WO2008151390A1

WO2008151390A1 - Differentiation of human embryonic stem cells

Info

Publication number: WO2008151390A1
Application number: PCT/AU2008/000865
Authority: WO
Inventors: Andrew Elefanty; Eduoard Stanley; Elizabeth Ng
Original assignee: Australian Stem Cell Centre Ltd
Priority date: 2007-06-15
Filing date: 2008-06-13
Publication date: 2008-12-18

Abstract

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 PDGFRα on the surface of cells in said population, wherein the presence of PDGFRα is indicative of hematopoietic progenitor cells.

Description

DIFFERENTIATION OF HUMAN EMBRYONIC STEM CELLS

FIELD OF THE INVENTION

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.

BACKGROUND OF THE INVENTION

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.

SUMMARY OF THE INVENTION

We have now demonstrated that expression of platelet derived growth factor (PDGFR)θ! is induced in hematopoietic progenitor cells produced by culturing pluripotent cells, e.g., hESCs under conditions sufficient to induce differentiation into hematopoietic progenitor cells, for example by culturing hESCs in the presence of BMP4, and that cells co- expressing PDGFRα are highly enriched in day 4 Blast colony forming cells (Bl-CFC), the earliest hematopoietic precursors. We have also demonstrated that PDGF, e.g., a PDGF capable of activating a PDGFRα is capable of inducing, stimulating or enhancing growth or survival of a hemtaopoietic cell in a population of differentiating pluripotent cells, e.g., hESCs. We have used hESCs, which are an established source of pluripotent cells as a model of pluripotent cells per se.

Accordingly, in a first aspect 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 PDGFRα on the surface of cells in said population, wherein the presence of PDGFRα is indicative of hematopoietic progenitor cells.

In a second aspect of the present invention there is provided 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 PDGFRa on its surface.

In a preferred embodiment of any aspect or embodiment of the present invention, the pluripotent cells are human pluripotent cells. In another preferred embodiment of any aspect or embodiment of the present invention, the pluripotent cells are embryonic stem cells (ESCs) or an induced pluripotent stem cell (iPSCs).

In a third aspect of the present invention there is provided 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:

(i) contacting a pluripotent cell with a compound for a time and under conditions sufficient for the compound to induce the pluripotent cell to produce a differentiated cell; and

(ii) detecting the expression of PDGFRα on the surface of the differentiated cell,

wherein the expression of PDGFRα 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. In a fourth aspect of the present invention there is provided 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, the method comprising obtaining a population of differentiating pluripotent cells and culturing the differentiating pluripotent cells in media comprising a PDGF.

In a fifth aspect of the present invention there is provided a method of 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.

In a sixth aspect of the present invention there is provided 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.

In a seventh aspect of the present invention there is provided 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.

In an eighth aspect of the present invention there is provided a method for producing a hematopoietic cell and/or a hematopoietic progenitor cell, the method 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. In 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.

In a tenth aspect of the present invention there is provided an isolated population of cells enriched for hematopoietic progenitor cells expressing PDGFRα on their surface.

In an eleventh aspect of the present invention there is provided a pharmaceutical composition comprising a cell or population of cells according to the present invention and a pharmaceutically acceptable carrier or excipient.

In a twelfth aspect of the present invention there is provided a cell or population of cells according to the present invention, or a pharmaceutical composition according to the present invention, for use in medicine

In a thirteenth aspect of the present invention there is provided 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.

In 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.

In a fifteenth aspect of the present invention there is provided 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, said method 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.

hi 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:

(ii) detecting the presence or absence of expression of PDGFRα on the surface of the differentiated cell,

wherein the presence of PDGFRα 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;

and selecting the compound which results in expression of PDGFRα on the surface of the differentiated cell.

In a seventeenth aspect, the present invention provides a kit for detecting a hematopoietic progenitor cell, said kit comprising a ligand that binds to a PDGFRα and, optionally, instructions for use in a method of the present invention.

In an eighteenth aspect, the present invention provides a kit comprising a PDGF packaged with instructions to use said PDGF in a method of the present invention. Optionally, the kit comprises additional components of a media for culturing a pluripotent cell, preferably for differentiating a pluripotent cell.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows generation and characterisation oi MIXLl^GFPAv HESCs

(A) Structure of the gene targeting vector used to insert sequences encoding GFP into exon 1 of the MIXLl locus using homologous recombination. Pad and iVotl are restriction enzyme sites used to linearize the vector prior to electroporation. 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. (B) Southern blot analysis of Mfel digested genomic DNA shows that a 5' external probe detects a fragment of 17 Kb representing the endogenous locus from both parental HES3 cells and HESCs with a targeted MIXLl locus. An additional fragment of 14.4 Kb is also detected in the genetically modified cells, representing the distance from the 5¹ external Mfel site to the 3' end of GFP.

(C) Probing this same DNA with GFP sequences indicates that these cells contain a single copy of the GFP gene consistent with a single genetic modification at the MIXLl locus.

(D) The integrity of sequences 3' of the GFP gene was validated using a PCR based approach (with primers a and b) to amplify DNA representing the junction of the targeting vector with the chromosomal DNA. Sequence analysis of this fragment confirmed that the relationship between the vector DNA and adjacent chromosomal sequences were as expected (data not shown). (E) PCR analysis indicates that GFP expression mirrors the wave of expression of endogenous MIXIl. 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.

