Transcription Factor Mediated Programming Towards Megakaryocytes

Abstract

This invention relates to the forward programming of pluripotent stem cells (PSCs) into megakaryocyte (MK) progenitor cells using the transcription factors GATA1, FLI1 and TAL1. Methods of producing megakaryocyte (MK) progenitor cells and subsequently differentiating them into mature megakaryocytes are provided.

Description

RELATED APPLICATIONS

[0001] This application is a continuation patent application of U.S. application Ser. No. 14/407,044 filed on Dec. 10, 2014, which is a national phase filing under 35 U.S.C. 371 of International Application No. PCT/GB2013/051600, filed on Jun. 19, 2013, which claims the benefit of and priority to GB Application No. 1210857.7, filed on Jun. 19, 2012, each of which are incorporated herein by reference in their entireties.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Sep. 12, 2018, is named 010600_Sequences_ST25.txt and is 70,865 bytes in size.

FIELD

[0003] This invention relates to the generation of megakaryocyte (MK) lineages from pluripotent stem cells (PSCs).

BACKGROUND

[0004] Megakaryocytes (MK) are blood cells which are uniquely responsible for formation of platelets regulating haemostasis and thrombosis (Kaushansky K. Blood. 2008; 111:981 986). Low blood platelet count, or thrombocytopenia, can originate from different conditions (including viral and bacterial infections, hereditary syndromes, cancers, medication induced) and may result in severe haemorrhages in extreme cases. In the UK, the National Health Service is delivering about 240,000 platelet concentrate units per year from voluntary donors for therapeutic and prophylactic purposes. The total platelet cost for NHS is evaluated at .English Pound.55m per year (.English Pound.230 per unit in 2009/10). In addition, platelet transfusion refractoriness due to alloimmune reactions is a serious issue in particular for patients with regular transfusion needs, which imposes additional biological characterization, i.e. HLA genotyping, of both donors and recipients. Eventually, as for other human biological products, donor platelet transfusion goes with a risk of transmission of contagious agents.

[0005] Human embryonic stem cells (hESC) can be maintained and expanded in culture indefinitely and are able to generate virtually all the different cell types of the organism (Keller G Genes Dev. 2005; 19:1129-1155). Indeed, they hold great promise for cell based therapies. Interestingly, it has been shown that the banking of a small number of carefully selected hESC lines to include the most common HLA haplotypes in the UK may be sufficient to offer practical benefits, i.e. matched or single mismatched tissues for a majority of the population (Taylor C J et al Lancet. 2005; 366:2019-2025). Moreover, the ability to derive pluripotent stem cells equivalent to hESCs--the so-called induced pluripotent stem cells (iPSCs)--from adult somatic cells like skin fibroblasts or circulating blood cells, opens new avenues for clinical applications with the opportunity of generating fully compatible tissues for virtually every single individual (Yamanaka S. Cell Stem Cell. 2010; 7:1-2; Takahashi K et al Cell. 2007; 131:861-872).

[0006] Human ESCs, iPSCs and other human stem cells (collectively human pluripotent stem cells, or hPSCs) may thus offer a valuable option for ex-vivo production of biocompatible plateleta. Up to now, generation of clinically relevant cell types from hPSCs has been essentially achieved by mimicking in vivo embryonic development based on scientific knowledge gathered from model vertebrate organisms (Murry C E, Keller G. Cell. 2008; 132:661-680). Existing protocols for generation of platelet precursors, the megakaryocytes, use poorly defined culture medium containing fetal calf serum and bone marrow derived murine stromal cell lines (OP9, C3H10T1/2) to support megakaryocyte differentiation and platelet production (Gaur M, et al J Thromb Haemost. (2006) 4:436-442; Takayama N, et al. Blood (2008) 111:5298-5306; Takayama N, et al. J Exp Med. (2010) 207:2817-2830). More recently, protocols using serum free culture conditions to differentiate megakaryocytes from hPSC were described but platelet production was still dependent on a co-culture step with the OP9 cell line (Lu S J, et al. Cell Res. 2011; Pick et al PLoS One February 2013 10.1371/journal.pone.0055530). These methods involve long-term culture of up to 26 days with complicated cell handling--as single haemangioblast colony picking in semi-solid medium--to produce mature megakaryocytes, yet showing low platelet release capacity. Noteworthy, as previously reported in the context of differentiation of other cell lineages, high variability has been found regarding megakaryocyte generation among different hPSC lines.

DESCRIPTION

[0007] This invention relates to the development of a process for the efficient forward programming of human pluripotent cells into megakaryocyte progenitor cells (MK-FoP). This may be useful, for example, in production of mature megakaryocytes and platelets; the modelling of thrombocytopenia and other platelet-associated conditions; and the development of therapeutics to these conditions.

[0008] An aspect of the invention provides a method of forward programming pluripotent cells into megakaryocyte progenitor cells; or producing megakaryocyte progenitor cells; the method comprising;

[0009] i) providing a population of isolated pluripotent stem cells (PSCs),

[0010] ii) introducing a combination of transcription factors (TFs) into the population of PSCs, said combination comprising GATA1, FLI1 and TAL1, and;

[0011] iii) culturing said population of cells.

[0012] The combination of transcription factors introduced into the cell population imposes a megakaryocyte progenitor phenotype i.e. one or more cells in the population are forward programmed by the transcription factor combination into megakaryocyte progenitor cells.

[0013] A method of producing mammalian cells with a megakaryocyte progenitor phenotype as described herein may comprise;

[0014] i) providing a population of isolated pluripotent stem cells (PSCs),

[0015] ii) introducing a combination of transcription factors into the population of PSCs, said combination comprising GATA1, FLI1 and TAL1, and;

[0016] iii) culturing said population of cells, such that one or more cells in the population displays a megakaryocyte progenitor phenotype.

[0017] The population may be cultured under suitable conditions and for a sufficient period of time, following introduction of the transcription factors, to allow one or more cells in the population to display a megakaryocyte progenitor phenotype, for example the stable expression of CD61, CD34 and CD41a; and/or the stable expression of CD61, CD235a and CD41a in said cells.

[0018] After programming, the megakaryocyte progenitor cells may be maintained in culture, expanded, stored, for example frozen using conventional techniques, or used in therapeutic or other applications as described herein.

[0019] Transcription factors are DNA binding proteins which regulate the expression of genes in cells. Preferably, the transcription factors introduced into the PSCs are human transcription factors.

[0020] The combination of transcription factors for programming PSCs to become megakaryocyte progenitors as described herein comprises GATA1 (SEQ ID NO: 1), FLI1 (SEQ ID NO: 2) and TAL1 (SEQ ID NO: 3). The amino acid sequences of GATA1, FLI1 and TAL1 are readily available on public databases. For example, the reference amino acid sequence of human GATA1 (GATA binding protein 1; also known as ERYF1: Gene ID 2623) has the NCBI database entry NP-002040.1 GI: 4503925 (SEQ ID NO: 1); the reference amino acid sequence of human FLI1 (Friend leukemia virus integration 1, also known as EWSR1, SIC-1 or ERGB; Gene ID 2313) has the NCBI database entry NP 002008.2 GI: 7110593--(SEQ ID NO: 2) and the reference amino acid sequence of human TAL1 (T cell acute lymphocytic leukemia protein 1; Gene No: 6886) has the NCBI database entry NP-003180.1 GI: 4507363 (SEQ ID NO: 3).

[0021] In some embodiments, the combination of transcription factors may lack TAL1. For example, a method of producing mammalian cells with a megakaryocyte progenitor phenotype as described herein may comprise;

[0022] i) providing a population of isolated pluripotent stem cells (PSCs),

[0023] ii) introducing a combination of transcription factors into the population of PSCs, said combination comprising GATA1 and FLI1, and;

[0024] iii) culturing said population of cells, such that one or more cells in the population displays a megakaryocyte progenitor phenotype.

[0025] GATA1, FLI1 and TAL1 may be produced using routine recombinant techniques or may be obtained from commercial suppliers (e.g. R&D Systems, Minneapolis, Minn., USA).

[0026] In some embodiments, the combination of transcription factors may consist of GATA1, FLI1 and TAL1 i.e. the only transcription factors in introduced into the PSCs are GATA1, FLI1 and TAL1.

[0027] In other embodiments, the combination of transcription factors may consist of GATA1, FLI1 and TAL1, with optionally, one, two, three or more, additional transcription factors. For example, additional transcription factors may include one or more of the transcription factors shown in Table 1 or one or more of IKZF1, HOXA5, RUNX1, ZFPM2, ZFPM1 and GATA2.

[0028] In some preferred embodiments, additional transcription factors may include one or more of ABLIM1, FHL1, RUNX3, NFIC, NFIL3, VDR, MESP1, BTBD11, APPL2, MICAL1, BATF, SCMH1 and MBP, as shown in table 2.

[0029] The amino acid sequences of IKZF1, HOXA5, RUNX1, ZFPM2, ZFPM1 and GATA2 and ABLIM1, FHL1, RUNX3, NFIC, NFIL3, VDR, MESP1, BTBD11, APPL2, MICAL1, BATF, SCMH1 and MBP are readily available on public databases.

[0030] Suitable transcription factor nucleic acids and proteins may be produced using routine recombinant techniques or obtained from commercial suppliers (e.g. R&D Systems, Minneapolis, Minn., USA; Cellgenix, DE; Life Technologies, USA).

[0031] Suitable transcription factors for use as described herein may comprise the reference database amino sequence or a variant thereof. A suitable variant may have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% sequence identity to the reference sequence.

[0032] Amino acid sequence identity is generally defined with reference to the algorithm GAP (GCG Wisconsin Package.TM., Accelrys, San Diego Calif.). GAP uses the Needleman & Wunsch algorithm (J. Mol. Biol. (48): 444-453 (1970)) to align two complete sequences that maximizes the number of matches and minimizes the number of gaps. Generally, the default parameters are used, with a gap creation penalty=12 and gap extension penalty=4. Use of GAP may be preferred but other algorithms may be used, e.g. BLAST or TBLASTN (which use the method of Altschul et al. (1990) J. Mol. Biol. 215: 405-410), FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448), or the Smith-Waterman algorithm (Smith and Waterman (1981) J. Mol Biol. 147: 195-197), generally employing default parameters.

[0033] Particular sequence variants may differ from a reference sequence by insertion, addition, substitution or deletion of 1 amino acid, 2, 3, 4, 5-10, 10-20 or 20-30 amino acids.

[0034] PSCs are unspecialized, undifferentiated cells that are capable of replicating or self-renewing themselves and developing into specialized cells of all three primary germ layers i.e. ectoderm, mesoderm and endoderm but are not able to develop into all embryonic and extra-embryonic tissues, including trophectoderm (i.e. not totipotent). Preferably, the PSCs are not committed to a haematopoietic lineage.

[0035] In preferred embodiments, the PSCs are human pluripotent stem cells.

[0036] PSCs include embryonic stem (ES) cells and non-embryonic stem cells, including foetal and adult somatic stem cells and stem cells derived from non-pluripotent cells, for example induced pluripotent (iPS) cells which are derived from non-pluripotent cells. iPS cells are described in more detail below.

[0037] PSCs may express one or more of the following pluripotency associated markers: Oct4, Sox2, Alkaline Phosphatase, SSEA-3, Nanog, SSEA-4 and Tra-1-60. Preferably, pluripotent stem cells express Oct4.

[0038] Human PSCs do not express haematopoietic cell or megakaryocyte markers, such as CD61, CD34, CD41, CD42a, CD42b and GPVI. For example, the pluripotent stem cells may have the phenotype CD61-, CD34-, CD41a, CD42a-, CD42b-, GPVI-.

[0039] Markers expressed by a cell, including pluripotency associated markers and haematopoietic cell markers, may be identified using standard techniques, such as flow cytometry, PCR, western blotting, immunocytochemistry and in situ hybridisation.

[0040] In some embodiments, the PSCs are ES cells, for example human ES cells and non-human ES cells. Suitable ES cells may be obtained from a cultured hES cell line, such as Edi2, H9 or hSF-6. Further examples of suitable human embryonic stem cells are described in (Thomson J A et al Science 282: 1145-1147 (1998); Reubinoff et al. Nat Biotechnol 18:399-404 (2000); Cowan, C. A. et al. N. Engl. J. Med. 350, 1353-1356 (2004), Gage, F. H., et al. Ann. Rev. Neurosci. 18 159-192 (1995); and Gotlieb (2002) Annu. Rev. Neurosci 25 381-407); Carpenter et al. Stem Cells. 5(1): 79-88 (2003); see also: the NIH stem cell registry which is accessible online. Potentially clinical grade hESCs are described in Klimanskaya, I. et al. Lancet 365, 1636-1641 (2005); and Ludwig, T. E. et al. Nat. Biotechnol. 24, 185-187 (2006).

[0041] In some embodiments, the PSCs are not hES cells.

[0042] In some embodiments, the ES cells may be obtained by methods which do not involve the destruction of a human embryo or the use of a human embryo for an industrial or commercial purpose. For example, hES cells may be obtained by blastomere biopsy techniques (Klimanskaya (2013) Semin Reprod Med. 31(1):49-55; Klimanskaya et al Nature (2006), 444(7118)481-5; Chung et al Cell Stem Cell 2008, 2(2), 113-117; U.S. Pat. No. 7,893,315).

[0043] In other embodiments, the pluripotent stem cells are iPS cells, for example human iPS cells.

[0044] iPS cells are pluripotent cells which are derived from non-pluripotent ancestor cells, for example somatic cells, such as fibroblasts. Ancestor cells are typically reprogrammed into iPS cells through the introduction of reprogramming factors Oct4, Sox2, Klf4 and c-Myc into the cell. Other suitable reprogramming factors and combinations of reprogramming factors for inducing pluripotency are known in the art. (see, for example, Yu et al Science 318 2007 1917-1920, Tesar, P. J. et al. Nature 448, 196-199 (2007); Nichols, J. & Smith, A. Cell Stem Cell 4, 487-492 (2009); Ying, Q. L. et al. Nature 453, 519-523 (2008), Hanna J, et al Proc Natl Acad Sci USA. 2010 May 18; 107(20):9222-7; Han D W, et al Nat Cell Biol. 2011 January; 13(1):66-71; Silva J et al Cell. 2009 Aug. 21; 138(4):722-37).

[0045] Reprogramming factors and techniques for the production of iPS cells are well-known in the art and include introducing reprogramming factors by plasmid or viral transfection, direct protein delivery or direct delivery of nucleic acid, such as mRNA. (Yamanaka et al Nature (2007); 448:313-7; Yamanaka 6 2007 Jun. 7; 1(1):39-49. Kim et al. Nature. 2008 Jul. 31; 454(7204):646-50; Takahashi Cell. 2007 Nov. 30; 131(5):861-72. Park et al Nature. 2008 Jan. 10; 451(7175):141-6; Kim et al Cell Stem Cell. 2009 Jun. 5; 4(6):472-6; Vallier, L., et al. (2009) Stem Cells 27, 2655-66.).

[0046] The non-pluripotent ancestor cells for use in the production of iPS cells may be obtained from an individual. The individual may be healthy (i.e. without any disease condition) or may have a disease condition. For example, iPS cells may be derived from a sample of cells obtained from an individual with a haematological condition, for example a thrombocytopenic or other platelet-related condition, including essential thrombocytosis and congenital amegakaryocytic thrombocytopenia (CAMT), Thrombocytopenia-absent radius syndrome (TAR), Bernard Soulier syndrome (BSS), Gray platelet syndrome (GPS), and Glanzmann thrombasthenia. IPS cells obtained from an individual with a haematological condition may be used to generate megakaryocyte progenitor cells or mature megakaryocytes using the methods described herein for modelling a haematological condition; for the treatment of an individual with a haematological condition or for the generation of platelets for the treatment of an individual with a haematological condition (Cell Mol Life Sci. 2012 Apr. 24).

