WO2008151390A1 - Différenciation de cellules souches embryonnaires humaines - Google Patents

Différenciation de cellules souches embryonnaires humaines Download PDF

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WO2008151390A1
WO2008151390A1 PCT/AU2008/000865 AU2008000865W WO2008151390A1 WO 2008151390 A1 WO2008151390 A1 WO 2008151390A1 AU 2008000865 W AU2008000865 W AU 2008000865W WO 2008151390 A1 WO2008151390 A1 WO 2008151390A1
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cell
cells
population
hematopoietic
pdgfrα
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PCT/AU2008/000865
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English (en)
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Andrew Elefanty
Eduoard Stanley
Elizabeth Ng
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Australian Stem Cell Centre Ltd
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Priority claimed from AU2007903225A external-priority patent/AU2007903225A0/en
Application filed by Australian Stem Cell Centre Ltd filed Critical Australian Stem Cell Centre Ltd
Publication of WO2008151390A1 publication Critical patent/WO2008151390A1/fr

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    • 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
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/105—Insulin-like growth factors [IGF]
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/135—Platelet-derived growth factor [PDGF]
    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/70—Polysaccharides
    • C12N2533/78—Cellulose

Definitions

  • the present invention relates generally to methods for identifying, detecting and isolating hematopoietic progenitor cells and hematopoietic cells in a population of cells comprising differentiating pluripotent cells, e.g., embryonic stem cells.
  • Human stem cell derived hematopoietic stem cell or mature cell therapies are routinely used to treat patients with cancers and other disorders of the blood and immune systems.
  • the efficient generation of hematopoietic stem cells and hematopoietic cells from differentiating human pluripotent cells, e.g., human embryonic stem cells (hESCs) is therefore desirable.
  • PDGFR platelet derived growth factor
  • PDGF e.g., a PDGF capable of activating a PDGFR ⁇ is capable of inducing, stimulating or enhancing growth or survival of a hemtaopoietic cell in a population of differentiating pluripotent cells, e.g., hESCs.
  • hESCs which are an established source of pluripotent cells as a model of pluripotent cells per se.
  • the present invention provides a method for detecting hematopoietic progenitor cells in a population of cells comprising differentiating pluripotent cells, the method comprising detecting the presence of PDGFR ⁇ on the surface of cells in said population, wherein the presence of PDGFR ⁇ is indicative of hematopoietic progenitor cells.
  • a method for isolating a hematopoietic progenitor cell in a population of cells comprising differentiating pluripotent cells comprising isolating a cell from said population expressing PDGFRa on its surface.
  • the pluripotent cells are human pluripotent cells.
  • the pluripotent cells are embryonic stem cells (ESCs) or an induced pluripotent stem cell (iPSCs).
  • a method for identifying a compound capable of inducing differentiation of a pluripotent cell into a hematopoietic cell or a hematopoietic progenitor cell and/or that is capable of inducing or enhancing or stimulating the growth or survival of hematopoietic cells and/or hematopoietic progenitor cells in a population of differentiating pluripotent cells comprising:
  • the expression of PDGFR ⁇ on the surface of the differentiated cell indicates that the compound induces differentiation of a pluripotent cell into a hematopoietic cell or a hematopoietic progenitor cell and/or that the compound induces or enhances or stimulates the growth or survival of hematopoietic cells and/or hematopoietic progenitor cells in a population of differentiating pluripotent cells.
  • a method of inducing or enhancing or stimulating the growth or survival of hematopoietic cells and/or hematopoietic progenitor cells from a population of differentiating pluripotent cells comprising obtaining a population of differentiating pluripotent cells and culturing the differentiating pluripotent cells in media comprising a PDGF.
  • a method of producing a hematopoietic progenitor cell or a hematopoietic cell comprising contacting a population comprising differentiating pluripotent cells and/or cells differentiated therefrom with a PDGF for a time and under conditions to produce a hematopoietic progenitor cell or a hematopoietic cell.
  • a culture medium for differentiating pluripotent cells into hematopoietic cells or hematopoietic progenitor cells and/or for inducing or enhancing or stimulating the growth or survival of hematopoietic cells and/or hematopoietic progenitor cells from a population of differentiating pluripotent cells said media comprising a PDGF and at least one factor selected from the group consisting of IGF2, FGF2 and combinations thereof.
  • a bioreactor for use in differentiating pluripotent cells into hematopoietic cells or hematopoietic progenitor cells and/or expanding populations of hematopoietic cells or hematopoietic progenitor cells, the bioreactor comprising a cell culture chamber in which at least one internal surface has immobilised thereon a PDGF.
  • a method for producing a hematopoietic cell and/or a hematopoietic progenitor cell comprising culturing a differentiating pluripotent cell in a culture medium according to the present invention for a time and under conditions sufficient for the differentiating pluripotent cell to differentiate into a hematopoietic cell and/or a hematopoietic progenitor cell.
  • a ninth aspect of the present invention there is provided an isolated hematopoietic progenitor cell or population thereof, or isolated hematopoietic cell or population thereof, produced by a method according to the present invention.
  • an isolated population of cells enriched for hematopoietic progenitor cells expressing PDGFR ⁇ on their surface In a tenth aspect of the present invention there is provided an isolated population of cells enriched for hematopoietic progenitor cells expressing PDGFR ⁇ on their surface.
  • a pharmaceutical composition comprising a cell or population of cells according to the present invention and a pharmaceutically acceptable carrier or excipient.
  • a cell or population of cells according to the present invention or a pharmaceutical composition according to the present invention, for use in medicine
  • a cell or population of cells according to the present invention or a pharmaceutical composition according to the present invention, for use in the treatment or prophylaxis of a disease or disorder resulting from a failure or a dysfunction of normal blood cell production and/or maturation or for the treatment of a subject in need of transfusion of blood or a cellular component thereof.