Figure 2 shows BMP4 induces a wave of GFP expression in differentiating

MIXL1^GFP/W HESCs

(A) Time course of GFP expression determined by flow cytometric analysis of differentiating MIXLl^GFP/w HESCs shows the transient appearance of mesendodermal progenitors in response to 50 ng/ml BMP4. Note the absence of GFP⁺ cells in cultures differentiated in CDM alone (upper panel). The proportion Of MIXLl⁺ cells for each time point is indicated. (B) GFP expression is also induced in d5 MIXLl^GFP/w EBs formed in CDM supplemented with either 100ng/ml BMP4 or 50 ng/ml Activin A but not with lOOng/ml FGF2. BF, bright field. (C) Time course analysis of GFP (MIXLl), E-CAD and PDGFRα expression in MIXLl^CFP/w HESCs differentiated in SFM containing BVS, shows the transit of cells from undifferentiated E-CAD⁺GFPTDGFRa^" HESCs towards GFP⁺PDGFRa⁺ mesoderm. This latter population gives rise to CD34⁺ hematopoietic/endothelial cells (lower panel). As expected, cells differentiated in FGF2 did not express GFP or PDGFRα, but retained E-CAD expression. The proportion of GFP⁺ cells at each time point is indicated in red (in the IgG control series), while the numbers in black indicate the fraction of cells present in the corresponding quadrant.

Figure 3 shows hematopoietic progenitors are enriched in the MIXLl⁺PDGFRa⁺ fraction of differentiating HESCs

(A) Cell sorting and re-culture experiment showing that d4 GFP⁺ PDGFRα^" cells give rise to GFP⁺PDGFRa⁺ cells when cultured in SFM supplemented with BVS. GFP⁺ cells can be recovered the GFP PDGFRα^" fraction, however they remained PDGFRα^" at the time points examined. The fraction Of MIXLl⁺ (red) cells is indicated, as is the proportion of cells in each quadrant (black text). (B) Results from five independent experiments (Expt. 1-5) showing the frequency of blast CFCs was highest in the GFP⁺(MIXLl⁺)PDGFRa⁺ fraction. The proportion of each sub-population present at the time of sorting is shown across the top of the panel. GT^", GFPTDGFRα^". G⁺P^", GFP⁺PDGFRa^". G⁺P⁺, GFP⁺PDGFRa⁺ GT⁺, GFPTDGFRa⁺. (C) Compared to the GFP⁺PDGFRa⁺ fraction, approximately 5 fold fewer CFCs were present in cell populations expressing only GFP (G⁺P^") or PDGFRα (GT⁺). (D) At early stages, colonies often contained a dense central core (white arrowhead) with a morphology distinct from the surrounding hematopoietic cells. (E, F) Over time, this feature was lost as cells within the colony underwent hemoglobinisation. (G) Some colonies also contained adherent cells (black arrowheads). (H-J) Colonies arising from the MIXLl⁺PDGFRa⁺ fraction displayed phenotypes indicative of erythroid, myeloid and bipotential progenitors. (K-P) Cytocentrifuge preparations of colonies from methylcellulose cultures confirmed the presence of nucleated primitive erythroid cells and cells with myeloid appearance. Enucleated erythroid cells were also observed (* in K) as well as cells with the morphological appearance of neutrophils (n), megakaryocytes (mk), macrophages (mø) and mast cells (m). Images N-P are derived from a cytocentrifuge preparation of a single erythroid colony similar to that shown in H.

Figure 4 shows flow cytometric analysis of the HES3 derivative MIXL1^GFP/W HESCs

(a) Flow cytometric analysis of the HES3 derivative MIXLl ^GFP/w 17.26 showing uniform expression of the surface markers E-CAD and TRA- 1-60 and (b) of the transcription factor OCT4. Numbers indicate the fraction of cells in the corresponding quadrant, (c) Chromosome preparation of the same clone showing a normal karyotype.

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). After 4d, cells were disaggregated and plated in serum- free methylcellulose (MC) (10k cells/well) in the presence of a blood growth factor cocktail alone (VEGF, SCF, TPO, IL3, IL6 and Epo; Blood GF) (A-F) or with addition of PDGF, IGF2 and FGF2 (G-L). 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.

DETAILED DESCRIPTION OF THE INVENTION Methods for detecting a hematopoietic cell or hematopoietic progenitor cell 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 PDGFRα on the surface of cells, wherein the presence of PDGFRα is indicative of hematopoietic progenitor cells.

As used herein the term "PDGFRα" or "platelet derived growth factor α" shall be understood to mean an alpha subunit of a PDGFR. In this respect, the skilled artisan will be aware that a PDGFR generally exists in nature as a dimer, e.g., comprising an α and a β chain or two α chains or two β chains, hi the context of the present invention, detection of PDGFRα shall be taken to include detection of a single α chain of a PDGFR and/or a dimeric receptor comprising two α chains.

For the purposes of nomenclature and not limitation an amino acid sequence of an α chain of a human PDGFR is set forth in SEQ ID NO: 1. The skilled artisan will be aware that the present invention extends to the detection of any form of an α chain of a PDGFR expressed on the surface of a cell, including an α 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 α chain.

hi one embodiment of the present invention, the presence of PDGFRα 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-PDGFRα 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. For example, the ligand is a PDGF that binds to a PDGFRα, preferably that binds to a PDGFRα. For example, the ligand is a PDGF-AA. Suitable commercial sources of PDGF will be apparent to the skilled artisan and include ProSpec- Tany TechnoGene Ltd. (Israel) or R&D Systems, Inc (MN, USA). Alternatively, 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).

