WO2011073517A1 - Method of distinguishing undifferentiated mesenchymal stem cells and differentiated mesenchymal stem cells from each other by using an antibody against the blood group i antigen - Google Patents

Method of distinguishing undifferentiated mesenchymal stem cells and differentiated mesenchymal stem cells from each other by using an antibody against the blood group i antigen Download PDF

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Publication number
WO2011073517A1
WO2011073517A1 PCT/FI2010/051032 FI2010051032W WO2011073517A1 WO 2011073517 A1 WO2011073517 A1 WO 2011073517A1 FI 2010051032 W FI2010051032 W FI 2010051032W WO 2011073517 A1 WO2011073517 A1 WO 2011073517A1
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population
msc
antigen
blood group
antibody against
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PCT/FI2010/051032
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French (fr)
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Annika Kotovuori
Suvi Natunen
Leena Valmu
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Suomen Punainen Risti Veripalvelu
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/34—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood group antigens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood

Definitions

  • the present invention relates to methods of distinguishing, and purifying or enriching, and screening mesenchymal stem cells using an antibody against the blood group i antigen.
  • the present invention also relates to a use of an antibody against the blood group i antigen in distinguishing and/or enriching mesenchymal stem cells.
  • the present invention further relates to a use of an antibody against the blood group i antigen in screening of mesenchymal stem cells.
  • the present invention relates to a cell population screened or enriched with an antibody against the blood group i antigen.
  • MSCs Mesenchymal stem cells
  • MSCs are multipotent stem cells that can differentiate into a variety of cell types, in particular to osteoblast, chondrocytes and adipocytes, both in vitro and in vivo.
  • MSCs can be obtained, for example, from the cord blood and bone marrow.
  • the exact definitions for MSC or cell lineages differentiated thereof are currently not finally established (Da Silva Meilleres et al., Stem Cells 2008; 26: 2287-99), but an example of a current set of criteria for undifferentiated MSC are described by Dominici et al., in Cytotherapy 2006; 8: 315-317.
  • MSCs have a large capacity for self-renewal while maintaining their multipotency.
  • a standard test to confirm multipotency is differentiation of the cells into osteoblasts, adipocytes, and chondrocytes, for example.
  • the degree to which the culture will differentiate varies depending on how differentiation is induced, e.g. chemically vs. mechanically.
  • the capacity of cells to differentiate and proliferate is known to decrease with the age of the donor, as well as the time cultivated in vitro. Whether this is due to a decrease in the number of MSCs or a change to the existing MSCs, is not known.
  • MSCs have the potential to differentiate into various cellular lineages and can be expanded in culture conditions without losing their multipotency, they present a valuable source for applications in cell therapy and tissue engineering.
  • MSC transplantation offers a promising approach for treating certain nonhematological malignant and nonmalignant diseases and for stem cell-mediated tissue regeneration. In particular, they can be applied to induce immunosuppression. This can be done as supportive therapy in hematological stem cell transplantations in which immunologically-mediated graft-versus-host disease is a major complication.
  • Bone marrow and cord blood have been studied, and also used in treating human patients, as mesenchymal stem cell sources.
  • stem cell transplantation suffer from several obstacles such as graft-versus-host disease and graft rejection.
  • MSCs represent a heterogeneous population of cells their actual number in a graft cannot be readily estimated.
  • Dominici et al. (2006) discloses that there are surface antigens that can be used to isolate a population of cells that have similar self-renewal and differentiation capacities, yet MSCs, as a population, typically do not all express the proposed markers; and it is not certain which ones must be expressed in order for that cell to be classified as a MSC.
  • antigenic structures detected by these antibodies are not exactly known and some variation in the structure can be assumed based on the above publications.
  • the antigenic structure is related to, or it is assumed to include as a shared feature a linear poly-N-acetyllactosamine having the following structure Gal(p1-4)GlcNAc( 1-3)Gal( 1-4)GlcNAc(p1-) linked to different molecules.
  • An additional object of the invention is to provide methods of distinguishing or purifying and/or enriching a native undifferentiated mesenchymal stem cell or a population thereof and a fibroblast or a population thereof from each other using an antibody against the blood group i antigen.
  • a further object of the present invention is to provide a use of an antibody against the blood group i antigen in distinguishing an undifferentiated MSC or a population thereof and a differentiated MSC or a population thereof from each other.
  • a still further object of the present invention is to provide a use of an antibody against the blood group i antigen in purifying and/or enriching a native undifferentiated mesenchymal stem cell or a population thereof and a differentiated MSC or a population thereof from each other.
  • An additional object of the invention is to provide uses of an antibody against the blood group i antigen in distinguishing or purifying and/or enriching a native undifferentiated mesenchymal stem cell or a population thereof and a fibroblast or a population thereof from each other.
  • a further object of the present invention is to provide a method of screening MSCs using an antibody against the blood group i antigen.
  • an object of the present invention is to provide a use of an antibody against the blood group i antigen in screening of mesenchymal stem cells.
  • Another object of the present invention is to provide a MSC population purified and/or enriched with an antibody against the blood group i antigen.
  • An additional object of the present invention is to provide a MSC population screened with an antibody against the blood group i antigen.
  • the present invention leads to an enriched and/or purified or more homogeneous preparation of MSCs useful for therapeutic purposes.
  • the invention is based on the observation that the target of the blood group i antibody on a mesenchymal stem cell lessens during the differentiation process of the cell.
  • the invention is based on a further observation that an antibody against the blood group i antigen may be used as a marker for an undifferentiated MSC and that an antibody against the blood group i antigen is able to distinguish differentiated MSCs and native, undifferentiated MSCs from each other.
  • the invention is based on an even further observation that an antibody against the blood group i antigen is able to distinguish native, undifferentiated MSCs from fibroblasts.
  • the current invention provides a novel and effective means for distinguishing, enriching and/or screening of MSC-populations and/or for assessing of the differential status of MSCs and/or quality of a MSC population.
  • synthetically-prepared target antigens or polymers or oligomers of lactose, lactosamine or acetyllac- tosamine structures, or their functional analogs are used to eluate the blood group i antibody from the purified MSC.
  • Figure 1 shows staining of mesenchymal stem cells (MSCs) and cells differentiated from MSCs by human serum containing anti-i antibodies. Positively stained cells are marked green in the dot plots, and negative cells are marked red. The percentage of positive cells was determined as the cell population with a higher fluorescence intensity than 99.5% of cells stained with secondary antibody only. Histogram overlays are shown with staining with anti-i serum in red fill and secondary controls in grey fill.
