WO2008151386A1 - Megakaryocyte differentiation - Google Patents

Abstract

The present invention provides a method for generating megakaryocytes and/or megakaryocyte precursors from a population of embryonic stem cells (ESCs), the method comprising: (i) culturing ESCs in a serum-free, stromal/feeder cell-free medium for a time and under conditions sufficient for formation of mesoderm and/or mesendoderm; and (ii) differentiating the cells cultured in step (i) in a medium comprising thrombopoietin (TPO), stem cell factor (SCF) and interleukin 3 (IL-3), or any functional fragment, variant or mimetic of TPO, SCF and/or IL-3, for a time and under conditions sufficient for formation of megakaryocytes and/or megakaryocyte precursors.

Description

MEGAKARYOCYTE DIFFERENTIATION

FIELD OF THE INVENTION

The present invention relates to methods for generating megakaryocytes and/or megakaryocyte progenitor cells from embryonic stem cells.

BACKGROUND OF THE INVENTION

Pancytopenia and prolonged thrombocytopenia remain significant clinical problems for patients undergoing chemotherapy and stem cell transplantation. Since finding suitable healthy donors to transfuse patients during this critical time is becoming increasingly difficult, alternative sources for blood products must be sought.

SUMMARY OF THE INVENTION

In a first aspect of the present invention there is provided a method for generating megakaryocytes and/or megakaryocyte precursors from a population of embryonic stem cells (ESCs), the method comprising:

(i) culturing the ESCs in a serum-free, stromal/feeder cell-free medium for a time sufficient and under conditions sufficient for formation of mesoderm and/or mesendoderm; and

(ii) differentiating the cells cultured in step (i) in a medium comprising thrombopoietin (TPO), stem cell factor (SCF) and interleukin 3 (IL-3), or any functional fragment, variant or mimetic of TPO, SCF and/or IL-3, for a time and under conditions sufficient for formation of megakaryocytes and/or megakaryocyte precursors.

In a second aspect of the present invention there is provided a megakaryocyte and/or megakaryocyte precursor, or a population of megakaryocytes and/or megakaryocyte precursors generated by performing the method of the first aspect of the invention. In a third aspect of the present invention there is provided a bioreactor for use in differentiating ESCs into megakaryocytes and/or megakaryocyte precursors under serum- free, stromal/feeder cell-free culture conditions, the bioreactor comprising a cell culture chamber in which at least one internal surface comprises thrombopoietin (TPO), stem cell factor (SCF) and interleukin 3 (IL-3), or any functional fragment, variant or mimetic of TPO, SCF and/or IL-3. Preferably, the cell culture chamber comprises a matrix suitable for supporting growth and/or proliferation and/or differentiation of an ESC. Preferably, the TPO, SCF and IL-3 or functional fragment, variant or mimetic thereof is immobilized on the matrix. In another embodiment, the TPO, SCF and IL-3 or functional fragment, variant or mimetic thereof is included in medium contained within the bioreactor.

Preferably, the bioreactor additionally comprises one or more growth factor(s) and/or cytokine(s) (e.g., bone morphogenetic protein (BMP-4) and/or vascular endothelial growth factor (VEGF) and/or stem cell factor (SCF) and/or fibroblast growth factor (FGF)-2) that induce differentiation of an ESC into mesoderm and/or mesendoderm. Preferably, the bioreactor comprises BMP-4 in combination with any one or more of VEGF, SCF or FGF- 2. This embodiment shall be taken to have disclosed every possible combination of BMP- 4, VEGF, SCF or FGF-2 as if each and every one of those combinations was individually recited herein, hi one embodiment, the growth factor(s) and/or cytokine(s) is(are) immobilized on a surface within a reaction chamber of the bioreactor and/or on the surface of a matrix within the bioreactor. In another embodiment, the cytokine(s) and/or growth factor(s) are included in medium within the bioreactor.

In a fourth aspect of the present invention there is provided a pharmaceutical composition comprising a megakaryocyte and/or megakaryocyte precursor, or a population of megakaryocytes and/or megakaryocyte precursors generated by performing the method of the first aspect of the invention.

In a fifth aspect of the present invention there is provided a megakaryocyte and/or megakaryocyte progenitor, or a population of megakaryocyte and/or megakaryocyte precursors generated by performing the method of the first aspect of the invention, or a pharmaceutical composition according to the fourth aspect of the invention, for use in human therapy.

In a sixth aspect of the present invention there is provided a method for treating or preventing a disorder caused by or associated with reduced platelet numbers or concentration or density of platelet numbers (e.g., thrombocytopenia) in a subject, said method comprising administering to the subject an effective amount of megakaryocytes and/or megakaryocyte precursors, or a population of megakaryocyte and/or megakaryocyte precursors generated by performing the method of the first aspect of the invention, or a pharmaceutical composition according to the fourth aspect of the invention. Exemplary disorders include, vitamin B12 or folic acid deficiency, leukemia, myelodysplastic syndrome, liver failure, sepsis, systemic viral or bacterial infection, chemotherapy-induced thrombocytopenia, 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. Preferably, a subject suffering from chemotherapy-induced thrombocytopenia is treated.

In a seventh aspect of the present invention there is provided a use of an effective amount of a megakaryocyte and/or megakaryocyte progenitor, or a population of megakaryocytes and/or megakaryocyte precursors generated by performing the method of the first aspect of the invention in the manufacture of a medicament for treating or preventing a disorder caused by or associated with reduced platelet numbers in a subject.

In an eighth aspect of the present invention there is provided a method of selecting a compound capable of inducing or enhancing proliferation of a megakaryocyte and/or megakaryocyte precursor, the method comprising: - A -

(i) contacting a megakaryocyte and/or megakaryocyte precursor produced by performing the method of the first aspect of the invention with a compound for a time and under conditions sufficient for the compound to induce the megakaryocyte and/or megakaryocyte precursor to proliferate; and

(ii) detecting the proliferation of the megakaryocyte and/or megakaryocyte precursor,

wherein enhanced proliferation of a megakaryocyte and/or megakaryocyte precursor contacted with the compound compared to a megakaryocyte and/or megakaryocyte precursor that has not been contacted with the compound indicates that the compound induces or enhances proliferation of a megakaryocyte and/or megakaryocyte precursor.

In a ninth aspect of the present invention there is provided a method of selecting a compound capable of inducing or enhancing differentiation of a megakaryocyte and/or megakaryocyte precursor into a platelet, the method comprising:

(i) contacting a megakaryocyte and/or megakaryocyte precursor produced by performing the method of the first aspect of the invention with a compound for a time and under conditions sufficient for the compound to induce the megakaryocyte and/or megakaryocyte precursor to differentiate into and/or produce a platelet; and

(ii) detecting platelet production,

wherein enhanced platelet production at (ii) compared to the platelet production from a megakaryocyte and/or megakaryocyte precursor that has not been contacted with the compound indicates that the compound induces or enhances differentiation of a megakaryocyte and/or megakaryocyte precursor into a platelet. The present invention clearly encompasses the direct product of any method described herein according to any aspect or embodiment of the invention.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows BMP4 induces MIXLl expression in differentiating HESCs. A) MIXLl expression detected by qRT-PCR in HESCs differentiated in serum-free cultures supplemented with no growth factors (No GF), lOng/ml VEGF, 25ng/ml SCF, lOng/ml FGF2 or 10ng/ml BMP4. MIXLl gene expression relative to GAPDH as a reference gene is shown on the vertical axis. Similar results were obtained using UBIQUITIN C or HPRT as reference genes (see Materials and Methods and Figure 7 for more details). Results shown are the mean ± SEM (n = 4, asterisk, p< 0.03 for BMP4 versus other conditions). B) HESC spin EBs at day 10 of differentiation demonstrate increased EB size and cyst formation in the presence of BMP4 (x50 magnification) and C) percentage of cells expressing OCT4 and both OCT4 and MIXLl proteins (OCT4/MIXL1) in day 6 HESC cultures supplemented with no GF, VEGF, SCF, FGF2 or BMP4. The results shown are the mean ± SD (n = 6, asterisk, p<0.01 for BMP4 versus other conditions). 97.5 ± 1.7% of the undifferentiated HESCs expressed OCT4 and 2.1 ± 0.2% also expressed MIXLl.

Figure 2 shows BMP4 induces primitive streak and hematopoietic mesoderm genes in a dose dependent manner.

Differentiating HESC cultures were supplemented with 1, 10 or 50ng/ml of BMP4 (Bl, BlO, B50) ± 10 ng/ml VEGF (VlO) and expression of the indicated genes relative to GAPDH (relative gene expression, see Material and Methods and Figure 7) was analyzed by qRT-PCR after 3 and 5 days. Expression profiles for undifferentiated cells (Undiff) and cells differentiated in VEGF alone (VlO) or in the absence of added growth factors (no GF) are also indicated. The experiment shown is representative of three independent experiments performed (see Figures 8A and 8B for additional experimental results). Figure 3 shows expression of hematopoietic genes in differentiating HESC.

Expression of GATA2, RUNXl, CD34 and SCL detected by qRT-PCR in undifferentiated HESCs and HESCs differentiated for 11 days in serum free culture supplemented with BMP4 (B) 10ng/ml, VEGF (V) lOng/ml, SCF (S) 25ng/ml, FGF2 (F) 10ng/ml singly or in the indicated combinations (mean ^SEM, n=3).

Figure 4 shows FGF2 increases cell yield during HESC differentiation.

