CN1228443C - 人胚胎干细胞的造血分化 - Google Patents

人胚胎干细胞的造血分化 Download PDF

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CN1228443C
CN1228443C CNB008153264A CN00815326A CN1228443C CN 1228443 C CN1228443 C CN 1228443C CN B008153264 A CNB008153264 A CN B008153264A CN 00815326 A CN00815326 A CN 00815326A CN 1228443 C CN1228443 C CN 1228443C
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D·S·考夫曼
J·A·汤姆森
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Abstract

本文公开了用哺乳动物基质细胞从人胚胎干细胞获得人造血细胞的方法。用本方法衍生的造血细胞可用于制备适于移植、输血和其它目的的细胞培养物。

Description

人胚胎干细胞的造血分化
与相关申请的相互参照：
没有适用的。
关于联邦政府赞助研究的声明
发明背景
本发明涉及用人胚胎干细胞来产生与血相关的细胞，和将这些与血相关的细胞用于各种目的。
最近我们实验室已描述了分离人胚胎干细胞稳定培养物的方法。参见美国专利No.5,843,780和J.Thomson等人，282 Science 1145-1147(1998)。这些出版物和以下所引用的所有出版物本文都全部纳入作为参考。
我们分别于1999年7月7日和1999年7月15日在美国典型培养物保藏中心(10801 University Boulevard，Manassas，Virginia 20110-2209，U.S.A)保藏了我们的两种人胚胎干细胞系。这两种保藏物的分类描述分别为人胚胎干细胞系H1和H9。在这些出版物中已指出这两种细胞系可与其它物质一起用来提供用于各种类型研究、移植和其它目的的特定的细胞系源。
在这些出版物所述的储存和培养条件下，这两种细胞系可以长期维持而不致分化成各种特定的细胞类型。当这两种细胞系被注射入免疫缺陷小鼠后，它们就可形成畸胎瘤，表明可分化成多样性组织类型。
当将胚胎干(ES)细胞用于产生所需的细胞时，往往侧重于优化向特定细胞类型的分化。对造血细胞而言，期望从而能得到可以分离并可用于形成多样性造血谱系的造血细胞。这些细胞包括(但不限制于)造血干细胞。
已从骨髓直接分离了若干造血干细胞群。参见C.Baum等人，89 PNAS USA2804-2808(1992)。但这依赖骨髓的供给来获得细胞。
人们已企图将鼠胚胎细胞群变成造血细胞。参见美国专利No.5,914,268；G.Keller，7 Current Opinion In Cell Biology，862-869(1995)；和T.Nakano等人，265Science 1098-1101(1994)。还可参见M.Weiss，11 Aplastic Anemia And StemCell Biology，1185-1195(1997)；和S.Morrison等人，11 Annu.Rev.CellDev.Biol.，35-71(1995)。
但将这些方法施用于灵长类动物就很困难。例如，在F.Li等人，92 Blood368a(1998)中讨论了用基质细胞系和外源细胞因子进行猕猴胚胎干细胞系的分化。但最近该试验组报道它们的方法不足以形成集落。