  • MSCs mesenchymal stem cells
  • Figure 2 shows the expression of the i-antigen in CB MSC lines and differentiated cells. The cells were stained with 10 % human anti-i serum (10%) and PE-conjugated donkey anti-human IgM.
  • Figure 3 shows the staining of cord blood mononuclear cell populations by anti-i serum.
  • Figure 4 shows the staining of juvenile and adult normal human dermal fibroblasts by anti-i serum.
  • An MSC-population is typically a heterogeneous population of cells.
  • a portion of the cell population presents native, undifferentiated cells and another portion presents cells that have been differentiated or have started to differentiate toward osteogenic, adipogenic and/or chondrogenic lineages, for example.
  • transplantation of MSC possesses promising approach for stem cell-mediated tissue regeneration, the heterogeneity within the differential status of the cells increases the probability and/or extent of ill effects. Furthermore, the cellular heterogeneity also leads to poor understanding of factors relevant in successful transplantation.
  • mesenchymal stem cells from bone marrow and cord blood, and cells differentiated from them toward adipogenic and osteogenic lineages were stained with human serum containing antibodies against the blood group i antigen.
  • Anti-i serum was bound to a high proportion of cord blood derived MSCs and a subpopulation of bone marrow derived MSCs.
  • the same analysis with cells differentiated into adipogenic and/or osteogenic directions resulted in a substantially lower staining, equivalent or close to that of nonspecific human serum.
  • the results indicate that the expression of the blood group i antigen is characteristic of proliferating MSCs and disappears upon differentiation, and could therefore be used as a marker for undifferentiated MSCs.
  • the present invention is thus based on the finding that the amount on a mesenchymal stem cell of the target antigen of anti-i antibodies becomes smaller and/or the presence of the blood group i antigen on a mesenchymal stem cell lessens during the differentation process of the cell.
  • the invention is based on a further finding that an antibody against blood group i antigen may be used as a marker for an undifferentiated MSC and that an an- tibody against blood group i antigen is able to distinguish differentiated MSCs and native, undifferentiated MSCs from each other.
  • the method is used in distinguishing a native, undifferentiated mesenchymal stem cell or a population thereof from a differentiated MSC or a population thereof.
  • the cell population so distinguished could more efficiently be used in stem cell therapy, such as stem cell mediated tissue regeneration.
  • the method is used in distinguishing a MSC differentiated toward osteogenic lineage or a population thereof from a native, undifferentiated MSC or a population thereof.
  • the cell population so obtained is capable of generating bone structures and bone- associated marrow elements more efficiently than a native or heterogeneous MCS-population. Further, the risk of cancer which could be related to the transplantation diminishes when the amount of the undifferentiated MSCs in the graft is diminished.
  • the method is used in distinguishing a MSC differentiated toward adipogenic lineage or a population thereof from a native, undifferentiated MSC or a population thereof.
  • the method is used in purifying and/or enriching a native, undifferentiated mesenchymal stem cell or a population thereof from a differentiated MSC or a population thereof.
  • the cell population so purified and/or enriched could more efficiently be used in stem cell therapy, such as stem cell mediated tissue regeneration.
  • the method is used in purifying and/or enriching a MSC differentiated toward osteogenic lineage or a population thereof from a native, undifferentiated MSC or a population thereof.
  • the cell population so enriched is capable of generating bone struc- tures and bone-associated marrow elements more efficiently than a native or heterogenous MCS-population. Further, the risk of cancer which could be related to the transplantation diminishes when the amount of the undifferentiated MSCs in the graft is diminished.
  • the method is used in purifying and/or enriching a MSC differentiated toward adipogenic lineage or a population thereof from a native, undifferentiated MSC or a population thereof.
  • one contaminating population can be fibroblasts that unlike T-cells and B-cells, but like MSCs, also are adherent to plastic.
  • fibroblasts were negative for the i-antigen. Accordingly, the expression of the i-antigen can be used to differentiate between mesenchymal stem cells and fibroblasts.
  • the method is used in distinguishing a native, undifferentiated mesenchymal stem cell or a population thereof from a fibroblast or a population thereof using an antibody against the i antigen.
  • the MSC population can further be purified by using the antibody against the i antigen; there are a number of different methods known in the art for purification of a particular cell population using antibodies detecting only the desired cell population.
  • the method is used in purifying and/or enriching a MSC or a population thereof from a fibroblast or a population thereof by using the antibody against the i antigen.
  • the cell population so enriched and/or purified is more homogeneous and requires less purification in later stages.
  • the present invention also relates to a method of isolating a mesenchymal stem cells or a population of MSCs with an antibody against the blood group i antigen. Further, the present invention relates to a method of assessing quality of a mesenchymal stem cell or a population thereof with an antibody against the blood group i antigen.
  • the quality can refer to, but is not limited to, estimation of the proportion in a clinical stem cell graft of cells detectable by anti-i antibody and hence assumed to be undifferentiated MSC, whereas those negative are differentiated MSCs.
  • the quality can be expressed e.g. by percentage of cells positive.
  • the methods of the present invention can be used in screening variation between the differential status of cells within a population of MSCs with an antibody against the blood group i antigen.
  • the methods of the invention may also contain additional and/or optional steps that are conventional to methods of distinguishing, screening, isolating and/or assessing cells, such as washing, incubating and dividing the cell populations.
  • the present invention further relates to a use of an antibody against the blood group i antigen in distinguishing and/or enriching native, undifferentiated mesenchymal stem cells and differentiated MSCs from each other.
  • the antibody against the blood group i antigen is used in distinguishing a native, undifferentiated MSC or a population thereof from a differentiated MSC or a population thereof.
  • the antibody is used in distinguishing and/or enriching a MSC differentiated toward osteogenic lineage or a population thereof from an undifferentiated MSC or a population thereof.
  • the cell population so obtained is capable of generating bone structures and bone-associated marrow elements more efficiently than a native MCS-population. Further, the risk of cancer which could be related to the transplantation diminishes when the amount of the undifferentiated MSCs in the graft is diminished.
  • the antibody is used in distinguishing and/or enriching a MSC differentiated toward adipogenic lineage or a population thereof from a native, undifferentiated MSC or a population thereof.