Viable cell counts of differentiating HESCs (using trypan blue exclusion) were calculated at the indicated day by harvesting EBs from a plate of 72 wells initially seeded with 2.5 x 10³ cells/well (i.e. a starting number of 180,000 cells per plate) in the presence of the indicated growth factors. A) Increased cell numbers were observed in BVSF media from day 2 of differentiation (n = 5, p values as shown for BVSF versus BV). B) Over a longer period of observation, the yield of differentiated cells was higher in factor combinations containing FGF2 (BVSF versus BV and BVS p < 0.05 from day 5, BVF versus BV and BVS p < 0.05 from day 10, n = 15).

Figure 5 shows the combination of BMP4, VEGF, SCF and FGF2 is required for efficient hematopoietic colony formation from HESC.

A and E) The frequency of hematopoietic CFCs (mean ± SEM, n = 5) in the presence of BMP4 and/or the indicated factor combinations from day 10 and day 20 cultures. C and

G) The total number of hematopoietic CFCs (mean ± SEM, n = 5) per plate (72 wells initially seeded at 2.5 x 10³ cells /well) generated from day 10 and day 20 cultures in the presence of BMP4 and/or the indicated factor combinations. B and D) The data from A) and C) respectively showing fold expansion, which represents the colony frequency for each factor combination normalized to the frequency (B) or number (D) of colonies counted at day 10 in the BMP4 alone cultures for each experiment. In B) p<0.05 for all combinations versus BMP4 and in D) p<0.05 for all combinations versus BMP4.

Asterisks denote p<0.05 for BVF and BVSF versus BV or BVS. F and H). The data from

E) and G) respectively showing fold expansion, which represents the colony frequency for each factor combination normalized to the frequency (F) or number (H) of colonies counted at day 20 in the BV cultures for each experiment. In H) p<0.03 for BVSF versus all other combinations. Examples of I) multi-lineage, J) myeloid and K) erythroid colonies from methylcellulose cultures established at day 10 of differentiation (x50 magnification) and cytospins of representative colonies stained with May-Griinwald-Giemsa (xlOOO magnification). Abbreviations: B - BMP4; V- VEGF; S- SCF; F -FGF2.

Figure 6 shows the combination of BMP4, VEGF, SCF and FGF2 is required for maximal generation of hematopoietic cells.

A-C) Flow cytometric analysis from a representative experiment at day 10 and day 20 of differentiating HESCs cultured with BMP4 or the indicated factor combinations, showing results of staining for CD34⁺, CD45⁺ and CD33⁺ cells. SSC, side scatter. The graphs at the end of each row indicate the percentage of cells expressing each antigen (mean +JSEM, n= 6) with the multifactor combinations (legend shown at the bottom of the Figure). D-G) Number of cells expressing D) CD34⁺, E) CD45⁺, F) CD33⁺ and G) CDl 17⁺, CD31⁺ and KDR⁺ as a function of time, in cultures supplemented with BMP4, VEGF, SCF and FGF2 combinations as indicated. The absolute cell numbers (viable cells) generated per plate containing 72 wells of differentiating HESC seeded at 2.5 x 10³ cells/well are shown on the Y axis (n=10). Values were determined by multiplying the number of viable cells per time point (determined by trypan blue exclusion) by the percentage of cells positive for each marker detected by flow cytometry. Asterisks denote p values, 0.01 < p < 0.05, compared to BV.

Figure 7A shows a comparison between GAPDH and alternative reference genes. GADPH expression is normalised to 1 for each sample. The mean ± standard deviation threshold cycle number (Ct) for each GADPH was 21.05 ± 2.53, for UBIQUITIN C was 24.58 ± 2.43 and for HRPT was 28.13 ± 2.26. The difference in the Ct (dCt) from GAPDH was 3.53 ± 0.48 for UBIQUITIN C and 7.08 ± 0.66 for HPRT. Therefore GAPDH is much more sensitive than UBIQUITIN C (~11.5 fold) or HPRT (-135 fold) for the detection of PCR amplifiable cDNA.

Figure 7B shows the correlation between threshold cycle number (Ct) for GAPDH, UBIQUITIN C and HPRT reference genes. Data set includes 38 independent samples that were used for the generation of Figures 1 and 2. Correlation is best between GAPDH and UBIQUITIN C (R² = 0.9638) and poorest between HPRT and UBIQUITIN C (R² = 0.8635).

Figure 7C shows the comparison of MIXLl gene expression for one of the experiments in Figure 1 related to GAPDH, UBIQUITIN C and HPRT reference genes.

This experiment shows that similar gene expression is observed for each sample independent of the reference gene used. The data has been corrected to take into account the differences in reference gene sensitivity by dividing the MIXLl /UBUITIN C gene expression by 10 and the MEXLl/HPRT gene expression by 100 (see Figure 7A).

Figure 7D shows the comparison of MIXLl relative gene expression shown in Figure 2 relative to GAPDH, UBIQUITIN C and HPRT reference genes. This experiment shows that similar gene expression is observed for each sample independent of the reference gene used. The data has been corrected to take into account the differences in reference gene sensitivity by dividing the MIXLl/UBIQUITIN C gene expression by 10 and the MIXLl/HPRT gene expression by 100 (see Fig 7A).

Figure 8A and Figure 8B shows BMP4 induces primitive streak and hematopoietic mesoderm genes in a dose dependent manager.

Differentiating HESC cultures were supplemented with 1.10 or 50mg/ml of BMP4 (Bl, BlO, B50) ± 10 ng/ml VEGF (VlO) and expression of the indicated genes relative to GAPDH (relative gene expression) was analysed by qRT-PCR after 3 and 5 days. Expression profiles for undifferentiated cells (undiff) and cells differentiated in VEGF alone (VlO) or in the absence of added growth factors (no GF) are also indicated. Data shown in Figures 8A and 8B are from two additional independent experiments to that shown as Figure 2. Figure 9 shows differentiation of HESCs supplemented with BMP4 and VEGF, and flow cytometric analysis.

Differentiating HESCs were supplemented with 1,10 or 50ng/ml of BMP4 (Bl, BlO, B50) ± 10 ng/ml VEGF (VlO) as described for Figure 2 and flow cytometric analysis to detect hematopoietic cells by staining with antibodies detecting CD34+, CD45+, CD33+ and CD34/CD45 double positive cells was performed. The percent of cell fractions increased in a BMP4 dose dependent manner but the inclusion of VEGF increased the cell differentiation greater than 4 fold. This is a result of a representative experiment at day 20 of differentiation.

Figure 10 shows qRT-PCR to detect expression of two trophectoderm genes. qRT-PCR was performed to detect expression of two trophectoderm genes, chorionic gonadotrophin (CGb) and leuteinizing hormone (LHb). Differentiating HESC cultures were supplemented with 1, 10 or 50ng/ml of BMP4 (Bl, BlO, B50) ± 10 ng/ml VEGF (VlO) and expression of the indicated genes relative to GAPDH was analysed by qRT-PCR after 3 and 5 days. Expression profiles for undifferentiated cells (DO) and cells differentiated in VEGF alone (VlO) or in the absence of added growth factors (no GF) are also indicated. The samples used to generate data in Figure 2 were also used to generate these data.

Figure 11 shows default expression of neural genes is inhibited by BMP4. Shown is the level of expression of PAX6 and SOXl in HESCs cultures differentiated for up to 10 days in serum- free cultures supplemented with no growth factors (no GF), lOng/ml VEGF (V), 25ng/ml SCF (S), lOng/ml FGF2 (F) or lOng/ml BMP4 (B) and combinations of these factors as indicated. Significant levels of PAX6 expression were not observed whilst the expression of SOXl was inhibited in BMP4 containing culture media.

Figure 12 shows immunophenotype of CD41 positive cells in human embryonic stem cell (hESC) differentiation cultures. hESC were cultured for 10 days in hu BMP4 (5-15ng/ml), hu VEGF (15ng/ml), hu SCF (25ng/ml) and hu bFGF (10ng/ml) and then for a further 3 or 10 days in hu TPO (20ng/ml), hu SCF (25ng/ml) and hu IL-3 (25ng/ml). Cells were dissociated into singles cells at day 13 or 20 of culture and stained with CD41 and combinations of directly conjugated monoclonal antibodies. Acquisition of samples was performed on FACSCalibur and analysis using CellQuest software with 50,000 events collected. A) CD41 expression on H3 hESC as compared to isotype control for cultures for 13 and 20 days. B) Gating strategy for analysis of CD41⁺ cells at day 13 and CD41^dim and CD41^bright cells at day 20 of hESC differentiation. C) Expression of hematopoietic - CD45, CD34, CD43; megakaryocyte - CDl 10, CD42b, CD61, myeloid - CD33 and hemato/endothelial - CDl 17 and KDR markers. Note the high expression of megakaryocyte markers in the Day 20 CD41 bright cells and not in the CD41 dim cells.

Figure 13 shows sorting strategy for day 13 and day 20 samples of differentiated Envy hESCs.

Cells were sorted according to their expression of CD41 and CD34 on day 13 and CD41, CD34 and CD45 on day 20. A) Sorting gates were designed to sort 4 fractions on day 13 and 5 fractions on day 20. CD41⁺CD34⁺ = megakaryocyte (Mks) progenitors, CD41⁺CD34^" and CD41⁺CD45⁺ = committed Mks, CD41^"CD34⁺ hematopoietic/endothelial progenitors, CD41^"CD34^" and CD41 CD34 CD45^" = non-hematopoietic, non-endothelial cells. B) Distribution of each fraction in 5 separate H3 and 5 separate Envy differentiations both at day 13 and day 20. C) May-Grunwald/Giemsa stain of some of the sorted fractions. Note the difference in morphology between the CD34⁺CD41^" and CD34⁺CD41⁺ cells and the presence of a mature granulocyte in the CD45⁺CD41^" fractions.

