1化g/mU的组织培养塑料组成)、聚赖氨酸/层粘连蛋白底物、或用纤粘连蛋白处理的表面。培养物然后每:Γ4天用生长培养基补料,培养基可W或可W不添加一种或多种增殖促进因子。当培养物不足50%汇合度时可W向生长培养基中添加增殖促进因子。 Preferably the substrate is a polyornithine / laminin substrate (by the packet is a poly-ornithine (of 1 g / mU and / or laminin (of 1 g / mU of plastic tissue culture), polylysine acid / laminin substrate or a surface treated with fibronectin each culture were then:. Γ4 days fed with growth medium, the medium may be W is not W or adding one or more growth-promoting factors as. W may be added to the growth medium to promote the growth factor on the culture was less than 50% confluence.

[0292] 条件性永生化的胎盘细胞系可W利用标准技术传代,例如在80-95%汇合时通过膜酶消化。 [0292] conditionally-immortalized placental cell lines can be passaged using standard techniques W, e.g. by membrane digestive enzymes when at 80-95% confluence. 在一些实施方案中,多达大约第二十几次传代有益于维持选择(通过例如,对含新霉素抗性基因的细胞添加G418)。 In some embodiments, up to about twenty several passages beneficial to maintain selection (by, for example, cells containing a neomycin resistance gene adding G418). 细胞还可W在液氮中冷冻进行长期储存。 W cells can also be frozen for long-term storage in liquid nitrogen.

[0293] 可W从如上文所述制备的条件性永生化人胎盘细胞系中分离克隆细胞系。 [0293] W may be immortalized human placenta from conditionally prepared as described above clonal cell lines isolated cell lines. 一般而言,所述克隆细胞系可W利用标准技术如通过有限稀释或利用克隆环分离并扩增。 Generally, the clonal cell lines may be W using standard techniques such as by limiting dilution or using cloning rings isolated and amplified. 克隆细胞系可W大体如上文所述进行补料和传代。 W clonal cell lines may generally be fed as described above and subculture.

[0294] 条件性永生化的人胎盘干细胞系可W是但不必须是克隆的,一般可W通过在有助于分化的培养条件下抑制生长促进性蛋白质的产量和/或活性来诱导分化。 [0294] conditionally-immortalized human placental stem cell lines may be W is cloned but not necessarily, generally W by inhibiting the growth under culture conditions conducive to the production promoting differentiation and / or activity of a protein to induce differentiation. 例如,如果编码生长促进性蛋白质的基因处于外部可调控启动子的控制之下,则可W改变条件例如溫度或培养基的组成,W抑制生长促进性基因的转录。 For example, if the gene encoding the growth-promoting protein is under the control of an externally regulatable promoter, can change the composition of W conditions such as temperature or culture media, W inhibit the growth promoting transcriptional gene. 对于如上文所讨论的四环素控制的基因表达系统,可W通过添加四环素抑制生长促进基因的转录来实现分化。 For the tetracycline-controlled gene expression system discussed above, W can inhibit the growth promoting transcription of the gene by addition of tetracycline to achieve differentiation. 通常,Uig/mL四环素在4-5天足W启动分化。 Typically, Uig / mL tetracycline promoter natural feet differentiation 4-5 W. 为促进进一步的分化,生长培养基中可W包含另外的试剂。 To promote further differentiation, growth medium may comprise additional agents W.

[0巧5] 5.11试剂盒 [Qiao 5 0] 5.11 kit

[0296]另一方面,此处提供试剂盒,适于治疗患有CNS中或周围血流破坏的个体,例如患有中风的个体,其在不同于装载试剂盒成分的另外容器中包含塑料贴壁组织培养的多能胎盘细胞,例如胎盘干细胞或胎盘多能细胞,和其分离的群,例如W上5.4.2节所述的细胞,W 及使用说明书。 [0296] On the other hand, provided herein is a kit suitable for treating an individual suffering from CNS disruption of blood flow in or around, for example, an individual suffering from a stroke, which comprises a plastic container is different from the load attached to the kit further ingredient wall tissue culture multipotent placental cells, e.g., placental stem cells or placental multipotent cells, isolated, and its population, such as cells described in section 5.4.2 of the W, W and instructions for use. 优选的,提供在药学可接受溶液中的胎盘干细胞,例如适于烦内施用的溶液或适于静脉注射施用的溶液。 Preferably, there is provided placental stem cells in a pharmaceutically acceptable solution such as a solution suitable for administration in trouble or a solution suitable for intravenous administration. 在某些实施方案中,胎盘干细胞或胎盘多能细胞为此处所述的任意CD10+、CD34-、CD105+胎盘细胞,例如〔010+、〔034-、〔0105+、〔0200+胎盘细胞。 In certain embodiments, placental stem cells or placental multipotent cells described herein any CD10 +, CD34-, CD105 + placental cells, e.g. 010 [+ [034--, 0105 [+ [0200+ placental cells.