[0047] A population of pluripotent stem cells for use in the present methods, for example human pluripotent stem cells, may be obtained by culturing cells from a pluripotent cell line, using conventional techniques (Vallier, L. et al Dev. Biol. 275, 403-421 (2004), Cowan, C. A. et al. N. Engl. J. Med. 350, 1353-1356 (2004), Joannides, A. et al. Stem Cells 24, 230-235 (2006) Klimanskaya, I. et al. Lancet 365, 1636-1641 (2005), Ludwig, T. E. et al. Nat. Biotechnol. 24, 185-187 (2006)). For example, human pluripotent cells suitable for use in the present methods may be conventionally cultured in a culture dish on a layer of feeder cells, such as irradiated mouse embryonic fibroblasts (MEF), at an appropriate density (e.g. 10.sup.5 to 10.sup.6 cells/60 mm dish), or on an appropriate substrate with feeder conditioned or defined medium. Human pluripotent cells for use in the present methods may be passaged by enzymatic or mechanical means. Suitable culture media for human pluripotent cells include SC medium (Knockout Dulbecco's Modified Eagle's Medium (KO-DMEM) supplemented with 20% Serum Replacement, 1% Non-Essential Amino Acids, 1 mM L-Glutamine, 0.1 mM .beta.-mercaptoethanol and 4 ng/ml to 10 ng/ml human bFGF) and ES medium (DMEM/F12 supplemented with 20% knockout serum replacement (KSR), 6 ng/ml FGF2 (PeproTech), 1 mM L-Gln, 100 .mu.m non-essential amino acids, 100 .mu.M 2-mercaptoethanol, 50 U/ml Penicillin and 50 mg/ml Streptomycin).

[0048] A population of pluripotent stem cells for use in the present methods, for example human pluripotent stem cells, is preferably substantially free from one or more other cell types.

[0049] Before introduction of feeder cells, the population of isolated pluripotent stem cells may be expanded. For example, the human pluripotent stem cells may be cultured in a monolayer under conditions that simulate FGF2 signalling. In some embodiments, the cells may be cultured in a culture medium supplemented with FGF2 (e.g. 5 to 20 ng/ml FGF2, preferably 10 ng/ml). Suitable culture media include the SC and ES media described above, which may be MEF-conditioned and supplemented with FGF2.

[0050] Any mammalian FGF2 may be employed, preferably human fibroblast growth factor 2(FGF2) (NCBI Gene ID: 2247, nucleic acid sequence NM_002006.3 GI: 41352694, amino acid sequence NP_001997.4 GI: 41352695). FGF2 may be produced using routine recombinant techniques or obtained from commercial suppliers (e.g. R&D, Minneapolis, Minn., USA).

[0051] As described above, pluripotent cells are typically cultured and maintained on MEF feeder cells and may be separated from the feeder cells by any suitable technique. For example, the cells may be briefly (e.g. one hour) cultured on gelatin, and then the human pluripotent cells, which do not adhere to the gelatin separated from the MEFs which do adhere to the gelatin. In most experimental settings, human pluripotent stem cells have been cultivated in CDM on gelatin coated dishes (Vallier et al Curr Protoc Stem Cell Biol. 2008 March; Chapter 1:Unit 1D.4.1-1D.4.7) in presence of FGF2 and Activin-A.

[0052] In some embodiments, the PSCs may have a defined genotype, such as a defined HLA haplotype. For example, the PSCs may be or have been subjected to biological characterisation, such as HLA genotyping. A method described herein may comprise providing a population of PSCs having a defined genotype, such as a defined HLA haplotype. For example, a population of individuals may be HLA/ABO typed and skin biopsy or peripheral blood from selected individuals in the population with particular HLA/ABO genotypes may be used to generate iPSC lines for forward programming as described herein. Suitable methods of HLA/ABO typing are well-known in the art.

[0053] Forward programming is the direct imposition of a more differentiated phenotype on a pluripotent stem cell or other precursor cell which bypasses normal differentiation pathway; i.e. the cell does not pass through intermediate stages of differentiation. For example, a PSC which is forward programmed into a megakaryocyte progenitor does not differentiate through all of the mesoderm progenitor, haemogenic endothelium progenitor and hematopoietic progenitor stages before displaying the megakaryocyte progenitor phenotype. In other words, during step (iii), the cells said population may not progressively display each of the mesoderm progenitor, haemogenic endothelium progenitor and hematopoietic progenitor phenotypes. In some embodiments, a PSC may be differentiated into a mesoderm progenitor cell as described herein and the mesoderm progenitor cell may be forward programmed into a megakaryocyte progenitor.

[0054] Mature megakaryocytes are non-proliferative bone marrow cells which are responsible for the production of platelets. Mature megakaryocytes may have the phenotype CD34+/-, CD61+, CD41a+, CD42a+, CD42b+, GPVI+ or CD61+, CD41a+, CD42a+, CD42b+, GPVI+, CD235a+/-. Mature megakaryocytes are large (20-100 um) polyploid cells (4-128N) which eventually produce platelets through pro-platelet formation. They include megakaryoblast, pro-megakaryocyte and megakaryocyte stages as described in Journal of Thrombosis and Haemostasis, 5 (Suppl. 1): 318-327.

[0055] Megakaryocyte progenitor cells are proliferative precursors of mature megakaryocytes which undergo a final differentiation step to form mature megakaryocytes. Megakaryocyte progenitors may have the phenotype CD235a+/-, CD34+/-, CD61+, CD41a+, CD42a-, CD42b-, GPVI-. They include BFU-MKs (burst forming units-megakaryocytic), CFU-MKs (colony forming units-megakaryocytic) and PMKBs (promegakaryoblasts) as described in Journal of Thrombosis and Haemostasis, 5 (Suppl. 1): 318-327.

[0056] A megakaryocyte progenitor phenotype may include surface expression of CD235a, CD34, CD61 and CD41a or CD34, CD61 and CD41a and may not include expression of CD42a, CD42b and GPVI.

[0057] PSCs are forward programmed to become megakaryocyte progenitors in the methods described herein through the introduction of a specific combination of transcription factors, which causes the intracellular levels of the transcription factors in the PSCs to be increased.

[0058] The combination of transcription factors may be introduced into the PSCs in the form of nucleic acids (Warren L et al. Cell Stem Cell. 2010 Nov. 5; 7(5):618-30) or proteins (Zhou H, et al Cell Stem Cell. 2009 May 8; 4(5):381-4) by any suitable technique, including plasmid or more preferably, viral transfection, direct protein delivery or direct delivery of nucleic acid, such as mRNA. Following introduction of the reprogramming nucleic acids or proteins, the population of treated cells may be cultured.

[0059] The combination of transcription factors, for example GATA1, FLI1 and TAL1 and optionally one or more additional transcription factors, may be introduced into the PSCs by expressing nucleic acid encoding the combination of transcription factors in the PSCs. For example, the nucleic acid may be operably linked to inducible or non-inducible regulatory elements within a suitable vector, for example a retroviral or lentiviral vector, for expression within the cells. Vectors containing the nucleic acid are then transfected into the PSCs. Any convenient technique for the transfection may be employed. Following transfection, the combination of transcription factors is expressed in the PSCs and programs the PSCs to become megakaryocyte progenitors.

[0060] In some embodiments, transposon-mediated or other random integration transgenesis techniques may be employed. Reprogramming cells through expression of nucleic acid encoding one or more transcription factors is well-known in the art (Takahashi et al 2007; Takahashi et al 2007; Seki et al 2010; Loh et al 2010; Staerk et al 2010).

[0061] In some preferred embodiments, the PSCs may be programmed to become megakaryocyte progenitors with minimal or no genetic modification to the cells. Suitable techniques are known in the art and include the use of excisable lentiviral and transposon vectors; repeated application of transient plasmid, episomal and adenovirus or adeno-associated vectors or; the use of small molecules, synthetic mRNA and/or microRNAs (Sidhu K S. Expert Opin Biol Ther. (2011) May; 11(5):569-79; Woltjen K et al (2009) Nature 458 (7239):766-70; Chou B K et al. Cell Res. 2011 21(3):518-29).

[0062] In other embodiments, the combination of transcription factors, for example GATA1, FLI1 and TAL1 and optionally one or more additional transcription factors, for example one or more transcription factors from Table 1 and/or Table 2, may be introduced into the PSCs by contacting transcription factor proteins or transcription factor nucleic acids, such as mRNAs encoding transcription factors, with the population of PSCs. Programming cells though contact with transcription factor nucleic acids (Warren L et al. Cell Stem Cell. 2010 Nov. 5; 7(5):618-30) or proteins (Zhou H, et al Cell Stem Cell. 2009 May 8; 4(5):381-4) is well-known in the art and any suitable technique may be employed. For example, the combination of transcription factor proteins or nucleic acids may be cultured in the presence of the PSCs under conditions which allow for entry of the proteins or nucleic acid into the cell. In some embodiments, entry of transcription factor proteins into the cell may be facilitated by a membrane penetrating peptide, which may be linked or attached to the transcription factor proteins. The combination of transcription factor proteins or nucleic acids may be introduced into the PSCs by traditional methods such as lipofection, electroporation, calcium phosphate precipitation, particle bombardment and/or microinjection, or may be delivered into cells by a protein delivery agent. For example, the combination of transcription factor proteins or nucleic acids can be introduced into cells by covalently or non-covalently attached lipids, e.g. a myristoyl group.

[0063] Transcription factor nucleic acids for direct delivery into PSCs may be translatable by endogenous translation factors within the cell. Suitable synthetic mRNAs may be modified. For example, 5-methylcytidine may be substituted for cytidine, and pseudouridine for uridine, followed by phosphatase treatment to produce the transcription factor nucleic acids (Zhou H, et al 2009).

[0064] In other embodiments, the combination of transcription factors, for example GATA1, FLI1 and TAL1 and optionally one or more additional transcription factors, for example one or more transcription factors from Table 1 and/or Table 2, may be introduced into the PSCs by activating expression of endogenous nucleic acid sequences encoding the transcription factors in the population of PSCs. Suitable techniques for endogenous gene activation include Zinc Finger or Transcription like Activator (TAL) techniques and are well established in the art (see for example Hum Gene Ther. 2012 May 15; Zhang P et al. Hum Gene Ther. 2012 November; 23(11):1186-99).

[0065] In preferred embodiments, the PSCs are forward programmed in a chemically defined medium (CDM). A CDM is a nutritive solution for culturing cells which contains only specified components, preferably components of known chemical structure. A CDM is devoid of components which are not fully defined, for example serum or proteins isolated therefrom, such as Foetal Bovine Serum (FBS), Bovine Serum Albumin (BSA), and feeder or other cells. In some embodiments, a CDM may be humanised and may be devoid of components from non-human animals. Proteins in the CDM may be recombinant human proteins Suitable CDMs are well known in the art and described in more detail below.

[0066] Media and ingredients thereof may be obtained from commercial sources (e.g. Gibco, Roche, Sigma, Europabioproducts, Cellgenix, Life Sciences). In a humanised CDM, for example BSA may be replaced in CDM by Polyvinyl alcohol (PVA), human serum albumin, Plasmanate.TM. (human albumin, alpha-globulin and beta globulin: Talecris Biotherapeutics NC USA) or Buminate.TM. (human albumin: Baxter Healthcare), all of which are available from commercial sources.

[0067] Suitable CDMs include Knockout (KS) medium supplemented with 4 ng/ml FGF.sub.2; Knockout Dulbecco's Modified Eagle's Medium (KO-DMEM) supplemented with 20% Serum Replacement, 1% Non-Essential Amino Acids, 1 mM L-Glutamine, 0.1 mM p-mercaptoethanol and 4 ng/ml to 10 ng/ml human FGF2; and DMEM/F12 supplemented with 20% knockout serum replacement (KSR), 6 ng/ml FGF2 (PeproTech), 1 mM L-Gln, 100 .mu.m non-essential amino acids, 100 .mu.M 2-mercaptoethanol, 50 U/ml penicillin and 50 mg/ml streptomycin and TeSR (Ludwig et al Nat Biotech 2006 24 185).

[0068] Other suitable CDM which may be used in accordance with the present methods are known in the art (e.g. N12 medium, Johansson and Wiles CDM; Johansson and Wiles (1995) Mol Cell Biol 15, 141-151). Suitable humanised CDMs may comprise a basal culture medium, such as IMDM and/or F12 supplemented with insulin, for example at 0.5 .mu.g/ml to 70 .mu.g/ml, transferin, for example at a concentration of 1.5 .mu.g/ml to 150 .mu.g/ml, an antioxidant, such as 1-thiolglycerol, for example at a concentration of 45 .mu.M to 4.5 mM, lipids, and one or more of human serum albumin, polyvinyl alcohol (PVA), Plasmanate.TM. (human albumin, alpha-globulin and beta globulin: Talecris Biotherapeutics NC USA) or Buminate.TM. (human albumin: Baxter Healthcare), for example at a concentration of 0.5 mg/ml to 50 mg/ml. For example, humanised CDM include humanised Johansson and Wiles CDM, which consists of: 50% IMDM (Gibco) plus 50% F12 NUT-MIX (Gibco); 7 .mu.g/ml insulin; 15 g/ml transferrin; 5 mg/ml human serum albumin, polyvinyl alcohol (PVA), Plasmanate.TM. or Buminate.TM.; 1% chemically defined lipid concentrate (Invitrogen); and 450 .mu.M 1-thiolglycerol. Another suitable chemically defined medium may comprise 50% IMDM, 50% F12 NUT-MIX, 7 .mu.g/ml insulin, 15 g/ml transferrin, 1% chemically defined lipid concentrate, 5 mg/ml human serum albumin or Polyvinyl Alcohol (PVA) and 450 .mu.M 1-thiolglycerol. Another suitable chemically defined medium is CellGRO SCGM.TM. which is commercially available (Cellgenix, DE).

[0069] Following the introduction of the combination of reprogramming factors into the PSCs, the cells may be cultured in a pluripotency cell culture medium, for example CDM with Activin-A and FGF2, or mesodermal cell culture medium, for example CDM supplemented with BMP4 (e.g. rh-BMP4 at 10 ng/ml) and/or FGF2 and/or LY294002 for 1 or more, 2 or more, 3 or more, 4 or more, or 5 or more days, preferably about 2 days.

[0070] The cells may then be cultured in an appropriate megakaryocyte (MK) programming medium. For example, population of cells may be cultured in a chemically defined medium (CDM) supplemented with TPO (Thrombopoietin) and/or SCF (Stem Cell Factor) and/or IL1B, (all preferably recombinant human proteins).

[0071] The amino acid sequences of TPO, SCF and IL1B are readily available on public databases. For example, the reference amino acid sequence of human TPO (Thrombopoietin: also known as THPO: Gene ID 7066) has the NCBI database entry NP_000451.1 GI:4507493; the reference amino acid sequence of human SCF (Stem Cell Factor: also known as KITLG: Gene ID 4254) has the NCBI database entry NP_000890.1 GI:4505175 and the reference amino acid sequence of human IL1B (Interleukin 1 beta: Gene ID 3553) has the NCBI database entry NP_000567.1 GI:10835145.

[0072] TPO, SCF and IL1B may be produced by synthetic or recombinant means or obtained available from commercial suppliers (e.g. R&D Systems, Minneapolis, Minn., USA; Sigma-Aldrich Co. LLC USA, EMD Millipore MA USA). For example, the pluripotent cells may be cultured by a method comprising;

[0073] (i) culturing said pluripotent cells in mesoderm medium comprising FGF2 and BMP4; and,

[0074] (ii) further culturing said cells in MK programming medium comprising TPO and/or SCF, preferably TPO and SCF.

[0075] Suitable media are known in the art (Cell Stem Cell. 2011 Aug. 5; 9(2):144-55; Dev Cell. 2011 May 17; 20(5):597-609; Blood. 2008 Jun. 1; 111(11):5298-306) and are described in more detail below.

[0076] Suitable cell culture conditions are well known in the art (Vallier, L. et al Dev. Biol. 275, 403-421 (2004), Cowan, C. A. et al. N. Engl. J. Med. 350, 1353-1356 (2004), Joannides, A. et al. Stem Cells 24, 230-235 (2006) Klimanskaya, I. et al. Lancet 365, 1636-1641 (2005), Ludwig, T. E. et al. Nat. Biotechnol. 24, 185-187 (2006)).