Figure 14 shows hematopoietic colonies generated in methylcellulose from sorted fractions.

10,000-sorted cells were placed in MethoCult™ containing SCF, GM-CSF, IL-3, IL-6 and EPO. Colonies were counted 14 days after plating and characterized as Colony forming unit (CFU)-GEMM, -GM, and E according to their morphology. A) The total number of colonies counted in unsorted and sorted fractions (n=5) for Envy and H3 cell lines. B). The distribution of CFU-GEMM, -GM and -E for each sorted fraction for both Envy and H3 cell lines. C-H") Photographs representing a typical colony found in MethoCult™ and cytocentrifuge preparations of the picked colony. Cytocentrifuge preparations were stained with May Grunwald/Giemsa to distinguish the morphology of the various hematopoietic cell types. C) Typical CFU-GM generated from H3 differentiations and C) cytospin of colony denoting the presence of both granulocytes and monocytes. D) Typical CFU-GM generated from Envy differentiations and D') cytospin of colony denoting the presence of both granulocytes and macrophages. E) Typical CFU-E generated from H3 differentiations and E') cytospin showing the presence of nucleated red blood cells (RBCs). Typical CFU-GEMM generated from F) H3 and G) Envy sorted cells. G') Cytospin of colony picked from envy CFU-GEMM. Note the presence of macrophage surrounded by nucleated RBCs, granulocytes and monocytes. G) A colony difficult identify when colony was picked and stained both nucleated RBCs and megakaryocytes (including polyploid nucleus) was detected.

Figure 15 shows Colonies of megakaryocyte origin generated in collagen-based cultures.

Unsorted and sorted cells were plated at 10,000 cells per well in duplicate for 14 days, fixed, dried and stained with antiCD41a indirectly conjugated to APAAP to detect nascent colonies. A) number of colonies generated by unsorted and sorted fractions. Recovery of colonies was calculated when number of colonies from sorted fractions was compared to unsorted cells. There was a tendency for more Mks colonies in the fractions containing CD41⁺ cells and Envy had a tendency to generate more Mks colonies. Since there was such a variation between experiments significant differences could not be determined. B) Number of cells present in each Mk colony generated. Note the more immature the sorted fractions the larger the colonies were. C) Distribution of CD41 negative, mixed positive and negative and CD41⁺ only colonies in each sorted fraction. Note that mixed colonies were mainly found in fractions that were CD34⁺. D) Colony seen before fixing and staining, E) Negative colony, F) Small positive colonies surrounded negative colonies, G) mixed colony seen typically in CD34⁺ sorted fractions. Arrows denoted single CD41⁺ cells within colony, H) single positive and negative cells seen within assay, I) Large CD41⁺ colony, G) Single polyploid CD41⁺ Mks (arrow) generating many red fragments believed to be platelets. H) CD41⁺ colony with 2 CD41⁺ (single nucleus) cells fragmenting (arrows), I) Smaller CD41 weak colony and J) CD41 bright colony.

Figure 16 shows quantitative RT-PCR performed on undifferentiated, unsorted and sorted fractions to detect efficiency of sorting and genes critical for megakaryocyte development. mRNA was collected, reverse transcribed and prepared for quantitative real time-PCR analysis using probe validated by ABI. CD34 gene expression was significantly higher in fractions containing CD34⁺ cells, CD41 gene expression was significantly higher in fractions containing CD41⁺ cells. Transcription factors such as GATAl and TALI (SCL) are induced during megakaryocyte differentiation. Higher levels of these genes were seen in CD41⁺ fractions. Megakaryocyte specific markers such as PF4 and MPL were significantly increased in fractions containing CD41⁺ cells. Levels of hematopoietic transcription factor, PUl, was significantly increased in all hematopoietic fractions while KDR a marker of hemato/endothelial cells was significantly higher in the CD34⁺CD41^" fraction containing these cells.

Figure 17 shows ploidy analysis of fractions containing CD41⁺ cells.

Once enough sorted cells could be collected flow cytometry and fluorescence in situ hybridization (FISH) analysis was performed to detect greater than 8N or polyploid megakaryocytes (Mks). Cells were fixed in ethanol overnight and then stained with propidium iodide and analysed by a FACSCalibur. A minimum of 20,000 cells was collected for ploidy analysis. A) Propidium iodide staining of CD45⁺ and CD34⁺CD41⁺ cells. Note the increased percent of 8N, 16N and >32N in the CD45⁺CD41^' fractions. B) 6 individual experiments analysing the ploidy of fractions containing CD41⁺ cells. Due to the limited number of sorted cells experiments could not be repeated for the day 13 differentiations. C) FISH analysis of single cells collected from sorted fractions.

Chromosome (Chm) 15 Fluoresce aqua, Chm 16 is red and Chm 22 green. Note two copies of each Chm in the CD34⁺ and CD45⁺ fraction while multiple copies of each Chm could be detected in some of the cells in the CD41⁺ fractions. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for generating megakaryocytes and/or megakaryocyte precursors from embryonic stem cells using serum-free and feeder- free/stromal cell-free culture conditions, and compositions of matter useful in such methods or produced by those methods. Preferably, these methods do not require use of conditioned medium, e.g., medium exposed to a feeder layer and/or a stromal cell population. Preferably, the methods make use of culture conditions wherein the biologically active compounds that induce or enhance differentiation of an ES cell into a megakaryocyte and/or megakaryocyte progenitor are known, e.g., the culture medium does not comprise uncharacterized mixtures of compounds at concentrations sufficient to exert a biological effect. Such culture conditions are desirable when producing cells useful in human therapy, e.g., there is a reduced risk that the cells have been exposed to an agent that result in a disease or disorder or undesirable condition in a human subject. These characterized culture conditions facilitate optimization compared to methods making use of uncharacterized biological agents, e.g., culture media comprising serum and/or stromal/feeder cells.

The present invention therefore provides a method for generating megakaryocytes and/or megakaryocyte precursors from a population of embryonic stem cells (ESCs), the method comprising:

(i) culturing the ESCs in a serum-free, stromal/feeder cell-free medium for a time and under conditions sufficient to form mesoderm and/or mesendoderm; and

(ii) differentiating the cells cultured in step (i) in a medium comprising thrombopoietin (TPO), stem cell factor (SCF) and interleukin 3 (IL-3), or any functional fragment, variant or mimetic of TPO, SCF and/or IL-3, for a time and under conditions sufficient to form megakaryocytes and/or megakaryocyte precursors. The term "serum-free, stromal/feeder cell-free medium" as used herein means a medium free of serum, such as fetal calf serum, bovine serum or ovine serum, and stromal/feeder cell layers, such as the murine bone marrow cell line S 17, the yolk sac endothelial cell line C 166, murine MCSF-null OP9 cells or human bone marrow stroma.

In one embodiment, ES cells are cultured in a so-called "flat culture" system in serum-free, stromal/feeder-cell free media e.g., essentially as described in (e.g. Nishikawa et al. (1998) Development 125:1747 and Nishikawa et al. (1998) Immunity 8:761, both of which are incorporated herein by reference).

In another example, ES cells are cultured under conditions to form embryoid bodies (EBs) and subsequently mesoderm and/or mesendoderm. Preferably, EBs are produced by performing a method essentially as described in International Patent Application No. PCT/AU2004/001593. In one embodiment, EBs are cultured in the presence of one or more growth factors or cytokines to induce or enhance formation of mesoderm and/or mesendoderm, e.g., as described in Ng et al. (2005) Blood. 106(5):1601-1603, the contents of which are also incorporated herein by reference. Exemplary cytokines include BMP-4 and/or VEGF and/or SCF and/or FGF2.

The formation of mesoderm and mesendoderm from embryonic stem cells results in the expression of cell surface markers including CD34, CD33, CD45 and/or PDGFRα, as well as the primitive streak genes MIXLl, BRACHYURY and/or GOOSECOID. Accordingly in an embodiment the method according to present invention involves identifying or detecting mesoderm and/or mesendoderm by detecting the expression of the cell surface markers CD34, CD33, CD45 and/or PDGFα and/or by detecting the expression of the primitive streak genes MIXLl, BRACHYURY and/or GOOSECOID. In accordance with this embodiment, expression of one or more of the foregoing genes can be detected in a subset of a population of cells, e.g., using a nucleic acid based assay (e.g., polymerase chain reaction) and considered indicative of expression of that(those) genes in the population as a whole. In another embodiment of the present invention, the method provides the step of isolating mesoderm and/or mesendoderm by contacting mesoderm and/or mesendoderm with a ligand that binds a marker expressed on the cell surface of mesoderm and/or mesendoderm for a time and under conditions sufficient to form a ligand-marker complex, and isolating a cell comprising the ligand-marker complex. According to this embodiment of the invention, the marker may be any marker expressed on the surface of a mesoderm or mesendoderm cell, e.g., a marker selected from the group consisting of CD34, CD33, CD45 and PDGFα and mixtures thereof.

In an embodiment of the present invention, the ligand is an antibody. In another embodiment of the present invention, the mesoderm and/or mesendoderm is isolated by fluorescence activated cell sorting (FACS) or by magnetic cell sorting.