[0297] 在某些实施方案中,试剂盒包含有助于递送胎盘细胞至个体的一种或多种组分。 [0297] In certain embodiments, the kit comprises placental cells to facilitate delivery of one or more individual components. 例如在某些实施方案中,试剂盒包含有助于烦内递送胎盘细胞至个体的组分。 For example, in certain embodiments, the kit comprises placental cells facilitate the delivery of the components subject to trouble. 在所述实施方案中,试剂盒可包含例如适于递送细胞至个体的注射器和针;能够显象受感染CNS组织的放射性或非放射性化合物(例如钻55)等等。 In such embodiments, the kit may comprise suitable for delivery of the cells to the subject, for example, syringes and needles; developer can be infected by radioactive or nonradioactive compound CNS tissue (e.g., drill 55) and the like. 在所述实施方案中,胎盘细胞可包含在袋中的试剂盒中或一个或多个小瓶中。 In such embodiments, the placental cells may be contained in the kit bag or one or more vials. 在某些其他的实施方案中,试剂盒包含有助于静脉内或动脉内递送胎盘细胞至个体的组分。 In certain other embodiments, the kit comprises placental cells facilitate delivery intravenously or intraarterially to individual components. 在所述实施方案中,胎盘细胞可包含在瓶或袋内(例如血袋或能包含多达约1.化包含所述细胞的溶液的类似袋),并且所述试剂盒另外包含适于递送细胞至个体的管和针。 In such embodiments, the placental cells may be contained in a bottle or bag (e.g., blood bag or can contain up to about 1 of said solution comprising a pouch-like cells), and said kit further comprises suitable for the delivery cells to the individual tubes and needles.

[0298] 另外,试剂盒可包括减少个体疼痛或炎症的一种或多种化合物(例如止痛剂、酱体或非-酱体抗炎化合物)等等。 [0298] Further, the kit may include reducing pain in an individual or one or more compounds of inflammation (e.g. analgesics, sauce or non - sauce antiinflammatory compound) and the like. 试剂盒还可W包括抗菌或抗病毒化合物(例如一种或多种抗生素)、减少个体焦虑的化合物(例如阿普挫仑)、减少个体免疫应答的化合物(例如环胞素A)、抗组胺药(苯海拉明、氯雷他定、地氯雷他定、奎替阿平、非索非那定、西替立嗦、异丙嗦、 扑尔敏、左旋西替利嗦、盐酸西咪替下、法莫替下、雷尼替下、尼扎替下、罗沙替下、拉巧替下等等)。 The kit may also include antibacterial or W antiviral compound (e.g., one or more antibiotics), reduce the anxiety of the individual compounds (e.g. alprazolam), reducing the compound (e.g., cyclosporine A) of the individual's immune response, an anti-group amine drugs (diphenhydramine, loratadine, desloratadine, Kui for Appin, fexofenadine, cetirizine stand winded, isopropyl winded, chlorpheniramine, winded Levocetirizine hydrochloride West, who replaced Mia, who replaced Farmer, who replaced Rainey, who replaced Nizar, who replaced Rocha, who replaced clever pull, etc.).

[0299] 另外,试剂盒可包括一次性用品,例如无菌擦、一次性纸品、手套等等,其有助于准备个体用于递送,或其降低个体中由于胎盘细胞的施用导致感染的可能性。 [0299] Further, the kit may include a disposable article, for example a sterile wipe, disposable paper products, gloves, etc., which contribute to an individual ready for delivery, or a decrease due to the administration of the placental cells in an individual results in infection possibility.

[0300] 6.实施例 [0300] 6. EXAMPLES

[0301] 6.1实施例1:烦内施用分离的胎盘细胞治疗中风 [0301] 6.1 Example 1: administering the isolated placental cells trouble treating stroke

[0302] 本实施例证实了在治疗与脑或CNS中或周围血流破坏有关的症状中施用分离的胎盘细胞的功效。 [0302] This example demonstrates the treatment of in or around the brain or CNS symptoms associated disruption of blood flow in the isolated placental cells are administered effect.