用组织移植治疗各种疾病已日益普遍。但需要排队获得天然捐赠的器官、细胞或组织。即使得到了天然供体的材料，通常还存在排斥的问题。抑制接受者免疫应答的传统方法还存在缺陷。例如，免疫抑制药价格昂贵且往往有副作用。
在WO 98/07841中，公开了衍生出与所选供体MHC相容的胚胎干细胞的方法(如将供体的细胞核移植入去核的卵母细胞，然后从其衍生出干细胞)。此申请建议可用如此得到的细胞获得MHC相容的造血干细胞，用于需要骨髓移植的医学处理中。
然而，一些疾病如1型糖尿病或多发性硬化都涉及自身免疫应答。例如，将朗格罕氏体(pancreatic islets)(与患者是MHC相容的)替代受损的朗格罕氏体，并不能为减轻I型糖尿病提供足够长时间，因为宿主的免疫系统最终将攻击移植入的胰岛。
因此可以看出需要一种方法能让人胚胎干细胞培养物分化成所需的造血细胞集落。另外，需要开发造血细胞的改进用途。
发明概述
本发明的一方面提供了获得人造血细胞的方法。将人胚胎干细胞培养物与哺乳动物造血基质细胞相接触，从而产生人造血细胞。至少部分如此产生的人造血细胞是CD34+和/或能在甲基纤维素培养基上形成造血细胞集落形成单位。
如C.Baum等人，89 PNAS USA 2804-2808(1992)和S.Morrison等人，11Annu.Rev.Cell Dev.Biol.，35-71(1995)所述，CD34是造血干细胞的标准标记物。形成集落形成单位能力的特性是细胞具有形成更多分化的造血谱系所需的特性。
较佳的是从骨髓细胞或胚胎卵黄囊细胞衍生的基质细胞。鼠基质细胞可用于此目的。而灵长类和其它哺乳动物的基质细胞也都适合。
本发明的另一方面提供了从体外培养的人胚胎干细胞衍生出的人造血细胞，能在甲基纤维素培养物中形成造血细胞集落形成单位。在本专利中，术语“衍生的”指直接或间接(如，通过一种或多种中间体或一次或多次传代)获得的。
在本发明在另一方面提供了一种将人细胞物质移植入人受体宿主的方法。获得了在体外从胚胎干细胞培养物衍生的人造血细胞。然后获得了造血细胞以外的所选的人细胞物质，这种所选的非造血系材料具有与造血细胞相容的主要组织相容性复合物。然后将造血细胞和所选的非造血细胞物质一起转移到人类宿主。
例如，可以获得在体外从胚胎干细胞培养物(如用以下方法)衍生的人造血细胞。也可获得对造血细胞具有MHC相容性的人朗格罕氏体。然后将造血细胞和朗格罕氏体一起移植入人体(较佳的是，当受体本身的骨髓已失活后)。
可以从供体(其细胞用于产生胚胎干细胞培养物)直接获得朗格罕氏体。另外，单一的胚胎干细胞培养物可以按两种不同的途径分化。一种过程，可以用所述方法来产生造血干细胞。随后，当移植入合适的宿主后，这些细胞可发育成多样化的造血谱系。这些谱系包括能耐受从同一亲代胚胎干细胞衍生的其它细胞的淋巴细胞。在另一种过程中，将干细胞直接转化成朗格罕氏体。
在另一实施例中，可以向患多发性硬化的人提供少突神经胶质细胞。获得在体外从胚胎干细胞培养物衍生的人造血细胞(如用以下方法)。也可获得与骨髓细胞具有MHC相容性的人少突神经胶质细胞，并可将骨髓细胞和少突神经胶质细胞一起移植入人体。
其遗传物质用于产生胚胎干细胞的同一人可用作少突神经胶质细胞的供体。另外，相同的胚胎干细胞培养物可通过两种途径分化，从而提供两种可移植的物质。
通过这种方法可以降低疾病(和潜在的其它自身免疫疾病)的免疫和自身免疫排斥问题。由此看来，在移植前可用辐射或化学方法使接受者本来的骨髓完全或部分失活。然后至少部分用移植入的造血细胞替代。自身骨髓的清除/减少降低了身体产生自体免疫反应的能力。替换的骨髓和后继的可移植物质的MHC匹配确保了第二次物质不会被已移植的骨髓排斥。
另外，可进行造血细胞和其它组织的一起移植来促进对后继组织的接受(如用心肌与造血细胞一起来治疗心脏病；用肝细胞与造血细胞一起来治疗肝病)。通过产生造血系嵌合体，可以获得类似匹配MHC类型的组织对移植的改善的接受性。