In another embodiment, the step of culturing ESCs in a serum-free, stromal/feeder cell-free medium for a time sufficient to observe formation of mesoderm and/or mesendoderm comprises culturing cells, preferably EBs under suitable conditions for between about 8 days and about 25 days, preferably between about 10 days and about 20 days, for example

10 days or 13 days or 15 days or 17 days or 20 days. Such a time is sufficient for formation of mesoderm or mesendoderm. Accordingly, following such a culture period it is sufficient to conclude that mesoderm or mesendoderm has been formed without actually detecting mesoderm or mesendoderm formation.

In an embodiment of the present invention the serum-free, stromal/feeder-cell free medium comprises bone morphogenic protein (BMP4) and/or vascular endothelial growth factor (VEGF), for example, BMP4 and VEGF, preferably human BMP4 (hu BMP4) and human VEGF (hu VEGF). Preferably, the bioreactor comprises BMP-4 in combination with any one or more of VEGF, SCF or FGF-2. The present embodiments shall be taken to have disclosed every possible combination of BMP-4, VEGF, SCF or FGF-2 as if each and every one of those combinations was individually recited herein, hi another embodiment of the present invention the serum-free, stromal/feeder-cell free medium comprises BMP4, VEGF, SCF and FGF2, preferably hu BMP4, hu VEGF, human SCF (hu SCF) and human FGF2 (hu FGF2).

In yet another embodiment of the present invention, the embryonic stem cells are human embryonic stem cells.

The addition of TPO, SCF and IL-3, or any functional fragment, variant or mimetic of TPO, SCF and IL-3 to culture media facilitates differentiation of ESCs to produce megakaryocytes and/or megakaryocyte precursors. Additional factors which may also be beneficial in directing differentiation of mesoderm and/or mesendoderm towards megakaryocytes and/or megakaryocyte precursors include, but are not limited to, IL-I l and/or IL-6 and/or IL-9.

It is to be appreciated that any functional fragment, variant or mimetic of TPO, SCF and IL-3 include functional fragments, variants or mimetics known in the art. These include, but are not limited to, mimetics of IL-3 (e.g., a protein IL-3 mimetic is described in

Thomas et al, Proc. Natl. Acad. ScL 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). 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.

Megakaryocytes may be characterized by expression of one or more of the cell surface markers CD34 and/or CD41 and/or CD45, and preferably all of these markers, optionally combined with other markers such as CD61, CDI lO and/or upregulation of certain genes including GATAl, PF4 and MPL. Megakaryocytes may also be characterized as being polyploid, and by their ability to make platelets. Accordingly in a preferred embodiment of the present invention, the formation of megakaryocytes and/or megakaryocyte precursors is determined by characterizing the expression of any combination of these markers and/or production of platelets and/or by detection of a polyploid cells, e.g., using microscopy techniques and/or fluorescence-mediated techniques using a fluorescent dye such as, for example, DAPI.

Media suitable for use in differentiating mesoderm and/or mesendoderm to megakaryocytes and/or megakaryocyte precursors include those media described in Ng et al. (2008) Nature Protocols 3:768-776, the contents of which are incorporated herein by reference, supplemented with suitable growth factors and/or cytokines and/or fragments, variants derivatives or mimetics as described herein.

In one embodiment, one or more of the culture steps described herein is performed in a bioreactor. Suitable bioreactors will be apparent to the skilled artisan. Exemplary bioreactors are described herein and shall be taken to apply mutatis mutandis to the present embodiment of the invention.

In an embodiment of the present invention, the method for generating a maegakaryocyte and/or megakaryocyte precursor comprises the step of isolating a megakaryocyte and/or megakaryocyte precursor. Isolation may be achieved by contacting the megakaryocyte and/or megakaryocyte precursor with a ligand that binds a marker expressed on the cell surface of the megakaryocyte and/or megakaryocyte precursor for a time and under conditions sufficient to form a ligand-marker complex, and isolating a cell comprising the ligand-marker complex. According to this embodiment of the invention, an exemplary marker is selected from any one or more of the group consisting of CD34, CD41, CD45, CD61 and CDl 10.

In one embodiment of the present invention, the ligand is an antibody. In another embodiment of the present invention, the megakaryocyte and/or megakaryocyte precursor is isolated by fluorescence activated cell sorting (FACS) or by magnetic cell sorting. In yet another embodiment of the present invention, the method comprises culturing a megakaryocyte and/or megakaryocyte precursor to obtain a cell population of megakaryocytes and/or megakaryocyte precursors.

In yet another embodiment of the present invention, the method comprises culturing megakaryocytes and/or megakaryocyte precursors to produce platelets. According to this embodiment of the invention, megakaryocytes and/or megakaryocyte precursors are cultured in media which comprises SCF and IL-9, and optionally TPO and erythropoietin (Epo), and is substantially free of IL-3. The presence of IL-3 can inhibit megakaryocyte maturation and prevent platelet release.

In yet another embodiment of the present invention, the method comprises formulating a megakaryocyte and/or megakaryocyte precursor, or a population of megakaryocytes and/or megakaryocyte precursors with a suitable carrier or excipient to produce a pharmaceutical composition.

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 population of megakaryocytes and/or megakaryocyte precursors cultured by performing the method of the present invention.

The present invention also provides a pharmaceutical composition comprising a population of megakaryocytes and/or megakaryocyte precursors obtained by performing any method of the present invention.

The present invention also provides a bioreactor for use in differentiating ESCs into megakaryocytes and/or megakaryocyte precursors under serum- free, stromal/feeder cell- free, the bioreactor comprising a cell culture chamber in which at least one internal surface comprises thrombopoietin (TPO), stem cell factor (SCF) and interleukin 3 (IL-3), or any functional fragment, variant or mimetic of TPO, SCF and/or IL-3. Preferably, the cell culture chamber comprises a matrix suitable for supporting growth and/or proliferation and/or differentiation of an ESC. Preferably, the TPO, SCF and IL-3 or functional fragment, variant or mimetic thereof is immobilized on the matrix. In another embodiment, the TPO, SCF and IL-3 or functional fragment, variant or mimetic thereof is included in medium contained within the bioreactor.

Preferably, the bioreactor additionally comprises one or more growth factor(s) and/or cytokine(s) (e.g., bone morphogenetic protein (BMP-4) and/or vascular endothelial growth factor (VEGF) and/or stem cell factor (SCF) and/or fibroblast growth factor (FGF)-2) that induce differentiation of an ESC into mesoderm and/or mesendoderm. Preferably, the bioreactor comprises BMP-4 in combination with any one or more of VEGF, SCF or FGF- 2. This embodiment shall be taken to have disclosed every possible combination of BMP- 4, VEGF, SCF or FGF-2 as if each and every one of those combinations was individually recited herein. In one embodiment, the growth factor(s) and/or cytokine(s) is(are) immobilized on a surface within a reaction chamber of the bioreactor and/or on the surface of a matrix within the bioreactor. In another embodiment, the cytokine(s) and/or growth factor(s) are included in medium within the bioreactor. 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.

In an embodiment of the present invention, the at least one internal surface comprises a matrix, wherein the matrix is comprised of cartilage, demineralised bone and a synthetic material. Preferably the matrix is comprised of demineralised bone.

The present invention also provides a pharmaceutical composition comprising a megakaryocyte and/or megakaryocyte precursor, or population of megakaryocyte and/or megakaryocyte precursors generated by performing the method of the present invention for use in human therapy.

The present invention also provides a method for treating or preventing a disorder caused by or associated with reduced platelet numbers or concentration or density (e.g., thrombocytopenia) in a subject said method comprising administering to the subject an effective amount of a population of megakaryocytes and/or megakaryocyte precursors according to the second aspect of the invention, or a pharmaceutical composition according to the fourth aspect of the invention. Suitable diseases and disorders are described supra and shall be taken to apply mutatis mutandis to the present embodiment of the invention.

In 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, or cannula.

The present invention also provides for use of an effective amount of a population of megakaryocytes and/or megakaryocyte precursors according to the second aspect of the invention, or a pharmaceutical composition according to the fourth aspect of the invention, in the manufacture of a medicament for treating or preventing a disorder caused by or associated with reduced platelet numbers or concentration or density (e.g., thrombocytopenia) in a subject in need thereof. Suitable diseases and disorders are described supra and shall be taken to apply mutatis mutandis to the present embodiment of the inventon.

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 non-limiting examples.

EXAMPLES

Example 1

Materials and Methods

HESC culture and differentiation

Human embryonic stem cells HES3 (Reubinoff et al. (2000) Nat Biotechnol. 18(4):399-404), Envy (Costa et al. (2005) Nat Methods. 2(4):259-60), and MELl (available from WiCeIl Research Institute, USA) were maintained on irradiated primary mouse embryonic fibroblast (PMEF) feeder cells in DMEM/Hams Fl 2 medium (Invitrogen, Corporation, Ca) supplemented with 20% Knock Out Serum Replacer (KOSR, Invitrogen) and recombinant human (r-hu) FGF2 (8 ng/ml, Peprotech, Haifa, Israel) by mechanical passaging. In order to generate cells for differentiation experiments, colonies were expanded onto tissue culture flasks pre-seeded with irradiated PMEFs at a density of 2 x 10⁴/cm² as previously described (Pick (2007), In press). On the day prior to differentiation, HESCs were passaged onto tissue culture flasks seeded with low-density irradiated PMEFs (Ix 10⁴/cm²) and the cultures were harvested with Tryple Select (Invitrogen). Following centrifugation, the pellet of single cells was resuspended in a serum-free chemically defined medium (CDM) initially described by Wiles et al. (1999) Exp Cell Res. 247(l):241-8, and modified as described in Ng et al. (2005) Blood. 106(5):1601-3. lOOμl of CDM containing 2,500 HESCs supplemented with growth factors was added per well in low attachment 96-well round-bottomed plates (Nunc A/S, Denmark) and centrifuged at 1500 rpm for 5 min to induce aggregation and placed in a humidified incubator (37⁰C in 5% CO₂). CDM was supplemented with the following recombinant human growth factors singly or in combination: r-hu BMP4 5-15 ng/ml (R&D systems, Inc., Mn), r-hu VEGF 10-15 ng/ml, r-hu SCF 25 ng/ml and r-hu FGF2 10 ng/ml (latter three from Peprotech).