  • the present invention also relates to a use of an antibody against the blood group i antigen in distinguishing or enriching and/or purifying a native, undifferentiated mesenchymal stem cell or a population thereof and a fibroblast or a population thereof from each other.
  • a further object of the present invention is to provide a use of an antibody against the blood group i in screening of mesenchymal stem cells.
  • the present invention also relates to use of an antibody against the blood group i antigen in isolating of mesenchymal stem cells or a population of MSCs. Further, the present invention relates to use of an antibody against the blood group i antigen in assessing quality of a mesenchymal stem cell or a population thereof. Thus, the uses of an antibody against the blood group i antigen according to the present invention can be applied in screening variation between the differential status of cells within a population of MSCs.
  • An additional object of the present invention is to provide a cell population screened and/or enriched with an antibody against the blood group i antigen.
  • a MSC or a population thereof is derived from bone marrow. In another embodiment, a MSC or a population thereof is derived from cord blood.
  • the antibody against the blood group i antigen is a monoclonal antibody or molecular fragment (e.g. fab fragment) or derivative of immunoglobulin (e.g. tetramer) prepared by methods known in the art.
  • the antibody against the blood group i antigen is a lectin or an inactivated enzyme or a nucleic acid molecule detecting the i antigen.
  • the term antibody here refers to any of these binders which effectively can detect the blood group i antigen as currently defined.
  • the invention relates to a method for producing antibodies against blood group antigen i or blood group antigen I by using an undifferentiated MSC or a population thereof and a differentiated MCS or a population thereof as immunisation antigens and antibody screening targets.
  • an undifferentiated MSC or a population thereof and a differentiated MCS or a population thereof are used in producing antibodies against blood group i antigen or blood group I antigen.
  • BM MSCs were obtained as described by Leskela et al. (Bio- chem. Biophys. Res. Commun. 2003; 311 : 1008- 3). Briefly, bone marrow obtained during orthopaedic surgery was cultured in Minimun Essential Alpha- Medium supplemented with 20 mM HEPES, 10 % fetal bovine serum, penicillin-streptomycin and 2 mM L-glutamine (all from Gibco). After a cell attachment period of 2 days the cells were washed with PBS, subcultured further by plating the cells at a density of 2000-3000 cells/cm 2 in the same media and replacing the medium twice a week. The cells used in the analyses were of passage 5-7.
  • MNCs Mononuclear cells
  • Ficoll-Paque Plus GE Healthcare Biosciences density gradient centrifu- gation.
  • the mononuclear cell fraction was plated on fibronectin (Sigma Aldrich) - coated 6-well plates (Nunc) at 10 6 cells/well in Minimun Essential Alpha-Medium supplemented with 10% fetal bovine serum, 50 nM Dexamethasone, 10 ng/ml EGF, 10 ng/ml rhPDGF-BB and penicillin-streptomycin.
  • BM and CB MSCs were analyzed by flow cytometry to be negative for CD14, CD34, CD45 and HLA-DR; and positive for CD 3, CD29, CD44, CD90, CD105 and H LA-ABC. The cells were shown to be able to differentiate toward osteogenic, adipogenic and chondrogenic lineages.
  • Adipogenic differentiation of BM and CB MSCs was induced by culturing the cells for 2-4 days in adipogenic induction medium: aMEM Glu- tamax (Gibco) supplemented with 20mM HEPES (Gibco), 0% fetal bovine serum (Gibco), penicillin-streptomycin (Gibco), 0, 1 mM Indomethasin (Sigma), 0,044 pg/ml IBMX-22 (PromoCell), 0,4 Mg/ml DM200 (PromoCell) and 0,5 pg/ml insulin (PromoCell).
  • the cells were changed into terminal differentiation medium: aMEM Glutamax (Gibco) supplemented with 20mM HEPES (Gibco), 10% fetal bovine serum (Gibco), penicillin-streptomycin (Gibco), 0,1 mM Indomethasin (Sigma), 0,5 pg/ml insulin and 3pg/ml Ciglita- zone (PromoCell). Cells were allowed to differentiate for 2.5-3 weeks. Adipogenic differentiation was verified by Sudan III staining.
  • Osteogenic differentiation of CB MSCs was induced by culturing the cells for 2-3 weeks in osteogenic induction medium: aMEM supplemented with 20 mM HEPES, 10% FCS, 2 mM glutamine, 0,1 ⁇ dexa- methasone, 10 mM ⁇ -glycerophosphate, 0,05 mM ascorbic acid-2-phosphate, and penicillin-streptomycin. Osteogenic differentiation was verified by von Kossa staining.
  • Cells were detached from culture plates by incubating with 0.25% trypsin in 1 mM EDTA-PBS for 5-7 min (MSCs and adipocytes) or 20 min (osteoblasts) at +37°C.
  • Cells (100 000) were incubated with 10% human serum serologically shown to contain anti-i antibodies or control serum with no anti-i antibodies (diluted in 0,3% BSA - PBS - 2 mM EDTA) for 30 min on ice and washed once before incubating with secondary antibody (PE-conjugated anti-human IgM, Rockland Immunochemicals; 1 :2000) for 30 min on ice. Control cells were treated similarly but without primary antibody.
  • the expression of the i-antigen is characteristic of proliferating MSCs and disappears upon differentiation, and could therefore be used as a marker for undifferentiated MSCs.
  • BALB/c mouse are immunized with human MSC by injecting with approximately 10 7 human bone marrow or cord blood derived MSC cells resuspended in less than 0.2 mL in volume of Complete Freund's Adjuvalent in physiological saline. The same animals are boosted after 10 to 14 days post- injection. The spleen cells of the immunized animals are harvested after sacri- fication and the spleen cells are fused with myeloma cell line SP2/0-Ag14 (ATCC#CRL 1581). All these protocols are described in the art, for example by Yokoyama et al (Production of monoclonal antibodies in Current Protocols in Immunology 2006; 2.5.1-2.5.25.
  • the hybridomas and cell clones produced using the above mentioned standard protocol by Yo- koyma et al (2006) are screened using undifferentiated MSC as a positive target and adipogenic and osteogenic lineages as a negative target.
  • the clones showing positive detection of undifferentiated MSC but not detecting the differentiated cells are potentially producing anti-i antibodies.