As used herein, the term "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. The skilled artisan will be aware that the nomenclature relating to a PDGF describes the two subunits included in that growth factor, e.g., 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.

In one embodiment the ligand is an antibody, e.g., an antibody that selectively or preferentially binds to a PDGFRα, e.g., as described herein below.

In one embodiment, the ligand is labeled with a detectable marker to facilitate detection. Suitable detectable markers will be apparent to the skilled artisan include, a fluorescent dye, a fluorescent nanocrystal (e.g., a Q-dot™), 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 PDGFRα on its surface.

In one embodiment according to the invention, 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 PDGFRα for a time and under conditions sufficient to form a ligand-PDGFRα complex; and

(i) isolating a cell comprising the ligand-PDGFRα! complex on its surface.

In a further embodiment according to the present invention, 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:

wherein 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.

hi an embodiment of the present invention, the method additionally comprises isolating the compound. In this respect, the compound may be isolated using conventional techniques known in the art. As discussed herein above, the present invention encompasses any pluripotent cell, preferably an embryonic stem cell (ESC) or inducible pluripotent stem cell (iPSC). Preferably, the cell is an ESC.

In one embodiment of any aspect of the present invention, it is preferred that the ligand is specific for PDGFRα, or a fragment of PDGFRα, e.g., an immunogenic fragment of a PDGFRα. Suitable ligands are described supra. In one embodiment of the present invention, 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 PDGFRα. As used herein, the terms "antibody" and "antibodies" include the entire antibody or any antigen binding fragment thereof. The terms "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 PDGFRα will be apparent to the skilled artisan and include, for example, BD Biosciences Pharmingen (CA, USA) or ABR Affinity Bioreagents (CO, USA).

Alternatively, an antibody that binds PDGFRα 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). For example, a monoclonal antibody against a PDGFRα is produced by immunizing an animal, e.g., a mouse or rat, with said protein or an immunogenic fragment thereof. Optionally, 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 PDGFRα 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 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 PDGFRα 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 PDGFRα or immunogenic fragment thereof to isolate an antibody capable of binding thereto. In addition, 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.

In another embodiment of the present invention, 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. These techniques are known in the art and described, for example, in Shapiro HM: Practical Flow Cytometry. 4th Edition. New York, Wiley-Liss, 2003.

In one embodiment of the present invention, the pluripotent cell is an embryonic stem cell, preferably a human embryonic stem cell, or inducible pluripotent stem cell (iPSC).

In another embodiment, 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).

Production of 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). The inclusion of a PDGF increased colony numbers by approximately 1.5 fold. These results indicate that 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 also demonstrate that the inclusion of insulin-like growth factor (IGF)-2 increased the frequency of Bl-CFCs ~2-fold. 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. Finally, the addition of IGF2 at d2 of differentiation with PDGF, IGF2 and FGF2 in the methylcellulose media at d4 resulted in the largest number of colonies. These results indicate that 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. These results provide the basis for methods and compositions of matter for producing or inducing or enhancing or stimulating the growth of hematopoietic cells or hematopoietic progenitor cells.

Accordingly, 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. In one embodiment, the PDGF is selected from the group consisting of PDGF-AA, PDGF-CC and PDGF-AB homo- and hetero- dimers. Preferably, the PDGF is PDGF-AA. In another embodiment the media further comprises IGF-2 and/or FGF-2.

The different isoforms of PDGF (e.g. PDGF-A, PDGF-B, PDGF-C and PDGF-D) form homo- and hetero-dimers in vivo which bind to and activate either the PDGFRα or PDGFRjS receptor. In the context of the present invention, it is preferred that the PDGF binds to and preferably activates a PDGFRα. For example, the PDGF is a PDGF-AA (i.e., comprising two PDGF-A isoforms). For the purposes of nomenclature, and not limitation, the amino acid sequence for human PDGF-A is listed in SEQ ID No:2 as follows:

1 MRTLACLLLL GCGYLAHVLA EEAEIPREVI ERLARSQIHS IRDLQRLLEI

51 DSVGSEDSLD TSLRAHGVHA TKHVPEKRPL PIRRKRSIEE AVPAVCKTRT

101 VIYEIPRSQV DPTSANFLIW PPCVEVKRCT GCCNTSSVKC QPSRVHHRSV 151 KVAKVEYVRK KPKLKEVQVR LEEHLECACA TTSLNPDYRE EDTGRPRESG

201 KKRKRKRLKP T (SEQ ID No: 2)

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.

In one embodiment, the method comprises the step of obtaining or producing pluripotent cells, e.g., ESCs or iPSCs or obtaining differentiating ESCs or iPSCs. For example, an

ESC is obtained from a source such as, for example, WiCeIl Research Institute (WI, USA);

Millipore Corporation (USA) or ES Cell International Pte Ltd (Singapore). Methods for producing human ES cells will be apparent to the skilled artisan and described, for example, in US Pat. Nos 5,843,780; 6,200,806; or 7,029,913. Methods for producing iPSC are also known in the art and described, for example, in Takahashi and Yamanaka Cell.

726:663-676, 2006 or Takahashi et al, Cell, 737:861-872, 2007

In another embodiment, the pluripotent cells (e.g., ESCs or iPSCs) 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. Optionally, the medium additionally comprises IGF2, and optionally FGF2. In yet another embodiment, 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.

In yet another embodiment according to the present invention, the differentiating pluripotent cells are directed to specific lineages of hematopoietic progenitor cells by the inclusion of specific growth factors in the culture medium. For example, 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; while a medium comprising SCF, TPO and IL3 will direct differentiation towards megakaryocytes.