[0303] 通过利用约1至约2mg/ml的胶原酶I酶促消化例如30分钟,随后用约0.25 %浓度的膜蛋白酶37C酶切消化10分钟,获得CD34-、CD10+、CD105\CD200+塑料贴壁组织培养的胎盘细胞。 [0303] By using collagenase from about 1 to about 2mg / ml of I enzymatic digestion such as 30 minutes, followed by digestion with a concentration of about 0.25% film protease digestion 37 C for 10 minutes, CD34-, CD10 +, CD105 \ CD200 + tissue culture plastic adherent placental cells. Sprague-Dawl巧大鼠用作中风模型。 Sprague-Dawl clever rat stroke model used. 大鼠为已建立的模型动物,其用于研究中风的结果和不同疗法对中风症状的效果。 Rats as an established animal model for stroke research results and the effect of different treatments for stroke symptoms. 参见例如,畑en等人,"A Model of Focal Ischemic Stroke in the Rat:Reproducible Extensive Cortical Infarction",Stroke 17(4): 738-743(1986)。 See, e.g., en Hata et al., "A Model of Focal Ischemic Stroke in the Rat: Reproducible Extensive Cortical Infarction", Stroke 17 (4): 738-743 (1986). 每种条件使用10只动物。 Each condition using 10 animals.

[0304] MCA中风手术:所有手术过程在无菌条件下进行。 [0304] MCA stroke Surgery: All surgical procedures performed under sterile conditions. 动物用氯化儀合剂麻醉(300mg/ kg,经腹膜)并且检查疼痛反射。 Animals with a chlorinating agent anesthesia instrument (300mg / kg, intraperitoneally) and checks pain reflex. 对深度麻醉下的动物进行MCA闭塞手术。 The animals under deep anesthesia MCA occlusion surgery. MCA缝合技术设及经过颈动脉插入细丝W实现MCA的接合,由此阻断来自总颈动脉W及来自Willis圆周的血流。 MCA suture techniques and provided W filament inserted through the carotid artery to achieve engagement of the MCA, thereby blocking W from the carotid artery and blood flow from the circumference of Willis. 确定且通过腹中线颈切口分离右总颈动脉。 Determining the right carotid artery and separated by a ventral midline cervical incision. 缝线大小为4-0,由无菌、非-可吸收缝线制成化thicon,Inc.,Somerville,NJ),缝线尖端利用橡胶胶水形成直径为24至26-量规大小的锥形。 4-0 size suture, a sterile, non - absorbable sutures made of thicon, Inc., Somerville, NJ), rubber cement formed using a suture tip having a diameter of 24 to 26- tapered gauge size. 约15至17mM的细丝从外部和内部颈动脉接合点插入W封闭MCA。 From about 15 to 17mM filament junctions inserted W A MCA from external and internal carotid arteries. 右MCA封闭一小时。 Right MCA blocked for one hour. 基于已公开的研究,一小时MCA封闭引起最大程度的梗塞。 Based on published studies, one hour to cause the greatest degree of closure MCA infarction. 参见Borlongan,Neurorep . 9 (16):3615-3621(1998);Borlongan等.Jharmacol.Biochem.Behav.52(l):225-229(1995); Borlongan等,Physiol.Behav.58(5) :909-917(1995)。 See Borlongan, Neurorep 9 (16): 3615-3621 (1998); Borlongan et .Jharmacol.Biochem.Behav.52 (l):. 225-229 (1995); Borlongan et, Physiol.Behav.58 (5): 909-917 (1995). 加热板和直肠溫度计用于维持体溫在正常限度内。 A rectal thermometer, and a heating plate for maintaining the temperature within normal limits. 利用激光多普勒测定成功的封闭和再灌注。 Doppler measurement of success and reperfusion closed with a laser. 激光多普勒探头置于MCA远端W 测定封闭之前、期间和之后的脑血流量。 Laser Doppler probe was placed closed prior to the assay, and cerebral blood flow during after MCA distal W.