Within 24 hours in culture, embryoid bodies (EBs) formed in each well. After 10 days, a 96 well plate of EBs (72 wells = 72 EBs) was transferred to 1 well of a 6 well adherent flat-bottom plates containing 7.2 ml of CDM supplemented with r-hu-thrombopoietin

(TPO) 20 ng/ml and r-hu-SCF 25 ng/ml and r-hu-interleukin (IL)-3 25 ng/ml (Peprotech).

EBs were harvested for analysis at day 13 and day 20, disaggregated with TrypleSelect

(Invitrogen) and passed through a 23-guage needle generating a single cell suspension. The hESC lines used in this experiment were constantly monitored for normal karyotype and at no point was an abnormal karyotype found.

Flow cytometric analysis of CD41+ differentiated HESCs

The following antibodies were used in combination for flow cytometric analysis of the harvested disaggregated EBs:

A) CD34-FITC, CD45-PE, CD4 Ia-APC

B) CD4 Ia-FITC, CDl 10-PE, CD61-PerCp, CD42b-APC

C) CD43-FITC, CD34-PE, CD41a-APC

D) CD4 Ia-FITC, CD 117-PE₅ KDR-APC E) CD34-FITC, CD33-PE, CD41a-APC AIl antibodies were purchased from BD Bioscience, IL USA. Antibodies were added according to predetermined optimal concentrations and incubated for 30 minutes at 4⁰C. Cells were washed in PBS (1500 rpm, 5 minutes, 4⁰C) and pellets were resuspended in PBS containing propidium iodide (PI) to access viability, and analyzed on a FACSCalibur (BD Biosciences). Overall, the viability of differentiating HESCs was above 70% in all experiments.

Preparation and sorting of populations

EBs generated from all three lines (H3, Envy and MeIl) were harvested, disaggregated and stained at day 13 with the following antibody combination for sorting: CD34-PE (BD

Biosciences) and CD4 Ia-APC (BD Biosciences). At day 20 H3 and MeIl were stained with CD34-FITC (BD Biosciences), CD45-PE (BD Biosciences) and CD41a-APC while

Envy cells were stained with CD45-PE, CD34-PerCP (BD Biosciences) and CD4 Ia-APC.

Staining was performed using the same protocol as above. To gate on viable cells DAPI was used and the UV laser was activated on a FACS vantage. Unstained and single color controls were used to set gates.

Hematopoietic colony assay of differentiated HESCs

Myeloid assay using methylcellulose Triplicate assays were performed in 24 well tissue culture treated plates (Nunc) with 10,000 sorted HESCs added per well in 0.5 mL of Methocult™ (Stem Cell Technologies, Canada) supplemented with the following recombinant human growth factors: Granulocyte-macrophage-colony stimulating factor (GM-CSF, 20ng/ml), SCF (50ng/ml), interleukin (IL)-3 (20ng/ml), erythropoietin (EPO, 3 U/ml) and IL-6 (20ng/ml) (all from Peprotech). Recombinant factor concentrations recommended by Stem Cell Technologies. Plates were incubated (5% CO₂, 37⁰C, 100% humidity) and scored for colony formation at 14 days.

Megakaryocyte assay using collagen Duplicate assays were performed according to instructions supplied with a kit (e.g., as commercially available from Stem Cell Technologies). Briefly 10,000 sorted HESC cells were added per well to a double chamber slide together with ImI of collagen based medium (MegaCult™, Stem Cell Technologies) supplemented with r-hu TPO 50 ng/ml and r-hu SCF 25 ng/ml and r-hu IL-3 10 ng/ml (all from Peprotech). Recombinant factor concentrations recommended by Stem Cell Technologies. Plates were incubated for 14 days (5% CO₂, 37⁰C, 100% humidity) then fixed dried and kept at 4⁰C till staining. Staining was performed essentially according to the kit instructions using, mouse anti- human GpI IbIIIa (CD41a), biotin conjugated goat anti-mouse IgG and Avidin-alkaline phosphatase conjugate with alkaline phosphatase as the substrate (Stem Cell Technologies).

Quantitative real time PCR (qRT PCR)

Total RNA from undifferentiated and differentiated HESCs was prepared using RNEasy reagents according to the manufacturer's instructions (Qiagen P/L, Australia). First-strand cDNA was reverse transcribed with random hexamer priming using Superscript III reagents (Invitrogen, country). qRT-PCR was performed using Taqman gene expression probes supplied by Applied Bioscience and Taqman reagents and the 7500 Fast Real-time PCR system absolute thermal cycler and software (Applied Bioscience, Ca). The comparative cycle threshold (CT) method was used to analyze data, with gene expression levels compared to GAPDH expression. In brief, the CT for expression was calculated for each gene and for GAPDH. Since gene expression is inversely proportional to the CT, the expression for a given target gene relative to GAPDH may be given by the formula:

GeneExpression ∞ _2CnCene__GAPDH)

In these samples, this calculated value was multiplied by 1000 for the purposes of presentation.

Ploidy analysis of sorted fractions

Sorted cells were pelleted (1500 rpm, 10 minutes, 4⁰C). To resuspended pellet 1 ml of cold ethanol is slowly added and incubated at 4⁰C overnight. Cells are pelleted (1500 rpm, 10 minutes, 4⁰C) and resuspended in 900 μL of cold PBS. 50 μL of RNase A (0.64 mg/ml, Sigma) is added and cells are incubated for 30 minutes at 37⁰C. 15 minutes before acquisition 50 μL of propidium iodide (45 //g/ml, Sigma). At least 50,000 events were collected per samples. Both logarithmic, to analysis ploidy, and linear mode, to discriminate doublets, for the fluorescent 3 (Propidium Iodide) channel was collected on a FACSCalibur. CellQuest was used to analysis ploidy of the samples.

Fluorescent In Situ Hybridisation (FISH)

The chromosomal status of each sorted fraction was investigated using FISH. Pelleted sorted ells were resuspended in fixative (3:1 methanol: glacial acetic acid). An aliquot of the cell suspension was dropped onto a clean glass slide and left to dry. Slides were then dehydrated through a series of ethanol solutions (75%, 90%, 100%) for two minutes each, dried and stored at -2O⁰C until required. Each sorted fraction was analysed using three FISH probes, namely CEP 15 (aqua) detecting chromosome 15, CEP 16 (orange) detecting chromosome 16 and LSI22(ql l.2)(green) dedtecting chromosome 22 (Vysis, Immunodiagnostics, Victoria, Australia). 1.5μl of probe mixture was applied to each slide and covered with a small circular coverslip. Slides were placed in a hybrite incubator (Vysis), denatured at 73⁰C for 5 minutes, and incubated at 37⁰C for approximately 3 hours. Following hybridisation, the coverslip was removed and the slides were washed in 0.4x Sodium Chloride Sodium Citrate (SSC) at 71⁰C for 30 seconds followed by 0.4xSSC at room temperature for two minutes. Slides were air dried and 5μl of DAPI was used as a counterstain (Vysis). Slides were analysed under 4Ox and 10Ox magnification using oil objective and an Olympus BX51 fluorescent microscope (Olympus, Victoria, Australia) with Quips Imaging Software, version 3.1.2 (Vysis) for taking photos.

Example 2

Differentiation of human embryonic stem cells in a serum free medium: roles for BMP4, VEGF, SCF and FGF2

BMP4 induces the primitive streak gene MIXLl (a marker of mesoderm and mesendoderm) in differentiating HESCs

Four growth factors (BMP4, FGF2, VEGF and SCF) were evaluated singly and in combination. Initial experiments focused on the ability of single factors to induce the transcription of MIXLl (Pearce et al. (1999) Mechanisms of Development. 87(1-2):189- 192; Robb et al. (2000) Developmental Dynamics. 219(4):497-504), a gene that is expressed in the embryonic primitive streak, a transient structure harboring precursors of mesodermal and endodermal lineages that is an obligate intermediate for blood cell formation (Loebel et al. (2003) Developmental Biology. 264(1): 1-14). In the absence of growth factors, or in media supplemented by VEGF, FGF2 or SCF, no increase in MIXLl transcription was observed during HESC differentiation (Figure IA and Figure 7). In contrast, supplementing the serum-free medium with BMP4 increased both the viability and number of cells in the EBs promoted cyst formation , consistent with its ability to improve viability in differentiating mouse ESCs (Ng et al. (2005) Development. 132(5):873-884; Figure IB) and led to the transient induction of MIXLl mRNA (Figure IA and Figure 7). Cells expressing MIXLl protein were detected by flow cytometry only in cultures containing BMP4. Furthermore, these cells co-expressed OCT4, consistent with the phenotype of a primitive streak-like population that we have described previously (Figure 1C; Mossman et al. (2005) Stem Cells and Development. 14(6):656-663).