  • a fourteenth aspect of the present invention there is provided a use of a cell or population of cells according to the present invention in the manufacture of a medicament for the treatment or prophylaxis of a disease or disorder resulting from a failure or a dysfunction of normal blood cell production and/or maturation or for the treatment of a subject in need of a transfusion of blood or a cellular component thereof.
  • a method for treating a subject suffering from or at risk of developing a disease or disorder resulting from a failure or a dysfunction of normal blood cell production and/or maturation or a subject in need of a transfusion of blood or a cellular component thereof comprising administering to the subject a cell or population of cells according to the present invention, or a pharmaceutical composition according to the present invention.
  • a sixteenth aspect of the present invention there is provided a method of selecting a compound capable of inducing differentiation of a pluripotent cell into a hematopoietic cell or a hematopoietic progenitor cell and/or that is capable of inducing or enhancing or stimulating the growth or survival of hematopoietic cells and/or hematopoietic progenitor cells in a population of differentiating pluripotent cells, the method comprising:
  • the presence of PDGFR ⁇ on the surface of the differentiated cell indicates differentiation of the pluripotent cell into a hematopoietic cell or a hematopoietic progenitor cell and/or that the compound induces or enhances or stimulates the growth or survival of hematopoietic cells and/or hematopoietic progenitor cells in a population of differentiating pluripotent cells;
  • the present invention provides a kit for detecting a hematopoietic progenitor cell, said kit comprising a ligand that binds to a PDGFR ⁇ and, optionally, instructions for use in a method of the present invention.
  • the present invention provides a kit comprising a PDGF packaged with instructions to use said PDGF in a method of the present invention.
  • the kit comprises additional components of a media for culturing a pluripotent cell, preferably for differentiating a pluripotent cell.
  • Figure 1 shows generation and characterisation oi MIXLl GFPAv HESCs
  • NeoR is the PGKNeo cassette encoding G418 resistance, flanked by loxP sites (black triangles). The positions of Mfel sites used to map the structure of the modified locus are shown, as are the position of primers (a, b) used to identify correctly targeted clones.
  • This analysis also shows the progressive downregulation of the stem cell marker, OCT4, the transient expression of the primitive streak genes, MIXLl and BRACHYURY, activation of genes diagnostic for endodermal (FOXA2, AFP-alpha fetoprotein, ALBUMIN) and mesodermal (GATA2, CDS4) cell types.
  • -RT - Reverse Transcriptase.
  • F Sorting and reanalysis experiments examining the relationship between expression of GFP and MDCLl protein.
  • the left panel shows the profile of GFP expressing cells in d5 MIXLl GFP/w EBs.
  • the vertical line indicates the division between GFP + and GFP ' cells based on gates set using MIXLl w/w (HES3) control EBs.
  • the middle panel shows the reanalysis of the sorted populations with the distribution of GFP + cells (red) and GFP " cells (white) indicated.
  • the right panel shows that endogenous MIXLl protein, as determined by intracellular flow cytometry with an anti- MIXLl antibody, is restricted to GFP + cells (red) and excluded from the GFP " cells (white).
  • the position of gates for intracellular flow cytometry were set with an appropriate isotype control antibody.
  • FIG. 2 shows BMP4 induces a wave of GFP expression in differentiating
  • Figure 3 shows hematopoietic progenitors are enriched in the MIXLl + PDGFRa + fraction of differentiating HESCs
  • Figure 4 shows flow cytometric analysis of the HES3 derivative MIXL1 GFP/W HESCs
  • Figure 5 shows enhanced formation of primitive hematopoietic colonies Enhanced formation of primitive hematopoietic colonies generated from d4 HESC spin EBs by PDGF, IGF2, and FGF2.
  • Envy cells were differentiated as spin EBs in BMP4, VEGF and SCF alone (BVS) (A,C,E,G,I,K) or with IGF2 addition at d2 as indicated (BVS d21GF2) (B,D,F,H,J,L).
  • MC serum- free methylcellulose
  • A-F blood growth factor cocktail alone
  • PDGF blood growth factor
  • IGF2 IGF2
  • FGF2 FGF2
  • Panels A,B,G,H show low power (xlO) darkfield images of representative fields from the MC cultures after 14 days and panels C,D,I,J show brigthfield images of different areas from the same experiment (magnification xlO).
  • Panels E,F,K,L show high power (x200) brigthfield images of representative hemoglobinised blast colonies after 1Od in MC.
  • the present invention provides a method for detecting hematopoietic progenitor cells in a population of cells comprising differentiating pluripotent cells, the method comprising detecting the presence of PDGFR ⁇ on the surface of cells, wherein the presence of PDGFR ⁇ is indicative of hematopoietic progenitor cells.
  • PDGFR ⁇ or "platelet derived growth factor ⁇ ” shall be understood to mean an alpha subunit of a PDGFR.
  • a PDGFR generally exists in nature as a dimer, e.g., comprising an ⁇ and a ⁇ chain or two ⁇ chains or two ⁇ chains, hi the context of the present invention, detection of PDGFR ⁇ shall be taken to include detection of a single ⁇ chain of a PDGFR and/or a dimeric receptor comprising two ⁇ chains.
  • an amino acid sequence of an ⁇ chain of a human PDGFR is set forth in SEQ ID NO: 1.
  • SEQ ID NO: 1 amino acid sequence of an ⁇ chain of a human PDGFR.
  • the present invention extends to the detection of any form of an ⁇ chain of a PDGFR expressed on the surface of a cell, including an ⁇ chain comprising a sequence having one or more conservative amino acids substitutions compared to the sequence set forth in SEQ ED NO: 1 or a deletion or addition that does not affect the function of a PDGFR comprising said ⁇ chain.