[0077] Methods for culturing mammalian cells are well-known in the art (see, for example, Basic Cell Culture Protocols, C. Helgason, Humana Press Inc. U.S. (15 Oct. 2004) ISBN: 1588295451; Human Cell Culture Protocols (Methods in Molecular Medicine S.) Humana Press Inc., U.S. (9 Dec. 2004) ISBN: 1588292223; Culture of Animal Cells: A Manual of Basic Technique, R. Freshney, John Wiley & Sons Inc (2 Aug. 2005) ISBN: 0471453293, Ho W Y et al J Immunol Methods. (2006) 310:40-52, Handbook of Stem Cells (ed. R. Lanza) ISBN: 0124366430). Media and ingredients thereof may be obtained from commercial sources (e.g. Gibco, Roche, Sigma, Europa bioproducts, R&D Systems). Standard mammalian cell culture conditions may be employed, for example 37.degree. C., 21% Oxygen, 5% Carbon Dioxide. Culture medium is preferably changed every two days and cells allowed to settle by gravity.

[0078] The population of pluripotent stem cells may be cultured for at least 7 days after introduction of the combination of transcription factors.

[0079] Megakaryocyte progenitors may be identified in the cell culture after at least 4, 5, 6, or 7 or more days.

[0080] In some embodiments, a method may comprise identifying or confirming the identity of the megakaryocyte progenitor cells or mature megakaryocytes in the culture.

[0081] In some embodiments, cells may be tested for presence of cell markers associated with the megakaryocyte progenitor cells, for example to identify or confirm their identity. Cells which express the markers may be identified as megakaryocyte progenitor cells. For example, megakaryocyte progenitor cells may identified by expression of CD34 and CD41a as described above but no expression of CD42a and CD42b.

[0082] Megakaryocyte progenitor cells do not express the pluripotency associated markers, such as Oct4, Sox2, Alkaline Phosphatase, SSEA-3, Nanog, SSEA-4 and Tra-1-60, which are expressed by PSCs or display reduced expression relative to PSCs.

[0083] A method may further comprise isolating and/or purifying the forward programmed megakaryocyte progenitor cells. Megakaryocyte progenitors may be separated from other cell types in the population using any technique known to those skilled in the art, including those based on the recognition of extracellular epitopes by antibodies and/or magnetic beads or fluorescence activated cell sorting (FACS), including the use of antibodies against extracellular regions of characteristic markers.

[0084] The megakaryocyte progenitor cells may be cultured and/or expanded to generate a homogenous or substantially homogenous population of cells. Suitable techniques for mammalian cell culture are well known in the art and described elsewhere herein.

[0085] A method may comprise monitoring or detecting the expression of one or more megakaryocyte progenitor cell markers and/or one or more pluripotent cell markers in cells in the population. This allows the extent of forward programming in the population to be determined as it is cultured.

[0086] At least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50% or at least 60% of the population of PSCs may become megakaryocyte progenitor cells following forward programming as described herein.

[0087] Megakaryocyte progenitor cells produced by the present methods may be substantially free from other cell types. For example, a population of megakaryocyte progenitors produced by a method described herein may contain 80% or more, 85% or more, 90% or more, or 95% or more megakaryocyte progenitor cells, following culture.

[0088] The population of megakaryocyte progenitor cells may be cultured and/or expanded and optionally stored.

[0089] Following the production of a population of megakaryocyte progenitor cells by forward programming, the methods described herein may further comprise allowing the population of megakaryocyte progenitor cells to differentiate into mature megakaryocyte cells, for example by culture in a megakaryocyte maturation medium.

[0090] For example, megakaryocyte progenitor cells may be passaged, for example from day 7 or day 10, into suspension culture plastic dishes in a megakaryocyte maturation medium comprising CDM (e.g. CellGRO SCGM.TM.) supplemented by TPO and/or IL1-beta and/or SCF; and then cultured for an additional 5-7 days or 5-15 days, up to 10-15 days.

[0091] In some embodiments, the megakaryocyte progenitor cells may be cultured in a megakaryocyte maturation medium comprising CDM (e.g. CellGRO SCGM.TM.) supplemented by TPO and/or IL1-beta and/or SCF, preferably TPO and IL1-beta, from day 10 onwards for an additional 5-7 days or 8-12 days, preferably 10 days.

[0092] Typically, the cells are cultured for 18 to 22 days from TF transduction, preferably about 20 days, to produce mature megakaryocyte cells.

[0093] The mature megakaryocyte cells may be isolated, purified and/or stored according to standard techniques.

[0094] In preferred embodiments, a population of mature megakaryocyte cells produced by a method described above may be pure or substantially pure and may not require further sorting or purification. For example, at least 80%, at least 90% or at least 95% of cells in the population of mature megakaryocyte cells may express CD41a (i.e. CD41a+ cells). At least 30%, at least 40% or at least 50% of cells in the population may express CD42a (i.e. CD42a+ cells). In some preferred embodiments, at least 95% of cells in the population express CD41a and at least 50% of cells in the population may express CD42a.

[0095] In some embodiments, mature megakaryocyte cells produced as described herein may be used in the production of platelets. For example, a method described herein may comprise allowing one or more of the mature megakaryocyte cells to produce platelets.

[0096] Another aspect of the invention provides a population of megakaryocyte progenitor cells or mature megakaryocytes produced as described herein.

[0097] Populations of megakaryocyte progenitor cells and mature megakaryocytes produced by forward programming as described herein are described in more detail above.

[0098] Megakaryocyte progenitor cells produced by forward programming as described herein may be highly proliferative (for example over 28 days or more).

[0099] Mature megakaryocyte cells produced by forward programming and differentiation as described herein may generate and/or release functional platelet like particles (PLPs) in vitro.

[0100] Another aspect of the invention provides the use of a population of megakaryocyte progenitor cells or mature megakaryocytes produced as described herein in the production of platelets.

[0101] Another aspect of the invention provides megakaryocyte progenitor cells or mature megakaryocytes produced as described herein for use in methods of treatment of haematological conditions as described herein and methods of treatment of haematological conditions which comprise administering megakaryocyte progenitor cells or mature megakaryocytes produced as described herein to an individual in need thereof.

[0102] Megakaryocyte progenitor cells or mature megakaryocytes produced as described herein may also be useful in screening. Screening may include drug or small molecule screening. For example, the isolated programmed cells may be contacted with a test compound and the effect of the test compound on the cells is determined. Screening may also include functional genomic screening. For example, a gene may be suppressed, knocked out or otherwise inactivated in the isolated reprogrammed cells and the effect of the inactivation on the cells determined.

[0103] In some embodiments, megakaryocyte progenitor or mature megakaryocytes cells may be produced from iPS cells as described herein. The iPS cells may be derived from normal differentiated cells or from differentiated cells having a disease phenotype or genotype, for example from an individual with a disease condition. After programming, the megakaryocyte progenitor cells or mature megakaryocytes may express a detectable reporter or display an observable cellular phenotype which differs between disease-affected cells and normal cells. The megakaryocyte progenitor cells or mature megakaryocytes may be exposed to test compounds and the effect of the test compound on the reporter expression or observable cellular phenotype determined. Compounds which cause the megakaryocyte progenitor cells or mature megakaryocytes to revert from disease cell state to the normal state may be identified. Alternatively, the one or more genes in the megakaryocyte progenitor cells or mature megakaryocytes may be inactivated, for example by targeted mutation or RNAi suppression, and the effect of the inactivation on the reporter expression or observable cellular phenotype determined. Genes whose inactivation causes the cells to revert from disease cell state to the normal state may be identified.

[0104] Screening may include toxicology screening. For example, the isolated megakaryocyte progenitor cells may be contacted with a test compound at various concentrations that mimic abnormal/normal concentrations in vivo. The effect of the test compound on the cells may be determined and toxic effects identified. Toxicology screening is well known in the art (see for example Barbaric I et al. Biochem Soc Trans. 2010 August; 38(4):1046-50).

[0105] Forward programmed megakaryocyte progenitor cells (and cells derived from the programmed cells, such as mature megakaryocytes and platelets) may also be used for the treatment of an individual, for example for the treatment of a platelet or megakaryocyte related condition. The individual may be the same individual from whom the original IPS cells were obtained.

[0106] In some embodiments, the forward programmed megakaryocyte progenitor cells or mature megakaryocytes may, for example, be admixed with a pharmaceutical acceptable carrier in a pharmaceutical composition. The composition may be administered to the individual ((Leukemia. 2008 January; 22(1):203-8).

[0107] Forward programmed megakaryocyte progenitor cells and cells derived from the programmed cells, such as mature megakaryocytes, may also be used for disease modelling. For example, cells may be programmed into megakaryocyte progenitors which are affected in a disease condition, either directly or by differentiating into the affected megakaryocytes or platelets. The effect of the mutation on the cellular phenotype may be studied and the genetic and/or biochemical interactors that contribute to the cellular pathology of the disease may be identified and/or characterised.

[0108] Another aspect of the invention provides a method of screening for a compound useful in the treatment of a disease condition, in particular a haematological condition, for example a thrombocytopenic or other platelet-related condition, including essential thrombocytosis, congenital amegakaryocytic thrombocytopenia (CAMT), Thrombocytopenia-absent radius syndrome (TAR), Bernard Soulier syndrome (BSS), Gray platelet syndrome (GPS) and Glanzmann thrombasthenia, comprising;

[0109] contacting a population of megakaryocyte progenitor cells produced by a method described above with a test compound, and;

[0110] determining the effect of the test compound on said cells and/or the effect of said reprogrammed cells on the test compound.

[0111] Suitable forward programmed megakaryocyte progenitor cells are described above.

[0112] The megakaryocyte progenitor cells may display a disease phenotype and the effect of the test compound on one or more disease pathologies in the reprogrammed cells may be determined. A decrease or amelioration of one or more disease pathologies in the reprogrammed cells in the presence, relative to the absence of test compound is indicative that the test compound may be useful in the treatment of the disease in the individual.

[0113] Suitable disease conditions and phenotypes are described above.

[0114] The forward programmed megakaryocyte progenitor cells may display a normal phenotype and the effect of the test compound on the growth, differentiation or viability of the reprogrammed cells or the ability of the reprogrammed cells to perform one or more cell functions may be determined. In some embodiments, cells may be modified to express reporters that can be used to measure particular cell functions or attributes. A decrease in growth, viability or ability to perform one or more cellular functions may be indicative that the compound has a cytotoxic effect (see for example, Barbaric I et al Biochem Soc Trans. 2010 August; 38(4):1046-50).

[0115] The data set out herein shows that GATA1 and FLI1 are also able to drive forward programming in the absence of TAL1. Other aspects and embodiments of the invention provide all of the aspects and embodiments described above with the transcription factor TAL1 omitted from the combination of transcription factors.

[0116] Other aspects and embodiments of the invention provide the aspects and embodiments described above with the term "comprising" replaced by the term "consisting of" and the aspects and embodiments described above with the term "comprising" replaced by the term "consisting essentially of".

[0117] Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

[0118] All documents and database entries which are mentioned in this specification are incorporated herein by reference in their entirety for all purposes.

[0119] The transcription factor symbols and names set out herein are the unique official HUGO Gene Nomenclature Committee (HGNC) symbols and names that have been assigned to that transcription factor (see Gray K A et al Nucleic Acids Res. 2013 Jan. 1; 41(D1):D545-52; and the HGNC Database, HUGO Gene Nomenclature Committee (HGNC), EMBL Outstation--Hinxton, European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire, CB10 1SD, UK).

[0120] "and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and/or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

[0121] Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments of the invention which are described. Thus, the features set out above are disclosed for use in the invention in all combinations and permutations.

[0122] Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the figures and tables described herein.

[0123] FIG. 1 shows the internal biological interactions of the top 20 TF candidates identified using VisANT, highlighting the 9 tested genes in this study (in dark) and the GATA1 centred network (grey shade). Tested combinations are indicated in the adjacent table.

[0124] FIG. 2 shows a comparison between 14TF and 9TF combinations for MK-FoP of the H9 ES line (hESC #1).

[0125] FIG. 3 shows transgene expression levels quantified by RT-QPCR in flow cytometry sorted CD41a+ cells generated 7 days after lentiviral transduction of the hESC #1 line with the 9 TFs on fibronectin coated plates and maintained in pluripotent medium (FGF2+Activin-A) for 2 days followed by MK medium (TPO+SCF) for 5 days.

[0126] FIG. 4 shows the fold increase of CD41a+ cells relative to the 9-TFs combination (mean.+-.sem, n=3/3/1/2 respectively) in hESC #1 cells transduced with different combinations of TFs using a 2 day mesoderm induction and measured by flow cytometry at day 7. 3TFs combination is shown as the most effective for MK-FoP.

[0127] FIG. 5 shows the fold increase of CD41a+ cells relative to the 9-TFs in hESC #1 cells transduced with different combinations of TFs under pluripotent conditions without mesoderm induction and measured by flow cytometry at day 7. 3TFs combination is shown as the most effective for MK-FoP.

[0128] FIG. 6 shows the fold increase of CD41a+ cells relative to the 3-TFs combination (mean.+-.sem, n=2) in hiPSC #1 cells transduced with all permutations of the 3-TFs combination using 2 days mesoderm induction and measured by flow cytometry at day 7.

[0129] FIG. 7 shows real time quantitative PCR analysis of MK gene expression at day 7 in an unsorted population: HES3 (hESC #2) ESC line before and after forward programming protocol (21% CD41a+ at day 7). ES: pluripotent ESC line; GFP: GFP transduced cells were cultivated in the same conditions as for 3TFs transduced cells.

[0130] FIG. 8 shows the megakaryocytic potential of cells sorted by flow cytometry based on CD41a expression at day 7 following transduction of hiPSC #2 line with the 3-TFs combination. The megakaryocytic potential of sorted cells was tested using the Megacult clonogenic assay (1,000 cells in duplicate). A representative megakaryocyte colony obtained from a CD41a+ cell and co-expressing CD41a and CD42b as detected by immunofluorescence is shown.

[0131] FIG. 9 shows real-time quantitative PCR analysis of MK gene expression at day 7 in sorted populations following forward programming of the hESC #1 line.

[0132] FIG. 10 shows a time course flow analysis of the mean values+/-SD of surface marker expression using forward programming protocol on the hiPSC #1 and #2 lines.

[0133] FIG. 11 shows the results of cytospin and Romanowsky staining: polyploid MK are produced from forward programmed hiPSC #1 and #2 lines (BOB and BBHX) and are similar to cord blood derived MKs.

[0134] FIG. 12 shows the percentages of CD41a expressing cells produced when human pluripotent stem cells sown on fibronectin coated plates were transduced in parallel with the 3-TFs and kept for 2 days in pluripotent (FGF2+Activin-A) or mesoderm (FGF2+BMP4+LY294002) medium followed by MK medium (TPO+SCF) for 6 days.

[0135] FIG. 13 shows histograms representing the CD41a+ cells fold increase relative to the hiPSC input (mean.+-.sem, n=4/3 respectively) when the hiPSC #2 line was sown in fibronectin coated plates (2-D culture) or induced to form embryoid bodies by forced aggregation of single cells and subsequently transduced with the 3-TFs. Cells were kept 2 days in mesoderm medium and 5 days in MK medium.

[0136] FIG. 14 shows the morphology of day 20 hiPSC-MKs and CB-MKs analysed after Romanowsky staining. Arrowheads point to cells showing several nuclei. Scale bars, 100 um.

[0137] FIG. 15 upper panel shows histograms representing the percentages of CD41a+ cells generated at day 10 (mean.+-.sem, n=11/6/2/1 respectively) from a variety of hiPSC lines using the optimised 3-TFs forward programming protocol. Lower panel shows cell fold expansion at day 20 for total and mature megakaryocytes (CD41a and CD42b positive cells respectively) relative to the hiPSC input (mean.+-.sem, n=6/6/2 respectively). ND not done.

[0138] FIG. 16 shows histograms representing the amount of PLPs generated per hiPSC-MK input after hiPSC #1 day 20 MKs were sowed in parallel on different stromal cell lines.

[0139] FIG. 17 presents Table 1, which shows 46 candidate TFs selected upon analysis of protein-protein interaction network using VisANT software, integrating interactions with chromatin remodelling factors and level of gene expression. 14 transcription factor candidates cloned into lentiviral vector backbones are highlighted.

[0140] Table 2 shows 13 TFs identified from differential expression between cord blood or peripheral blood derived megakaryocytes and hiPSC-MKs

[0141] Table 3 shows an enrichment analysis for biological processes using the DAVID bioinformatics resource which indicates significant enrichment for megakaryocyte/platelet function genes in hiPSC-MKs compared to the starting hiPSCs. Input gene expression dataset from CD42b sorted hiPSC-MKs (line #1 and #2) and hISPC pluripotnet culture analysed on an Illumina Human HT-12 v4 BeadArray (hiPSC #1-MK, n=4; hiPSC #2-MK, n=2; hiPSC #1, n=2).