本发明适用于获得广范围的有价值的造血细胞，如红细胞、粒细胞、巨噬细胞、淋巴细胞前体、单核细胞、B细胞、T细胞等。由此看来，在收获了分成单一类型细胞和接种于合适的培养基时，分化的ES细胞集落就可长成造血集落。这些集落表明集落形成细胞的发展可增殖成集落形成单位(包括集落形成单位-红细胞(CFU-E)、母细胞形成单位-红细胞(BFU-E)、集落形成单位-巨噬细胞(CFU-M)、集落形成单位-粒细胞/巨噬细胞(CFU-GM)和集落形成单位-高增殖潜能(CFU-HPP))。集落形成细胞的鉴定表明，胚胎干细胞分化成造血细胞，在限定条件下能扩大发展成确定的造血细胞谱系。
因此本发明的目的包括提供：
(a)上述获得造血细胞的各种方法；
(b)用这些方法衍生的各种细胞；和
(c)将这些衍生的细胞用于移植、输血和其它目的的各种方法。
在以下的优选实施例中，本发明的这些及其它一些目的和优点就更加显然。但为了对本发明更全面地进行评价，就应当关注这些权利要求。
发明详述
胚胎干细胞的培养
大部分试验都是用上述人ES细胞系H1，但以下的一些研究则是用上述ES细胞系H9(或H9.2)进行的，得到的结果类似。从初始分离和传代的细胞系衍生的细胞冷冻(液氮)原液取出这些细胞。在6孔培育板(Nunclon，Fisher)上培养H1 ES细胞。
首先用0.1％明胶溶液(Sigma)覆盖该培育板在37℃/5％ CO2培养箱中放置1天或多天。此后，除去明胶溶液，再用辐照过的小鼠胚胎成纤维细胞(MEF)覆盖此培育板孔。这些MEF细胞是从12-13日胚龄小鼠胚胎(在由补充有10％胎牛血清(Hyclone或Harlan)、2mM 1-谷氨酰胺(GibcoBRL)和100单位/ml青霉素、100mg/ml链霉素(Sigma)的DMEM(GibcoBRL)构成的培养基中)衍生的。
在接种培养孔之前，用铯源发出的5500cGy辐照MEF细胞。以5×104细胞/ml、2.5ml/孔的密度添加MEF。然后将涂敷有MEF的平板在37℃/5％CO2培养箱中培养1天或多天，然后添加ES细胞。
以约5-8天的间隔将ES细胞在新MEF上传代。时间间隔取决于细胞密度和分化的形态表现。在传代时，除去ES细胞孔中的培养基，加入1-2ml含有1mg/ml胶原酶IV的DMEM(GibcoBRL)培养基。然后将此平板置于37℃/5％CO2中5-20分钟，直到开始形成ES细胞的集落。
然后用5ml移液管刮取平板孔上的ES细胞。将收获孔的内含物置于15ml锥形管(Fisher)中，以1000rpm离心5分钟。除去培养基，加入10ml新鲜培养基。ES细胞培养基由补充有20％血清替代物培养基(GibcoBRL)、8ng/mlbFGF(GibcoBRL)、1％非必要氨基酸溶液(GibcoBRL)、1mM 1-谷氨酰胺(GibcoBRL)和0.1Mβ-巯基乙醇的F12/DMEM(GibcoBRL)构成。
再次离心细胞(5分钟/1000rpm)，除去培养基，以每孔2.5ml培养基的浓度悬浮(通常将15ml培养基接种于6个新孔中，1∶6传代)。然后用移液管将细胞移入上述用MEF覆盖的平板孔中。将细胞均匀地分布在各孔中，将平板置于37℃/5％CO2的培养箱中。
如果在细胞传代前就有表现分化形态的ES细胞集落，就用玻璃吸液管轻柔地将这些集落取出。操作是在解剖显微镜下观察进行的。除去已分化的细胞后，将剩下的集落进行上述传代。
传代后，对各孔的ES细胞以24-48小时间隔补加新鲜培养基培养。除去各孔的培养基，分别加入2.5ml新鲜的ES培养基。ES细胞的所有加料和传代都是在无菌环境下进行的。
ES细胞的分化