[0305] 在局部缺血后2天时,烦内直接注射至局部缺血部位而施用胎盘贴壁细胞(大约5 微升中400,000个细胞)。 [0305] On day 2, the direct injection trouble in post-ischemic administration to the ischemic site and placental adherent cells (approximately 400,000 cells in 5 [mu] l). 大鼠仅给予赋形剂(10 %葡聚糖和5 %人血清白蛋白)、4 X 105个活细胞、1 Omg/kg环胞素A与4 X 105个活细胞、或4 X 105个无活力的细胞和环胞素A。 Rats were given only vehicle (10% dextran and 5% human serum albumin), 4 X 105 viable cells, 1 Omg / kg cyclosporine A with 4 X 105 viable cells, or 4 X 105 th non-viable cells and cyclosporine A.

[0306] 在局部缺血后第7和14天,测定动物中风诱发的运动不平衡。 [0306] In the post-ischemic 7 and 14 days, and the animals induced movement stroke imbalance. 利用提高身体摆动检测化BST)或Bederson检测评估运动不平衡。 Improve the detection of the use of body sway BST) or Bederson motion detection and evaluation imbalance. 参见Borlongan&Sanberg,J.化urosci 15(7): 5372-5378(1995)。 See Borlongan & Sanberg, J of urosci 15 (7): 5372-5378 (1995). 邸ST中,通过持起尾部升高动物,并且记录摆动习性的频率和方向。 Di ST by holding the tail from animals raised, and the recording frequency and direction of the swing habits. 大鼠放入有机玻璃框中并且允许其适应约2分钟。 Rats were placed Plexiglass box and allowed to adapt to about 2 minutes. 握住大鼠尾根部大约1英寸,使得大鼠的鼻子离表面大约1英寸。 Holding the base of the tail of rats approximately 1 inch from the surface so that the nose of the rat about 1 inch. 大鼠保持垂直或中线位置,定义为头部向右或左摆动不超过10。 Rats maintain the vertical or neutral position, is defined as the head to the right or left swing is not more than 10 . 每当大鼠从垂直移动其头部至右或左时记录摆动;当所述动物的头部从垂直分别向右或左移动10或W上时计数右摆动或左摆动。 Whenever the rat moves his head from a vertical recording is swung to the right or left; when the head of the animal to the right or left, respectively, from a vertical count left or right swing when swing W or 10 . 其中所述大鼠重复努力至特定侧时,仅记录单次摆动。 Wherein said duplication of effort in rats to a particular side, only a single recording wobble. 单次摆动后,动物躺于有机玻璃框中放置,且允许在重复试验之前自由移动30秒。 After a single swing, the animal lying in the plexiglass box placed for 30 seconds and allowed to move freely before repeating the test. 每只动物反复运些步骤20次(每种情形10只动物)。 Each animal repeated delivery of these steps 20 times (10 animals in each case).

[0307] 计数摆动总数,W及右摆动和左摆动的次数。 [0307] Count the total number of swings, W, and the number of left and right swing swing. 当摆动次数相等或超过70%时摆动习性被认为有偏差。 When equal to or more than 70% the number of swings is considered biased swing habit. 利用二-因子方差分析(AN0VA)分析结果,并且利用Tukey HSD (honestly显著差异)检验进行hoc后检验。 Factor analysis of variance (AN0VA) analysis, and using the Tukey HSD (honestly significant difference) post hoc test test - using two.

[0308] 还利用测定感觉运动作业的Bederson化urological检验评估大鼠中胎盘细胞诱导的神经性缺损修复。 [0308] also measured by sensory nerve defect Bederson placental cells induced motion of urological assessment test job rats. 参见Bederson等.,Stroke 17:472-6( 1986) ;Altumbabic,Stroke 29:1917_22(1998)。 See, Bederson et, Stroke 17: 472-6 (1986); Altumbabic, Stroke 29:. 1917_22 (1998).