It is to be understood that active fragments, mimetics or variants of the various growth factors used in accordance with the invention are to be considered to form part of the scope of the invention. For example, mimetics of IL-3 (e.g., a protein IL-3 mimetic is described in Thomas et al, Proc. Natl. Acad. Sd. USA, 92: 3779-3783, 1995) and TPO (e.g., a non- peptide mimetic of TPO is described in US Pat. No. 6,875,786; 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.

It is preferred that 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. In one embodiment, the PDGF is selected from the group consisting of PDGF-AA, PDGF-CC and PDGF-AB homo- and hetero-dimers. Preferably, the PDGF is a PDGF-AA homodimer. hi yet another embodiment, 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, hi another embodiment, 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.

hi one embodiment, 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.

Examples of 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. Methods for determining production of a hematopoietic cell or a hematopoietic progenitor cell will be apparent to the skilled artisan based on the exemplified subject matter herein and/or described in Sullivan, Cowan and Kevin (Eds) Human Embryonic Stem Cells The Practical Handbook, Wiley, 2007.

In one embodiment, the pluripotent cells are cultured in or on methylcellulose.

In one embodiment, 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 PDGFRα on their surface. In one embodiment, 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 PDGFRα on their cell surface.

The present invention also provides 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. Examples of 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 TWEEN™, polyethylene glycol (PEG), and PLURONICS™. Additional suitable pharmaceutically acceptable carriers or excipients will be apparent to the skilled artisan and/or described in U.S. Pharmacopeia, or the European Pharmacopeia or "Remington's Pharmaceutical Sciences" by E. W. Martin. Pharmaceutical carriers suitable for use in a composition of the present invention should not be toxic to a cell of the present invention The 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.

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. In one embodiment, the pharmaceutical composition comprises between about 1 x 10⁵ to about 1 x 10¹³ cells, e.g., between about 2 x 10⁵ to about 8 x 10¹² cells.

The pharmaceutical composition of the invention is formulated according to the chosen form of administration. The formulation should suit the mode of administration. In a particular embodiment, 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. Illustrative, non limiting examples, include formulating the cells of the invention in a sterile suspension with 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). The skilled artisan will be aware that several of the previously discussed disorders may be a result of a congenital defect or may be induced by chemical means (e.g., chemotherapy) or 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 Hypersplenism. Alternatively, a subject suffering from chemotherapy- induced thrombocytopenia is treated.

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. Alternatively, 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.

hi one example, the cells are autologous, i.e., derived from the subject being treated. In another embodiment, 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. In one embodiment, 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:

In this embodiment, the pluripotent cell is an embryonic stem cell (ESC), preferably a human embryonic stem cell, or an inducible pluripotent stem cell (iPSC).

Although it is preferred that the embryonic stem cells are human embryonic stem cells, it will be appreciated that embryonic stem cells from mammals other than humans could be used to practice the invention. In order that the nature of the present invention may be more clearly understood, 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 posterior⁵'⁶. Epiblast cells ingressing through the streak emerge as either definitive endoderm or mesoderm, the latter including the progenitors of the hematopoietic system⁷, 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 mesoderm⁸ and endoderm⁹ and that mouse embryos deficient in Mixll display multiple defects in the formation of mesodermal and endodermal derived structures¹⁰. Analysis of genetically tagged Mixll^GFPΛv mouse embryonic stem cells (ESCs) showed that Mixlf cells contained hematopoietic precursors and that Mixlϊ'^' ESCs had a reduced ability to generate hematopoietic mesoderm⁸. Induction of Mixll expression during the differentiation of mouse ESCs in serum free medium (SFM) is dependent on the presence of Bone Morphogenetic Protein (BMP) 4, Activin A⁸'¹¹ or Wnt3a.

hi order to facilitate the analysis of early hESC differentiation, we targeted sequences encoding green fluorescent protein (GFP) to the human MIXLl locus using homologous recombination. We demonstrate that GFP fluorescence faithfully reported expression of the endogenous MIXLl gene and that a mesodermal cell population defined by co- expression of GFP (MIXLl) and the platelet derived growth factor receptor α (PDGFRα) was highly enriched in primitive hematopoietic precursors, the earliest derivatives of the primitive streak. EXAMPLES

Materials and Methods

Generation and identification of targeted MIXL1^GFP/W HESCs 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 3¹ homology arm. The homology arms were derived from previously described genomic clones of the human MIXLl locus⁴ 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 iVσtl prior to electroporation into HESCs as described elsewhere¹². 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 2μg/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 5¹ 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. CeII culture and differentiation

HESC lines were passaged as reported elsewhere ¹²'²³ and differentiated as spin EBs according to previously established protocols¹³. SFM was supplemented with the following growth factors at the concentrations indicated: 10-lOOng/ml BMP4, 50 ng/ml Activin A

(R&D Systems), 50-100ng/ml FGF2, 10-50 ng/ml VEGF, 20-lOOng/ml SCF, 30ng/ml IL3,

30 ng/ml IL6, 30ng/ml TPO, 3U/ml EPO (PeproTech). EBs were dissociated using either

0.25% w/v Trypsin-EDTA (Invitrogen) or TrypLE select™ (Invitrogen). Preparation and analysis of methylcellulose cultures and cytocentrifuge preparations were conducted according to Ng et al (2005)¹³. Karyotype analysis and teratoma assays were performed as described previously²³.

