Classifications

• • 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.

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