  • the presence of PDGFR ⁇ on the surface of the cells is detected by contacting the population of cells with a ligand that binds PDGFRa for a time and under conditions sufficient to form a ligand-PDGFR ⁇ complex, and detecting the complex to thereby detect expression of the PDGFR on the surface of the cell.
  • Suitable ligands will be apparent to the skilled artisan and include, for example a peptide or a small molecule or an antibody.
  • the ligand is a PDGF that binds to a PDGFR ⁇ , preferably that binds to a PDGFR ⁇ .
  • the ligand is a PDGF-AA.
  • PDGF PDGF-like protein
  • a PDGF is produced using recombinant or synthetic techniques known in the art, e.g., as described in Ausubel et al (In: Current Protocols in Molecular Biology. Wiley Interscience, ISBN 047 150338, 1987) and/or Sambrook et al (In: Molecular Cloning: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Third Edition 2001).
  • PDGF shall be taken to mean a growth factor comprising two subunits, e.g., disulphide linked subunits that is capable of binding to a PDGFR, and preferably activating signal transduction mediated by the PDGFR.
  • PDGF-AA is a growth factor comprising two A subunits
  • PDGF- AC comprises an A subunit and a C subunit
  • PDGF-AB comprises an A subunit and a C subunit.
  • the structure of a PDGF is described in more detail infra.
  • the ligand is an antibody, e.g., an antibody that selectively or preferentially binds to a PDGFR ⁇ , e.g., as described herein below.
  • the ligand is labeled with a detectable marker to facilitate detection.
  • detectable markers will be apparent to the skilled artisan include, a fluorescent dye, a fluorescent nanocrystal (e.g., a Q-dotTM), a radioactive moiety or an enzyme.
  • the present invention also provides a method for isolating a hematopoietic progenitor cell in a population of cells comprising differentiating pluripotent cells, said method comprising isolating a cell from said population expressing PDGFR ⁇ on its surface.
  • the method for isolating a hematopoietic progenitor cell in a population of cells comprises the steps of: (i) contacting the population of differentiating pluripotent cells with a ligand that binds PDGFR ⁇ for a time and under conditions sufficient to form a ligand-PDGFR ⁇ complex; and
  • the ligand is an antibody.
  • the present invention also provides a method for identifying a compound capable of inducing differentiation of a pluripotent cell into a hematopoietic cell or a hematopoietic progenitor cell and/or that is capable of inducing or enhancing or stimulating the growth or survival of hematopoietic cells and/or hematopoietic progenitor cells in a population of differentiating pluripotent cells, the method comprising:
  • the expression of PDGFRa on the surface of the differentiated cell indicates that the compound induces differentiation of a pluripotent cell into a hematopoietic cell or a hematopoietic progenitor cell and/or that the compound induces or enhances or stimulates the growth or survival of hematopoietic cells and/or hematopoietic progenitor cells in a population of differentiating pluripotent cells.
  • the method additionally comprises isolating the compound.
  • the compound may be isolated using conventional techniques known in the art.
  • the present invention encompasses any pluripotent cell, preferably an embryonic stem cell (ESC) or inducible pluripotent stem cell (iPSC).
  • ESC embryonic stem cell
  • iPSC inducible pluripotent stem cell
  • the cell is an ESC.
  • the ligand is specific for PDGFR ⁇ , or a fragment of PDGFR ⁇ , e.g., an immunogenic fragment of a PDGFR ⁇ . Suitable ligands are described supra.
  • the ligand is an antibody.
  • the antibody used in the present invention may encompass any antibody or antigen binding fragment thereof, either native or recombinant, synthetic or naturally derived, monoclonal or polyclonal which retains sufficient specificity to bind PDGFR ⁇ .
  • the terms "antibody” and “antibodies” include the entire antibody or any antigen binding fragment thereof.
  • antibody and “antibodies” also include any monospecific or bispecific compound comprised of a sufficient portion of the light chain variable region and/or heavy chain variable region to effect binding to an epitope to which the antibody or antigen binding fragment has specificity.
  • the antigen binding fragments include the variable region of at least one heavy or light chain immunoglobulin polypeptide, and include but are not limited to, dAb, Fab, F(ab') 2 , and Fv fragments.
  • Suitable commercial sources of antibodies that bind PDGFR ⁇ will be apparent to the skilled artisan and include, for example, BD Biosciences Pharmingen (CA, USA) or ABR Affinity Bioreagents (CO, USA).
  • an antibody that binds PDGFR ⁇ is produced using a method known in the art, and described, for example, in Harlow and Lane (In: Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988).
  • a monoclonal antibody against a PDGFR ⁇ is produced by immunizing an animal, e.g., a mouse or rat, with said protein or an immunogenic fragment thereof.
  • the protein or fragment is injected in the presence of an adjuvant, such as, for example Freund's complete or incomplete adjuvant, lysolecithin and/or dinitrophenol to enhance the immune response to the PDGFR ⁇ or immunogenic fragment thereof.
  • an adjuvant such as, for example Freund's complete or incomplete adjuvant, lysolecithin and/or dinitrophenol to enhance the immune response to the PDGFR ⁇ or immunogenic fragment thereof.
  • Spleen cells are then obtained from the immunized animal.
  • the spleen cells are then immortalized by, for example, fusion with a myeloma cell fusion partner, preferably one that is syngenic with the immunized animal.
  • a myeloma cell fusion partner preferably one that is syngenic with the immunized animal.
  • a variety of fusion techniques may be employed, for example, the spleen cells and myeloma cells can be combined with a nonionic detergent or electrofused and then grown in a selective medium that supports the growth of hybrid cells, but not myeloma cells.
  • a preferred selection technique uses HAT (hypoxanthine, aminopterin, thymidine) selection. After a sufficient time, usually about 1 to 2 weeks, colonies of hybrids are observed. Single colonies are selected and growth media in which the cells have been grown is tested for the presence of binding activity against the PDGFR ⁇ or immunogenic fragment thereof. Hybridomas having high reactivity and specificity are preferred.