Flow cytometry

Intracellular flow cytometry with anti-Mixll and anti-Oct4 antibodies was performed as described previously¹⁴. 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 PDGFRα (BD Biosciences, cat #556001), and mouse anti-human Tra-1-60 (Chemicon, cat #MAB4360) was performed as described previously¹³. Unconjugated primary antibodies were detected with either PE or allophycocyanin (APC)-conjugated goat anti-mouse IgG (BD Biosciences, cat #550589 and #550826). For flow cytometric analysis and sorting, gates were set using control cells (HES3) and 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 media²³. For re-aggregation/re-culture experiments, cells obtained from flow cytometric sorting were forcibly aggregated using the spin EB protocol (10⁴/well), in SFM supplemented with 30ng/ml BMP4, 30ng/ml VEGF and 40ng/ml SCF.

Gene expression analysis

RNA was prepared using RNAeasy™ according to manufacturers instructions (Qiagen). RNA samples were reverse transcribed and normalised as described previously¹³. PCR was performed under standard conditions (30 cycles of 94⁰C, 20 seconds; 60⁰C, 30 seconds; 68⁰C, 60 seconds) using the primer sets listed in supplementary Table 2. For PCR with PAX6 specific primers an annealing temperature of 55°C was used.

Results

The MIXLl-GFP targeting vector (Figure IA) was electroporated into HESCs and G418 resistant colonies isolated as described elsewhere¹². 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%). The parental line and subclones were phenotypically indistinguishable (data not shown). MIXLl^GFP/w HESCs had normal karyotypes, formed teratomas and expressed markers of undifferentiated HESCs (Supplementary Figure 1 and data not shown).

In order to examine the temporal association between expression of GFP and the endogenous MIXLl allele, the gene expression profile of MIXL1^GFP/W HESCs differentiating in response to BMP4 was analysed over a 12 day period (Figure IE). The expression profile of GFP transcripts mirrored that of MIXLl, and, as previously reported,

MIXLl expression was contemporaneous with that of BRACHYURY, a transcription factor also present in the primitive streak¹³. The decline in the level of expression of these primitive streak genes between days (d) 6-8, overlapped with the expression of mesodermal (GATA2, CD34) and endodermal (FOXA2, ALPHA FETOPROTEIN,

ALBUMIN) genes. This transient wave of MIXLl expression was consistent with our previous data demonstrating the kinetics of differentiation using the 'spin embryoid body'

(spin EB) system in SFM supplemented with BMP4¹³. These conditions do not promote neurectodermal differentiation, as evidenced by the weak expression of PAX6. To further investigate the relationship between MIXLl expression and GFP, MIXLl^GFP/w HESCs were differentiated for 5 days and the GFP⁺ and GFP^" fractions analysed by intracellular flow cytometry using MIXLl antibodies¹⁴. This analysis demonstrated that the expression of MIXLl protein was restricted to the GFP⁺ fraction (Figure IF). Collectively, these analyses indicate that GFP and MIXLl showed co-ordinated expression during the course of HESC differentiation and GFP fluorescence can be used as surrogate marker to identify MIXLl⁺ cells.

Flow cytometric analysis of MIXLl ^GFP/w HESCs differentiated in SFM supplemented with BMP4 revealed a wave of GFP expression that mirrored that of MIXLl and GFP RNA

(Figure 2A). GFP expression was absolutely dependent on the inclusion of an inducing growth factor, in this case BMP4 or Activin A (Figure 2 A, B and data not shown). In this experiment, the frequency of GFP⁺ cells was maximal at d4-6 (-50%) and declined gradually thereafter, becoming negligible by dl2. Apart from the prolonged kinetics of differentiation, these results are analogous to those obtained with mouse Mixll^GFP/w ESCs differentiated under similar conditions⁸. The enhanced viability associated with BMP4 treatment seen in differentiating mouse ESCs⁸ also occurred with human ESCs (data not shown). Like mouse Mixll^GFP/w ESCs, GFP expression was induced with Activin A but not

FGF2, indicating that the signalling pathways for mesendoderm formation are likely to be generally conserved between these two species (Figure 2B and C).

During mouse embryogenesis, epiblast cells entering the primitive streak retain E-cadherin (E-cad) before passing through a transition during which E-cad expression is down regulated and expression of early mesodermal genes, including the receptors for vascular endothelial growth factor (FIk 1) and platelet derived growth factor (PDRFRα) are increased¹⁵'¹⁶. We have previously shown that in differentiating mouse Mixll^GFP/w ESCs, GFP expression spanned the interval during which cells transit from E-cad⁺ epiblast to E-cad^"Flkl⁺ mesoderm⁸. We sought to determine if a similar transition period could be discerned during the course of HESC differentiation. However, because we and others have observed that the human homologue of Flkl, KDR, is expressed on undifferentiated HESCs (ref¹⁷ and ESN, EGS and AGE, unpublished data), we instead examined the relationship between the expression of GFP, E-CAD and PDGFRα (Figure 2C). Flow cytometric analysis of MIXL1^GFP/W HESCs differentiated in BMP4, VEGF and SCF (BVS) showed that the GFP⁺ (MIXLl⁺) cells were regularly seen from d3. These cells were E- CAD⁺ and some cells already expressed PDGFRα. By d5, the proportion Of E-CAD⁺ cells started to fall whilst the frequency of cells expressing PDGFRα had increased to 40%. In this experiment, the peak in the frequency Of GFP⁺ (MIXLl⁺) cells occurred at d7 (35%) and the majority of these were PDGFRa⁺. At dlO, the proportion of E-CAD⁺ cells had fallen to below 40% and almost all of the GFP⁺ cells were also PDRFRa⁺. From d8 onwards, a population of cells emerged which expressed the hematopoietic and endothelial marker, CD34. This population, which was absent prior to d6 (data not shown), appeared to derive predominantly from the pre-existing GFP⁺PDGFRa⁺ fraction, as evidenced by the initial co-expression of these markers on the CD34⁺ cells (Figure 2C).