[0309] 对于Bederson检验,利用4种检测获得每只大鼠的神经学得分,其包括:(a)自发同侧绕圈的观察,从〇(不绕圈)至3(连续绕圈)分级;(b)对侧后肢缩回,其测定动物横向移动2 至3cm后交替后肢的能力,从0(立即交替)至3(数分钟后交替或不交替)分级;(C)圆木行走能力,从〇(大鼠很容易穿越2.4cm宽80cm长的圆木)至3(大鼠不能停留在圆木上10秒)分级; 和(d)双侧前爪紧握,其测定握在2mM直径钢杆上的能力,从0(大鼠具有正常的前爪紧握习性)至3(大鼠不能用前爪紧握)分级。 [0309] For the Bederson test, using the obtained four kinds neurological score of each rat, which comprises: (a) spontaneous ipsilateral turns was observed from the square (not windings) to 3 (continuous windings) rating ; (b) contralateral hindlimb retraction, which measures the animal 2 to 3cm lateral movement ability of hind limb after alternately to 3 (or alternatively after a few minutes without alternation) graded from 0 (immediate alternation); (C) walking ability log from square (rat 80cm wide 2.4cm easily through the length of logs) to 3 (rat can not remain in log 10 seconds) classification; and (d) bilateral forepaw grip, which is held in the measured 2mM diameter steel rods on the ability, from 0 (normal forepaw of rats having gripping habit) to 3 (not grip front paws of rats) rating. 增加在第7和14天评估约15分钟时间内进行的所有4种检测的得分,W给出0至12的神经性缺损得分,得分越低表示大鼠神经学上越正常。 Increasing the evaluation score of all four detection performed within about 15 minutes and 14 days 7, W is given 0-12 neurological deficit score, the lower the score the more neurological normal rat.

[0310] 结果 [0310] results

[0311] 第0天,就在局部缺血诱导之前进行,所有动物呈现大致正常的(无偏差的)摆动习性(图1,基线)。 [0311] On day 0, just before the induction of ischemia for all animals exhibit substantially normal (unbiased) swing habit (FIG. 1, base). 局部缺血后第2天,给予胎盘细胞,所有动物呈现几乎100%局部缺血诱发的摆动习性偏差(图1)。 2 days after ischemia, administration of placental cells, all animals exhibit nearly 100% of the wobble deviation habits ischemia-induced (FIG. 1). 第7和14天,接受有活力胎盘细胞的动物显示显著的改善,分别表现大致65%和60%的摆动偏差(图1,第7和14天)。 7 and 14 days, placental cells receiving viable animals showed significant improvement, respectively, showed 65% and 60% approximately of the wobble deviation (FIG. 1, 7 and 14 days). 接受无活力胎盘细胞和环胞素A或仅赋形剂的动物显示无统计上的显著改善。 Receiving nonviable placental cells and cyclosporine A, or vehicle only animals showed no statistically significant improvement.

[0312] 在局部缺血诱导之前第0天评估的大鼠全部为神经学上正常的,并且在Bederson 检验中缺损得分为零(图2,基线)。 [0312] Before ischemia-induced rat assessed on Day 0 all neurologically normal, and the defect in the Bederson test score of zero (FIG. 2, the baseline). 局部缺血诱导后第2天,给予分离的胎盘细胞,仅接受赋形剂的大鼠显示总计平均缺损得分为大约2.5(图2,第2天)。 2 days after the induction of ischemia, administration of isolated placental cells, rats receiving only vehicle display defect average total score of about 2.5 (FIG. 2, Day 2). 第7和14天,接受胎盘细胞的大鼠显示神经性缺损得分统计学上显著改善,分别从大约2.5的得分改善至约1.7和1.1。 7 and 14 days in rats receiving placental cells displaying neurological deficit score statistically significant improvement were improved score from about 2.5 to about 1.7 and 1.1. 正如EBST的情况,仅接受赋形剂或无活力胎盘细胞和环胞素A的大鼠不显示神经性缺损得分在统计学上的显著改善。 As in the case of EBST, only receiving vehicle or nonviable placental cells and cyclosporine A in rats do not show significant improvement in the neurological deficit score statistically.

[0313] 总之,通过两种神经性缺损检测表明,在局部缺血动物模型中,局部缺血诱导后第2天给予4X105个人胎盘细胞明显改善了所述模式动物的神经学功能。 [0313] In summary, the two test showed neurological deficit in an animal model of ischemia, day 2 after induction of local ischemic administered 4X105 individual placental cells significantly improved neurologic the pattern animal. 给予环胞素AW抑制任何针对所述胎盘细胞的宿主免疫反应并不是胎盘细胞引起神经改善所必需的。 Administration of cyclosporine suppressed AW host immune response against any of the placental cells, placental cells that are not required for causing neurological improvement.