1. Materials and Methods

1.1 Cell Lines

[0142] The human embryonic stem cell lines HES3 and H9 (from ES Cell International, Singapore and Wicell, Madison respectively) and the human iPSC lines #1-4 (A1ATD1, BBHX8, iPS40 and S4-SF5 respectively, obtained from the Cambridge Biomedical Research Centre iPSC Core Facility) are cultivated at 37 C/5% CO2 in chemically defined culture conditions as described previously (Curr Protoc Stem Cell Biol. 2008 March; Chapter 1:Unit 1D.4.1-1D.4.7). Briefly, cells are maintained in a chemically defined basal medium (CDM) supplemented with recombinant human FGF2 (12 ng/ml, University of Cambridge) and Activin-A (15 ng/ml, University of Cambridge) on feeder free gelatin coated wells as previously described (Curr Protoc Stem Cell Biol. 2008 March; Chapter 1:Unit 1D.4.1-1D.4.7) with medium changes daily. Subculture is performed every 5-7 days by detaching pluripotent colonies by incubation in a dispase/collagenase-IV mix (1 mg/ml, Sigma Aldrich) for 45 minutes at 37.degree. C., collecting detached colonies, breaking them down into small clumps carefully pipetting up and down with a P1000 tip and plating them onto new gelatin coated plastic dishes.

[0143] Human iPSC line derivation has been performed under appropriate ethical approval and volunteer consent were obtained (Ethics reference no. 08/H0311/201; R&D no. A091485). The iPSC lines have been derived from adult dermal fibroblasts using murine oncoretroviral vectors (hiPSC #1-3) or Sendai vectors (hiPSC #4) expressing the human OCT4, SOX2, KLF4 and MYC reprogramming factors and following subculture steps on irradiated mouse embryonic feeder cells in 20% KSR medium supplemented by rh-FGF2 as previously described.

1.2 Transcription Factor Candidate Selection

[0144] We performed a differential expression analysis focused on DNA binding protein coding genes (PANTHER) from whole genome expression data generated in the H9 hESC line (internal data, Illumina HumanWG-6 v3) and human cord blood derived MKs (HaemAtlas, Blood. 2009; 113:e1-9, Illumina HumanWG-6 v2). The list of 116 MK specific genes generated was further refined by removal of 21 histone coding genes and addition of 6 genes with known or potential roles in megakaryopoiesis. Using the VisANT web-based software (Nucleic Acids Res. 2005; 33:W352-357) for analysing networks of biological interactions, the resulting 101 candidate genes were subsequently ranked based on number of 1) internal protein interactions, 2) interaction with epigenome modifiers (HAC, HDAC, DNMT) and 3) differential expression levels. Weakly differentially expressed genes (log 2(MK-ESC)<1) were excluded from the candidate list. The final gene candidate list is eventually made of 46 factors ranked (from highest to lowest value) on 1) number of internal interactions 2) number of nodes with chromatin remodelling factors 3) differential expression value. Exceptions in the final list are the MLL and MLL3 genes which were removed since they have coding sequences of over 10 kb and are incompatible with lentiviral vectors. MCM7 was also removed since it is not known to act as a transcription factor (mini-chromosome maintenance complex). CLOCK has been added despite a low expression value (0.89) because it has reported Histone Acetyl Transferase activity.

[0145] A heatmap of differentially expressed transcription factors between cord blood or peripheral blood derived megakaryocytes and hiPSC-MKs was generated using Heatmap Builder v1.1 and a row normalised sorting algorithm (cut-off >2-fold increase, FDR1%). Each line of the heatmap represented a gene specific probe on the Illumina Human HT-12 v4 Beadarrays. Mean values of CB-MK (n=4), PB-MK (n=2), hiPSC #1 (n=4) and hiPSC #2 (n=2). The candidate list was then reduced to the 13 TF genes showing very low/null expression in hiPSC-MKs (log 2<8). These factors may play an important role in further maturation of hiPSC-MKs

1.3 Transcription Factor Cloning into Lentiviral Backbone

[0146] The full human coding sequences of the 9 candidate genes (CDS of transcript variants 1, NCBI Refseq) including the 5' Kozak concensus sequence were generated by PCR using cDNA from cord blood derived MKs as template. PCR fragments were subsequently individually cloned into the pWPT lentiviral vector backbone (Trono laboratory, Addgene 12255) in place of the eGFP coding sequence in-between the MluI and SalI restriction sites, downstream of the human EF1-alpha ubiquitous promoter. Final constructs has been checked for sequence integrity against the NCBI Refseq library before use for lentiviral vector production. Replication deficient lentiviral vector particles (LVPs) were produced by transient co-transfection of HEK 293T/17 cells (ATCC CRL-11268) with the generated pWPT constructs along with the psPAX2 and pMD2.G helper plasmids (Trono laboratory, Addgene 12260 and 12259) using TranslT-LT1 transfection reagent (MirusBIO). Crude supernatants containing LVPs were collected 48 hours after transfection, filtered through a 0.45 um membrane and DNaseI treated before concentration by ultracentrifugation or PEG-based precipitation (LentiX-concentrator, Clontech). Functional LVPs titres were determined by QPCR measurement of provirus copy number in genomic DNA of transduced HCT116 cells (ATCC CCL-247) and were in the range of 1-10E+8 TU/ml for all vectors. Human PSC lines were routinely transduced using multiplicity of infection of 20 in presence of 10 micrograms Protamine Sulfate (Sigma, P4505) per millilitre of culture medium leading to 60-80% transduction efficiencies through experiments.

1.4 MK Forward Programming

Optimised Embryoid Body Based Protocol:

[0147] on transduction day (day 0), sub confluent (50-80%) human pluripotent stem cells were dissociated to single cells using TrypLE (Life Technologies) for 5 min at 37 C and viable cells counted on a haemocytometer. Embryoid body (EB) formation was initiated with 5.times.10.sup.5 to 1.times.10.sup.6 viable cells per well of an Aggrewell.TM.400 plate (Stemcell Technologies, France) in order to obtain embryoid bodies (EB) of 400 to 800 cells per EB following spin aggregation (detailed protocol in Aggrewell.TM. Technical manual).

[0148] Importantly, lentiviral transduction was performed concomitantly to the aggregation phase. Briefly, cells were added to the well in CDM supplemented by Y-27632 (10 microM, Sigma Aldrich), rh-BMP4 (10 ng/ml, R&D) and protamine sulfate (10 .mu.g/ml, Sigma). Concentrated lentiviral vectors individually coding for each forward programming factor were added to the well to MOI20 (multiplicity of infection). Subsequently, Aggrewell.TM. plates were centrifuged at 100 g for 3 minutes and put into the incubator (37 C/5% CO2) for 24 hours.

[0149] After 24 h (day 1), transduced EBs were collected and sowed in ultralow adherent cell culture plates (Corning) at a density of 600 EB per 10 cm2 dish in CDM supplemented with s rh-BMP4 (10 ng/ml, R&D) and rh-FGF2 (5 ng/ml, University of Cambridge).

[0150] EBs were collected 24 hours later (day 2) and further cultivated in ultralow adherent plates in Cellgro SCGM medium (Cellgenix, Germany) supplemented with rh-TPO (100 ng/ml, Cellgenix) and rh-SCF (25 ng/ml, Life Technologies).

[0151] At day 10, EBs were dissociated to single cells using CollagenaseIV and Dispasell (1 mg/ml, Gibco) followed by enzyme free cell dissociation buffer treatment (Gibco). Collected cells were cultivated on suspension culture plates (Grenier) for an additional 10 days in Cellgro SCGM supplemented with TPO (100 ng/ml) and IL1-beta (10 ng/ml, Miltenyi Biotec) for further MK maturation.

[0152] Culture medium is changed every three days by aspirating half of the volume and adding 2 times cytokine concentrated fresh medium on top.

Adherent Cell Protocol:

[0153] Small cell clumps were generated from sub-confluent hPSC cultures using a CollagenaseIV/Dispasell mix (1 mg/ml) and sowed on human fibronectin coated (50 ug/ml, Millipore) tissue culture plates in CDM with FGF (12 ng/ml) and Activin-A (15 ng/ml) at an approximated density of 2-5E+5 cells/10 cm2. Cells were transduced the day after with MOI20. The culture media used for the first two days were devised for pluripotency maintenance (as above) or mesoderm induction (FLyB; Bernardo et al. Cell Stem Cell. 2011 Aug. 5; 9(2):144-55) depending on experiments. The following days, cells were maintained in Cellgro SCGM supplemented with TPO (100 ng/ml) and SCF (25 ng/ml) until analysis.

1.5 Cord Blood Derived Megakaryocytes

[0154] Cord blood was obtained after informed consent under a protocol approved by the National Research Ethics Service. CD34-positive cells (298%) isolated by magnetic cell sorting (Miltenyi Biotec) were seeded at 1E+5 cells/ml in Cellgro SCGM with TPO (100 ng/ml) and IL1-beta (10 ng/ml) and incubated for 10 days. We routinely obtained 70-90% CD41a+ and 20-60% CD42a+ cells by the end of the culture.

1.6 Flow Cytometry Analysis

[0155] Flow cytometry experiments were performed on a CyAn ADP (Beckman Coulter). Single cell suspensions were generated using CollagenaseIV/Dispasell and/or enzyme free dissociation buffer when needed. Cells were stained for 20-30' at room temperature in PBS 0.5% BSA 2 mM EDTA using combinations of FITC, PE and APC conjugated antibodies (all from BD Pharmingen except anti-GP6 antibody from NHSBT-Bristol). Background fluorescence was set against matched isotype control antibodies and compensation matrix defined using single-colour stained cells. Flow count beads (Flow count fluorospheres, Beckman Coulter) and DAPI were used to determine viable cell count in samples.

1.7 Immunofluorescence Analysis

[0156] MKs were cultivated on human fibrinogen coated (50 ug/ml, Millipore) tissue culture plates for 48 hours to monitor proplatelet formation. Cells were fixed with 2% PFA and permeabilised/blocked with 0.1% Saponin/0.2% Gelatin. Cells were incubated with primary antibodies (anti alpha-Tubulin, Sigma; anti vwf, Dako; anti P-selectin, NHSBT-Bristol) at room temperature for 2 hours and secondary antibodies conjugated with Cy3 or Alexa-488 fluorochromes (Invitrogen Molecular Probes) for 45 minutes. Cell nuclei were stained with DAPI before image acquisition on a confocal Zeiss Axiovert 200M microscope.

1.8 Cell Morphology Analysis

[0157] Cells were spun on a glass slide using cytofunnels at 400 g for 5', methanol fixed and Romanowsky stained (eosin and methylene blue). Cells were observed on a phase contrast Axiovert Zeiss microscope (630.times. magnification).

1.9 Transmission Electron Microscopy

[0158] MKs were fixed in 2% glutaraldehyde 0.1M phosphate buffer for 60' at room temperature. After washing with phosphate buffer, the samples were post-fixed with 1% osmium tetroxide in phosphate buffer for 60' on ice, ethanol dehydrated and infiltrated with and embedded in Epoxy resin. Ultrathin sections (50 nm) were cut and stained with 2% uranyl acetate in methanol and Reynolds' lead citrate. Samples were read using a FEI Tecnai 12 (Philips) transmission electron microscope.

1.10 Gene Expression Analysis by RT-QPCR

[0159] Total RNA was extracted with RNeasy kits according to the manufacturer's instructions (Qiagen) including DNase treatment. cDNA was prepared from 250-500 ng RNA using Maxima First Strand cDNA Synthesis Kit and random hexamers (Fermentas). QPCR reactions were performed in duplicates using recommended SYBR green based PCR mixes on ABI 7500HT or Mx3000P real time thermal cyclers using 2-step amplification protocols (Applied Biosystems, Agilent Technologies). Relative gene expression was calculated with the 2.sup.-deltaCt method using HMBS for normalisation. Primer pairs were designed to amplify cDNA only, have no reported off targets after blasting against human Refseq and showed 80-120% PCR efficiencies. Endogene specific primers were designed to amplify UTR regions absent from transgene sequences while transgene specific primer pairs have 3' primers binding to viral sequences.

1.11 Whole Genome Expression Study

[0160] DNA free total RNA (RNeasy, Qiagen) was extracted from sorted CD42b+ cells (EasySEP, Stem cell Technologies; >95% purity) and 500 ng were hybridized to Illumina Human HT-12 v4 BeadArrays. Data import. Raw Illumina bead-level output was imported to the R statistical programming environment using functions of the beadarray package for the Bioconductor software suite. Data processing. Signal intensities were background corrected, summarized and converted to log 2 expression units using functionality of the beadarray package. Probe-sets without signal deemed significantly above background level in all profiles of at least one sample group (Illumina signal detection statistic p<0.01) were removed. Quantile normalization, implemented in the limma package for Bioconductor, was employed to equalize summarized expression intensity distributions across all sample profiles. Probe sets were annotated to gene targets using information available from the manufacturer. Data analysis. The statistical overrepresentation of gene categories among genes deemed differentially expressed between sample group profiles was assessed using the DAVID bioinformatics resource. Gene set enrichment analyses were performed using web tools from the Broad Institute using HaemAtlas data as input gene set. Differential gene expression between two sample groups was assessed through the output of a moderated t-test and significance P-values obtained converted to corrected q-values using the FDR method.

1.12 In Vitro Platelet Study.

Co-Culture on Feeder Cells.

[0161] In order to promote platelet like particle (PLP) production, day 10 CB and day 20 hiPSC derived MKs were further cultivated for 48 h in Cellgro SCGM on gamma-irradiated stromal cells (OP9, ATCC CRL2749; C3H10T1/2, Riken Institute; HBMEC, courtesy of Dr. Weksler) sowed on gelatine coated tissue culture plates at 1E+4 cells/cm.sup.2.

PLP Flow Analysis.

[0162] Crude supernatant containing the PLPs were analysed by flow cytometry after addition of 1/9 volume of acid citrate dextrose (ACD, Sigma Aldrich) and cell removal by centrifugation at 150 g for 10'. Antibodies against human CD41a and CD42a were added directly to the medium (BD Pharmingen, APC/PE conjugated respectively, used at 1:50 dilution) and flow count fluorospheres used for quantification. Human platelets were analysed from fresh whole blood collected in citrate buffer.

Washed Platelet Preparation.

[0163] Human platelet rich plasma (PRP) and hiPSC PLPs collected as above were washed twice in pH7.4 modified Tyrode-HEPES buffer (10 mM HEPES, 12 mM NaHCO.sub.3, 138 mM NaCl, 5.5 mM glucose, 2.9 mM KCl, and 1 mM MgCl2) using 800 g/10' centrifugation steps after an initial addition of prostaglandin E1 (1.quadrature.M) and apyrase (1 U/ml) to prevent activation. Washed platelet counts were subsequently determined by flow cytometry.

Thrombus Formation in Laminar Flow.

[0164] A defined amount of washed platelets or PLPs are mixed with 1 ml of mouse blood collected in ACD and the participation of human platelets to collagen induced mouse thrombi subsequently monitored by immunofluorescence. The procedure was modified from Auger J M, ATVB, 2008. Briefly, glass slides were locally coated with Horm collagen spots (100 ug/ml) and mounted into a flow chamber placed under a fluorescent microscope (EVOS system, Advanced Microscopy Group). The blood was then perfused through the chamber at 1600 s-1 (7.2 ml/hr) for 3 minutes allowing thrombi formation on collagen spots. A perfusion of 1:100 diluted anti CD41a-FITC (BD Pharmingen) and anti P-selectin-PE (internal NHSBT-Bristol) was then run on the clots for 2 minutes and then washed before pictures were taken.