  • Monoclonal antibodies are isolated from the supernatants of growing hybridoma colonies using methods such as, for example, affinity purification using the PDGFR ⁇ or immunogenic fragment thereof to isolate an antibody capable of binding thereto.
  • various techniques may be employed to enhance the yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse.
  • Monoclonal antibodies are then harvested from the ascites fluid or the blood of such an animal subject. Contaminants are removed from the antibodies by conventional techniques, such as chromatography, gel filtration, precipitation, and/or extraction.
  • the hematopoietic cell or hematopoietic progenitor cell is isolated using fluorescence activated cell sorting (FACS) or magnetic cell sorting, thereby isolating the hematopoietic progenitor cell.
  • FACS fluorescence activated cell sorting
  • magnetic cell sorting thereby isolating the hematopoietic progenitor cell.
  • the pluripotent cell is an embryonic stem cell, preferably a human embryonic stem cell, or inducible pluripotent stem cell (iPSC).
  • iPSC inducible pluripotent stem cell
  • the compound is part of a library of compounds, and the method further comprises the step of isolating the compound from the library. In yet a further embodiment, the method further comprises the step of providing or producing a library of compounds to be screened.
  • Suitable compounds and libraries thereof will be apparent to the skilled artisan and include, for example, Representative libraries include but are not limited to a peptide or peptide library (e.g., as described in U.S. Patent Nos 6,156,511; 6,107,059; 5,922,545; and 5,223,409), an aptamer or aptamer library (e.g., as described in U.S.
  • Patent Nos 6,180,348 and 5,756,291 a small molecule or small molecule library (e.g., as described in U.S. Patent Nos 6,168,912 and 5,738,996), or an antibody or antibody fragment or library thereof (e.g., as described in U.S. Patent Nos 6,174,708; 6,057,098; 5,922,254; 5,840,479; 5,780,225; 5,702,892; and 5,667988).
  • hematopoietic cells and/or hematopoietic precursor cells As exemplified herein, functional analysis of single cells, cultured in serum free methylcellulose supplemented with blood growth factor cocktail (VEGF, SCF, TPO, IL3, IL6 and Epo) with or without a PDGF resulted in the development of hematopoietic Bl -colony forming cells (CFCs).
  • VEGF blood growth factor cocktail
  • SCF serum free methylcellulose
  • TPO blood growth factor cocktail
  • IL3 IL3
  • Epo blood growth factor cocktail
  • CFCs hematopoietic Bl -colony forming cells
  • contacting a pluripotent cell with a PDGF increases the production of hematopoietic cells and/or hematopoietic progenitor cells, e.g., by inducing or enhancing differentiation of pluripotent cells into hematopoietic cells and/or hematopoietic progenitor cells and/or by enhancing survival of hematopoietic cells and/or hematopoietic progenitor cells.
  • results presented herein demonstrate that the inclusion of insulin-like growth factor (IGF)-2 increased the frequency of Bl-CFCs ⁇ 2-fold.
  • IGF insulin-like growth factor
  • the combination of PDGF, IGF2 and fibroblast growth factor (FGF) 2 enhanced CFC numbers, resulting in an approximately 10 fold increase in CFC numbers compared to media that is not supplemented with these growth factors alone.
  • FGF fibroblast growth factor
  • growth factors such as IGF2 and/or FGF2 synergize with a PDGF and induce or enhance differentiation of pluripotent cells into hematopoietic cells and/or hematopoietic progenitor cells and/or by enhancing survival of hematopoietic cells and/or hematopoietic progenitor cells.
  • IGF2 and/or FGF2 synergize with a PDGF and induce or enhance differentiation of pluripotent cells into hematopoietic cells and/or hematopoietic progenitor cells and/or by enhancing survival of hematopoietic cells and/or hematopoietic progenitor cells.
  • the present invention also provides a method of method of inducing or enhancing or stimulating the growth of hematopoietic cells or hematopoietic progenitor cells from a population of differentiating pluripotent cells, the method comprising obtaining a population of differentiating pluripotent cells and culturing the differentiating pluripotent cells in media comprising a PDGF.
  • the PDGF is selected from the group consisting of PDGF-AA, PDGF-CC and PDGF-AB homo- and hetero- dimers.
  • the PDGF is PDGF-AA.
  • the media further comprises IGF-2 and/or FGF-2.
  • the different isoforms of PDGF form homo- and hetero-dimers in vivo which bind to and activate either the PDGFR ⁇ or PDGFRjS receptor.
  • the PDGF binds to and preferably activates a PDGFR ⁇ .
  • the PDGF is a PDGF-AA (i.e., comprising two PDGF-A isoforms).
  • the amino acid sequence for human PDGF-A is listed in SEQ ID No:2 as follows:
  • the present invention also provides a method for producing a hematopoietic progenitor cell or a hematopoietic cell, the method comprising contacting a population comprising differentiating pluripotent cells and/or cells differentiated therefrom with a PDGF for a time and under conditions to produce a hematopoietic progenitor cell or a hematopoietic cell.
  • the method comprises the step of obtaining or producing pluripotent cells, e.g., ESCs or iPSCs or obtaining differentiating ESCs or iPSCs.
  • pluripotent cells e.g., ESCs or iPSCs or obtaining differentiating ESCs or iPSCs.
  • ESC is obtained from a source such as, for example, WiCeIl Research Institute (WI, USA);
  • the pluripotent cells are initially differentiated by forming embryoid bodies (described in Ng et al. (2005) Blood 106(5): 1601 -1603) and/or by culturing in the presence of BMP -4.