[0314] 6.2实施例2:利用静脉内给予的分离的胎盘细胞治疗中风 [0314] 6.2 Example 2: use of isolated placental cells intravenously administered treatment of stroke

[0315] 本实施例证实了在治疗与脑或CNS中或周围血流破坏有关的症状,例如治疗低氧损伤或缺氧损伤中静脉内给予分离的胎盘细胞的功效。 [0,315] This example demonstrates the efficacy of the treatment in or around the brain or CNS symptoms related to disruption of blood flow, for example, treating hypoxic injury or anoxic injury intravenous administration of the isolated placental cells. 例如,此处呈现的结果表明,相比接受无活力人胎盘细胞的动物,有活力的人胎盘细胞的静脉内给予促进给予了胎盘细胞的动物在运动和神经检测中剂量-依赖性的行为康复。 For example, the results presented here indicate that, compared with vigor accepted without human placenta animal cells, promote the intravenous administration of human placental cells viable given the animal placental cells and nerves in motion detection dose - dependent behavior rehabilitation .

[0316] 如本发明其它地方所述的,通过酶促消化获得分离的CD34-、CD10+、CD105+、CD200+ 塑料贴壁组织培养的胎盘细胞。 [0316] As described elsewhere in this disclosure, CD34- isolated by enzymatic digestion, CD10 +, CD105 +, CD200 + tissue culture plastic-adherent placental cells. Sprague-Dawl巧大鼠用作中风模型,并且如W上实施例1所述封闭中动脉封闭。 Sprague-Dawl clever rat stroke model used, and the W as described in Example 1 closed artery closure. 封闭后第2天,受者大鼠给予大约扣L赋形剂(10%葡聚糖和5%人血清白蛋白)中的4X 105、1 X 106、4X 106或8X 106个胎盘细胞,或作为对照的赋形剂中无活力的细胞。 2 days after the closure, snap recipient rats were given approximately L vehicle (10% dextran and 5% human serum albumin) in 4X 105,1 X 106,4X 106 or 8X 106 placental cells, or as a control excipient nonviable cells. 如W上实施例1所述的,在手术之前第0天评估大鼠,并且在手术后第2、14、28、56和84 天经过提高的身体摆动检测化BST)。 As described in Example 1, the W, the evaluation day 0 prior to surgery rats, and after surgery 2,14,28,56 and 84 days after the detection of elevated body swing BST). 进行改进的Bederson检测,其中受者大鼠评估(1)前肢缩回,其测定动物在横向移动2至3cm后前肢交替的能力,从0(立即交替)至3(数秒后交替或不交替)分级,(2)圆木行走能力,从0(大鼠很容易穿越2.4cm宽,80cm长的圆木)至3(大鼠不能在所述圆木上停留10秒)分级;和(3)双侧前爪紧握,其测定握在2mM直径钢杆上的能力, 从〇(大鼠具有正常的前爪紧握习性)至3(大鼠不能用前爪紧握)分级。 Improved detection of Bederson, wherein assess recipient rats (1) forelimb retraction, which was measured after the animals ability to 3cm 2 forelimb alternating transverse movement, from 0 (immediate alternation) to three (or alternatively after several seconds alternating) grade, (2) ability to walk the log, from 0 (2.4cm wide rats through easily, 80cm long logs) to 3 (rat can not remain in the log 10 seconds) classification; and (3) bilateral forepaw grip, its ability to grip on a steel rod measuring 2mM diameter, from square (rats with normal forepaw grasping habits) to 3 (not grip front paws of rats) rating. 计算在每个评估天约15分钟时间内进行的所有巧中检测的得分之和,W给出神经性缺损得分(最大可能得分,9分除W3种检测=3) dEBST和Bederson检测的结果通过W上实施例1所述的二-因子方差分析(ANOVA)T址巧服D检验评估。 Calculated for each evaluation day in about 15 minutes all of the coincidence detection scores and, W is given neurological deficit score (maximum possible score of 9 points W3 = 3 other species detected) results of the detection by Bederson and dEBST described in Example 1, the two W - factor analysis of variance (ANOVA) T D serving access coincidence evaluation test.