1.13 Gene Expression Analysis by Quantitative PCR

[0165] For gene expression analysis, cultivated cells are treated by TRIzol and RNA extracted from the aqueous phase following published protocol (Life Technologies). Alternatively, RNA is extracted and purified using Qiaprep RNeasy mini columns (Qiagen) including an on column DNA digestion step. Subsequently, cDNA is synthesized from 250-500 ng of purified RNA using the Maxima Reverse Transcriptase kit and random hexamers (Fermentas). The PCR reaction is performed using a SYBR green based PCR mix (Applied Biosystems, FastSYBR green) on a real time thermal cycler analyser (ABI 7500HT) following a fast 2-step amplification protocol. Relative gene expression quantification is calculated using the 2.sup.-.quadrature.ct method using HMBS endogene expression as a reference. Primer pairs specific for any given gene has been carefully design using the NCBI primer design website in order to be separated by at least one intron on the corresponding gDNA and with no identified potential off target after a BLAST on the human RefSeq repository. Furthermore, all primer pairs have been tested for PCR efficiency between 80-120%. Primer pair specific for endogene expression has been designed in the 5' or 3'UTR of corresponding transcript. Primer pair specific for transgene expression are made of a reverse primer specific for the lentiviral backbone and a forward primer specific for a given transgene.

1.14 Cell Morphology Analysis

[0166] Cell morphology is observed after sedimentation on a glass slide by cytospin (400 g/5 min, 2,000-20,000 cells per slide) and Romanowsky staining (Eosin/Methylene blue) after Methanol fixation.

2. Results

[0167] 2.1 a Combination of GATA1, FLI1 and TAL1 Induces Megakaryocyte Differentiation from Human Pluripotent Stem Cells

[0168] We used a rational whole genome expression data driven process to select a list of candidate genes to test in the context of megakaryocyte forward programming (MK-FoP).

[0169] Briefly, from the initial set of 116 genes coding for DNA binding proteins specifically expressed in cord-blood derived megakaryocytes (CB-MKs) compared to the H9 hESC line (hESC #1), 101 genes were retained after histone gene removal. 46 candidates were then selected upon analysis of protein-protein interaction network using VisANT software, integrating interactions with chromatin remodelling factors and level of gene expression. The visualization notably showed GATA1 as a core factor for 11 interacting partners (FIG. 1).

[0170] 14 transcription factor candidates were then cloned into lentiviral vectors for experimental assessment of their MK-FoP potential (Table 1 of FIG. 17). All 14 factors together were tested in parallel to a combination of 9 factors (higher rank factors) (FIG. 1). The 9TFs combination was found to be better than the 14TFs combination (FIG. 2).

[0171] The transduction of the hESC #1 line with the 9 TFs concurrently generated a well-defined population of CD41a positive cells (integrin alpha-IIb, megakaryocyte marker) identified by flow cytometry at day 7 (1.7.+-.0.8%).

[0172] The CD41a+ population (9TFs forward-programmed cells) was then flow sorted at day 7 and individual transgene expression measured by RT-QPCR. Expression of all 9 TFs was detected in the CD41a negative cell population.

[0173] CD41a expressing cells showed a clear dominance in GATA1, FLI1 and TAL1 transgene expression providing indication that the combined expression of these 3 TFs was instrumental in the differentiation process (FIG. 3). Indeed, the 3-TFs combination showed a better CD41a+ cells outcome when compared with the 9-TFs or a reduced 5-TFs combination including GATA1 interactors (FIG. 4)

[0174] The potential of the GATA1+FLI1 combination was further explored by systematic addition of one of the remaining 7 factors (from 9TFs combination). The 14TFs, 9TFs and 5TFs (top ranked core network factors: GATA1+FLI1+TAL1+SPI1+ZBTB16 were tested in parallel in hESCs cells under pluripotent conditions without mesoderm induction (FIG. 5). The 3TFs combination was identified as the best one to drive forward programming. However, the GATA1+TAL1 combination was tested in a separate experiment and was not able to drive forward programming.

[0175] We validated further the requirement for a concurrent expression of the 3 TFs by testing all permutations of the latter (FIG. 6). Interestingly, the combination of GATA1 and FLI1 also generated a significant amount of CD41a+ cells under some conditions, although with a reduced efficiency providing indication that TAL1 might act more as a promoter than a primary inducer of forward programming.

[0176] In an initial MK-FoP protocol, hPSCs were dissociated with Collagenase IV in order to generate small cell clumps subsequently seeded on human fibronectin coated plates (around 5E+5 cells per 10 cm2). The day after (day 0), cell clumps were transduced by lentiviral vectors expressing t GATA1, TAL1 and FLI1, using MOI20 in presence of protamine sulfate. Cells were kept in pluripotency medium (chemically defined with Activin-A and FGF2) for two days, then in MK medium (chemically defined with TPO and SCF) for five days.

[0177] In a modified MK-FoP protocol, hPSCs were seeded as single cells after dissociation by TrypLE and allowed to attach on fibronectin coated plates in pluripotency medium supplemented with rock inhibitor Y-27632 (inhibition apoptosis) for 24 hours before transduction. In addition, cells were cultivated in mesoderm inducing conditions (FGF2, BMP4 and LY294002) for the first two days, then in MK medium (chemically defined with TPO and SCF) for five days.

[0178] Another modified MK-FoP protocol, which achieved the best cell yield, used an embryoid body culture approach in chemically defined conditions (FIG. 9). On transduction day (day 0), sub-confluent (50-80%) human pluripotent stem cells in pluripotency medium are dissociated to single cells using TrypLE (Life Technologies) for 5 min at 37.degree. C. and viable cells counted on a haemocytometer. Desired amount of cells (e.g. 1E+6 cells) is added to Aggrewell.TM.400 plates (Stemcell Technologies, France) in order to obtain embryoid bodies (EB) of 300 cells each following spin aggregation (detailed protocol in Aggrewell.TM. Technical manual). Importantly, lentiviral transduction is performed concomitantly to the aggregation phase. Briefly, cells are added to the well in CDM supplemented by Y-27632 (10 uM, Sigma Aldrich), rh-BMP4 (10 ng/ml, R&D) and protamine sulfate (8 ug/ml, Sigma). Concentrated lentiviral vectors individually coding for each forward programming factor are added to the well to MOI20 (multiplicity of infection) (2E+7 TU). Subsequently, Aggrewell.TM. plates are centrifuged at 100 g for 3 minutes and put into the incubator (37 C/5% CO2) for 24 hours. Transduced EB are collected the day after and rinse twice with PBS before being seeded in ultralow adherent cell culture plates (Corning) at a density of 600 EB per 10 cm2 dish in CDM plus rh-BMP4 (10 ng/ml, R&D) and rh-FGF2 (5 ng/ml, University of Cambridge) (i.e. mesoderm induction medium). Twenty four hours later, EB are collected, rinse with PBS and further cultivated in ultralow adherent plates in Cellgro SCGM medium (Cellgenix, Germany) supplemented with rh-TPO (100 ng/ml, Cellgenix) and rh-SCF (25 ng/ml, Life Technologies) (MK programming medium) until collection for analysis. Culture medium was changed every three days by aspirating half of the volume and adding 2 times cytokine concentrated fresh medium on top.

[0179] We performed additional analyses to confirm the megakaryocyte identity of the emerging CD41a+ population. The expression of key megakaryocyte genes was measured by QPCR at day 7. Forward programming of the hESC #2 line using the 3TF combination (FLI1, GATA1 and TAL1) was shown to induce expression of the megakaryocyte genes MPL (coding for the thrombopoietin (TPO) receptor), ZFPM1, RUNX1 and late differentiation markers like NFE2, MEIS1 and MEF2C, as well as endogenous expression of GATA1, TAL1 and FLI1 (FIG. 7). Moreover, functional megakaryocyte progenitors were limited to the CD41a+ population at day 7 as demonstrated by clonogenic colony forming assays (FIG. 8). Indeed, these cells were found to be able to form mature MK colonies expressing CD41a and CD42b in semi-solid collagen cultures; interestingly, CD41a- cells did not show such potential. Importantly, key MK gene expression was shown to be restricted to the CD41+ cell population at day 7 (FIG. 9).

[0180] Altogether, we identified GATA1, FLI1 and TAL1 as a minimal combination of TFs inducing efficient megakaryocyte forward programming from hPSCs, TAL1 acting as an enhancer while GATA1 and FLI1 were instrumental to the programming process

[0181] Time course analysis of surface marker expression by flow cytometry showed that human iPSC lines forward programmed using the 3TFs started to express CD41a from day 4 and CD42a from day 7 (FIG. 10). By day 14, cells kept in the MK culture medium TPO+SCF have reached near homogeneity for CD41a expression (91%) and a clear CD42a+ population has developed (22%), generating on average 13+/-7 millions mature MK per million iPSC input. The morphology of day 14 cells obtained by 3TF forward programming as observed by Romanowsky staining is very similar to in vitro cord blood derived MK regarding cell size, cytoplasm and nuclei content (FIG. 11). Mature MKs co-expressed CD41a, CD42b, GPVI, CD61 and CD42a.

[0182] To improve forward programming efficiency, we tested different chemically defined in vitro culture condition settings following 3-TF transduction of various hPSC lines. We measured the effect of commitment to mesoderm--the embryonic germ lineage from which the haematopoietic system originates--when induced simultaneously to 3-TF expression. We observed that mesoderm induction by exposition of hPSCs to BMP4, FGF2 and LY294002 for two days after viral transduction significantly increased the number of CD41a+ cells at day 7 in a variety of cell lines (FIG. 12). Importantly, the transduction efficiencies were similar for hPSCs maintained in pluripotent or mesoderm conditions indicating that mesoderm cells are indeed more responsive to the 3-TF driven forward programming. Additionally, we tested different hPSCs sowing techniques for their impact on forward programming at day 7. When comparing hPSCs sowed and transduced as clumps or single cells in tissue culture plates with those induced to form embryoid bodies (EB) by forced aggregation, we observed a significant improvement of the CD41a+ cell number with the latter (FIG. 13). Overall, we showed that the 3-TF driven MK-FoP efficiency was significantly improved by mesoderm commitment and cultivation as spin aggregated EB. The latter also offered the advantage of a standardised compact suspension culture and had been consequently used for the remaining experiments of this study.

2.2 A Chemically Defined Optimised Protocol Generates High Number of Mature Megakaryocytes

[0183] Using the optimised chemically defined protocol comprising viral transduction of spin aggregated EBs at day 0, culture in mesoderm medium comprising FGF2 and BMP4 and LY294002 for two days after viral transduction; culture in MK medium comprising TPO and SCF until day 10 and dissociation of embryoid bodies showing cystic structures and actively growing cell aggregates to single cells at day 10 and further cultivated in MK maturation medium (TPO+IL1b) for an additional 10 days, we analysed megakaryocyte maturation of 3-TF forward programmed hiPSCs compared to cord blood derived megakaryocytes. We observed a gradual increase of CD41a+ megakaryocyte progenitor cells followed by the progressive acquisition of the maturation marker CD42b (glycoprotein Ib, part of the MK specific GPIb/V/IX receptor complex) over cultivation time mimicking normal megakaryocyte differentiation.

[0184] Interestingly, the culture reached megakaryocyte purity (95.+-.2% CD41a+, n=12) with more than half mature cells (56.+-.4% CD42b+, n=12) by day 20 post-transduction without additional sorting procedure. Further maturation could be obtained by maintaining cells in culture for longer periods (>80% CD42b+) but the cell yield is reduced by higher cell death as observed in prolonged cord blood cultures. In addition to CD41a and CD42b, we also observed expression of additional key surface proteins involved in megakaryocyte and platelet functions (itgb3 (CD61), gp6 and gp9 (CD42a)) by flow cytometry on hiPSC forward programmed day 20 cells and cord blood derived megakaryocytes. Similar expression profiles between cord blood and hiPSC derived megakaryocytes were observed.

[0185] Moreover, forward programmed cells showed typical size, morphological and ultrastructural features of megakaryocytes. We observed numerous polyploid cells with sizes ranging from 15 to 30 um and large cytoplasms (FIG. 14) and using transmission electron microscopy, cells displaying characteristic structures of maturing megakaryocytes as multi vesicular bodies--precursors of platelet granules--and developing demarcation membrane systems. Importantly, we showed that the 3-TF forward programming protocol efficiently generated mature megakaryocytes from different hiPSC lines with up to 33 fold increase in mature CD42b+ cell number compared to the hiPSCs input (FIG. 15).

[0186] We further characterised the megakaryocyte identity of the forward programmed cells by whole genome expression analysis. The tissue enrichment analysis between the parental hiPSCs and derived 3-TF forward programmed cells showed most significance for platelets (P=1.03E-62) while the top 5 enriched biological processes are related to haemostasis and platelet activity confirming on a genome wide level the megakaryocyte phenotype (Table 3). A gene set enrichment analysis ((GSEA web tools, Broad Institute) on the genes differentially expressed between hiPSC-MKs and the parental pluripotent stem cells showed a significant enrichment for megakaryocyte specific genes (dataset generated from HaemAtlas, MK compared to other blood lineages) and confirmed further the MK identity of forward programmed cells amongst other blood cell types (NES=1.47, data not shown). Intriguingly, we observed that MK-FoP applied to two different hiPSC lines, although showing variability in cell number outcome (FIG. 15), generated highly similar expression profiles in the differentiated mature MKs (R2=0.99).

[0187] We identified 13 TFs whose expression is missing in hiPSC-MKs compared to CB or peripheral blood differentiated MKs (Table 2). These genes may be determinants for post-natal phenotype acquisition and may further improve the maturation of hiPSC derived MKs.

[0188] In summary, we demonstrated that the 3-TF forward programming approach was able to efficiently generate genuine megakaryocytes sharing key features with their cord blood derived counterpart.

2.3 Forward Programmed Megakaryocytes Produce Functional Platelet-Like Particles In Vitro

[0189] We tested the ability of forward programmed megakaryocytes to generate functional platelet like particles (PLPs) in vitro. Platelet production happens by a process of proplatelet formation by mature MKs. Immunofluorescence analysis of hiPSC forward programmed day 20 megakaryocytes cultivated for an additional 48 hours on fibrinogen coated plates showed pro-platelet like cytoplasmic protrusions in vitro showing bulbous structures expressing von Willebrand factor (vwf) and P-selectin along alpha-tubulin positive cytoplasmic filaments. von Willebrand factor (vwf) and P-selectin which are key proteins embedded in platelet granules.

[0190] The release of platelet-like particles (PLPs) in the culture supernatant was highly improved by co-culture of hiPSC derived MKs on the OP9 stromal cell line for 48 hours. In such conditions, we detected significant amount of PLPs by flow cytometry identified as human platelet size particles co-expressing CD41a and CD42a. They were produced at an average of 1.+-.0.3 million per million hiPSC-MK input after 48 hours of co-culture on various feeder cells (FIG. 16).

[0191] We explored further the functionality of the generated PLPs by monitoring their contribution to in vitro collagen induced mouse thrombi under flow. Human particles were detected by immunostaining after clot formation on collagen fibres following mixing of PLPs or human platelets with mouse blood (3E+6 and 5E+6 per ml respectively). Activated platelets co-expressing CD41a and P-selectin on their surface were also identified. This shows that the hiPSC derived PLPs in formed mouse platelet clots displayed granule content on their surface, demonstrating functional activation.

[0192] We describe above a method for generating megakaryocytes from hPSCs using a forward programming strategy based on combined forced expression of the three transcription factors GATA1, FLI1 and TAL1. The generated cells were genuine megakaryocytes able to release functional platelet like particles in vitro.

[0193] Indeed, this method offers several advantages compared to previously described methods. First, the differentiation and maturation of megakaryocytes is achieved in chemically defined conditions without supporting stromal cells. This improves reproducibility and will greatly help the transition to clinically compatible procedures. By acting directly at the level of the gene regulatory network, the forward programming strategy allows an efficient megakaryocyte differentiation using a reduced cytokine combination and minimal cell handling. The cell yield of forward programming matches the best described approaches so far; leading in 20 days to up to 50 megakaryocytes per hiPSC input and provides a pure megakaryocyte population without the need for sorting (>95% CD41a+ and >50% CD42a+). An additional benefit of the protocol described above is the use of suspension culture only (embryoid bodies followed by single cells) which greatly reduces the footprint of the experiment and should facilitate its transfer to large scale production systems.

[0194] A mesoderm inducing treatment concomitant to transgene expression was found to be beneficial to forward programming, providing indication that the epigenome and/or transcriptional profile of mesoderm cells were more amenable to respond to the programming factors. The forward programming happens very rapidly since markers of MK commitment are detected as early as four days after 3 TFs transduction, the MK potential is restricted from day 7 to the CD41a+ population and cells show an early dependency to haematopoietic cytokines.