BMP4 is sufficient to induce primitive streak and early hematopoietic mesoderm gene expression

We extended these studies to examine whether other primitive streak or hematopoietic genes were also induced by BMP4 stimulation. Cultures of differentiating HESCs were exposed to increasing concentrations of BMP4 in the presence or absence of the KDR ligand, VEGF, and gene expression was examined after 3 and 5 days. VEGF was chosen because it is absolutely required for blood cell formation in the mouse embryo Carmeliet et al. (1996) Nature. 380(6573):435-439; Hoang T. (2004) Oncogene. 23(43):7188-7198) and we had previously documented that BMP4 induced Flkl (the mouse orthologue of KDR) in mouse ESC (Ng et al. (2005) Development. 132(5):873-884). These experiments confirmed that BMP4 induced expression of the primitive streak genes MIXLl, BRACHYURY, and GOOSECOID (Figure 2A-C and Figure 8) and that the levels of expression and kinetics of induction were not influenced by concomitant exposure to VEGF. Furthermore, the expression of two genes associated with development of hematopoietic mesoderm, GATA2 and RUNXl, was also induced by BMP4 and was independent of VEGF (Figure 2D, E). As described for differentiating mouse ESCs, BMP4 also induced expression of KDR (Figure 2F). High levels of KDR expression during the early phases of differentiation were consistent with expression of KDR protein detected by flow cytometry on undifferentiated HESC (data not shown). However, in the case of the stem cell marker, CD34 and the hematopoietic transcription factor, SCL/TAL 1, the addition of VEGF to BMP4 cultures enhanced gene expression at day 5 of differentiation (Figure 2G, H). These data argued that VEGF contributed to the survival, expansion and/or further differentiation of the early hematopoietic mesoderm induced by BMP4. Consistent with this hypothesis, immunophenotypic analysis of EBs differentiated for 20 days revealed that the percentage of hematopoietic cells increased in a BMP4 dose dependent manner, and that the inclusion of VEGF acted synergistically to further increase hematopoietic cell frequency by greater than 4- fold (Figure 9).

Consistent with a prior study that reported the induction of trophectoderm by BMP4 (Xu et al. (2002) Nature Biotechnology. 20(12): 1261-1264), we also observed BMP4 dependent induction of expression for chorionic gonadoptrophin (CGb) after 5 days (Figure 10). Expression of another trophectoderm gene, luteinizing hormone (LH), also increased slightly with differentiation but this was not BMP4 dependent. Since expression of both trophectoderm genes clearly postdated the induction of primitive streak genes (compare with Figure 2, expression of BRACHYURY, MIXLl and GOOSECOID), it is likely that the primitive streak inducing effects of BMP4 were direct, and not via a trophectoderm intermediate.

We subsequently compared the expression of GATA2, RUNXl, CD34 and SCL in HESCs when the cells were differentiated in cultures supplemented with single factors or combinations of BMP4 (B), VEGF (V), SCF (S) and FGF2 (F). Analysis of gene expression by real time PCR showed very low levels of GATA2, RUNXl, CD34 or SCL in undifferentiated HESCs (Figure 3). In experiment analyzed at day 11 of differentiation, similar levels of GATA2 and RUNXl expression were observed in cultures supplemented either by BMP4 or by any of the BMP4-containing factor combinations tested (Figure 3B). In contrast, much lower levels were observed in the cultures differentiated in the other single factors. As anticipated by the studies shown in Figure 2, levels of CD34 and SCL in the presence of BMP4 were intermediate between the very low levels seen in VEGF, SCF or FGF2 stimulated cultures and the higher expression in the BMP4-containing multi- factor combinations (Figure 3). When we examined early neuroectodermal gene expression, we observed significant induction of SOXl expression by dlO in the absence of growth factor stimulation, consistent with the default neural differentiation of ES cells (Figure 11). Whilst similar levels of SOXl induction were observed in cultures supplemented with VEGF, SCF or FGF2, the inclusion of BMP4 overrode the neural induction and high levels of SOXl were not observed in any BMP4 containing media. In our cultures, PAX6 was only expressed at very low levels in all the factor combinations.

FGF2 increases cell yield during HESC differentiation

The influence of different cytokine combinations on cell yield during differentiation was examined. After 2 days in culture, cell numbers were already significantly higher in BVSF medium compared to BV medium (p<0.02 at day 2, p<0.03 at day 3, Figure 4A). Analysis after 5 days of differentiation, showed that both BVSF and BVF media generated over twice as many cells as BV or BVS media (BVSF versus BV and BVS, p< 0.05 from day 5; BVF versus BV and BVS p < 0.05 from day 10, Figure 4B). There was a tendency for the BVSF combination to give higher total cell counts than BVF, but this did not reach statistical significance. There was no difference in viability observed between the cytokine combinations tested (data not shown), raising the possibility that the differences in final cell number reflected differences in cell proliferation rather than cell survival.

The combination of BMP4, VEGF, SCF and FGF2 generated the greatest number of hematopoietic cells from differentiating HESCs In order to correlate the growth factors required for expression of hematopoietic genes with those required for the generation of hematopoietic progenitors, the frequency of colony forming cells (CFCs) in cultures supplemented with BMP4 alone was compared to the CFC frequency observed in the factor combinations. Whilst HESC differentiated for 10 days in the presence of BMP4 formed colonies in methylcellulose at a frequency of 29.5 ± 11 (mean ± SEM) CFC per 10⁵ cells, differentiating HESCs cultured in VEGF-containing combinations consistently generated colonies at a 3- to 4-fold higher frequency (Figure 5A, B). When the total yield of CFCs obtained per plate of EBs was compared for the various growth factor combinations, it was evident that the inclusion of FGF2 in the BVF and BVSF resulted in a greater total number of colonies than was observed in the BV and BVS conditions at day 10 (Figure 5C, D) and day 20 of differentiation (Figure 5E-H). The hematopoietic colony yield was always higher in BVSF than in BVF media, but this difference did not reach significance until day 20 of differentiation (Figure 5H). Colony morphology and stained cytospin preparations of individual picked colonies indicated the presence of similar proportions of mixed, macrophage and erythroid colony types cultured under all permissive conditions (Figure 5 I-K and Table 1).

Table 1 : The percent distribution of hematopoietic colonies generated in the HESC differentiations

GEMM E GM

DlO

B 0.0 12.5 87.5

BV 8.4 25.7 65.9

BVS 1 1.9 37.2 50.9

BVF¹ 9.1 28.5 62.4

BVSF 27.1 22.5 50.4

D20

B 5.0 50.0 45.0

BV 44.4 11.1 44.4

BVS 39.7 6.7 53.5

BVF' 46.3 14.0 39.7

BVSF 49.5 32.0 18.5

These colony distribution data were derived from experiments (n=5) used to generate Figure 11. The data are shown as the percentage of erythroid (E), granulocyte/macrophage (GM) and mixed lineage (GEMM) colonies observed under each set of differentiation growth factor conditions when assayed at dlO and d20. A reduced proportion of multilineage colonies (as well as a reduction of CFC numbers) was observed in cultures differentiated in BMP4 alone. Abbreviations: B, BMP4; V, VEGF; S, SCF; F, FGF2. In order to further dissect the role of BMP4 alone versus the BV factor combinations in the generation of hematopoietic cells, flow cytometry was used to characterize the hematopoietic composition of the differentiating HESC populations. Cultures supplemented with BMP4 alone (or any other single cytokine tested) failed to produce a substantial percentage of CD34⁺ (progenitor and endothelial cell marker) cells at day 10 or CD45⁺ (leukocyte marker) or CD33⁺ (myeloid cell marker) cells by day 20 (Figure 6A-C and Figure 9). On average, a 10-fold increase in the proportion of CD34⁺, CD45⁺ and CD33⁺ cells were observed in all media combinations containing BMP4 and VEGF (Figure 6A-C). Although, in the example shown, BV medium did not increase the percentage of CD45⁺ and CD33⁺ cells to the same extent as the other cytokine combinations, over a number of experiments there was no statistically significant difference in the percent of CD45⁺ and CD33⁺ cells generated between the four multi- factor combinations (Figure 6A-C). When the total number of CD34⁺, CD45⁺ and CD33⁺ cells generated was calculated, it was observed that the combination of BVSF in the culture medium yielded the greatest number of hematopoietic cells, with an average of over 800 CD45⁺ cells generated per EB (Figure 6D-F).

We also examined the differentiating cultures for expression of CDl 17 (c-KIT), the receptor for the cytokine SCF, and CD31 and KDR, proteins present on both early hematopoietic progenitors and endothelial cells (Figure 6G). CDl 17 was expressed on approximately 32% of undifferentiated HESCs (data not shown) and a second wave of expression was observed at day 10, when most of the CD117⁺ cells also co-expressed CD34 (data not shown). The greatest yield of CDl 17⁺ cells was in cultures supplemented with BVSF (Figure 6G) and similar results were observed for CD31 and KDR (Figure 6G). Consistent with the observations of others (Wang et al. (2004). Immunity. 21(1):31-41), we found that CD34, CD31 and KDR were frequently co-expressed (data not shown). These experiments demonstrate that BMP4 was sufficient to initiate hematopoietic gene expression and permit a proportion of cells to develop to the progenitor cell stage, but was unable to support the generation of significant numbers of mature blood cells. This work also showed that VEGF was necessary for robust generation of CFCs and the combination of four factors that included FGF2 and SCF increased the total yield of hematopoietic progenitors and mature cells.