[0317] 在第84天杀死大鼠用于尸检和评估胎盘细胞的移植和分化。 [0317] On day 84 the rats were sacrificed for necropsy and evaluation of placental cells transplantation and differentiation. 简而言之,在40X放大倍数下检查20WI1低溫恒溫器切片组织,并且利用基于PC图像工具的计算机程序数字化。 Briefly, check 20WI1 cryostat tissue at 40X magnification, using a computer program and a PC-based digital image tool. 脑切片经盲-编码。 Brain sections were blind - encoded. 利用标准的ABC方法处理组织切片。 Processing of tissue sections using standard ABC method. 利用人特异性抗体化Nu,其不与晒齿类细胞表面标记或其他的晒齿类蛋白质交叉反应,评估人胎盘来源的干细胞移植物存活的细胞移植和分化指数。 Using human specific antibodies of Nu, which is not labeled with a sun tooth surface or other type of cell-based protein cross-reactive sun gear, the assessor placenta-derived stem cell transplant survival and differentiation of cell transplantation index. 为了检测细胞移植物中神经元表型的表达,利用使用神经元标记MAP2的免疫组织化学分析。 To detect expression of neuronal cell graft phenotype, analysis using immunohistochemistry using neuronal marker MAP2 is. 处理另外的脑切片用于GFAP和04, W掲示所移植细胞的神经胶质和少突神经胶质细胞表型的表达。 Further processing of brain sections for GFAP and 04, W kei shown glial expression of transplanted cells and oligodendrocytes glial cell phenotype.

[0318] 人胎盘来源的细胞静脉内移植不需要免疫抑制。 [0318] Transplantation does not require immunosuppression human placenta-derived cells within the vein.

[0319] 本研究中的所有动物呈现基线的正常习性,并且达到在中风后第2天移植之前大脑局部缺血诱导成功的标准。 [0319] All animals in this study presented normal habits baseline, day 2 before reaching and transplantation after stroke induced by cerebral ischemia success. 从中风后第7天时移植后最早时间点检验开始,行为检验掲示相比接受无活力人胎盘来源细胞的中风动物,接受有活力人胎盘来源细胞的中风动物在运动和神经学功能上呈现剂量-依赖的显著改善。 After the first 7 days after transplantation wind from the earliest point of time testing began, conduct inspection kei show compared to accept the stroke animal nonviable human placenta-derived cells, accept the energetic human placenta-derived cells in stroke animals a dose on the movement and neurological function - dependent significantly improved. 直至中风后第84天,在接受有活力人胎盘来源细胞的中风动物中,存在上述两项进一步改善增强的趋势。 Until 84 days after the stroke, in accepting energetic human placenta-derived cells in an animal stroke, there is further improvement to enhance these two trends. 在任何移植动物中,没有检测到中风诱发的行为缺损加剧,包括那些接受非活细胞移植的动物。 Any animals in transplantation, not detected increased stroke-induced behavioral defects, including those animals receiving non-viable cell transplantation. 在移植成熟期间,在接受非活细胞的动物中存在自发行为康复的一般趋势,但运种非特异性的、非移植介导的功能改善相比移植手术前(即中风后第2天)的动物没有达到统计学上的显著性。 During migration maturation, the presence of a general trend in the spontaneous behavior recovery animals receiving non-viable cells but Nonspecific transport, non-transplant-mediated functional improvement compared to the previous grafting (i.e., post-stroke day 2) Animals It did not reach statistical significance.

[0320] 在移植后早期,接受800万高剂量活细胞的中风动物清楚地表现出运动和神经学任务的实际改善。 [0320] In the early post-transplantation, stroke the animals receiving high doses of 8 million live cells shows clearly the actual exercise and improve neurological tasks. 然而随着时间的过去,接受较低剂量活细胞的中风动物也呈现很强的行为缺陷减弱,其大致可与高剂量的800万活细胞相比。 However, over time, to accept a lower dose of stroke animal living cells also showed a strong behavioral deficits weaken, which can be roughly compared with 8 million live cell high doses.

[0321] 在局部缺血诱导之前第0天评估的大鼠全部为神经学上正常的,并且缺损得分为零(图3,基线)。 [0321] Before ischemia-induced rat assessed on Day 0 all neurologically normal, and the defect score of zero (FIG. 3, the baseline). 局部缺血后第2天给予胎盘干细胞,所有动物呈现几乎100%局部缺血诱发的摆动习性偏差(图3)。 Day 2 post-ischemic administration of placental stem cells, all animals exhibit nearly 100% of the wobble deviation habits ischemia-induced (FIG. 3). 相比对照,在第7、14、28、56和84天的邸ST中,接受有活力的分离胎盘细胞的动物显示显著的、剂量-依赖的改善,并且还显示剂量之间的显著改善(P<〇.01), 但所有天数中4 X 106对比8 X 106个分离的胎盘细胞W及第7天4 X 105对比1 X 106个分离的胎盘细胞除外。 Compared to the control, and in the first 84 days 7,14,28,56 Di ST, accept animal viable isolated placental cells showed significant, dose - dependent improvement, and also showed significant improvement between dose ( P <〇.01), but in contrast to all days 4 X 106 8 X 106 W isolated placental cells and 7 days 4 X 105 1 X 106 except Comparative isolated placental cells. 参见图3。 See Figure 3. 接受无活力的分离胎盘细胞的动物相比对照不显示统计学上显著的改善。 Isolated placental cells in animals receiving non-viable compared to the control statistically significant improvement is not displayed.