  • the specificity of the immunoglobulin so produced can be confirmed by using synthetic linear poly- N-acetyllactosamine structures as a target and additionally, they can be compared with conventional anti-i antisera in blood grouping settings.
  • Mononuclear cells were isolated from fresh cord blood units as described in Example 1. The cells were stained with anti-i serum as described in Example 1 , and with following APC-conjugated antibodies for hematopoietic cell types: anti-CD3 (T-cells), anti-CD14 (monocytes), anti-CD20 (B- cells), anti-CD34 (hematopoietic progenitors) and anti-CD56 (NK cells) (all from BD Biosciences). Appropriate isotype controls (from BD Biosciences) were used. Flow cytometry analysis was carried out as described in Example 1.
  • Human adult and juvenile fibroblasts (Normal Human Dermal Fibroblasts, adult donor and juvenile foreskin; PromoCell) were cultured in Fibroblast Growth Medium 2 (PromoCell) and Fibroblast Growth Medium (Pro- mocell), respectively, with Supplement Mix (PromoCell). The cells were stained with anti-i serum and analyzed by flow cytometry as described in Example 1.

Abstract

The present invention relates to a method of distinguishing and/or screening mesenchymal stem cells using an antibody against the blood group i antigen. The present invention also relates to a use of an antibody against the blood group i antigen in distinguishing mesenchymal stem cells. The present invention also relates to a use of an antibody against the blood group i antigen in screening of mesenchymal stem cells. Further, the present invention relates to a cell population screened with an antibody against the blood group i antigen.

Description

USE OF AN ANTIBODY
FIELD OF THE INVENTION
[0001] The present invention relates to methods of distinguishing, and purifying or enriching, and screening mesenchymal stem cells using an antibody against the blood group i antigen. The present invention also relates to a use of an antibody against the blood group i antigen in distinguishing and/or enriching mesenchymal stem cells. The present invention further relates to a use of an antibody against the blood group i antigen in screening of mesenchymal stem cells. In addition, the present invention relates to a cell population screened or enriched with an antibody against the blood group i antigen.
BACKGROUND OF THE INVENTION
[0002] Mesenchymal stem cells (MSCs) are multipotent stem cells that can differentiate into a variety of cell types, in particular to osteoblast, chondrocytes and adipocytes, both in vitro and in vivo. MSCs can be obtained, for example, from the cord blood and bone marrow. The exact definitions for MSC or cell lineages differentiated thereof are currently not finally established (Da Silva Meilleres et al., Stem Cells 2008; 26: 2287-99), but an example of a current set of criteria for undifferentiated MSC are described by Dominici et al., in Cytotherapy 2006; 8: 315-317.
[0003] MSCs have a large capacity for self-renewal while maintaining their multipotency. A standard test to confirm multipotency is differentiation of the cells into osteoblasts, adipocytes, and chondrocytes, for example. However, the degree to which the culture will differentiate varies depending on how differentiation is induced, e.g. chemically vs. mechanically. The capacity of cells to differentiate and proliferate is known to decrease with the age of the donor, as well as the time cultivated in vitro. Whether this is due to a decrease in the number of MSCs or a change to the existing MSCs, is not known.
[0004] Since MSCs have the potential to differentiate into various cellular lineages and can be expanded in culture conditions without losing their multipotency, they present a valuable source for applications in cell therapy and tissue engineering.
[0005] MSC transplantation offers a promising approach for treating certain nonhematological malignant and nonmalignant diseases and for stem cell-mediated tissue regeneration. In particular, they can be applied to induce immunosuppression. This can be done as supportive therapy in hematological stem cell transplantations in which immunologically-mediated graft-versus-host disease is a major complication.
[0006] Bone marrow and cord blood have been studied, and also used in treating human patients, as mesenchymal stem cell sources. Unfortunately, utilization and success of stem cell transplantation suffer from several obstacles such as graft-versus-host disease and graft rejection.
[0007] A critical factor with regard to stem cell transplantation in general, is the dose or number of stem cells in the graft. As MSCs represent a heterogeneous population of cells their actual number in a graft cannot be readily estimated. Currently, no test exists that can be performed on a single cell to determine whether that cell is an MSC and further whether the cell is a native, undifferentiated MSC or a differentiated MSC. Dominici et al. (2006) discloses that there are surface antigens that can be used to isolate a population of cells that have similar self-renewal and differentiation capacities, yet MSCs, as a population, typically do not all express the proposed markers; and it is not certain which ones must be expressed in order for that cell to be classified as a MSC.
[0008] Purified or partially purified antibodies or antisera - i.e. samples of serum containing effective levels of anti-i antibodies - especially targeted for i/l blood grouping, have been described in some details (Gooi et al., Molecular Immunology 1984; 21 : 1099-1104; Niemann et al., Biochemical and Biophysical Research Communications 1978; 81 : 1286-1293; Feizi et al., Carbohydrate Research 1992; 228: 289-297). Mouse monoclonal antibodies against the blood group I antigen have been prepared using the standard hy- bridoma method described in Gooi et al. (1984). The antigenic structures detected by these antibodies, either anti-l or anti-i, are not exactly known and some variation in the structure can be assumed based on the above publications. However, the antigenic structure is related to, or it is assumed to include as a shared feature a linear poly-N-acetyllactosamine having the following structure Gal(p1-4)GlcNAc( 1-3)Gal( 1-4)GlcNAc(p1-) linked to different molecules.
[0009] It has been described in WO 2008/087260 that certain poly- N-acetyllactosamines are found on human MSCs, but the usefulness of blood group i antigen as a differentiation marker has not been disclosed therein. BRIEF DESCRIPTION OF THE INVENTION
[0010] An object of the present invention is to provide a method of distinguishing a native undifferentiated mesenchymal stem cell or a population thereof and a differentiated MSC or a population thereof from each other using an antibody against the blood group i antigen. Another object of the present invention is to provide a method of purifying and/or enriching a native undifferentiated mesenchymal stem cell or a population thereof and a differentiated MSC or a population thereof from each other using an antibody against the blood group i antigen. An additional object of the invention is to provide methods of distinguishing or purifying and/or enriching a native undifferentiated mesenchymal stem cell or a population thereof and a fibroblast or a population thereof from each other using an antibody against the blood group i antigen.