To document the relationship between GFP and PDGFRα expressing cells, we isolated cells expressing combinations of these markers by flow cytometric sorting and re-cultured each fraction for 3 days in SFM supplemented with BVS (Figure 3A). This analysis demonstrated that GFP⁺PDGFRa⁺ cells arose from the GFP⁺PDGFRa^" population and implied that cells sequentially acquired the expression of GFP (MDCLl), PDGFRα and subsequently CD34, reflecting the sequential commitment to primitive streak, mesoderm and hematopoietic development. PDGFRα expression was quite stable, as evidenced by the high proportion of cells (-85-95%) from both GFP⁺PDGFRa^" and GFP⁺PDGFRa⁺ populations that expressed PDGFRα 3 days after sorting and re-culturing. Conversely,

GFP expression was only retained in -40-60% of cells in both populations over this time period. These experiments also showed that little new GFP expression was induced after d4 under these conditions and that GFP PDGFRa⁺ cells did not give rise to GFP- expressing cells (Figure 3A).

We and others have observed that the earliest hematopoietic precursors that develop from hESCs, termed blast colony forming cells (Bl-CFCs), are seen after 3-4 days of differentiation (ref¹⁷ and ESN, EGS and AGE, unpublished data). In order to investigate the phenotype of these cells, we compared the methylcellulose colony forming ability of d4 sub-populations isolated on the basis of their GFP and PDGFRα expression. In the five consecutive experiments shown in figure 3B, the first two utilised a batch of BMP4 with a 3- to 5-fold lower specific activity (batch 1) than the batch used in the last three experiments (batch 2). This led to a lower percentage of GFP⁺ and PDGFRa⁺ expressing cells in the first two experiments (similar to the experiment shown in Figure 2c) that could be correlated with the low frequency of Bl-CFCs in the unsorted d4 EBs. For example, 7- 12 Bl-CFC/2 x 10⁴ cells were observed in the d4 EBs differentiated in BMP4 from batch 1, whilst 112-349 Bl-CFC/2 x 10⁴ cells were seen in the d4 EBs cultured in BMP4 from batch 2. Despite these differences, 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 . Although hematopoietic CFCs were also present in the GFP⁺PDGFRa^" and GFPTDGFRa⁺ populations, 84-97% of CFCs were present in the GFP⁺PDGF⁺ fraction. Indeed, 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^'PDGFRα^" populations.

Although the frequency of hematopoietic colonies varied between the different sorted populations, a similar spectrum of colony morphologies was detected in each case. At early stages of blast colony formation, blood cells emerged from a dense core of cells that was morphologically similar to the mesodermal core observed in mouse hemangioblast colonies¹⁹ (Figure 3D). In the presence of erythropoietin (EPO), developing blast colonies became overtly hemoglobinised (Figure 3E, F). Where colonies contacted the plate surface, hematopoietic cells developed in association with adherent cells with a morphology resembling hemangioblast derived endothelial cells recently reported by Keller and colleagues¹⁷ (Figure 3G). Although erythroid colonies, occasionally with a halo of migrating myeloid cells comprised the most frequent colony types (-95%) (Figure 3H, I and data not shown), colonies wholly composed of migrating myeloid cells were also routinely observed (~5%)(Figure 3 J). Examination of May-Grunwald-Giemsa stained cytospin preparations revealed that most colonies were composed of primitive nucleated erythrocytes, although the presence of enucleated erythrocytes was frequently observed (Figure 3K). Myeloid colonies contained macrophages, often in combination with mast cells or neutrophils (Figure 31-M). Many erythroid colonies, even those without an obvious myeloid component (such as Figure 3H), contained macrophages, mast cells, neutrophils and megakaryocytes at a low frequency, indicating that these CFCs were multipotent (Figure 3N-P).

Discussion 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 locus¹, or methods that rely on drug resistance resulting from disruption of the targeted gene². 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 ESCs¹². 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 (ref¹² and data not shown).

In this study we examined the relationship between expression of the primitive streak marker, MIXLl, and the commitment of cells to hematopoiesis in differentiating HESCs. MIXLl is of particular relevance, not only because of the population it marks, but because analogous targeted mouse ESC lines exist¹³, enabling comparisons between Mixll -expressing cells derived from two different species. This comparison demonstrates that fundamental aspects of early ESC differentiation are conserved between mouse and human. In both species, SFM supplemented by BMP4 induces Mixll⁺ cells that give rise to a mesoderm-committed sub-population that harbours progenitors of primitive hematopoiesis⁸'¹³'²⁰. Thus, despite basic differences in the self-renewal mechanisms between mouse and human ESCs²¹, our study indicates the conservation of critical aspects of early development between the two species, a finding that bodes well for the translation of differentiation protocols from mouse to human systems and the eventual development of cell based therapies.

PDGF and IGF2 stimulate the growth of progenitor cells from differentiating human

ESC

HESC carrying a GFP reporter gene at the MIXLl locus (M/A£7^GFP/w) 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 10³ 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. "^""

The results from one representative experiment are shown in Table 1.