  • These initial steps and or a subsequent step comprises culturing differentiating pluripotent cells (e.g., ESCs or iPSCs) in a medium comprising a PDGF, preferably PDGF-AA and/or PDGF-CC and/or PDGF-AB, and most preferably PDGF-AA.
  • the medium additionally comprises IGF2, and optionally FGF2.
  • the differentiating pluripotent cells are cultured in a serum-free medium comprising vascular endothelial growth factor (VEGF), stem cell factor (SCF), thrombopoietin (TPO), interleukin (IL) 3, PDGF-AA, IGF2 and/or FGF2, and optionally erythropoietic (EPO), or any other growth factor required for differentiation of a specific hematopoietic lineage ⁇ e.g. megakaryocyte, erythroid, myeloid or lymphoid lineages). Suitable growth factors will be apparent to the skilled artisan.
  • the differentiating pluripotent cells are directed to specific lineages of hematopoietic progenitor cells by the inclusion of specific growth factors in the culture medium.
  • a medium comprising EPO will direct differentiation towards red blood cells
  • a medium comprising G-CSF, GM-CSF or thrombopoietin will direct differentiation towards neutrophils
  • a medium comprising SCF, TPO and IL3 will direct differentiation towards megakaryocytes.
  • IL-3 e.g., a protein IL-3 mimetic is described in Thomas et al, Proc. Natl. Acad. Sd. USA, 92: 3779-3783, 1995
  • TPO e.g., a non- peptide mimetic of TPO is described in US Pat. No.
  • a peptide mimetic of TPO comprising an amino acid sequence Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala- Arg-Ala (SEQ ID NO: 3) is described in Cwirla et al, Science, 276: 1696-1699, 1997, and a variant of SCF comprising an extracellular domain fused to an immunoglobulin domain (Erben et al, Caner Res., 59: 2924-2930, 1999) are known in the art.
  • the present invention also encompasses methods making use of any two or more of the growth factors described herein fused to form a single protein, e.g., an fusion of TPO an IL-3 is described on US Pat. No. 6,254,870.
  • the pluripotent cells used in accordance with the present invention are human pluripotent cells, preferably, human embryonic stem cells, although it will be appreciated that embryonic stem cells from mammals other than humans may be used to practice the invention described herein.
  • the present invention also provides a culture medium for differentiating pluripotent cells into hematopoietic cells or hematopoietic progenitor cells comprising a PDGF and at least one factor selected from the group consisting of IGF2, FGF2 and combinations thereof.
  • the PDGF is selected from the group consisting of PDGF-AA, PDGF-CC and PDGF-AB homo- and hetero-dimers.
  • the PDGF is a PDGF-AA homodimer.
  • the medium comprises PDGF-AA and VEGF and/or SCF and/or TPO and/or IL3 and/or IGF-2 and/or FGF2 and, optionally, EPO, or any other growth factor required for differentiation of a specific hematopoietic lineage
  • the medium comprises PDGF-AA and VEGF and SCF and TPO and IL3 and IGF-2 and FGF2 and, optionally, EPO, or any other growth factor required for differentiation of a specific hematopoietic lineage.
  • the medium according to the present invention may also comprise any other combination of growth factors so as to direct differentiation of pluripotent cells, e.g. ESCs or iPSCs towards a specific hematopoietic cell lineage. While it is preferable to culture pluripotent cells in a serum-free medium that is free from animal or human contaminants, it is not essential.
  • An exemplary medium to which the various growth factors are added include those media described in Ng et al. (2008) Nature Protocols 3:768-776, the contents of which are incorporated herein by reference.
  • the present invention also provides a bioreactor for use in differentiating pluripotent cells into hematopoietic cells or hematopoietic progenitor cells and/or expanding populations of hematopoietic cells or hematopoietic progenitor cells, the bioreactor comprising a cell culture chamber in which at least one internal surface has immobilised thereon PDGF.
  • the PDGF is selected from the group consisting of PDGF-AA, PDGF- CC and PDGF-AB homo- and hetero-dimers and mixtures thereof; and is preferably PDGF-AA.
  • a suitable bioreactors and membrane bioreactors are known in the art and are described in, for example, WO 2008/011664; United States Patent No. 6,190,193 and United States Patent No. 6,544,788, the contents of which are incorporated herein by reference.
  • the present invention also provides a method for producing a hematopoietic cell and/or a hematopoietic progenitor cell, the method comprising culturing differentiating pluripotent cells in a culture medium according to the present invention for a time and under conditions sufficient for the differentiating embryonic stem cell to differentiate into a hematopoietic cell and/or a hematopoietic progenitor cell.
  • the pluripotent cells are cultured in or on methylcellulose.
  • the pluripotent cells are cultured as an embryoid body.
  • the present invention also provides an isolated hematopoietic progenitor cell or population thereof or isolated hematopoietic cell or population thereof isolated or produced by a method as described herein according to any embodiment.
  • the present invention also provides an isolated population of cells enriched for hematopoietic progenitor cells expressing PDGFR ⁇ on their surface.
  • the population of cells comprise at least about 50%, preferably at least about 60%, 70%, 80%, 90%, 95% and most preferably at least about 99% of cells which express PDGFR ⁇ on their cell surface.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a cell or population of cells according to the present invention and a pharmaceutically acceptable carrier or excipient and/or a medium.
  • An exemplary carrier is an aqueous pH buffered solution.
  • pharmaceutically acceptable carriers include, but are not limited to, saline, solvents, dispersion media, cell culture media, aqueous buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt- forming counterions such as sodium; and/or nonionic surfactants such as TWEENTM, polyethylene glycol (PEG), and PLURONICSTM.
  • compositions of the present invention should not be toxic to a cell of the present invention
  • pharmaceutical composition of the invention can also contain an additive to enhance, control, or otherwise direct the intended therapeutic effect of the cells comprising said pharmaceutical composition, and/or auxiliary substances or pharmaceutically acceptable substances, such as minor amounts of pH buffering agents, tensioactives, co-solvents, preservatives, etc.