[0011] A further object of the present invention is to provide a use of an antibody against the blood group i antigen in distinguishing an undifferentiated MSC or a population thereof and a differentiated MSC or a population thereof from each other. A still further object of the present invention is to provide a use of an antibody against the blood group i antigen in purifying and/or enriching a native undifferentiated mesenchymal stem cell or a population thereof and a differentiated MSC or a population thereof from each other. An additional object of the invention is to provide uses of an antibody against the blood group i antigen in distinguishing or purifying and/or enriching a native undifferentiated mesenchymal stem cell or a population thereof and a fibroblast or a population thereof from each other.
[0012] A further object of the present invention is to provide a method of screening MSCs using an antibody against the blood group i antigen. In addition, an object of the present invention is to provide a use of an antibody against the blood group i antigen in screening of mesenchymal stem cells. Another object of the present invention is to provide a MSC population purified and/or enriched with an antibody against the blood group i antigen. An additional object of the present invention is to provide a MSC population screened with an antibody against the blood group i antigen. Thus, the present invention leads to an enriched and/or purified or more homogeneous preparation of MSCs useful for therapeutic purposes.
[0013] The invention is based on the observation that the target of the blood group i antibody on a mesenchymal stem cell lessens during the differentiation process of the cell. The invention is based on a further observation that an antibody against the blood group i antigen may be used as a marker for an undifferentiated MSC and that an antibody against the blood group i antigen is able to distinguish differentiated MSCs and native, undifferentiated MSCs from each other. The invention is based on an even further observation that an antibody against the blood group i antigen is able to distinguish native, undifferentiated MSCs from fibroblasts.
[0014] Accordingly, the current invention provides a novel and effective means for distinguishing, enriching and/or screening of MSC-populations and/or for assessing of the differential status of MSCs and/or quality of a MSC population.
[0015] In the present invention it was also found that monoclonal antibodies against blood group i and I antigens could be produced using human MSCs as antigens. Thus, the present invention can also be applied for production of antibodies against blood group i antigen and blood group I antigen.
[0016] In one embodiment of the invention, synthetically-prepared target antigens, or polymers or oligomers of lactose, lactosamine or acetyllac- tosamine structures, or their functional analogs are used to eluate the blood group i antibody from the purified MSC.
[0017] The objects of the invention are achieved by the methods, uses and cell populations set forth in the independent claims. Preferred embodiments of the invention are described in the dependent claims.
[0018] Other objects, details and advantages of the present invention will become apparent from the following drawings, detailed description and examples.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 shows staining of mesenchymal stem cells (MSCs) and cells differentiated from MSCs by human serum containing anti-i antibodies. Positively stained cells are marked green in the dot plots, and negative cells are marked red. The percentage of positive cells was determined as the cell population with a higher fluorescence intensity than 99.5% of cells stained with secondary antibody only. Histogram overlays are shown with staining with anti-i serum in red fill and secondary controls in grey fill.
a) undifferentiated bone marrow MSCs,
b) adipocytes differentiated from the bone marrow MSCs of the a), c) cord blood MSCs,
d) adipocytes differentiated from the cord blood MSCs of the c), e) cord blood MSCs,
f) osteoblastic cells differentiated from the cord blood MSCs of e).
[0020] Figure 2 shows the expression of the i-antigen in CB MSC lines and differentiated cells. The cells were stained with 10 % human anti-i serum (10%) and PE-conjugated donkey anti-human IgM.
[0021] Figure 3 shows the staining of cord blood mononuclear cell populations by anti-i serum.
[0022] Figure 4 shows the staining of juvenile and adult normal human dermal fibroblasts by anti-i serum.
DETAILED DESCRIPTION OF THE INVENTION
[0023] An MSC-population is typically a heterogeneous population of cells. A portion of the cell population presents native, undifferentiated cells and another portion presents cells that have been differentiated or have started to differentiate toward osteogenic, adipogenic and/or chondrogenic lineages, for example. Although transplantation of MSC possesses promising approach for stem cell-mediated tissue regeneration, the heterogeneity within the differential status of the cells increases the probability and/or extent of ill effects. Furthermore, the cellular heterogeneity also leads to poor understanding of factors relevant in successful transplantation.
[0024] In the present invention, mesenchymal stem cells from bone marrow and cord blood, and cells differentiated from them toward adipogenic and osteogenic lineages were stained with human serum containing antibodies against the blood group i antigen. Anti-i serum was bound to a high proportion of cord blood derived MSCs and a subpopulation of bone marrow derived MSCs. The same analysis with cells differentiated into adipogenic and/or osteogenic directions resulted in a substantially lower staining, equivalent or close to that of nonspecific human serum. The results indicate that the expression of the blood group i antigen is characteristic of proliferating MSCs and disappears upon differentiation, and could therefore be used as a marker for undifferentiated MSCs.
[0025] The present invention is thus based on the finding that the amount on a mesenchymal stem cell of the target antigen of anti-i antibodies becomes smaller and/or the presence of the blood group i antigen on a mesenchymal stem cell lessens during the differentation process of the cell. The invention is based on a further finding that an antibody against blood group i antigen may be used as a marker for an undifferentiated MSC and that an an- tibody against blood group i antigen is able to distinguish differentiated MSCs and native, undifferentiated MSCs from each other.
[0026] On the basis of these findings, a method of distinguishing a native, undifferentiated mesenchymal stem cell or a population thereof and a differentiated MSC or a population thereof from each other by an antibody against the blood group i antigen has been developed.
[0027] In one embodiment of the invention, the method is used in distinguishing a native, undifferentiated mesenchymal stem cell or a population thereof from a differentiated MSC or a population thereof. The cell population so distinguished could more efficiently be used in stem cell therapy, such as stem cell mediated tissue regeneration.
[0028] In another embodiment of the invention, the method is used in distinguishing a MSC differentiated toward osteogenic lineage or a population thereof from a native, undifferentiated MSC or a population thereof. The cell population so obtained is capable of generating bone structures and bone- associated marrow elements more efficiently than a native or heterogeneous MCS-population. Further, the risk of cancer which could be related to the transplantation diminishes when the amount of the undifferentiated MSCs in the graft is diminished.
[0029] In a further embodiment of the invention, the method is used in distinguishing a MSC differentiated toward adipogenic lineage or a population thereof from a native, undifferentiated MSC or a population thereof.
[0030] On the basis of the findings of the present invention, a method of purifying and/or enriching a native undifferentiated mesenchymal stem cell or a population thereof and a differentiated MSC or a population thereof from each other using an antibody against the blood group i antigen has been developed.