[0195] In conclusion, our study demonstrates forward programming to generate MKs from hiPSCs. The generated hiPSC-megakaryocytes shared key features with their cord blood derived counterpart at the genetic and ultrastructural levels, and are able to release functional platelet like particles in vitro. This novel differentiation approach will have broad applications in both basic research and clinical development of hiPSC derived transfusion products.

TABLE-US-00001 TABLE 2 Factor Name Gene ID Reference Sequence 1 ABLIM1 Actin binding LIM 3983 NP_001003407.1 protein 1 GI: 51173713 (SEQ ID NO: 4) 2 FNL1 Four and a half 2272 NP_001153174.1 LIM GI: 228460211 domains 1 (SEQ ID NO: 5) 3 RUNX3 Runt-related 864 NP_001026850.1 transcription GI: 72534652 factor 3 (SEQ ID NO: 6) 4 NFIC Nuclear factor 4782 NP_001231931.1 I/C (CCAAT- GI: 350529396 binding (SEQ ID NO: 7) transcription factor) 5 NFIL3 Nuclear factor, 4783 [[N2]]NP_005375.2 interleukin 3 GI: 52630429 regulated (SEQ ID NO: 8) 6 VDR Vitamin D (1,25- 7421 NP_000367.1 dihydroxyvitamin GI: 4507883 D3) receptor (SEQ ID NO: 9) 7 MESP1 Mesoderm 55897 NP_061140.1 posterior 1 GI: 14149724 (SEQ ID NO: 10) 8 BTBDII TB (P03) domain 121551 NP_001018082.1 containing 11 GI: 65786661 (SEQ ID NO: 11) 9 APPL2 Adaptor protein, 55198 NP_060641.2 phosphotyrosine GI: 24586663 Interaction, PH (SEQ ID NO: 12) domain and leucine zipper containing 2 10 MICAL1 Microtubule 64780 NP_073602.3 associated GI: 205360947 monooxygenase, (SEQ ID NO: 13) calponin and LIM domain containing 1 11 BATF Basic leucine 10538 NP_006390.1 zipper GI: 5453563 transcription (SEQ ID NO: 14) factor, ATF-like 12 SCMH1 Sex comb on 22955 NP_001026864.1 midleg homolog 1 GI: 72534680 (SEQ ID NO: 15) 13 MBP Myelin basic 4155 NP_001020252.1 protein GI: 66509930 (SEQ ID NO: 16)

TABLE-US-00002 TABLE 3 Top 5 enriched Biological Processes (hiPSC-MK vs. hiPSC) Significant Total Term p-value Genes No. Genes No. hemostasis 1.48E-25 191 501 coagulation 3.15E-25 190 500 platelet activation 3.33E-25 107 216 blood coagulation 1.08E-24 188 497 vesicle- 5.19E-22 284 903 mediated transport

Sequence CWU 1

1

161413PRTHomo sapiens 1Met Glu Phe Pro Gly Leu Gly Ser Leu Gly Thr Ser Glu Pro Leu Pro1 5 10 15Gln Phe Val Asp Pro Ala Leu Val Ser Ser Thr Pro Glu Ser Gly Val 20 25 30Phe Phe Pro Ser Gly Pro Glu Gly Leu Asp Ala Ala Ala Ser Ser Thr 35 40 45Ala Pro Ser Thr Ala Thr Ala Ala Ala Ala Ala Leu Ala Tyr Tyr Arg 50 55 60Asp Ala Glu Ala Tyr Arg His Ser Pro Val Phe Gln Val Tyr Pro Leu65 70 75 80Leu Asn Cys Met Glu Gly Ile Pro Gly Gly Ser Pro Tyr Ala Gly Trp 85 90 95Ala Tyr Gly Lys Thr Gly Leu Tyr Pro Ala Ser Thr Val Cys Pro Thr 100 105 110Arg Glu Asp Ser Pro Pro Gln Ala Val Glu Asp Leu Asp Gly Lys Gly 115 120 125Ser Thr Ser Phe Leu Glu Thr Leu Lys Thr Glu Arg Leu Ser Pro Asp 130 135 140Leu Leu Thr Leu Gly Pro Ala Leu Pro Ser Ser Leu Pro Val Pro Asn145 150 155 160Ser Ala Tyr Gly Gly Pro Asp Phe Ser Ser Thr Phe Phe Ser Pro Thr 165 170 175Gly Ser Pro Leu Asn Ser Ala Ala Tyr Ser Ser Pro Lys Leu Arg Gly 180 185 190Thr Leu Pro Leu Pro Pro Cys Glu Ala Arg Glu Cys Val Asn Cys Gly 195 200 205Ala Thr Ala Thr Pro Leu Trp Arg Arg Asp Arg Thr Gly His Tyr Leu 210 215 220Cys Asn Ala Cys Gly Leu Tyr His Lys Met Asn Gly Gln Asn Arg Pro225 230 235 240Leu Ile Arg Pro Lys Lys Arg Leu Ile Val Ser Lys Arg Ala Gly Thr 245 250 255Gln Cys Thr Asn Cys Gln Thr Thr Thr Thr Thr Leu Trp Arg Arg Asn 260 265 270Ala Ser Gly Asp Pro Val Cys Asn Ala Cys Gly Leu Tyr Tyr Lys Leu 275 280 285His Gln Val Asn Arg Pro Leu Thr Met Arg Lys Asp Gly Ile Gln Thr 290 295 300Arg Asn Arg Lys Ala Ser Gly Lys Gly Lys Lys Lys Arg Gly Ser Ser305 310 315 320Leu Gly Gly Thr Gly Ala Ala Glu Gly Pro Ala Gly Gly Phe Met Val 325 330 335Val Ala Gly Gly Ser Gly Ser Gly Asn Cys Gly Glu Val Ala Ser Gly 340 345 350Leu Thr Leu Gly Pro Pro Gly Thr Ala His Leu Tyr Gln Gly Leu Gly 355 360 365Pro Val Val Leu Ser Gly Pro Val Ser His Leu Met Pro Phe Pro Gly 370 375 380Pro Leu Leu Gly Ser Pro Thr Gly Ser Phe Pro Thr Gly Pro Met Pro385 390 395 400Pro Thr Thr Ser Thr Thr Val Val Ala Pro Leu Ser Ser 405 4102452PRTHomo sapiens 2Met Asp Gly Thr Ile Lys Glu Ala Leu Ser Val Val Ser Asp Asp Gln1 5 10 15Ser Leu Phe Asp Ser Ala Tyr Gly Ala Ala Ala His Leu Pro Lys Ala 20 25 30Asp Met Thr Ala Ser Gly Ser Pro Asp Tyr Gly Gln Pro His Lys Ile 35 40 45Asn Pro Leu Pro Pro Gln Gln Glu Trp Ile Asn Gln Pro Val Arg Val 50 55 60Asn Val Lys Arg Glu Tyr Asp His Met Asn Gly Ser Arg Glu Ser Pro65 70 75 80Val Asp Cys Ser Val Ser Lys Cys Ser Lys Leu Val Gly Gly Gly Glu 85 90 95Ser Asn Pro Met Asn Tyr Asn Ser Tyr Met Asp Glu Lys Asn Gly Pro 100 105 110Pro Pro Pro Asn Met Thr Thr Asn Glu Arg Arg Val Ile Val Pro Ala 115 120 125Asp Pro Thr Leu Trp Thr Gln Glu His Val Arg Gln Trp Leu Glu Trp 130 135 140Ala Ile Lys Glu Tyr Ser Leu Met Glu Ile Asp Thr Ser Phe Phe Gln145 150 155 160Asn Met Asp Gly Lys Glu Leu Cys Lys Met Asn Lys Glu Asp Phe Leu 165 170 175Arg Ala Thr Thr Leu Tyr Asn Thr Glu Val Leu Leu Ser His Leu Ser 180 185 190Tyr Leu Arg Glu Ser Ser Leu Leu Ala Tyr Asn Thr Thr Ser His Thr 195 200 205Asp Gln Ser Ser Arg Leu Ser Val Lys Glu Asp Pro Ser Tyr Asp Ser 210 215 220Val Arg Arg Gly Ala Trp Gly Asn Asn Met Asn Ser Gly Leu Asn Lys225 230 235 240Ser Pro Pro Leu Gly Gly Ala Gln Thr Ile Ser Lys Asn Thr Glu Gln 245 250 255Arg Pro Gln Pro Asp Pro Tyr Gln Ile Leu Gly Pro Thr Ser Ser Arg 260 265 270Leu Ala Asn Pro Gly Ser Gly Gln Ile Gln Leu Trp Gln Phe Leu Leu 275 280 285Glu Leu Leu Ser Asp Ser Ala Asn Ala Ser Cys Ile Thr Trp Glu Gly 290 295 300Thr Asn Gly Glu Phe Lys Met Thr Asp Pro Asp Glu Val Ala Arg Arg305 310 315 320Trp Gly Glu Arg Lys Ser Lys Pro Asn Met Asn Tyr Asp Lys Leu Ser 325 330 335Arg Ala Leu Arg Tyr Tyr Tyr Asp Lys Asn Ile Met Thr Lys Val His 340 345 350Gly Lys Arg Tyr Ala Tyr Lys Phe Asp Phe His Gly Ile Ala Gln Ala 355 360 365Leu Gln Pro His Pro Thr Glu Ser Ser Met Tyr Lys Tyr Pro Ser Asp 370 375 380Ile Ser Tyr Met Pro Ser Tyr His Ala His Gln Gln Lys Val Asn Phe385 390 395 400Val Pro Pro His Pro Ser Ser Met Pro Val Thr Ser Ser Ser Phe Phe 405 410 415Gly Ala Ala Ser Gln Tyr Trp Thr Ser Pro Thr Gly Gly Ile Tyr Pro 420 425 430Asn Pro Asn Val Pro Arg His Pro Asn Thr His Val Pro Ser His Leu 435 440 445Gly Ser Tyr Tyr 4503331PRTHomo sapiens 3Met Thr Glu Arg Pro Pro Ser Glu Ala Ala Arg Ser Asp Pro Gln Leu1 5 10 15Glu Gly Arg Asp Ala Ala Glu Ala Ser Met Ala Pro Pro His Leu Val 20 25 30Leu Leu Asn Gly Val Ala Lys Glu Thr Ser Arg Ala Ala Ala Ala Glu 35 40 45Pro Pro Val Ile Glu Leu Gly Ala Arg Gly Gly Pro Gly Gly Gly Pro 50 55 60Ala Gly Gly Gly Gly Ala Ala Arg Asp Leu Lys Gly Arg Asp Ala Ala65 70 75 80Thr Ala Glu Ala Arg His Arg Val Pro Thr Thr Glu Leu Cys Arg Pro 85 90 95Pro Gly Pro Ala Pro Ala Pro Ala Pro Ala Ser Val Thr Ala Glu Leu 100 105 110Pro Gly Asp Gly Arg Met Val Gln Leu Ser Pro Pro Ala Leu Ala Ala 115 120 125Pro Ala Ala Pro Gly Arg Ala Leu Leu Tyr Ser Leu Ser Gln Pro Leu 130 135 140Ala Ser Leu Gly Ser Gly Phe Phe Gly Glu Pro Asp Ala Phe Pro Met145 150 155 160Phe Thr Thr Asn Asn Arg Val Lys Arg Arg Pro Ser Pro Tyr Glu Met 165 170 175Glu Ile Thr Asp Gly Pro His Thr Lys Val Val Arg Arg Ile Phe Thr 180 185 190Asn Ser Arg Glu Arg Trp Arg Gln Gln Asn Val Asn Gly Ala Phe Ala 195 200 205Glu Leu Arg Lys Leu Ile Pro Thr His Pro Pro Asp Lys Lys Leu Ser 210 215 220Lys Asn Glu Ile Leu Arg Leu Ala Met Lys Tyr Ile Asn Phe Leu Ala225 230 235 240Lys Leu Leu Asn Asp Gln Glu Glu Glu Gly Thr Gln Arg Ala Lys Thr 245 250 255Gly Lys Asp Pro Val Val Gly Ala Gly Gly Gly Gly Gly Gly Gly Gly 260 265 270Gly Gly Ala Pro Pro Asp Asp Leu Leu Gln Asp Val Leu Ser Pro Asn 275 280 285Ser Ser Cys Gly Ser Ser Leu Asp Gly Ala Ala Ser Pro Asp Ser Tyr 290 295 300Thr Glu Glu Pro Ala Pro Lys His Thr Ala Arg Ser Leu His Pro Ala305 310 315 320Met Leu Pro Ala Ala Asp Gly Ala Gly Pro Arg 325 3304718PRTHomo sapiens 4Met Leu Met Thr Leu Glu Met Thr Glu Leu Thr Asp Pro His His Thr1 5 10 15Met Gly Asp Tyr Lys Val Ala His Pro Gln Asp Pro His His Pro Ser 20 25 30Glu Lys Pro Val Ile His Cys His Lys Cys Gly Glu Pro Cys Lys Gly 35 40 45Glu Val Leu Arg Val Gln Thr Lys His Phe His Ile Lys Cys Phe Thr 50 55 60Cys Lys Val Cys Gly Cys Asp Leu Ala Gln Gly Gly Phe Phe Ile Lys65 70 75 80Asn Gly Glu Tyr Leu Cys Thr Leu Asp Tyr Gln Arg Met Tyr Gly Thr 85 90 95Arg Cys His Gly Cys Gly Glu Phe Val Glu Gly Glu Val Val Thr Ala 100 105 110Leu Gly Lys Thr Tyr His Pro Asn Cys Phe Ala Cys Thr Ile Cys Lys 115 120 125Arg Pro Phe Pro Pro Gly Asp Arg Val Thr Phe Asn Gly Arg Asp Cys 130 135 140Leu Cys Gln Leu Cys Ala Gln Pro Met Ser Ser Ser Pro Lys Glu Thr145 150 155 160Thr Phe Ser Ser Asn Cys Ala Gly Cys Gly Arg Asp Ile Lys Asn Gly 165 170 175Gln Ala Leu Leu Ala Leu Asp Lys Gln Trp His Leu Gly Cys Phe Lys 180 185 190Cys Lys Ser Cys Gly Lys Val Leu Thr Gly Glu Tyr Ile Ser Lys Asp 195 200 205Gly Ala Pro Tyr Cys Glu Lys Asp Tyr Gln Gly Leu Phe Gly Val Lys 210 215 220Cys Glu Ala Cys His Gln Phe Ile Thr Gly Lys Val Leu Glu Ala Gly225 230 235 240Asp Lys His Tyr His Pro Ser Cys Ala Arg Cys Ser Arg Cys Asn Gln 245 250 255Met Phe Thr Glu Gly Glu Glu Met Tyr Leu Gln Gly Ser Thr Val Trp 260 265 270His Pro Asp Cys Lys Gln Ser Thr Lys Thr Glu Glu Lys Leu Arg Pro 275 280 285Thr Arg Thr Ser Ser Glu Ser Ile Tyr Ser Arg Pro Gly Ser Ser Ile 290 295 300Pro Gly Ser Pro Gly His Thr Ile Tyr Ala Lys Val Asp Asn Glu Ile305 310 315 320Leu Asp Tyr Lys Asp Leu Ala Ala Ile Pro Lys Val Lys Ala Ile Tyr 325 330 335Asp Ile Glu Arg Pro Asp Leu Ile Thr Tyr Glu Pro Phe Tyr Thr Ser 340 345 350Gly Tyr Asp Asp Lys Gln Glu Arg Gln Ser Leu Gly Glu Ser Pro Arg 355 360 365Thr Leu Ser Pro Thr Pro Ser Ala Glu Gly Tyr Gln Asp Val Arg Asp 370 375 380Arg Met Ile His Arg Ser Thr Ser Gln Gly Ser Ile Asn Ser Pro Val385 390 395 400Tyr Ser Arg His Ser Tyr Thr Pro Thr Thr Ser Arg Ser Pro Gln His 405 410 415Phe His Arg Pro Gly Asn Glu Pro Ser Ser Gly Arg Asn Ser Pro Leu 420 425 430Pro Tyr Arg Pro Asp Ser Arg Pro Leu Thr Pro Thr Tyr Ala Gln Ala 435 440 445Pro Lys His Phe His Val Pro Asp Gln Gly Ile Asn Ile Tyr Arg Lys 450 455 460Pro Pro Ile Tyr Lys Gln His Ala Ala Leu Ala Ala Gln Ser Lys Ser465 470 475 480Ser Glu Asp Ile Ile Lys Phe Ser Lys Phe Pro Ala Ala Gln Ala Pro 485 490 495Asp Pro Ser Glu Thr Pro Lys Ile Glu Thr Asp His Trp Pro Gly Pro 500 505 510Pro Ser Phe Ala Val Val Gly Pro Asp Met Lys Arg Arg Ser Ser Gly 515 520 525Arg Glu Glu Asp Asp Glu Glu Leu Leu Arg Arg Arg Gln Leu Gln Glu 530 535 540Glu Gln Leu Met Lys Leu Asn Ser Gly Leu Gly Gln Leu Ile Leu Lys545 550 555 560Glu Glu Met Glu Lys Glu Ser Arg Glu Arg Ser Ser Leu Leu Ala Ser 565 570 575Arg Tyr Asp Ser Pro Ile Asn Ser Ala Ser His Ile Pro Ser Ser Lys 580 585 590Thr Ala Ser Leu Pro Gly Tyr Gly Arg Asn Gly Leu His Arg Pro Val 595 600 605Ser Thr Asp Phe Ala Gln Tyr Asn Ser Tyr Gly Asp Val Ser Gly Gly 610 615 620Val Arg Asp Tyr Gln Thr Leu Pro Asp Gly His Met Pro Ala Met Arg625 630 635 640Met Asp Arg Gly Val Ser Met Pro Asn Met Leu Glu Pro Lys Ile Phe 645 650 655Pro Tyr Glu Met Leu Met Val Thr Asn Arg Gly Arg Asn Lys Ile Leu 660 665 670Arg Glu Val Asp Arg Thr Arg Leu Glu Arg His Leu Ala Pro Glu Val 675 680 685Phe Arg Glu Ile Phe Gly Met Ser Ile Gln Glu Phe Asp Arg Leu Pro 690 695 700Leu Trp Arg Arg Asn Asp Met Lys Lys Lys Ala Lys Leu Phe705 710 7155323PRTHomo sapiens 5Met Ala Glu Lys Phe Asp Cys His Tyr Cys Arg Asp Pro Leu Gln Gly1 5 10 15Lys Lys Tyr Val Gln Lys Asp Gly His His Cys Cys Leu Lys Cys Phe 20 25 30Asp Lys Phe Cys Ala Asn Thr Cys Val Glu Cys Arg Lys Pro Ile Gly 35 40 45Ala Asp Ser Lys Glu Val His Tyr Lys Asn Arg Phe Trp His Asp Thr 50 55 60Cys Phe Arg Cys Ala Lys Cys Leu His Pro Leu Ala Asn Glu Thr Phe65 70 75 80Val Ala Lys Asp Asn Lys Ile Leu Cys Asn Lys Cys Thr Thr Arg Glu 85 90 95Asp Ser Pro Lys Cys Lys Gly Cys Phe Lys Ala Ile Val Ala Gly Asp 100 105 110Gln Asn Val Glu Tyr Lys Gly Thr Val Trp His Lys Asp Cys Phe Thr 115 120 125Cys Ser Asn Cys Lys Gln Val Ile Gly Thr Gly Ser Phe Phe Pro Lys 130 135 140Gly Glu Asp Phe Tyr Cys Val Thr Cys His Glu Thr Lys Phe Ala Lys145 150 155 160His Cys Val Lys Cys Asn Lys Ala Ile Thr Ser Gly Gly Ile Thr Tyr 165 170 175Gln Asp Gln Pro Trp His Ala Asp Cys Phe Val Cys Val Thr Cys Ser 180 185 190Lys Lys Leu Ala Gly Gln Arg Phe Thr Ala Val Glu Asp Gln Tyr Tyr 195 200 205Cys Val Asp Cys Tyr Lys Asn Phe Val Ala Lys Lys Cys Ala Gly Cys 210 215 220Lys Asn Pro Ile Thr Gly Lys Arg Thr Val Ser Arg Val Ser His Pro225 230 235 240Val Ser Lys Ala Arg Lys Pro Pro Val Cys His Gly Lys Arg Leu Pro 245 250 255Leu Thr Leu Phe Pro Ser Ala Asn Leu Arg Gly Arg His Pro Gly Gly 260 265 270Glu Arg Thr Cys Pro Ser Trp Val Val Val Leu Tyr Arg Lys Asn Arg 275 280 285Ser Leu Ala Ala Pro Arg Gly Pro Gly Leu Val Lys Ala Pro Val Trp 290 295 300Trp Pro Met Lys Asp Asn Pro Gly Thr Thr Thr Ala Ser Thr Ala Lys305 310 315 320Asn Ala Pro6429PRTHomo sapiens 6Met Ala Ser Asn Ser Ile Phe Asp Ser Phe Pro Thr Tyr Ser Pro Thr1 5 10 15Phe Ile Arg Asp Pro Ser Thr Ser Arg Arg Phe Thr Pro Pro Ser Pro 20 25 30Ala Phe Pro Cys Gly Gly Gly Gly Gly Lys Met Gly Glu Asn Ser Gly 35 40 45Ala Leu Ser Ala Gln Ala Ala Val Gly Pro Gly Gly Arg Ala Arg Pro 50 55 60Glu Val Arg Ser Met Val Asp Val Leu Ala Asp His Ala Gly Glu Leu65 70 75 80Val Arg Thr Asp Ser Pro Asn Phe Leu Cys Ser Val Leu Pro Ser His 85 90 95Trp Arg Cys Asn Lys Thr Leu Pro Val Ala Phe Lys Val Val Ala Leu 100 105 110Gly Asp Val Pro Asp Gly Thr Val Val Thr Val Met Ala Gly Asn Asp 115 120 125Glu Asn Tyr Ser Ala Glu Leu Arg Asn Ala Ser Ala Val Met Lys Asn 130 135 140Gln Val Ala Arg Phe Asn Asp Leu Arg Phe Val Gly Arg Ser Gly Arg145 150 155 160Gly Lys Ser Phe Thr Leu Thr Ile Thr Val Phe Thr Asn Pro Thr Gln 165 170 175Val Ala Thr Tyr His Arg Ala Ile Lys Val Thr Val Asp Gly Pro Arg 180 185 190Glu Pro Arg Arg His Arg Gln Lys Leu Glu Asp Gln Thr Lys Pro Phe 195 200 205Pro Asp Arg Phe Gly Asp Leu Glu Arg Leu Arg Met Arg Val Thr Pro 210 215 220Ser Thr Pro