  • a pharmaceutical composition of the invention can additionally or alternatively comprise a metal chelating agent and/or an amino acid such as aspartic acid, glutamic acid, etc.
  • a pharmaceutical composition of the present invention can also comprise an agent to facilitate storage of the composition and cells therein, e.g., a cryopreservative.
  • Illustrative, non limiting, examples of carriers for the administration of the cells contained in the pharmaceutical composition of the invention include, for example, a sterile saline solution (0.9% NaCl), PBS.
  • a pharmaceutical composition of the present invention can also comprise a bioactive agent (such as, for example, a growth factor) to reduce or prevent cell death and/or to enhance cell survival and/or to enhance cell differenitation and/or proliferation.
  • a bioactive agent such as, for example, a growth factor
  • the pharmaceutical composition of the invention will contain a prophylactically or therapeutically effective amount of the cells of the invention, preferably in a substantially purified form, together with the suitable carrier or excipient.
  • the pharmaceutical composition comprises between about 1 x 10 5 to about 1 x 10 13 cells, e.g., between about 2 x 10 5 to about 8 x 10 12 cells.
  • the pharmaceutical composition of the invention is formulated according to the chosen form of administration.
  • the formulation should suit the mode of administration.
  • the pharmaceutical composition is prepared in a liquid dosage form, e.g., as a suspension, to be injected into a subject in need of treatment.
  • a pharmaceutically acceptable carrier or excipient such as saline solution, phosphate buffered saline solution (PBS), or any other suitable pharmaceutically acceptable carrier, for parenteral administration to a subject, e.g., a human being, e.g., intravenously, intraperitonealy, subcutaneously, etc.
  • the present invention also provides a cell or population of cells according to the present invention, or a pharmaceutical composition according to the present invention, for use in medicine.
  • the present invention also provides a cell or population of cells according to the present invention, or a pharmaceutical composition according to the present invention, for use in the treatment of a disease or disorder resulting from a failure or a dysfunction of normal blood cell production and/or maturation.
  • Exemplary disorders include pancytopenia, thrombocytopenia, anaemia (including drug induced anaemia, hypoplastic anemia, Fanconi anemia or Diamond-Blackfan anemia), leukopenia, neutropenia or a bone marrow defect (e.g., acquired bone marrow failure or inherited bone marrow failure).
  • anaemia including drug induced anaemia, hypoplastic anemia, Fanconi anemia or Diamond-Blackfan anemia
  • leukopenia e.g., neutropenia or a bone marrow defect
  • a bone marrow defect e.g., acquired bone marrow failure or inherited bone marrow failure.
  • chemical means e.g., chemotherapy
  • radiation e.g., radiation therapy
  • a preferred disorder is caused by, causes or is associated with reduced platelet numbers in a subject, e.g., vitamin B 12 or folic acid deficiency, leukemia, myelodysplastic syndrome, liver failure, sepsis, systemic viral or bacterial infection, Congenital Amegakaryocytic Thrombocytopenia (CAMT), Thrombocytopenia absent radius syndrome, Fanconi anemia, Grey platelet syndrome, Alport syndrome, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), hemolytic-uremic syndrome (HUS), disseminated intravascular coagulation (DIC), paroxysmal nocturnal hemoglobinuria (PNH), antiphospholipid syndrome, systemic lupus erythematosus (SLE), post transfusion purpura, neonatal alloimmune thrombocytopenia (NAITP) or splenic sequestration of platelets due to Hypers
  • Another preferred disorder is caused by, causes or is associated with reduced neutrophil numbers in a subject, e.g. congenital neutropenia, cyclic neutropenia, cancer, Vitamin B12 or folate deficiency, aplastic anemia or autoimmune neutropenia.
  • a subject suffering from radiation-induced neutropenia or chemotherapy-induced neutropenia is treated.
  • the present invention also provides a use of a cell or population of cells according to the present invention in the manufacture of a medicament for the treatment of a disease or disorder resulting from a failure or a dysfunction of normal blood cell production and/or maturation or for the treatment of a subject in need of a transfusion of blood or a cellular component thereof.
  • the present invention also provides a method for treating suffering from or at risk of developing a disease or disorder resulting from a failure or a dysfunction of normal blood cell production and/or maturation or a subject in need of a transfusion of blood or a cellular component thereof comprising administering to the subject a cell or population of cells according to the present invention, or a pharmaceutical composition according to the present invention.
  • the cells are autologous, i.e., derived from the subject being treated.
  • the cells are allogenic, preferably being derived from a subject having the same blood group and/or HLA type as the subject to be treated or from a subject having a blood group and/or HLA type that is unlikely to induce an immune response when administered to the subject being treated.
  • the administration of the cells or pharmaceutical composition of the invention to the subject can be carried out by any conventional means.
  • the cells or pharmaceutical composition is administered to the subject in need by intravenous administration using a device such as a syringe, catheter, trocar, cannula.
  • the present invention also provides a method of selecting a compound capable of inducing differentiation of a pluripotent cell into a hematopoietic cell or a hematopoietic progenitor cell pluripotent cell into a hematopoietic cell or a hematopoietic progenitor cell and/or that is capable of inducing or enhancing or stimulating the growth or survival of hematopoietic cells and/or hematopoietic progenitor cells in a population of differentiating pluripotent cells, the method comprising:
  • the presence of PDGFR ⁇ on the surface of the differentiated cell indicates differentiation of the pluripotent cell into a hematopoietic cell or a hematopoietic progenitor cell and/or that the compound induces or enhances or stimulates the growth or survival of hematopoietic cells and/or hematopoietic progenitor cells in a population of differentiating pluripotent cells;
  • the pluripotent cell is an embryonic stem cell (ESC), preferably a human embryonic stem cell, or an inducible pluripotent stem cell (iPSC).