[0031] In one embodiment of the invention, the method is used in purifying and/or enriching a native, undifferentiated mesenchymal stem cell or a population thereof from a differentiated MSC or a population thereof. The cell population so purified and/or enriched could more efficiently be used in stem cell therapy, such as stem cell mediated tissue regeneration.
[0032] In another embodiment of the invention, the method is used in purifying and/or enriching a MSC differentiated toward osteogenic lineage or a population thereof from a native, undifferentiated MSC or a population thereof. The cell population so enriched is capable of generating bone struc- tures and bone-associated marrow elements more efficiently than a native or heterogenous MCS-population. Further, the risk of cancer which could be related to the transplantation diminishes when the amount of the undifferentiated MSCs in the graft is diminished.
[0033] In a further embodiment of the invention, the method is used in purifying and/or enriching a MSC differentiated toward adipogenic lineage or a population thereof from a native, undifferentiated MSC or a population thereof.
[0034] While purifying a MSC population, one contaminating population can be fibroblasts that unlike T-cells and B-cells, but like MSCs, also are adherent to plastic. In the present invention it was found that fibroblasts were negative for the i-antigen. Accordingly, the expression of the i-antigen can be used to differentiate between mesenchymal stem cells and fibroblasts.
[0035] On the basis of this finding, a method of distinguishing a native, undifferentiated mesenchymal stem cell or a population thereof and a fibroblast or a population thereof from each other by an antibody against the blood group i antigen has been developed.
[0036] Thus, in one embodiment the method is used in distinguishing a native, undifferentiated mesenchymal stem cell or a population thereof from a fibroblast or a population thereof using an antibody against the i antigen. The MSC population can further be purified by using the antibody against the i antigen; there are a number of different methods known in the art for purification of a particular cell population using antibodies detecting only the desired cell population.
[0037] In another embodiment of the invention, the method is used in purifying and/or enriching a MSC or a population thereof from a fibroblast or a population thereof by using the antibody against the i antigen. The cell population so enriched and/or purified is more homogeneous and requires less purification in later stages.
[0038] The present invention also relates to a method of isolating a mesenchymal stem cells or a population of MSCs with an antibody against the blood group i antigen. Further, the present invention relates to a method of assessing quality of a mesenchymal stem cell or a population thereof with an antibody against the blood group i antigen. Here, the quality can refer to, but is not limited to, estimation of the proportion in a clinical stem cell graft of cells detectable by anti-i antibody and hence assumed to be undifferentiated MSC, whereas those negative are differentiated MSCs. The quality can be expressed e.g. by percentage of cells positive. Thus, the methods of the present invention can be used in screening variation between the differential status of cells within a population of MSCs with an antibody against the blood group i antigen.
[0039] The methods of the invention may also contain additional and/or optional steps that are conventional to methods of distinguishing, screening, isolating and/or assessing cells, such as washing, incubating and dividing the cell populations.
[0040] The present invention further relates to a use of an antibody against the blood group i antigen in distinguishing and/or enriching native, undifferentiated mesenchymal stem cells and differentiated MSCs from each other.
[0041] In one embodiment of the invention, the antibody against the blood group i antigen is used in distinguishing a native, undifferentiated MSC or a population thereof from a differentiated MSC or a population thereof.
[0042] In another embodiment of the invention, the antibody is used in distinguishing and/or enriching a MSC differentiated toward osteogenic lineage or a population thereof from an undifferentiated MSC or a population thereof. The cell population so obtained is capable of generating bone structures and bone-associated marrow elements more efficiently than a native MCS-population. Further, the risk of cancer which could be related to the transplantation diminishes when the amount of the undifferentiated MSCs in the graft is diminished.
[0043] In a further embodiment of the invention, the antibody is used in distinguishing and/or enriching a MSC differentiated toward adipogenic lineage or a population thereof from a native, undifferentiated MSC or a population thereof.
[0044] The present invention also relates to a use of an antibody against the blood group i antigen in distinguishing or enriching and/or purifying a native, undifferentiated mesenchymal stem cell or a population thereof and a fibroblast or a population thereof from each other.
[0045] A further object of the present invention is to provide a use of an antibody against the blood group i in screening of mesenchymal stem cells.
[0046] The present invention also relates to use of an antibody against the blood group i antigen in isolating of mesenchymal stem cells or a population of MSCs. Further, the present invention relates to use of an antibody against the blood group i antigen in assessing quality of a mesenchymal stem cell or a population thereof. Thus, the uses of an antibody against the blood group i antigen according to the present invention can be applied in screening variation between the differential status of cells within a population of MSCs.
[0047] An additional object of the present invention is to provide a cell population screened and/or enriched with an antibody against the blood group i antigen.
[0048] The observed expression of the i antigen in proliferating MSCs and its disappearing upon differentiation provides a valuable means for enriching and screening MSCs, especially distinguishing undifferentiated MSCs and differentiated ones from each others.
[0049] In one embodiment of the invention, a MSC or a population thereof is derived from bone marrow. In another embodiment, a MSC or a population thereof is derived from cord blood.
[0050] In one embodiment of the invention, the antibody against the blood group i antigen is a monoclonal antibody or molecular fragment (e.g. fab fragment) or derivative of immunoglobulin (e.g. tetramer) prepared by methods known in the art. In another embodiment, the antibody against the blood group i antigen is a lectin or an inactivated enzyme or a nucleic acid molecule detecting the i antigen. The term antibody here refers to any of these binders which effectively can detect the blood group i antigen as currently defined.
[0051] In the present invention it was also found that monoclonal antibodies against blood group i and I antigens could be produced using human MSCs as antigens. Due to problems not understood, it has been surprisingly difficult to raise anti-i antibodies for blood grouping purposes. Hence, there is a constant need for the anti-i and anti-l antibodies by blood banks. Immunization using an undifferentiated MSC or a population thereof and subsequent screening with an undifferentiated and a differentiated MSC or populations thereof provides a novel way to produce anti-i antibodies and/or anti-l antibodies. Accordingly in one embodiment, the invention relates to a method for producing antibodies against blood group antigen i or blood group antigen I by using an undifferentiated MSC or a population thereof and a differentiated MCS or a population thereof as immunisation antigens and antibody screening targets. [0052] In another embodiment of the invention, an undifferentiated MSC or a population thereof and a differentiated MCS or a population thereof are used in producing antibodies against blood group i antigen or blood group I antigen.