Ser Pro Arg Gly Ser Leu Ser Thr Thr Ser His Phe Ser225 230 235 240Ser Gln Pro Gln Thr Pro Ile Gln Gly Thr Ser Glu Leu Asn Pro Phe 245 250 255Ser Asp Pro Arg Gln Phe Asp Arg Ser Phe Pro Thr Leu Pro Thr Leu 260 265 270Thr Glu Ser Arg Phe Pro Asp Pro Arg Met His Tyr Pro Gly Ala Met 275 280 285Ser Ala Ala Phe Pro Tyr Ser Ala Thr Pro Ser Gly Thr Ser Ile Ser 290 295 300Ser Leu Ser Val Ala Gly Met Pro Ala Thr Ser Arg Phe His His Thr305 310 315 320Tyr Leu Pro Pro Pro Tyr Pro Gly Ala Pro Gln Asn Gln Ser Gly Pro 325 330 335Phe Gln Ala Asn Pro Ser Pro Tyr His Leu Tyr Tyr Gly Thr Ser Ser 340 345 350Gly Ser Tyr Gln Phe Ser Met Val Ala Gly Ser Ser Ser Gly Gly Asp 355 360 365Arg Ser Pro Thr Arg Met Leu Ala Ser Cys Thr Ser Ser Ala Ala Ser 370 375 380Val Ala Ala Gly Asn Leu Met Asn Pro Ser Leu Gly Gly Gln Ser Asp385 390 395 400Gly Val Glu Ala Asp Gly Ser His Ser Asn Ser Pro Thr Ala Leu Ser 405 410 415Thr Pro Gly Arg Met Asp Glu Ala Val Trp Arg Pro Tyr 420 4257508PRTHomo sapiens 7Met Tyr Ser Ser Pro Leu Cys Leu Thr Gln Asp Glu Phe His Pro Phe1 5 10 15Ile Glu Ala Leu Leu Pro His Val Arg Ala Phe Ala Tyr Thr Trp Phe 20 25 30Asn Leu Gln Ala Arg Lys Arg Lys Tyr Phe Lys Lys His Glu Lys Arg 35 40 45Met Ser Lys Asp Glu Glu Arg Ala Val Lys Asp Glu Leu Leu Gly Glu 50 55 60Lys Pro Glu Val Lys Gln Lys Trp Ala Ser Arg Leu Leu Ala Lys Leu65 70 75 80Arg Lys Asp Ile Arg Pro Glu Cys Arg Glu Asp Phe Val Leu Ser Ile 85 90 95Thr Gly Lys Lys Ala Pro Gly Cys Val Leu Ser Asn Pro Asp Gln Lys 100 105 110Gly Lys Met Arg Arg Ile Asp Cys Leu Arg Gln Ala Asp Lys Val Trp 115 120 125Arg Leu Asp Leu Val Met Val Ile Leu Phe Lys Gly Ile Pro Leu Glu 130 135 140Ser Thr Asp Gly Glu Arg Leu Val Lys Ala Ala Gln Cys Gly His Pro145 150 155 160Val Leu Cys Val Gln Pro His His Ile Gly Val Ala Val Lys Glu Leu 165 170 175Asp Leu Tyr Leu Ala Tyr Phe Val Arg Glu Arg Asp Ala Glu Gln Ser 180 185 190Gly Ser Pro Arg Thr Gly Met Gly Ser Asp Gln Glu Asp Ser Lys Pro 195 200 205Ile Thr Leu Asp Thr Thr Asp Phe Gln Glu Ser Phe Val Thr Ser Gly 210 215 220Val Phe Ser Val Thr Glu Leu Ile Gln Val Ser Arg Thr Pro Val Val225 230 235 240Thr Gly Thr Gly Pro Asn Phe Ser Leu Gly Glu Leu Gln Gly His Leu 245 250 255Ala Tyr Asp Leu Asn Pro Ala Ser Thr Gly Leu Arg Arg Thr Leu Pro 260 265 270Ser Thr Ser Ser Ser Gly Ser Lys Arg His Lys Ser Gly Ser Met Glu 275 280 285Glu Asp Val Asp Thr Ser Pro Gly Gly Asp Tyr Tyr Thr Ser Pro Ser 290 295 300Ser Pro Thr Ser Ser Ser Arg Asn Trp Thr Glu Asp Met Glu Gly Gly305 310 315 320Ile Ser Ser Pro Val Lys Lys Thr Glu Met Asp Lys Ser Pro Phe Asn 325 330 335Ser Pro Ser Pro Gln Asp Ser Pro Arg Leu Ser Ser Phe Thr Gln His 340 345 350His Arg Pro Val Ile Ala Val His Ser Gly Ile Ala Arg Ser Pro His 355 360 365Pro Ser Ser Ala Leu His Phe Pro Thr Thr Ser Ile Leu Pro Gln Thr 370 375 380Ala Ser Thr Tyr Phe Pro His Thr Ala Ile Arg Tyr Pro Pro His Leu385 390 395 400Asn Pro Gln Asp Pro Leu Lys Asp Leu Val Ser Leu Ala Cys Asp Pro 405 410 415Ala Ser Gln Gln Pro Gly Pro Leu Asn Gly Ser Gly Gln Leu Lys Met 420 425 430Pro Ser His Cys Leu Ser Ala Gln Met Leu Ala Pro Pro Pro Pro Gly 435 440 445Leu Pro Arg Leu Ala Leu Pro Pro Ala Thr Lys Pro Ala Thr Thr Ser 450 455 460Glu Gly Gly Ala Thr Ser Pro Thr Ser Pro Ser Tyr Ser Pro Pro Asp465 470 475 480Thr Ser Pro Ala Asn Arg Ser Phe Val Gly Leu Gly Pro Arg Asp Pro 485 490 495Ala Gly Ile Tyr Gln Ala Gln Ser Trp Tyr Leu Gly 500 5058462PRTHomo sapiens 8Met Gln Leu Arg Lys Met Gln Thr Val Lys Lys Glu Gln Ala Ser Leu1 5 10 15Asp Ala Ser Ser Asn Val Asp Lys Met Met Val Leu Asn Ser Ala Leu 20 25 30Thr Glu Val Ser Glu Asp Ser Thr Thr Gly Glu Glu Leu Leu Leu Ser 35 40 45Glu Gly Ser Val Gly Lys Asn Lys Ser Ser Ala Cys Arg Arg Lys Arg 50 55 60Glu Phe Ile Pro Asp Glu Lys Lys Asp Ala Met Tyr Trp Glu Lys Arg65 70 75 80Arg Lys Asn Asn Glu Ala Ala Lys Arg Ser Arg Glu Lys Arg Arg Leu 85 90 95Asn Asp Leu Val Leu Glu Asn Lys Leu Ile Ala Leu Gly Glu Glu Asn 100 105 110Ala Thr Leu Lys Ala Glu Leu Leu Ser Leu Lys Leu Lys Phe Gly Leu 115 120 125Ile Ser Ser Thr Ala Tyr Ala Gln Glu Ile Gln Lys Leu Ser Asn Ser 130 135 140Thr Ala Val Tyr Phe Gln Asp Tyr Gln Thr Ser Lys Ser Asn Val Ser145 150 155 160Ser Phe Val Asp Glu His Glu Pro Ser Met Val Ser Ser Ser Cys Ile 165 170 175Ser Val Ile Lys His Ser Pro Gln Ser Ser Leu Ser Asp Val Ser Glu 180 185 190Val Ser Ser Val Glu His Thr Gln Glu Ser Ser Val Gln Gly Ser Cys 195 200 205Arg Ser Pro Glu Asn Lys Phe Gln Ile Ile Lys Gln Glu Pro Met Glu 210 215 220Leu Glu Ser Tyr Thr Arg Glu Pro Arg Asp Asp Arg Gly Ser Tyr Thr225 230 235 240Ala Ser Ile Tyr Gln Asn Tyr Met Gly Asn Ser Phe Ser Gly Tyr Ser 245 250 255His Ser Pro Pro Leu Leu Gln Val Asn Arg Ser Ser Ser Asn Ser Pro 260 265 270Arg Thr Ser Glu Thr Asp Asp Gly Val Val Gly Lys Ser Ser Asp Gly 275 280 285Glu Asp Glu Gln Gln Val Pro Lys Gly Pro Ile His Ser Pro Val Glu 290 295 300Leu Lys His Val His Ala Thr Val Val Lys Val Pro Glu Val Asn Ser305 310 315 320Ser Ala Leu Pro His Lys Leu Arg Ile Lys Ala Lys Ala Met Gln Ile 325 330 335Lys Val Glu Ala Phe Asp Asn Glu Phe Glu Ala Thr Gln Lys Leu Ser 340 345 350Ser Pro Ile Asp Met Thr Ser Lys Arg His Phe Glu Leu Glu Lys His 355 360 365Ser Ala Pro Ser Met Val His Ser Ser Leu Thr Pro Phe Ser Val Gln 370 375 380Val Thr Asn Ile Gln Asp Trp Ser Leu Lys Ser Glu His Trp His Gln385 390 395 400Lys Glu Leu Ser Gly Lys Thr Gln Asn Ser Phe Lys Thr Gly Val Val 405 410 415Glu Met Lys Asp Ser Gly Tyr Lys Val Ser Asp Pro Glu Asn Leu Tyr 420 425 430Leu Lys Gln Gly Ile Ala Asn Leu Ser Ala Glu Val Val Ser Leu Lys 435 440 445Arg Leu Ile Ala Thr Gln Pro Ile Ser Ala Ser Asp Ser Gly 450 455 4609427PRTHomo sapiens 9Met Glu Ala Met Ala Ala Ser Thr Ser Leu Pro Asp Pro Gly Asp Phe1 5 10 15Asp Arg Asn Val Pro Arg Ile Cys Gly Val Cys Gly Asp Arg Ala Thr 20 25 30Gly Phe His Phe Asn Ala Met Thr Cys Glu Gly Cys Lys Gly Phe Phe 35 40 45Arg Arg Ser Met Lys Arg Lys Ala Leu Phe Thr Cys Pro Phe Asn Gly 50 55 60Asp Cys Arg Ile Thr Lys Asp Asn Arg Arg His Cys Gln Ala Cys Arg65 70 75 80Leu Lys Arg Cys Val Asp Ile Gly Met Met Lys Glu Phe Ile Leu Thr 85 90 95Asp Glu Glu Val Gln Arg Lys Arg Glu Met Ile Leu Lys Arg Lys Glu 100 105 110Glu Glu Ala Leu Lys Asp Ser Leu Arg Pro Lys Leu Ser Glu Glu Gln 115 120 125Gln Arg Ile Ile Ala Ile Leu Leu Asp Ala His His Lys Thr Tyr Asp 130 135 140Pro Thr Tyr Ser Asp Phe Cys Gln Phe Arg Pro Pro Val Arg Val Asn145 150 155 160Asp Gly Gly Gly Ser His Pro Ser Arg Pro Asn Ser Arg His Thr Pro 165 170 175Ser Phe Ser Gly Asp Ser Ser Ser Ser Cys Ser Asp His Cys Ile Thr 180 185 190Ser Ser Asp Met Met Asp Ser Ser Ser Phe Ser Asn Leu Asp Leu Ser 195 200 205Glu Glu Asp Ser Asp Asp Pro Ser Val Thr Leu Glu Leu Ser Gln Leu 210 215 220Ser Met Leu Pro His Leu Ala Asp Leu Val Ser Tyr Ser Ile Gln Lys225 230 235 240Val Ile Gly Phe Ala Lys Met Ile Pro Gly Phe Arg Asp Leu Thr Ser 245 250 255Glu Asp Gln Ile Val Leu Leu Lys Ser Ser Ala Ile Glu Val Ile Met 260 265 270Leu Arg Ser Asn Glu Ser Phe Thr Met Asp Asp Met Ser Trp Thr Cys 275 280 285Gly Asn Gln Asp Tyr Lys Tyr Arg Val Ser Asp Val Thr Lys Ala Gly 290 295 300His Ser Leu Glu Leu Ile Glu Pro Leu Ile Lys Phe Gln Val Gly Leu305 310 315 320Lys Lys Leu Asn Leu His Glu Glu Glu His Val Leu Leu Met Ala Ile 325 330 335Cys Ile Val Ser Pro Asp Arg Pro Gly Val Gln Asp Ala Ala Leu Ile 340 345 350Glu Ala Ile Gln Asp Arg Leu Ser Asn Thr Leu Gln Thr Tyr Ile Arg 355 360 365Cys Arg His Pro Pro Pro Gly Ser His Leu Leu Tyr Ala Lys Met Ile 370 375 380Gln Lys Leu Ala Asp Leu Arg Ser Leu Asn Glu Glu His Ser Lys Gln385 390 395 400Tyr Arg Cys Leu Ser Phe Gln Pro Glu Cys Ser Met Lys Leu Thr Pro 405 410 415Leu Val Leu Glu Val Phe Gly Asn Glu Ile Ser 420 42510268PRTHomo sapiens 10Met Ala Gln Pro Leu Cys Pro Pro Leu Ser Glu Ser Trp Met Leu Ser1 5 10 15Ala Ala Trp Gly Pro Thr Arg Arg Pro Pro Pro Ser Asp Lys Asp Cys 20 25 30Gly Arg Ser Leu Val Ser Ser Pro Asp Ser Trp Gly Ser Thr Pro Ala 35 40 45Asp Ser Pro Val Ala Ser Pro Ala Arg Pro Gly Thr Leu Arg Asp Pro 50 55 60Arg Ala Pro Ser Val Gly Arg Arg Gly Ala Arg Ser Ser Arg Leu Gly65 70 75 80Ser Gly Gln Arg Gln Ser Ala Ser Glu Arg Glu Lys Leu Arg Met Arg 85 90 95Thr Leu Ala Arg Ala Leu His Glu Leu Arg Arg Phe Leu Pro Pro Ser 100 105 110Val Ala Pro Ala Gly Gln Ser Leu Thr Lys Ile Glu Thr Leu Arg Leu 115 120 125Ala Ile Arg Tyr Ile Gly His Leu Ser Ala Val Leu Gly Leu Ser Glu 130 135 140Glu Ser Leu Gln Arg Arg Cys Arg Gln Arg Gly Asp Ala Gly Ser Pro145 150 155 160Arg Gly Cys Pro Leu Cys Pro Asp Asp Cys Pro Ala Gln Met Gln Thr 165 170 175Arg Thr Gln Ala Glu Gly Gln Gly Gln Gly Arg Gly Leu Gly Leu Val 180 185 190Ser Ala Val Arg Ala Gly Ala Ser Trp Gly Ser Pro Pro Ala Cys Pro 195 200 205Gly Ala Arg Ala Ala Pro Glu Pro Arg Asp Pro Pro Ala Leu Phe Ala 210 215 220Glu Ala Ala Cys Pro Glu Gly Gln Ala Met Glu Pro Ser Pro Pro Ser225 230 235 240Pro Leu Leu Pro Gly Asp Val Leu Ala Leu Leu Glu Thr Trp Met Pro 245 250 255Leu Ser Pro Leu Glu Trp Leu Pro Glu Glu Pro Lys 260 265111104PRTHomo sapiens 11Met Ala Arg Arg Gly Lys Lys Pro Val Val Arg Thr Leu Glu Asp Leu1 5 10 15Thr Leu Asp Ser Gly Tyr Gly Gly Ala Ala Asp Ser Val Arg Ser Ser 20 25 30Asn Leu Ser Leu Cys Cys Ser Asp Ser His Pro Ala Ser Pro Tyr Gly 35 40 45Gly Ser Cys Trp Pro Pro Leu Ala Asp Ser Met His Ser Arg His Asn 50 55 60Ser Phe Asp Thr Val Asn Thr Ala Leu Val Glu Asp Ser Glu Gly Leu65 70 75 80Asp Cys Ala Gly Gln His Cys Ser Arg Leu Leu Pro Asp Leu Asp Glu 85 90 95Val Pro Trp Thr Leu Gln Glu Leu Glu Ala Leu Leu Leu Arg Ser Arg 100 105 110Asp Pro Arg Ala Gly Pro Ala Val Pro Gly Gly Leu Pro Lys Asp Ala 115 120 125Leu Ala Lys Leu Ser Thr Leu Val Ser Arg Ala Leu Val Arg Ile Ala 130 135 140Lys Glu Ala Gln Arg Leu Ser Leu Arg Phe Ala Lys Cys Thr Lys Tyr145 150 155 160Glu Ile Gln Ser Ala Met Glu Ile Val Leu Ser Trp Gly Leu Ala Ala 165 170 175His Cys Thr Ala Ala Ala Leu Ala Ala Leu Ser Leu Tyr Asn Met Ser 180 185 190Ser Ala Gly Gly Asp Arg Leu Gly Arg Gly Lys Ser Ala Arg Cys Gly 195 200 205Leu Thr Phe Ser Val Gly Arg Val Tyr Arg Trp Met Val Asp Ser Arg 210 215 220Val Ala Leu Arg Ile His Glu His Ala Ala Ile Tyr Leu Thr Ala Cys225 230 235 240Met Glu Ser Leu Phe Arg Asp Ile Tyr Ser Arg Val Val Ala Ser Gly 245 250 255Val Pro Arg Ser Cys Ser Gly Pro Gly Ser Gly Ser Gly Ser Gly Pro 260 265 270Gly Pro Ser Ser Gly Pro Gly Ala Ala Pro Ala Ala Asp Lys Glu Arg 275 280 285Glu Ala Pro Gly Gly Gly Ala Ala Ser Gly Gly Ala Cys Ser Ala Ala 290 295 300Ser Ser Ala Ser Gly Gly Ser Ser Cys Cys Ala Pro Pro Ala Ala Ala305 310 315 320Ala Ala Ala Val Pro Pro Ala Ala Ala Ala Asn His His His His His 325 330 335His His Ala Leu His Glu Ala Pro Lys Phe Thr Val Glu Thr Leu Glu 340 345 350His Thr Val Asn Asn Asp Ser Glu Ile Trp Gly Leu Leu Gln Pro Tyr 355 360 365Gln His Leu Ile Cys Gly Lys Asn Ala Ser Gly Val Leu Cys Leu Pro 370 375 380Asp Ser Leu Asn Leu His Arg Asp Pro Gln Arg Ser Asn Lys Pro Gly385 390 395 400Glu Leu Pro Met Phe Ser Gln Ser Glu Leu Arg Thr Ile Glu Gln Ser 405 410 415Leu Leu Ala Thr Arg Val Gly Ser Ile Ala Glu Leu Ser Asp Leu Val 420 425 430Ser Arg Ala Met His His Leu Gln Pro Leu Asn Ala Lys His His Gly 435 440 445Asn Gly Thr Pro Leu His His Lys Gln Gly Ala Leu Tyr Trp Glu Pro 450 455 460Glu Ala Leu Tyr Thr Leu Cys Tyr Phe Met His Cys Pro Gln Met Glu465 470 475 480Trp Glu Asn Pro Asn Val Glu Pro Ser Lys Val Asn Leu Gln Val Glu 485 490 495Arg Pro Phe Leu Val Leu Pro Pro Leu Met Glu Trp Ile Arg Val Ala 500 505 510Val Ala His Ala Gly His Arg Arg Ser Phe Ser Met Asp Ser Asp Asp 515 520 525Val Arg Gln Ala Ala Arg Leu Leu Leu Pro Gly Val Asp Cys Glu Pro 530 535 540Arg Gln Leu Arg Ala Asp Asp Cys Phe Cys Ala Ser Arg Lys Leu Asp545 550 555 560Ala Val Ala Ile Glu Ala Lys Phe Lys Gln Asp Leu Gly Phe Arg Met 565 570 575Leu Asn Cys Gly Arg Thr Asp Leu Val Lys Gln Ala Val Ser Leu Leu 580 585 590Gly