  • ESC embryonic stem cell
  • iPSC inducible pluripotent stem cell
  • embryonic stem cells are human embryonic stem cells
  • embryonic stem cells from mammals other than humans could be used to practice the invention.
  • preferred forms thereof will now be described with reference to the following definitions and non- limiting examples.
  • a prerequisite for the development of the primary germ layers is the commitment of epiblast cells to gastrulation, a process accompanied by the formation of the primitive streak, a morphological structure at the prospective embryonic posterior 5 ' 6 .
  • Epiblast cells ingressing through the streak emerge as either definitive endoderm or mesoderm, the latter including the progenitors of the hematopoietic system 7 , hi the mouse, primitive streak cells are marked by expression of the transcription factor Mixll*' 4 . Consistent with this, recent studies have confirmed that Mixll expression marks precursors of both mesoderm 8 and endoderm 9 and that mouse embryos deficient in Mixll display multiple defects in the formation of mesodermal and endodermal derived structures 10 .
  • GFP green fluorescent protein
  • the MIXLl targeting vector comprised a 9.4 Kb 5' homology arm, GFP, loxP flanked PGK-promoter-neomycin resistance gene and a 1.9 Kb 3 1 homology arm.
  • the homology arms were derived from previously described genomic clones of the human MIXLl locus 4 and spanned sequences from a Pad site situated 9466 bp 5' of the ATG to an Hpal site located 2242 bp 3' of the ATG.
  • the vector was digested with the restriction enzymes Pad and iV ⁇ tl prior to electroporation into HESCs as described elsewhere 12 .
  • HESC clones with a putative targeted MIXLl allele were identified using a PCR based screening strategy utilising the primer, Neo4, in conjunction with MIXLl ScreenRev (primer b in Figure Ia), a primer located immediately 3' of the genomic sequences encompassed by the targeting vector (see supplementary Table 1 for primer details). Using this criterion, a number of clones were identified in which the vector appeared to be correctly integrated into the MIXLl locus. Two HES3 clones were expanded and transiently transfected with a pEFBOS-cre-ERESPuro vector using Fugene 6 transfection reagent according to the manufacturers instructions (Invitrogen).
  • This vector was designed to express a single transcript encoding ere recombinase and puromycin resistance, the latter translated from an internal ribosomal entry site (IRES). 24-32 hours post transfection, cells were selected in 2 ⁇ g/ml puromycin for 2 days and subsequently allowed to form colonies for a further 7 days. Several colonies representing each primary clone were picked and screened for the loss of the neomycin resistance cassette and for the absence of the ere expression plasmid using a PCR based approach (see supplementary Table 1 for primer details and PCR conditions). Southern blot analysis was performed as described elsewhere 22 . The 5 1 external DNA probe included a mixture of fragments corresponding to human genomic sequences flanked by primer pairs listed in supplementary Table 1.
  • the GFP probe used to verify the presence of a single integration event encompassed the coding sequences of EGFP (Invitrogen).
  • the DNA fragment generated by PCR using the primers GFPl (primer a in Figure IA) and MIXLl 3' probe #1 was cloned and sequenced to establish that the 3' arm of the targeting vector had correctly integrated into the locus.
  • HESC lines were passaged as reported elsewhere 12 ' 23 and differentiated as spin EBs according to previously established protocols 13 .
  • SFM was supplemented with the following growth factors at the concentrations indicated: 10-lOOng/ml BMP4, 50 ng/ml Activin A
  • Intracellular flow cytometry with anti-Mixll and anti-Oct4 antibodies was performed as described previously 14 .
  • Dissociation of HESCs to single cell suspension and labeling with phycoerythrin (PE) -conjugated mouse anti-human CD34 (BD Biosciences, cat #348057), mouse anti-human E-CADHERJN (Zymed, cat #13-1700), mouse anti-human PDGFR ⁇ (BD Biosciences, cat #556001), and mouse anti-human Tra-1-60 (Chemicon, cat #MAB4360) was performed as described previously 13 .
  • Unconjugated primary antibodies were detected with either PE or allophycocyanin (APC)-conjugated goat anti-mouse IgG (BD Biosciences, cat #550589 and #550826).
  • APC allophycocyanin
  • FACS allophycocyanin
  • MIXLl GFP/w HESCs labelled with the appropriate isotype control antibody.
  • Single cell cloning was performed using the single cell deposition function of a FACSaria FACS station to place single cells into each well of 10 96 well trays pre-seeded with irradiated primary mouse embryonic fibroblasts (PMEFs) and containing HESC culture media 23 .
  • PMEFs irradiated primary mouse embryonic fibroblasts
  • cells obtained from flow cytometric sorting were forcibly aggregated using the spin EB protocol (10 4 /well), in SFM supplemented with 30ng/ml BMP4, 30ng/ml VEGF and 40ng/ml SCF.
  • the MIXLl-GFP targeting vector ( Figure IA) was electroporated into HESCs and G418 resistant colonies isolated as described elsewhere 12 . Correctly targeted clones were identified using a PCR based strategy with the primers indicated (Supplementary Table 1). Following removal of the G418 resistance cassette (see Methods), the structural integrity of the targeted locus was verified by Southern blot analysis ( Figure IB, C) and sequencing of the PCR product representing the 3' junction between the vector and flanking genomic DNA ( Figure ID and data not shown). In addition, one MIXLl GFP/w HESC line was cloned by single cell deposition into 96 well trays using flow cytometry (cloning efficiency of ⁇ 5%).
  • MIXLl GFP/w HESCs had normal karyotypes, formed teratomas and expressed markers of undifferentiated HESCs (Supplementary Figure 1 and data not shown).
  • MIXLl expression was contemporaneous with that of BRACHYURY, a transcription factor also present in the primitive streak 13 .