[0053] The invention will be described in more detail by means of the following examples. These examples are not to be construed to limit the claims in any manner whatsoever.
EXAMPLE 1
[0054] Mesenchymal stem cells from cord blood and bone marrow, and cells differentiated from them toward osteogenic and adipogenic lingeages, were stained with human serum containing antibodies against the blood group i antigen.
BONE MARROW (BM) DERIVED MESENCHYMAL STEM CELLS (MSCS)
[0055] BM MSCs were obtained as described by Leskela et al. (Bio- chem. Biophys. Res. Commun. 2003; 311 : 1008- 3). Briefly, bone marrow obtained during orthopaedic surgery was cultured in Minimun Essential Alpha- Medium supplemented with 20 mM HEPES, 10 % fetal bovine serum, penicillin-streptomycin and 2 mM L-glutamine (all from Gibco). After a cell attachment period of 2 days the cells were washed with PBS, subcultured further by plating the cells at a density of 2000-3000 cells/cm2 in the same media and replacing the medium twice a week. The cells used in the analyses were of passage 5-7.
CORD BLOOD (CB) DERIVED MSCS
[0056] Human term umbilical cord blood units were collected after delivery with informed consent of the mothers and the cord blood was processed within 24 hours of collection. Mononuclear cells (MNCs) were isolated from each unit by Ficoll-Paque Plus (GE Healthcare Biosciences) density gradient centrifu- gation. The mononuclear cell fraction was plated on fibronectin (Sigma Aldrich) - coated 6-well plates (Nunc) at 106 cells/well in Minimun Essential Alpha-Medium supplemented with 10% fetal bovine serum, 50 nM Dexamethasone, 10 ng/ml EGF, 10 ng/ml rhPDGF-BB and penicillin-streptomycin. Most of the non-adherent cells were removed as the medium was replaced the next day. The cells were subcultured essentially as described for BM MSCs above. The CB MSCs used in the analyses were of passage 4-6. [0057] Both BM and CB MSCs were analyzed by flow cytometry to be negative for CD14, CD34, CD45 and HLA-DR; and positive for CD 3, CD29, CD44, CD90, CD105 and H LA-ABC. The cells were shown to be able to differentiate toward osteogenic, adipogenic and chondrogenic lineages.
ADIPOGENIC DIFFERENTIATION
[0058] Adipogenic differentiation of BM and CB MSCs was induced by culturing the cells for 2-4 days in adipogenic induction medium: aMEM Glu- tamax (Gibco) supplemented with 20mM HEPES (Gibco), 0% fetal bovine serum (Gibco), penicillin-streptomycin (Gibco), 0, 1 mM Indomethasin (Sigma), 0,044 pg/ml IBMX-22 (PromoCell), 0,4 Mg/ml DM200 (PromoCell) and 0,5 pg/ml insulin (PromoCell). After a few days the cells were changed into terminal differentiation medium: aMEM Glutamax (Gibco) supplemented with 20mM HEPES (Gibco), 10% fetal bovine serum (Gibco), penicillin-streptomycin (Gibco), 0,1 mM Indomethasin (Sigma), 0,5 pg/ml insulin and 3pg/ml Ciglita- zone (PromoCell). Cells were allowed to differentiate for 2.5-3 weeks. Adipogenic differentiation was verified by Sudan III staining.
OSTEOGENIC DIFFERENTIATION
[0059] Osteogenic differentiation of CB MSCs was induced by culturing the cells for 2-3 weeks in osteogenic induction medium: aMEM supplemented with 20 mM HEPES, 10% FCS, 2 mM glutamine, 0,1 μΜ dexa- methasone, 10 mM β-glycerophosphate, 0,05 mM ascorbic acid-2-phosphate, and penicillin-streptomycin. Osteogenic differentiation was verified by von Kossa staining.
FLOW CYTOMETRY
[0060] Cells were detached from culture plates by incubating with 0.25% trypsin in 1 mM EDTA-PBS for 5-7 min (MSCs and adipocytes) or 20 min (osteoblasts) at +37°C. Cells (100 000) were incubated with 10% human serum serologically shown to contain anti-i antibodies or control serum with no anti-i antibodies (diluted in 0,3% BSA - PBS - 2 mM EDTA) for 30 min on ice and washed once before incubating with secondary antibody (PE-conjugated anti-human IgM, Rockland Immunochemicals; 1 :2000) for 30 min on ice. Control cells were treated similarly but without primary antibody. Cells were analyzed with BD FACSAria (Becton Dickinson). Results were analyzed with BD FACSDiva software version 5.0.1 (Becton Dickinson). [0061] The results are presented in Figure 1. Anti-i serum stained most of cord blood derived MSCs (c and e) and a subpopulation of bone marrow derived MSCs (a). When the cells were differentiated into adipogenic (b and d) or osteogenic (f) directions, the staining declined into the level of staining by nonspecific human serum.
[0062] The cells that stain positive for anti-i among the differentiated cells, as well as cells that stained positive for control sera among both MSCs and differentiated cells, were situated in a position in the scatter indicative of necrotic morphology, while the MSCs that stained positive for anti-i serum were distributed evenly within the scatter. Therefore it seems that the background staining by human sera is due to nonspecific binding of antibodies to necrotic cells, while the specific binding anti-i antibodies is evenly distributed over the whole population of undifferentiated MSCs.
[0063] Based on these results the expression of the i-antigen is characteristic of proliferating MSCs and disappears upon differentiation, and could therefore be used as a marker for undifferentiated MSCs.
EXAMPLE 2
PRODUCTION OF MONOCLONAL ANTIBODIES AGAINST i/l ANTIGENS USING HUMAN MSC AS ANTIGENS
[0064] BALB/c mouse are immunized with human MSC by injecting with approximately 107 human bone marrow or cord blood derived MSC cells resuspended in less than 0.2 mL in volume of Complete Freund's Adjuvalent in physiological saline. The same animals are boosted after 10 to 14 days post- injection. The spleen cells of the immunized animals are harvested after sacri- fication and the spleen cells are fused with myeloma cell line SP2/0-Ag14 (ATCC#CRL 1581). All these protocols are described in the art, for example by Yokoyama et al (Production of monoclonal antibodies in Current Protocols in Immunology 2006; 2.5.1-2.5.25. John Wiley & Sons, Inc). The hybridomas and cell clones produced using the above mentioned standard protocol by Yo- koyma et al (2006) are screened using undifferentiated MSC as a positive target and adipogenic and osteogenic lineages as a negative target. The clones showing positive detection of undifferentiated MSC but not detecting the differentiated cells are potentially producing anti-i antibodies. The specificity of the immunoglobulin so produced can be confirmed by using synthetic linear poly- N-acetyllactosamine structures as a target and additionally, they can be compared with conventional anti-i antisera in blood grouping settings.