Table 1

Blood GF Blood GF+PDGF 20 Colony Ratio

Table 1. Hematopoietic Blast colonies generated from d4 spin EBs. Colony numbers in each well of triplicates containing 2O x IO³ single cells in methylcellulose are shown. The colony ratio between MC cultures with or without PDGF supplementation is shown.

In this experiment, inclusion of PDGF at the time of methylcellulose culture increased colony numbers by approximately 1.5 fold. A similar enhancement of colony numbers had been observed in previous experiments.

In similar experiments using Envy cells, we have demonstrated that the inclusion of 40ng/ml IGF2 at d2 of the differentiation culture increased the frequency of Bl-CFCs ~2-fold (Table 2).

The combination of 20ng/ml PDGF, 40ng/ml IGF2 and lOng/ml FGF2 in the methylcellulose medium at d4 synergistically enhanced CFC numbers, resulting in an approximately 10 fold increase in CFC numbers compared to BVS alone (Table 2). Finally, combining the addition of IGF2 at d2 of differentiation with PDGF, IGF2 and FGF2 in the methylcellulose at d4 gave the largest number of colonies (Table 2). Images reflecting data from Table 2 are shown in Figure 5.

Table 2

Table 2. Hematopoietic blast colonies generated from d4 spin EBs differentiated in BVS with or without 40ng/ml IGF 2 added at d2. In both cases, disaggregated cells (10 x 10³ well) were cultured at d4 in methylcellulose supplemented with Blood GF alone or Blood GF plus PDGF, FGF2 and IGF2 (PIF2). Colony ratios for IGF2 and for PIF2 supplementation are shown, demonstrating up to 14-fold greater colony numbers in samples differentiated in BVS plus IGF 2 followed by Blood GF plus PFI2 in methylcellulose. Conclusions

The results from these experiments suggest that the frequency and size of early hematopoietic colonies derived from differentiating hESCs is enhanced by the inclusion of PDGF optinally supplemented with IGF2 and/or FGF2 at the time of or prior to culture in a media comprising methylcellulose. The inclusion of these growth factors in the cocktail used to drive hematopoietic differentiation appears instrumental in optimising culture conditions for the most efficient generation of human hematopoietic cells from hESCs.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

All publications mentioned in this specification are herein incorporated by reference. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia or elsewhere before the priority date of each claim of this application.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. REFERENCES

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Claims

1. A method for detecting hematopoietic progenitor cells in a population of cells comprising differentiating pluripotent cells, the method comprising detecting the presence of PDGFRα on the surface of cells in said population, wherein the presence of PDGFRα is indicative of hematopoietic progenitor cells.

2. The method according to claim 1 wherein the presence of PDGFRα on the surface of cells is detected by contacting the population of cells with a ligand that binds PDGFRα for a time and under conditions sufficient to form a ligand- PDGFRα complex and detecting the complex to thereby detect expression of the PDGFRα on the surface of the cell.

3. The method according to claim 2 wherein the ligand is an antibody.

4. 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 PDGFRα on its surface.

5. The method according to claim 4 wherein the method comprises the steps of:

(i) contacting the population of differentiating pluripotent cells with a ligand that binds PDGFRα for a time and under conditions sufficient to form a ligand-PDGFRα complex; and

(ii) isolating a cell comprising the ligand-PDGFRα complex on its surface.

6. The method according to claim 5 wherein the ligand is an antibody.

7. The method according to any one of claims 4 to 6 wherein the hematopoietic progenitor cell is isolated by fluorescence activated cell sorting (FACS) or magnetic cell sorting.

8. A method of inducing or enhancing or stimulating the growth of hematopoietic 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.

9. The method according to claim 8 wherein the media further comprises IGF-2 and/or FGF-2.

10. The method according to claim 8 or claim 9 wherein the PDGF is selected from the group consisting of PDGF-AA, PDGF-CC and PDGF-AB.

11. The method according to any one of claims 8 to 10 wherein the PDGF is PDGF-AA.

12. A method for identifying a compound capable of inducing differentiation of a pluripotent 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:

wherein the expression of PDGFRα 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.

13. The method according to claim 12 comprising identifying a compound capable of inducing differentiation of a pluripotent cell.

14. The method according to claim 12 or claim 13 wherein the method further comprises isolating the compound capable of inducing differentiation of a pluripotent cell into a hematopoietic cell or a hematopoietic progenitor cell.

15. The method according to any one of claims 12 to 14 wherein the compound is part of a library of compounds and the method further comprises the step of isolating the compound from the library.

16. The method according claim 15 wherein the method further comprises the step of providing or producing a library of compounds to be screened.

17. 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.

18. The method of claim 17 wherein the pluripotent cells are differentiated by inclusion in embryoid bodies and/or by culturing in the presence of BMP-4.

19. The method according to any one of claims 1 to 18 wherein the pluripotent cell is an embryonic stem cell (ESC) or an inducible pluripotent stem cell (iPSC).

20. The method according to claim 19 wherein the ESC is a human embryonic stem cell (hESC).

21. A culture medium for differentiating pluripotent cells into hematopoietic cells or a hematopoietic progenitor cells and/or for 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 media comprising a PDGF and at least one factor selected from the group consisting of IGF2, FGF2 and combinations thereof.

22. The culture medium according to claim 21 wherein the medium is serum- free.

23. The culture medium according to claims 21 or 22 wherein the culture medium further comprises methylcellulose and one or more of the growth factors selected from the group consisting of VEGF, SCF, TPO, IL3 and active fragments, variants and mimetics thereof.