Example 3

Generation of Megakaryocytes

Cell Type Day 13 and Day 20 of differentiation CD34⁺CD41^bπghtCD45^+Λ cells

Aim 1

Since there were a few published papers discussing the specificity of CD41 as a maker of megakaryocytes experiments were performed to determine the correct megakaryocyte population.

Method

Human embryonic cell lines - H3, Envy and MELl were differentiated in serum- and feeder-free "Spin EB" method for 13 and 20 days (Ng et al. (2005) Blood. 106(5):1601-1603).

Cytokines supplemented between day 0 and day 10: were BMP4, VEGF, SCF and bFGF (FGF2) and then from day 1 1 till either dl3 or d20: were TPO, SCF and IL3. A single cell suspension of differentiation HESCs was prepared to allow immunophenotyping of the cells present at two time points of differentiation. Cells were identified with the used of a flow cytometer and various anti-human monoclonal antibodies. Gating of cells at analysis was performed to allow characterization of the day 13 CD41⁺ and day 20 CD41^bπght and CD41^dim fractions.

Results

The immunophenotype analysis demonstrated that a low percent of day 13 CD41⁺ expressed CD45, however, expression increased at day 20 and was found on the majority of CD41^bπght and CD41^dim cells (Figure 12C). This is in agreement with the observation made that with time CD45 expression increases as more cells in the culture become hematopoietic. CD34 is expressed on the majority of day 13 CD41⁺ cells and day 20 CD41^bπght cells but on only a few of the CD41^dim cells, thus the CD41^dιm cells are likely to be more mature and less hematopoietic "progenitor" like (Figure 12C). CD33, a myeloid marker is expressed on over 80% of day 13 CD41⁺ and on day 20 CD41^dim but only about 40% of day 20 CD41^bπght cells, these cells are therefore likely to have a myeloid potential although this potential could be decreased in the CD41^bπght cells (Figure 12C). CDl 17 (or kit ligand receptor) and KDR (or VEGF receptor) expression levels was less then 10% for all cells expression CD41 (Figure 12C). Megakaryocyte markers, such as CDI lO, CD41b and CD61, are expressed on a small percent of day 13 CD41⁺ and day 20 CD41^bπght cells but not on day 20 CD41^dim cells therefore, in the future sorting experiments day 13 CD41⁺ and day 20 CD41^bπght were sorted to enrich from megakaryocyte populations (Figure 12C).

Aim 2

To sort populations generated in HESCs differentiation cultures and enrich for megakaryocytes.

Method 2.1

A single cell suspension was prepared and cells generated from day 13 and day 20 cultures were stained in the following combinations and then sorted:

Cells were then sorted on their expression of the above markers into 4 populations for day 13 and 5 populations for day 20 in the following manner: Dl 3 fractions were:

CD34⁺CD41^" = HSC and Endothelial stem cells CD34⁺CD41 ⁺ = Mks progenitors

CD34-CD41 ⁺ = Committed Mks CD34^'CD4r = Non hematopoietic compartment D20 fractions were:

CD34⁺CD41^" = HSC and Endothelial stem cells

CD34⁺CD41^Bπght = Mks progenitors

CD45⁺CD41^Bπght = Committed Mks

CD45⁺CD41^" = Hematopoietic cells

CD34^"CD45^'CD41^" = Non hematopoietic compartment DAPI was added to all samples to enable only viable cells to be sorted. Various tests were performed to determine which sorted fraction was enriched for megakaryocytes. 1) qRT-PCR for genes important for megakaryocyte markers and 2) colony assay to quantitate the number of megakaryocytes progenitor cells.

Results As megakaryocytes mature the expression of GATAl and MPL (the receptor for thrombopoietin) increase, while, TAL1/SCL, although not a specific gene for megakaryocytes is critical for platelet production. Low but increasing levels of genes, GATAl, MPL (CDIlO) and TAL1/SCL, was detected in unsorted fractions of differentiated cells. Expression levels of GATAl, MPL and TALI /SCL were highest in the sorted fractions that contained CD41 positive cells both in day 13 and day 20 differentiations (Figure 16A). Levels of these genes in fractions containing no hematopoietic cells, that is day 13 CD34^"CD41^" and day 20 CD34^"CD45^"CD41\ were similar to that seen in unsorted cells (Figure 16A). Gene expression of day 13 and day 20 CD34⁺ (CD41 ) fractions was higher then in the unsorted cells, but at least 3-fold lower then in fractions containing CD41 positive cells (Figure 16A). Day 20 CD45⁺ (CD41^") sorted fraction showed gene expression levels similar to CD34⁺ sorted fractions (Figure 16A).

PF4, is a protein secreted by platelets that binds to and neutralize heparin and is a critical player in coagulation and a protein exclusive to megakaryocyte. The relative gene expression levels of platelet factor 4 (PF4) were significantly higher then the other megakaryocyte genes and were 10-fold higher in the day 20 unsorted cells compared to undifferentiated HESCs (Figure 16B). PF4 levels were generally higher in all day 20 sorted fractions then day 13 (Figure 16B). Again fractions containing CD41⁺ cells contained higher levels of PF4 then the sorted fractions that were CD4T in some cases up to 100-fold higher (Figure 16B).

Method 2b

Sorted fractions were analysed for the ability to generate megakaryocyte colonies in a collagen-based system. 10,000 cell were plated per well in duplicate, cultured for a further 14 days, fixed and dried then stained with mouse anti-human CD41 and detected using fast red and anti mouse streptavidin biotin conjugated to alkaline phosphatase.

Results 2b

From the day 13 differentiations, the number of megakaryocyte colonies generated from both Envy and H3 hESC lines are about the same. Once cells are sorted into the fraction the potential of the Envy line to generated megakaryocyte colonies increases and both fractions that contain CD41 positive cells generate a 2-fold higher number of colonies than the fraction that does not contain these cells (Figure 15A). In addition, the CD34⁺CD41⁺ fraction sorted from Envy lines seems to have a tendency to generate the most number of megakaryocyte colonies compared to the other sorted fractions but this difference is not significant. In the H3 line the sorted fractions containing CD34⁺ cells have the highest potential to generated megakaryocyte colonies and sorting on CD41 does not seem to enrich from megakaryocyte progenitors.

From the day 20 differentiations, the number of megakaryocyte colonies generated from both Envy and H3 HESC lines is about 2-fold lower then day 13 differentiated cells (Figure 15A). The Envy line generated more colonies in all sorted fractions and even in the unsorted fraction compared to H3 line. There seems to be no enrichment of megakaryocyte colonies in the CD41 positive fractions with one exception, the Envy CD45⁺CD41⁺ fraction generated significantly more colonies then the CD45⁺CD41^" fraction.

Example 4

In vivo administration of megakaryocytes/megakaryocyte progenitors

Cell fractions produced essentially as described in Example 2 (e.g., Method 2.1) using

ENVY cells are intravenously injected into mice, e.g., NOD/SCID mice. These cells are fluorescently labelled to facilitate detection in mice. Preferred cell populations are CD34⁺CD41⁺(MkS progenitors) and CD34^"CD41 ^""(Committed Mk). Approximately 5 or 15 hours post transplant (injection) the number of cells that have homed to the bone marrow are assessed by determining the number of fluorescent cells present in the bone marrow compartment of femurs of treated mice. These assays demonstrate that megakaryocytes and/or megakaryocyte precursors produced by a method of the invention are capable of homing to and/or populating bone marrow of a subject, e.g., to facilitate production of platelets.

The spatial distribution of megakaryocyte cells and/or megakaryocyte precursor cells is determined by perfusion fixing recipient mice, removing femurs and cutting sections of fixed femurs. These sections are then analysed using microscopy to determine the anatomical location of transplanted cells that home to the marrow. This method is described in more detail in S. K. Nilsson, H.M. Johnston, J.A. Coverdale. (2001) Spatial localisation of transplanted hemopoietic stem cells: inferences for the localization of stem cell niches. Blood 97:2293-2299.

Engraftment and platelet production from transplanted megakaryocyte cells and/or megakaryocyte precursor cells is assessed by transplanting the cells (e.g., as described in S. K. Nilsson et al. (2005) Osteopontin, a Key Component of the Hematopoietic Stem Cell Niche and Negative Regulator of Primitive Hematopoietic Progenitor Cells. Blood 106:1232-1239. DN Haylock et al. (2007) HSC with higher hemopoietic potential reside at the bone marrow endosteum. Stem Cells 25:1062-9), and determining human platelet numbers in the peripheral blood of mice essentially as described in Mattia et al. "Long- term platelet production assessed in NOD/SCDD mice injected with cord blood CD34+ cells, thrombopoietin-amplified in clinical grade serum-free culture, Exp Hematol. 36:244-252, 2008 and/or Tijssen et al., Leukemia 22: 203-208, 2008..

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.

Claims

1. A method for generating megakaryocytes and/or megakaryocyte precursors from a population of embryonic stem cells (ESCs), the method comprising:

(i) culturing ESCs in a serum-free, stromal/feeder cell-free medium for a time and under conditions sufficient for formation of mesoderm and/or mesendoderm; and

(ii) differentiating the cells cultured in step (i) in a medium comprising thrombopoietin (TPO), stem cell factor (SCF) and interleukin 3 (IL-3), or any functional fragment, variant or mimetic of TPO, SCF and/or IL-3, for a time and under conditions sufficient for formation of megakaryocytes and/or megakaryocyte precursors.