[0322] 对于Bederson检验,在局部缺血诱导之前第0天评估的大鼠全部为神经学上正常的,并且缺损得分为零(图4,基线)。 [0322] For the Bederson test, prior to ischemia-induced rat evaluation day 0 all neurologically normal, and the defect score of zero (FIG. 4 baseline). 局部缺血诱导后第2天,给予分离的胎盘细胞,仅接受无活力细胞的大鼠显示总计平均缺损得分为大约2.5Ί3.0(图2,第2天)。 2 days after induction of ischemia, isolated placental cells administered, rats receiving only nonviable cells showed an average total of about deficit score 2.5Ί3.0 (FIG. 2, Day 2). 在第7、14、28、56和84 天,相比接受无活力细胞的大鼠,接受分离的胎盘细胞的大鼠显示统计学上显著的神经性缺损得分的改善(在所有情况下Ρ<〇.〇1)。 At 7,14,28,56 and 84 days, as compared to rats that received non-viable cells, a statistically significant improvement in the neurological deficit score accepted rat isolated placental cells displayed (in each case Ρ < 〇.〇1). 第7天(4 X 105对比8Χ 106,1 X 106对比8 X 106) W 及第14和28天(4Χ105对比4Χ106,4Χ105对比8Χ10 6)的改善呈现特别显著的(ρ<0.01)剂量-依赖性。 Day 7 (4 X 105 Comparative Comparative 8Χ 106,1 X 106 8 X 106) W and 14 and 28 days (4Χ105 4Χ106,4Χ105 Comparative Comparative 8Χ10 6) exhibit particularly improved significantly (ρ <0.01) dose - dependent sex. 正如EBST的情况,仅接受赋形剂或无活力分离的胎盘细胞和环胞素A的大鼠不显示神经性缺损得分在统计学上显著的改善。 Placental cells and cyclosporine A in rats As in the case of EBST, only receiving vehicle or nonviable isolated not show neurological deficit score in a statistically significant improvement.

[0323] 因此,静脉内给予分离的胎盘细胞促进运动和神经学检验中剂量-依赖性的行为康复。 [0323] Thus, intravenous administration of isolated placental cells facilitate the movement and neurological examination dose - dependent behavior recovery. 早在给予后第7天功能即有明显改善,并具有在移植后3个月期间更好康复的增加趋势。 As early as day 7 after administration function that is a significant improvement, and has a tendency to increase in the period of 3 months after transplantation better rehabilitation. 在任何受者大鼠中没有观察到明显有害的行为副作用。 No significant adverse side effects were observed in the behavior of any recipient rats.

[0324] 因此,此处呈现的结果表明静脉内给予用于治疗与脑中或周围血流破坏有关的症状的分离的胎盘细胞的安全性和功效。 [0324] Thus, the results presented herein indicate that intravenous administration for treating safety and efficacy of blood flow in the brain or peripheral damage related symptoms of isolated placental cells. 例如,没有移植的动物显示中风诱发的行为异常的加剧。 For example, there is no transplanted animals showed increased stroke-induced abnormal behavior. 相比接受无活力人胎盘来源的细胞的动物,那些接受活细胞的动物呈现中风诱发的行为缺损的显著改善W及局部缺血边缘部分宿主细胞的显著恢复。 Compared to accept nonviable cells derived from human placental animals, those animals receiving living cells showed a significant recovery significantly improve ischemic W and edge portions of the host cell's behavior stroke-induced defects. 此外,在任何移植动物中没有检测到形成肿瘤或异位组织。 In addition, the tumor is not detected or ectopic form any tissue transplanted animal.

[0325] 6.3实施例3:利用胎盘细胞经静脉内途径治疗中风一其他神经学检验评估 [0325] 6.3 Example 3: using placental cells intravenously route a treatment of stroke other neurological assessment tests

[0326] 本实施例证实了利用胎盘干细胞治疗中风的有效性,如๩