  • MIXLl GFP/w HESCs were differentiated for 5 days and the GFP + and GFP " fractions analysed by intracellular flow cytometry using MIXLl antibodies 14 . This analysis demonstrated that the expression of MIXLl protein was restricted to the GFP + fraction ( Figure IF).
  • E-cad E-cadherin
  • FIk 1 vascular endothelial growth factor
  • PDRFR ⁇ platelet derived growth factor
  • blast colony forming cells Bl-CFCs
  • ESN EGS and AGE
  • unpublished data we compared the methylcellulose colony forming ability of d4 sub-populations isolated on the basis of their GFP and PDGFR ⁇ expression.
  • hematopoietic Bl-CFCs were highly enriched in the GFP + PDGFRa + fraction in all 5 experiments ( Figure 3B and C), demonstrating that, as in the mouse, the earliest human hematopoietic progenitors arose within the primitive streak and nascent mesoderm .
  • hematopoietic CFCs were also present in the GFP + PDGFRa " and GFPTDGFRa + populations, 84-97% of CFCs were present in the GFP + PDGF + fraction.
  • the GFP + (MDCLl + ) population as a whole contained 90- 99% of Bl-CFCs (Supplementary Tables 3-6). Bl-CFCs were essentially absent from the GFP ' PDGFR ⁇ " populations.
  • Gene targeting is a critical technology for the analysis of gene function and for genetic tagging experiments that enable the real time monitoring of gene expression in viable cells during the course of ESC differentiation in vitro.
  • Previous reports of gene targeting in HESCs have used a promoter trapping approach that takes advantage of expression from the target locus 1 , or methods that rely on drug resistance resulting from disruption of the targeted gene 2 . Since most genes are not amenable to such approaches, we developed a generic strategy to target human MIXLl, utilizing conventional gene targeting in which the selectable marker is driven from a promoter within the vector and that does not require expression of the target locus in undifferentiated ESCs 12 . Targeted clones were obtained from a number of different HESC lines and the generality of this approach has been confirmed with the targeting of 2 other loci using vectors of similar configuration (ref 12 and data not shown).
  • MIXLl the primitive streak marker
  • MIXLl is of particular relevance, not only because of the population it marks, but because analogous targeted mouse ESC lines exist 13 , enabling comparisons between Mixll -expressing cells derived from two different species.
  • SFM supplemented by BMP4 induces Mixll + cells that give rise to a mesoderm-committed sub-population that harbours progenitors of primitive hematopoiesis 8 ' 13 ' 20 .
  • PDGF and IGF2 stimulate the growth of progenitor cells from differentiating human
  • HESC carrying a GFP reporter gene at the MIXLl locus were differentiated as spin EBs in a medium supplemented with BVS at 2500 cells per well. After 4d, EBs were disaggregated with TRYPLE Select and GFP and PDGFR expressing cell populations were sorted by flow cytometry. 10-20 x 10 3 single cells were cultured in serum free methyl cellulose supplemented with Blood growth factor cocktail (VEGF, SCF, TPO, IL3, IL6 and Epo) with or without PDGF at 20ng/ml. The development of hematopoietic Bl-CFCs was observed and colonies were counted after 10-14 days. " ""
  • Table 1 Hematopoietic Blast colonies generated from d4 spin EBs. Colony numbers in each well of triplicates containing 2O x IO 3 single cells in methylcellulose are shown. The colony ratio between MC cultures with or without PDGF supplementation is shown.

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Abstract

La présente invention concerne une méthode de détection de cellules progénitrices hématopoïétiques dans une population de cellules comprenant des cellules pluripotentes en différenciation. Cette méthode consiste à détecter la présence de PDGFRα sur la surface des cellules de cette population, la présence de PDGFRα indiquant que les cellules sont des cellules progénitrices hématopoïétiques.
PCT/AU2008/000865 2007-06-15 2008-06-13 Différenciation de cellules souches embryonnaires humaines WO2008151390A1 (fr)

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US10604738B2 (en) 2012-06-19 2020-03-31 Cambridge Enterprise Limited Transcription factor mediated programming towards megakaryocytes
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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11566228B2 (en) 2006-04-14 2023-01-31 Astellas Institute For Regenerative Medicine Hemangio-colony forming cells
US10604738B2 (en) 2012-06-19 2020-03-31 Cambridge Enterprise Limited Transcription factor mediated programming towards megakaryocytes
US9993503B2 (en) 2012-12-21 2018-06-12 Astellas Institute For Regenerative Medicine Methods for production of platelets from pluripotent stem cells and compositions thereof
US10426799B2 (en) 2012-12-21 2019-10-01 Astellas Institute For Regenerative Medicine Methods for production of platelets from pluripotent stem cells and compositions thereof
US10894065B2 (en) 2012-12-21 2021-01-19 Astellas Institute For Regenerative Medicine Methods for production of platelets from pluripotent stem cells and compositions thereof
US11400118B2 (en) 2012-12-21 2022-08-02 Astellas Institute For Regenerative Medicine Methods for production of platelets from pluripotent stem cells and compositions thereof
US12076347B2 (en) 2012-12-21 2024-09-03 Astellas Institute For Regenerative Medicine Methods for production of platelets from pluripotent stem cells and compositions thereof
WO2014200030A1 (fr) * 2013-06-12 2014-12-18 国立大学法人京都大学 Méthode de sélection de cellules souches pluripotentes induites et méthode d'induction de différentiation en cellules sanguines
JPWO2014200030A1 (ja) * 2013-06-12 2017-02-23 国立大学法人京都大学 人工多能性幹細胞の選別方法および血球への分化誘導方法
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CN114269906A (zh) * 2019-08-20 2022-04-01 艾达普特免疫有限公司 从多能干细胞产生造血祖细胞的方法

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