EXAMPLE 3
THE i-ANTIGEN AS A MARKER FOR CORD BLOOD MESENCHYMAL STEM CELLS
[0065] 3 different cord blood mesenchymal stem cell lines were stained with anti-i serum in their proliferating state as well as differentiated along osteogenic and adipogenic lineages.
[0066] Generation, culture, characterization and differentiation of cells, staining with anti-i serum and flow cytometry analyses were carried out as described in Example 1.
[0067] Anti-i serum stained the three different cord blood mesenchymal stem cell lines (391 P, 588P, 594P) in their proliferating state (80-90 % of cells positive for the i-antigen; Figure 2). Cells of all three cell lines were essentially negative for the i-antigen when differentiated along osteogenic and adipogenic lineages (see Figure 2).
[0068] The expression of the i-antigen in proliferating but not differentiated lines was characteristic for cord blood derived mesenchymal stem cell lines.
EXAMPLE 4
CELL TYPES CARRYING THE i-ANTIGEN WITHIN THE CORD BLOOD MONONUCLEAR CELL POPULATION
[0069] Mononuclear cells were isolated from fresh cord blood units as described in Example 1. The cells were stained with anti-i serum as described in Example 1 , and with following APC-conjugated antibodies for hematopoietic cell types: anti-CD3 (T-cells), anti-CD14 (monocytes), anti-CD20 (B- cells), anti-CD34 (hematopoietic progenitors) and anti-CD56 (NK cells) (all from BD Biosciences). Appropriate isotype controls (from BD Biosciences) were used. Flow cytometry analysis was carried out as described in Example 1.
[0070] Cord blood T-cells and B-cells, and a small subpopulation of NK cells and CD34 positive cells, expressed the i-antigen, whereas monocytes were negative for the i-antigen (Figure 3). Cells of the monocyte-macrophage lineage typically contaminate the early passages of mesenchymal stem cell cultures. If mesenchymal stem cells were pre-selected with an anti-i antibody before plating, the culture would be more homogeneous in the early passages. T-cells and B-cells do not adhere to plastic, so that even if they were enriched by the selection with anti-i, they would disappear in the first medium exchange.
EXAMPLE 5
FIBROBLASTS DO NOT EXPRESS THE i-ANTIGEN
[0071] Human adult and juvenile fibroblasts (Normal Human Dermal Fibroblasts, adult donor and juvenile foreskin; PromoCell) were cultured in Fibroblast Growth Medium 2 (PromoCell) and Fibroblast Growth Medium (Pro- mocell), respectively, with Supplement Mix (PromoCell). The cells were stained with anti-i serum and analyzed by flow cytometry as described in Example 1.
[0072] Human fibroblasts were negative for the i-antigen (see Figure 4). The staining of the juvenile fibroblasts (15.9% of cells positive) is likely be nonspecific background, because a control serum devoid of anti-i antibodies stained 20.4% of the cells (data not shown). Fibroblasts typically carry many of the same antigens as mesenchymal stem cells, thus necessitating a functional characterization (tri-lineage differentiation) to define mesenchymal stem cells. Since fibroblasts were negative for the i-antigen, expression of the i-antigen can be used to differentiate between mesenchymal stem cells and fibroblasts.

Claims

1. A method of distinguishing a native undifferentiated MSC or a population thereof and a differentiated MSC or a population thereof from each other by using an antibody against blood group i antigen.
2. A method of purifying and/or enriching a native undifferentiated MSC or a population thereof and a differentiated MSC or a population thereof from each other by using an antibody against the blood group i antigen.
3. The method according to claim 1 or claim 2, wherein a MSC differentiated toward osteogenic lineage or a population thereof is distinguished or purified and/or enriched from a native, undifferentiated MSC or a population thereof.
4. The method according to claim 1 or claim 2, wherein a MSC differentiated toward adipogenic lineage or a population thereof is distinguished or purified and/or enriched from a native, undifferentiated MSC or a population thereof.
5. A use of an antibody against blood group i in distinguishing a differentiated MSC or a population thereof and a native, undifferentiated MSC or a population thereof from each other.
6. A use of an antibody against blood group i in purifying and/or enriching a differentiated MSC or a population thereof and a native, undifferentiated MSC or a population thereof from each other.
7. The use according to claim 5 or claim 6, wherein a MSC differentiated toward osteogenic lineage or a population thereof is distinguished or purified and/or enriched from a native, undifferentiated MSC or a population thereof.
8. The use according to claim 5 or claim 6, wherein a MSC differentiated toward adipogenic lineage or a population thereof is distinguished or purified and/or enriched from a native, undifferentiated MSC or a population thereof.
9. A method of screening MSCs using an antibody against blood group i for enrichment or quantitation of MSC.
10. A use of an antibody against blood group i antigen in screening of MSCs.
1 1. A population of MSCs produced by the method of any one of claims 1 to 4.
12. A population of MSCs purified and/or enriched with an antibody against the blood group i antigen.
13. A method for production of antibodies against blood group antigen i by using an undifferentiated MSC as immunisation antigens and antibody screening targets.
14. A use of an undifferentiated MSC in producing an antibody against blood group i antigen.
15. A method of assessing the quality of MSC population using an antibody against blood group i.
16. A use of an antibody against blood group i antigen in assessing the quality of MSC population.
17. A method of distinguishing a native undifferentiated MSC or a population thereof and a fibroblast or a population thereof from each other by using an antibody against blood group i antigen.
18. A method of purifying and/or enriching a native undifferentiated MSC or a population thereof and a fibroblast or a population thereof from each other by using an antibody against blood group i antigen.
19. A use of an antibody against blood group i in distinguishing a native, undifferentiated MSC or a population thereof and a fibroblast or a population thereof from each other.
20. A use of an antibody against blood group i in purifying and/or enriching a native, undifferentiated MSC or a population thereof and a fibroblast or a population thereof from each other.
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