24. The culture medium according to claim 23 further comprising EPO.

25. The culture medium according to any one of claims 21 to 24 wherein the PDGF is selected from the group consisting of PDGF-AA, PDGF-CC and PDGF-AB.

26. The culture medium according to any one of claims 21 to 25 wherein the PDGF is PDGF-AA.

27. The culture medium according to any one of claims 21 to 26 wherein the pluripotent cell is an embryonic stem cell (ESC) or an inducible pluripotent stem cell (iPSC).

28. The culture medium according to claim 27 wherein the ESC is a human embryonic stem cell (hESC).

29. A bioreactor for use in differentiating pluripotent cells into hematopoietic cells or a hematopoietic progenitor cells and/or expanding populations of haematopoietic progenitor cells, the bioreactor comprising a cell culture chamber in which at least one internal surface has immobilised thereon a PDGF.

30. The bioreactor according to claim 29 wherein the PDGF is PDGF-AA.

31. A method for producing a hematopoietic cell and/or a hematopoietic progenitor cell, the method comprising culturing a differentiating pluripotent cells in a culture medium according to any one of claims 21 to 28 for a time and under conditions sufficient for the differentiating pluripotent cells to differentiate into a hematopoietic cell and/or a hematopoietic progenitor cell.

32. An isolated hematopoietic progenitor cell or population thereof or isolated hematopoietic cell or population thereof produced by a method according to any one of claims 17 to 20.

33. An isolated population of cells enriched for hematopoietic progenitor cells expressing PDGFRα on their surface.

34. The population according to claim 33 wherein at least about 50% of cells express PDGFRa on their surface.

35. The population according to claim 33 or claim 34 wherein at least about 60% of cells express PDGFRα on their surface.

36. The population according to any one of claims 33 to 35 wherein at least about 70% of cells express PDGFRα on their surface.

37. The population according to any one of claims 33 to 36 wherein at least about 80% of cells express PDGFRα on their surface.

38. The population according to any one of claims 33 to 37 wherein at least about 90% of cells express PDGFRα on their surface.

39. The population according to any one of claims 33 to 38 wherein at least about

95% of cells express PDGFRα on their surface.

40. The population according to any one of claims 33 to 39 wherein at least about 99% of cells express PDGFRα on their surface.

41. A pharmaceutical composition comprising a cell or population of cells according to any one of claims 33 to 40 and a pharmaceutically acceptable carrier or excipient and/or a medium.

42. The cell or population of cells according to any one of claims 33 to 40 or a pharmaceutical composition according to claim 41 for use in medicine.

43. The cell or population of cells according to any one of claims 33 to 40 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.

44. The cell or population of cells according to claim 43 wherein the disease or disorder resulting from a failure or a dysfunction of normal blood cell production and/or maturation is selected from the group consisting of pancytopenia, thrombocytopenia, anaemia including drug induced anaemia, hypoplastic anemia, Fanconi anemia or Diamond-Blackfan anemia, leukopenia, neutropenia or a bone marrow defect including acquired bone marrow failure and inherited bone marrow failure.

45. The cell or population of cells according to claim 43 or claim 44 wherein the disorder is caused by, causes or is associated with reduced platelet numbers in a subject.

46. The cell or population of cells according to claim 45 wherein the disorder caused by, which causes or is associated with reduced platelet numbers is selected from the group consisting of 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) and splenic sequestration of platelets due to hypersplenism.

47. The cell or population of cells according to claim 43 or claim 44 wherein the disorder is caused by, causes or is associated with reduced neutrophil numbers in a subject.

48. The cell or population of cells according to claim 47 wherein the disorder caused by, which causes or is associated with reduced neutrophil numbers is selected from the group consisting of congenital neutropenia, cyclic neutropenia, cancer, Vitamin B12 or folate deficiency, aplastic anemia and autoimmune neutropenia.

49. Use of a cell or population of cells according to any one of claims 33 to 40 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.

50. 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, the method comprising administering to the subject a cell or population of cells according to any one of 33 to 40 or a pharmaceutical composition according to claim 41.

51. A method of selecting a compound capable of inducing differentiation of a pluripotent cell into a hematopoietic cell and/or a hematopoietic progenitor cell and/or that is capable of inducing or enhancing or stimulating the growth or survival of hematopoietic cell and/or a hematopoietic progenitor cell in a population of differentiating pluripotent cells, the method comprising:

wherein the presence of PDGFRα 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 cell and/or a hematopoietic progenitor cell in a population of differentiating pluripotent cells;

52. The method according to claim 51 or claim 52 wherein the method comprises selecting a compound capable of inducing differentiation of a pluripotent cell into a hematopoietic cell and/or a hematopoietic progenitor cell.

53. The method according to claim 51 or claim 52 wherein the pluripotent cell is an embryonic stem cell (ESC) or an inducible pluripotent stem cell (iPSC).

54. The method according to claim 53 wherein the ESC is a human embryonic stem cell (hESC).

55. A kit for detecting and/or isolating a hematopoietic cell and/or a hematopoietic progenitor cell, the kit comprising a ligand that binds to PDGFRα.

56. The kit according to claim 55 further comprising instructions for use in a method according to any one of claims 1 to 7.

57. A kit comprising a PDGF packaged with instructions to use the PDGF in a method according to any one of claims 8 to 11 and 17 to 20.

58. The kit according to claim 57 wherein the kit comprises components of a media for culturing a pluripotent cell.

59. The kit according to claim 58 wherein the pluripotent cell is a differentiating pluripotent cell.