2. A method for generating megakaryocytes from a population of embryonic stem cells (ESCs), the method comprising:

(i) culturing ESCs in a serum-free, stromal/feeder cell-free medium for a time sufficient to observe formation of mesendoderm characterized by expression of MIKLl; and

(ii) differentiating the cells cultured in step (i) in a medium comprising thrombopoietin (TPO), stem cell factor (SCF) and interleukin 3 (IL-3), or any functional fragment, variant or mimetic of TPO, SCF or IL3, for a time sufficient to observe the formation of megakaryocytes and/or megakaryocyte precursors.

3. The method according to claim 1 or claim 2 wherein the formation of mesoderm and/or mesendoderm is characterised by detecting expression of one or more cell surface markers selected from the group consisting of CD34, CD33, CD45 and PDGFRα and mixtures thereof and/or detecting the expression of one or more primitive streak genes selected from the group consisting of MIXLl, BRACHYURY, and GOOSECOID and mixtures thereof.

4. The method according to any one of claims 1 to 3 wherein the serum-free, stromal/feeder cell-free medium comprises bone morphogenic protein (BMP4).

5. The method according to claim 4 wherein the serum- free, stromal/feeder cell-free medium further comprises vascular endothelial growth factor (VEGF).

6. The method according to claim 5 wherein the serum-free, stromal/feeder cell-free medium further comprises stem cell factor (SCF) and fibroblast growth factor (FGF) 2.

7. The method according to any one of claims 4 to 6 wherein BMP4, VEGF, SCF and FGF2 are human BMP4 (hu BMP4), human VEGF (hu VEGF), human SCF (hu SCF) and human FGF2 (hu FGF2), respectively.

8. The method according to any one of claims 1 to 7 wherein the ESCs are cultured for between about 8 days and about 25 days.

9. The method according to any one of claims 1 to 8 wherein the medium comprising thrombopoietin (TPO), stem cell factor (SCF) and interleukin 3 (IL-3), or any functional fragment, variant or mimetic of TPO, SCF and/or IL-3 further comprises any one or more of IL- 11 , IL-6 and IL-9.

10. The method according to any one of claims 1 to 9 wherein the formation of megakaryocytes and/or megakaryocyte precursors is characterised by detecting expression of one or more cell surface markers selected from the group consisting of CD34, CD41, CD45.

11. The method according to claim 10 wherein the method further comprises detecting expression of the cell surface markers CD61 and/or CDI lO and/or detecting the presence of one or more primitive streak genes selected from the group consisting of GATAl, PF4 and MPL.

12. The method according to any one of claims 1 to 11 wherein the method further comprises isolating mesoderm and/or mesendoderm or isolating a megakaryocyte and/or a megakaryocyte precursor by performing a method comprising:

(i) contacting mesoderm and/or mesendoderm or megakaryocyte and/or a megakaryocyte precursor with a ligand that binds a marker expressed on the cell surface for a time and under conditions sufficient to form a ligand- marker complex; and

(ii) isolating a cell comprising the ligand-marker complex.

13. The method according to claim 12 wherein the marker present on the cell surface of mesoderm and/or mesendoderm is selected from the group consisting of CD34,

CD33, CD45, PDGFRαand mixtures thereof.

14. The method according to claim 12 wherein the marker present on the cell surface of a megakaryocyte and/or a megakaryocyte precursor is selected from the group consisting of CD34, CD41, CD45, CD61, CDI lO and mixtures thereof.

15. The method according to any one of claims 12 to 14 wherein the ligand is an antibody.

16. The method according to any one of claims 12 to 15 wherein the mesoderm and/or mesendoderm or the megakaryocyte and/or the megakaryocyte precursor is isolated by fluorescence activated cell sorting (FACS) or by magnetic cell sorting.

17. The method according to any one of claims 1 to 16 wherein the ESC is a human embryonic stem cell.

18. The method according to any one of claims 1 to 17 wherein the method further comprises culturing megakaryocytes and/or megakaryocyte precursors to produce platelets.

19. The method according to claim 18 wherein the megakaryocytes and/or megakaryocyte precursors are cultured in media comprising SCF and IL-9, and wherein the media is substantially free of IL-3.

20. The method according to claim 18 wherein the media further comprises TPO and erythropoietin (Epo).

21. The method according to any one of claims 12 to 20 wherein the method comprises culturing the generated megakaryocyte and/or megakaryocyte precursor to form a population of megakaryocytes or megakaryocyte precursors.

22. The method according to any one of claims 12 to 21 wherein the method additionally comprises formulating a megakaryocyte and/or megakaryocyte precursor or population of megakaryocytes or megakaryocyte precursors with a suitable carrier or excipient to produce a pharmaceutical composition.

23. A megakaryocyte and/or megakaryocyte precursor, or population of megakaryocyte and/or megakaryocyte precursors generated by performing the method of any one of claims 1 to 22.

24. A bioreactor for use in differentiating embryonic stem cells (ESCs) into megakaryocytes and/or megakaryocyte precursors under serum-free, stromal/feeder cell-free conditions, the bioreactor comprising a cell culture chamber in which at least one internal surface comprises thrombopoietin (TPO), stem cell factor (SCF) and interleukin 3 (IL-3), or any functional fragment, variant or mimetic of TPO,

SCF and/or IL-3.

25. The bioreactor according to claim 24 wherein the cell culture chamber comprises a matrix suitable for supporting growth and/or proliferation and/or differentiation of an ESC.

26. The bioreactor according to claim 25 wherein the matrix is comprised of a material selected from the group consisting of cartilage, demineralised bone and a synthetic material.

27. The bioreactor according to any one of claims 24 to 26 wherein the TPO, SCF and IL-3 or functional fragment, variant or mimetic thereof is immobilized on the matrix.

28. The bioreactor according to any one of claims 24 to 26 wherein the TPO, SCF and IL-3 or functional fragment, variant or mimetic thereof is included in a medium contained within the bioreactor.

29. The bioreactor according to any one of claims 24 to 28 wherein the bioreactor additionally comprises one or more growth factor(s) and/or cytokine(s).

30. The bioreactor according to claim 29 wherein the growth factor(s) and/or cytokine(s) is selected from the group consisting of bone morphogenic protein (BMP4), and/or vascular endothelial growth factor (VEGF) and/or stem cell factor (SCF) and/or fibroblast growth factor (FGF) 2.

31. The bioreactor according to claim 30 wherein the bioreactor comprises BMP4 in combination with any one or more of VEGF, SCF and FGF2.

32. The bioreactor according to any one of claims 29 to 31 wherein the growth factor(s) and/or cytokine(s) is immobilized on the internal surface within the bioreactor and/or on the surface of the matrix within the bioreactor.

33. The bioreactor according to any one of claims 29 to 31 wherein the growth factor(s) and/or cytokine(s) is included within the culture medium within the bioreactor.

34. A pharmaceutical composition comprising a megakaryocyte and/or megakaryocyte precursor, or population of megakaryocyte and/or megakaryocyte precursors generated by performing the method of any one of claims 1 to 22.

35. A megakaryocyte and/or megakaryocyte precursor, or population of megakaryocyte and/or megakaryocyte precursors generated by performing the method of any one of claims 1 to 22 or a pharmaceutical composition according to claim 34 for use in human therapy.

36. A method for treating or preventing a disorder caused by or associated with reduced platelet numbers in a subject comprising administering to the subject an effective amount of a megakaryocyte and/or megakaryocyte precμrsor, or population of megakaryocyte and/or megakaryocyte precursors generated according to the method of any one of claims 1 to 22 or a pharmaceutical composition according to claim 34.

37. The method according to claim 36 wherein the disorder caused by or 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, chemotherapy-induced thrombocytopenia, 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.

38. The method according to claim 36 or claim 37 wherein chemotherapy-induced thrombocytopenia is treated.

39. Use of an effective amount of megakaryocyte and/or megakaryocyte precursor, or population of megakaryocytes and/or megakaryocyte precursors generated according to the method of any one of claims 1 to 22 in the manufacture of a medicament for treating or preventing a disorder caused by or associated with reduced platelet numbers in a subject.

40. A method of selecting a compound capable of inducing or enhancing proliferation of a megakaryocyte and/or megakaryocyte precursor, the method comprising:

(i) contacting a megakaryocyte and/or megakaryocyte precursor produced by performing the method of any one of claims 1 to 22 with a compound for a time and under conditions sufficient for the compound to induce the megakaryocyte and/or megakaryocyte precursor to proliferate; and

(ii) detecting the proliferation of the megakaryocyte and/or megakaryocyte precursor.

wherein enhanced proliferation of wherein enhanced proliferation of a megakaryocyte and/or megakaryocyte precursor contacted with the compound compared to a megakaryocyte and/or megakaryocyte precursor that has not been contacted with the compound indicates that the compound induces or enhances proliferation of a megakaryocyte and/or megakaryocyte precursor.

41. A method of selecting a compound capable of inducing or enhancing differentiation of a megakaryocyte and/or megakaryocyte precursor into a platelet, the method comprising:

(i) contacting a megakaryocyte and/or megakaryocyte precursor produced by performing the method according to any one of claims 1 to 22 with a compound for a time and under conditions sufficient for the compound to induce the megakaryocyte and/or megakaryocyte precursor to differentiate into and/or produce a platelet; and

(ii) detecting platelet production,

wherein enhanced platelet production at (ii) compared to the platelet production from a megakaryocyte and/or megakaryocyte precursor that has not been contacted with the compound indicates that the compound induces or enhances differentiation of a megakaryocyte and/or megakaryocyte precursor into a platelet.

42. The method of any one of claims 1 to 22 wherein the cells are cultured and/or differentiated in the bioreactor of any one of claims 24 to 33.