为了促进人ES细胞向造血系分化，按以上所述的方法收获ES细胞。然后将细胞接种于涂有哺乳动物基质细胞的6孔平板上。在一项试验中，我们采用上述经2500cGy辐射过的C166细胞。C166细胞最初是从12日胚龄的小鼠胚胎卵黄囊获得的，由Dr.Robert Auerbach(UW-Madison)惠赠。
在另一试验中，是用S17细胞。它们最初是从小鼠骨髓得到的，由Dr.Kenneth Dorshkind(当时在UC-Riverside，现在在UCLA)惠赠。
以1×105个细胞/ml，2.5ml/孔的密度接种C166或S17细胞。然后让接种S17或C166细胞的ES细胞，在补充有20％胎牛血清(Hyclone)、1％非必要氨基酸溶液、0.1Mβ-巯基乙醇和1mM 1-谷氨酰胺的DMEM(GibcoBRL)构成的培养基中生长。以24-72小时的间隔，将新鲜的这种培养基置于各孔中。对合适培养基的选择而言，虽然补充有特定的基质细胞，也只需要提供细胞生长所需的常规条件。
在接种S17或C166细胞上的3-7天后，ES细胞开始表现出分化，它们没有与在MEF喂养细胞上维持的未分化ES细胞相同均匀的外表。ES细胞的集落开始形成多种不同的细胞类型。这些集落中的一些区域表现为由卵石形态的细胞构成，表明是早期祖代造血细胞。
与血液相关的细胞的确定
一种确定有合适造血细胞存在与否的方法是在含有半固体甲基纤维素的培养基中分析造血集落形成细胞(CFC)。此时，让ES细胞在C166或S17细胞上分化2-3星期(如上所述)。然后除去培养基，加入2.5ml无钙和镁的磷酸盐缓冲盐水(PBS)，2-5分钟后除去，加入1.5ml胰蛋白酶(0.125％)-EDTA(1mM)培养基。
将细胞置于37℃/5％CO2中10分钟。此后，集落开始分离。用移液管吸取和刮擦孔进一步将这些细胞分离。将这些细胞置于15ml锥形管中，在1000rpm离心5分钟，除去培养基，加入10ml新鲜培养基(DMEM+10％FBS+1-谷氨酰胺+青霉素/链霉素)，再次离心。然后将细胞悬浮于5ml培养基中，用100mMnytex过滤器过滤除去聚团细胞。
另用5ml培养基洗涤此过滤器。然后将分离的/过滤的细胞在血细胞计数器上计数，并将1×106(通常，但不必总是如此多)细胞置于新的15ml锥形管中。然后将这些细胞离心，除去培养基，加入由IMDM(GibcoBRL)(补充有2％胎牛血清(Hyclone))构成的培养基。将细胞离心，除去培养基再加入250μl培养基(IMDM+2％FBS)。
根据特定的测试条件，将这些细胞加入2.5mlMethocult GF+H4435培养基(StemCell Technologies)中。该培养基由1.0％甲基纤维素构成，补充有30％FBS、20ng/ml IL-3、20ng/ml IL-6、50ng/ml干细胞因子、3单位/ml红细胞生成素、20ng/ml GM-CSF、20ng/ml G-CSF、2mM 1-谷氨酰胺、0.1mM b-巯基乙醇、1％胎牛血清白蛋白。然后剧烈旋转搅拌甲基纤维素中的细胞，然后将1.1ml此混合物接种于P35塑料皿(Stem Cell Technologies)上，在此皿上均匀涂布，并置于37℃/5％CO2中。
各样品一式两份按标准涂板，通常为4×105个细胞/平板。14-21天后，在显微镜下分析平板上造血集落的存在与否。通过与集落图谱(StemCellTechnologies)或书(造血细胞的培养，RI Freshney，IB Pragnell，MG Freshney编辑，Wiley-Liss，Inc，1994)比较来鉴定集落。将集落鉴定为以下情况之一：集落形成单位-红细胞(CFU-E)、母细胞形成单位-红细胞(BFU-E)、集落形成单位-巨噬细胞(CFU-M)、集落形成单位-粒细胞/巨噬细胞(CFU-GM)或集落形成单位-高增殖潜能(CFU-HPP)。
也可用流式细胞术鉴定是否存在所选的造血细胞。通过流式细胞计术可以寻找特定的细胞表面抗原。如上所述，用胰蛋白酶/EDTA收获在S17细胞或C166细胞上分化14-21天的ES细胞(如上所述)，并通过100mM nytex滤器。在血细胞计数器上计数过滤的细胞，然后等份地加入15×75塑料管(Fisher)中，约1×105个细胞/管。然后离心细胞，除去培养基，加入2-3mlFACS培养基。(FACS培养基为含有0.5％BSA(Sigma)、0.1％叠氮化钠(Sigma)的PBS)。
再次离心细胞，并除去培养基。然后，以供应商推荐的浓度在这些孔中加入与荧光标记物(FITC或PE)直接连接的抗体。用以下抗体分析细胞：CD34-FITC(Immunotech)、CD45-PE(Pharmingen)。将IgG1-FITC和IgG1-PE用作细胞非特异性染色的同型对照。在冰浴上用合适的抗体培养这些细胞约30分钟，用2-3ml FACS培养基洗涤1-2次，然后悬浮于约0.5ml FACS培养基中。
然后按制造商说明用FACScan(Becton Dickinson)分析标记有抗体的细胞。加入二碘化丙锭(1mg/ml溶液，每管添加5μl)或7-AAD(Calbiochem)(0.2mg/ml，5μl/管)，来测定死细胞的存在。用PC Lysis或Cellquest软件进行分析。
用以下试验方法来分析由免疫组织化学(IHC)的抗原表达。此时，如上述用胰蛋白酶/EDTA收获分化的与C166或S17(如上述)共培养的ES细胞。以约1×104-1×105浓度，将细胞悬浮于补充有10％FBS的DMEM培养基中。然后用Cytospin II离心机(Shanndon)将1×103-1×104个细胞离心到载玻片(Superfrost/plus，Fisher)上，得到这些细胞的“Cytospin”制备物。
然后用冷丙酮固定这些载玻片，在-20℃冷冻保藏。载玻片经室温解冻后进行IHC染色，用蜡笔(DAKO)勾画出细胞团。然后如下用Vectastain ABC试剂盒(Vector Laboratories，Burlingame，CA)将细胞染色，所有培养都是在室温中进行的。在细胞上加入100-200μl PBS，5分钟后除去。然后在细胞上加入Vectastain封阻抗体溶液(马血清)15分钟。然后将这些细胞印吸干燥，加入100-200μl初级抗体溶液。初级抗体是：IgG1(1μg/样品，Sigma)，抗-CD34(0.5μg/样品，Immunotech)、抗-CD45(1μg/样品，DAKO)、抗-I类(1μg/样品，Dr.Paul Leibson，Mayo Clinic馈赠)、抗-CD14(1μg/样品，Pharmingen)、抗-CD31(1μg/样品，Pharmingen)。
加入初级抗体30分钟，然后加入PBS 10分钟。随后，加入经生物素酰化的抗-IgG抗体(Vectastain试剂盒，溶液B)30分钟，随后加入PBS10分钟。室温加入Vectastain ABC溶液30分钟后，加入PBS10分钟。然后加入DAB溶液(Vectastain)5分钟，然后用自来水流冲洗10分钟。在一些试验中，随后用Gill苏木精溶液(Vector labs)复染色这些载玻片3分钟，再用自来水冲洗10分钟。然后将这些载玻片风干。阳性细胞染色为褐色。
2-3周后，在混合的细胞群体中证明有CD34+(约1％)。更重要的是，当收获、分离细胞并接种于甲基纤维素(半-固体)培养物时，分化的ES细胞显示生长成造血集落。
移植
目前造血细胞移植在临床上主要用于已接受高剂量化疗(治疗恶性肿瘤)的患者。这些患者通常接受自身或异体来源的造血细胞的多相混合物。人ES衍生的造血干细胞则至少可提供更同质性的细胞群体，用于造血细胞的移植。
另外，如上所述，如今可以控制移植的MHC特性，从而就有可能治疗各种自体免疫疾病。例如，造血干细胞(HSCs)和次级谱系(如用于糖尿病的朗格罕氏体(pancreatic islet)或用于多发性硬化的少突神经胶质细胞)都可以从同一亲代ES细胞系衍生。有了这两种细胞谱系，就可通过完全同种异体的造血细胞移植(HCT)首先产生造血嵌合体。已建立的嵌合状态可以让接受者的免疫系统将以后移植的次级细胞类型(如朗格罕氏体或少突神经胶质细胞)“视为”“自身”的，而不会被排斥。
例如，从小鼠ES细胞已获得少突神经胶质细胞(O.Brustle等人，285 Science754-6(1999))作为心肌细胞(M.Klug等人，98 J.Clin.Invest.216-224(1996))。
这种产生造血嵌合体的方法也能促进对移植组织的接受(除自身免疫以外的原因)。此时，接受同种异体移植造血干细胞的小鼠不会排斥具有相同遗传背景的其它组织(作为造血细胞)，但依旧排斥第三方的移植物。参见K.Gandy等人，65 Transplantation 295-304(1998)。
除动物研究以外，现有一个临床案例报道了先前接受造血细胞移植的患者后来需要实体器官(肾脏)移植。在这些情况中，由先前提供骨髓移植的同一人的肾脏移植，在免疫学上是可接受的，不会再发生免疫抑制作用，参见T.Spitzer等人，68 Transplantation 480-484(1999)。
对犬类模型进行的研究和最近对人体作的一些临床试验表明可用更温和的不施行骨髓部分切除术(myeloablative)的条件疗法(conditioning regimen)更好地让宿主作好准备以接受同种异体的HCT。此时，只有中等剂量的全身辐照和短程的免疫抑制可用于制备接受同种异体HCT的宿主。
虽然以上已描述了一些优选的实施例，但本领域技术人员可以正确评价在本发明的范围内还可进行其它一些改变。例如，虽选择采用两种特定基质类型的细胞，但还有许多其它细胞也是适用的。例如，一种已公开可获得的基质细胞系是M2-10B4细胞系，其ATCC登录号为CRL-1972。
另外，以上描述侧重于产生红细胞和骨髓的前体，但采用上述各种方法还可以批量获得其它多种多样的感兴趣的与血液相关的细胞。参见美国专利No.5,914,268。因此，只有权利要求可以用来判断本发明的范围。
工业应用
本发明提供可用于移植、研究和其它目的的与血液相关的细胞。

Claims (9)

1.一种获得人造血细胞的方法，其特征在于，所述的方法包括将人胚胎干细胞培养物与哺乳动物的造血基质细胞体外接触，从而制备人造血细胞。
2.如权利要求1所述的方法，其特征在于，至少部分用所述方法产生的人造血细胞是CD34+。
3.如权利要求1所述的方法，其特征在于，至少有部分用所述方法产生的人造血细胞可在甲基纤维素培养物上形成造血细胞集落形成单位。
4.如权利要求1所述的方法，其特征在于，所述的基质细胞选自骨髓细胞和胚胎卵黄囊细胞。
5.一种获得人造血细胞的方法，其特征在于，所述的方法包括体外将人胚胎干细胞培养物与哺乳动物基质细胞接触，从而产生人造血细胞，其中至少部分用所述方法产生的人造血细胞可在甲基纤维素培养物上形成红细胞系母细胞形成单位。
6.如权利要求5所述的方法，其特征在于，至少部分用所述方法产生的人造血细胞是CD34+。
7.在体外从胚胎干细胞培养物衍生的人造血细胞和除造血细胞以外的所选人细胞在制备移植物中的用途，其中，所选的非造血系细胞具有与造血细胞相容的主要组织相容性复合物。
8.如权利要求7所述的用途，其特征在于，所述的所选人细胞是人朗格罕氏体。
9.如权利要求7所述的用途，其特征在于，所述的所选人细胞是人少突神经胶质细胞。
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