Pro Asp Gly Ile Asn Thr Met Ser Glu Gln Gly Met Thr Pro Leu 595 600 605Met Tyr Ala Cys Val Arg Gly Asp Glu Ala Met Val Gln Met Leu Leu 610 615 620Asp Ala Gly Ala Asp Leu Asn Val Glu Val Val Ser Thr Pro His Lys625 630 635 640Tyr Pro Ser Val His Pro Glu Thr Arg His Trp Thr Ala Leu Thr Phe 645 650 655Ala Val Leu His Gly His Ile Pro Val Val Gln Leu Leu Leu Asp Ala 660 665 670Gly Ala Lys Val Glu Gly Ser Val Glu His Gly Glu Glu Asn Tyr Ser 675 680 685Glu Thr Pro Leu Gln Leu Ala Ala Ala Val Gly Asn Phe Glu Leu Val 690 695 700Ser Leu Leu Leu Glu Arg Gly Ala Asp Pro Leu Ile Gly Thr Met Tyr705 710 715 720Arg Asn Gly Ile Ser Thr Thr Pro Gln Gly Asp Met Asn Ser Phe Ser 725 730 735Gln Ala Ala Ala His Gly His Arg Asn Val Phe Arg Lys Leu Leu Ala 740 745 750Gln Pro Glu Lys Glu Lys Ser Asp Ile Leu Ser Leu Glu Glu Ile Leu 755 760 765Ala Glu Gly Thr Asp Leu Ala Glu Thr Ala Pro Pro Pro Leu Cys Ala 770 775 780Ser Arg Asn Ser Lys Ala Lys Leu Arg Ala Leu Arg Glu Ala Met Tyr785 790 795 800His Ser Ala Glu His Gly Tyr Val Asp Val Thr Ile Asp Ile Arg Ser 805 810 815Ile Gly Val Pro Trp Thr Leu His Thr Trp Leu Glu Ser Leu Arg Ile 820 825 830Ala Phe Gln Gln His Arg Arg Pro Leu Ile Gln Cys Leu Leu Lys Glu 835 840 845Phe Lys Thr Ile Gln Glu Glu Glu Tyr Thr Glu Glu Leu Val Thr Gln 850 855 860Gly Leu Pro Leu Met Phe Glu Ile Leu Lys Ala Ser Lys Asn Glu Val865 870 875 880Ile Ser Gln Gln Leu Cys Val Ile Phe Thr His Cys Tyr Gly Pro Tyr 885 890 895Pro Ile Pro Lys Leu Thr Glu Ile Lys Arg Lys Gln Thr Ser Arg Leu 900 905 910Asp Pro His Phe Leu Asn Asn Lys Glu Met Ser Asp Val Thr Phe Leu 915 920 925Val Glu Gly Arg Pro Phe Tyr Ala His Lys Val Leu Leu Phe Thr Ala 930 935 940Ser Pro Arg Phe Lys Ala Leu Leu Ser Ser Lys Pro Thr Asn Asp Gly945 950 955 960Thr Cys Ile Glu Ile Gly Tyr Val Lys Tyr Ser Ile Phe Gln Leu Val 965 970 975Met Gln Tyr Leu Tyr Tyr Gly Gly Pro Glu Ser Leu Leu Ile Lys Asn 980 985 990Asn Glu Ile Met Glu Leu Leu Ser Ala Ala Lys Phe Phe Gln Leu Glu 995 1000 1005Ala Leu Gln Arg His Cys Glu Ile Ile Cys Ala Lys Ser Ile Asn 1010 1015 1020Thr Asp Asn Cys Val Asp Ile Tyr Asn His Ala Lys Phe Leu Gly 1025 1030 1035Val Thr Glu Leu Ser Ala Tyr Cys Glu Gly Tyr Phe Leu Lys Asn 1040 1045 1050Met Met Val Leu Ile Glu Asn Glu Ala Phe Lys Gln Leu Leu Tyr 1055 1060 1065Asp Lys Asn Gly Glu Gly Thr Gly Gln Asp Val Leu Gln Asp Leu 1070 1075 1080Gln Arg Thr Leu Ala Ile Arg Ile Gln Ser Ile His Leu Ser Ser 1085 1090 1095Ser Lys Gly Ser Val Val 110012664PRTHomo sapiens 12Met Pro Ala Val Asp Lys Leu Leu Leu Glu Glu Ala Leu Gln Asp Ser1 5 10 15Pro Gln Thr Arg Ser Leu Leu Ser Val Phe Glu Glu Asp Ala Gly Thr 20 25 30Leu Thr Asp Tyr Thr Asn Gln Leu Leu Gln Ala Met Gln Arg Val Tyr 35 40 45Gly Ala Gln Asn Glu Met Cys Leu Ala Thr Gln Gln Leu Ser Lys Gln 50 55 60Leu Leu Ala Tyr Glu Lys Gln Asn Phe Ala Leu Gly Lys Gly Asp Glu65 70 75 80Glu Val Ile Ser Thr Leu His Tyr Phe Ser Lys Val Val Asp Glu Leu 85 90 95Asn Leu Leu His Thr Glu Leu Ala Lys Gln Leu Ala Asp Thr Met Val 100 105 110Leu Pro Ile Ile Gln Phe Arg Glu Lys Asp Leu Thr Glu Val Ser Thr 115 120 125Leu Lys Asp Leu Phe Gly Leu Ala Ser Asn Glu His Asp Leu Ser Met 130 135 140Ala Lys Tyr Ser Arg Leu Pro Lys Lys Lys Glu Asn Glu Lys Val Lys145 150 155 160Thr Glu Val Gly Lys Glu Val Ala Ala Ala Arg Arg Lys Gln His Leu 165 170 175Ser Ser Leu Gln Tyr Tyr Cys Ala Leu Asn Ala Leu Gln Tyr Arg Lys 180 185 190Gln Met Ala Met Met Glu Pro Met Ile Gly Phe Ala His Gly Gln Ile 195 200 205Asn Phe Phe Lys Lys Gly Ala Glu Met Phe Ser Lys Arg Met Asp Ser 210 215 220Phe Leu Ser Ser Val Ala Asp Met Val Gln Ser Ile Gln Val Glu Leu225 230 235 240Glu Ala Glu Ala Glu Lys Met Arg Val Ser Gln Gln Glu Leu Leu Ser 245 250 255Val Asp Glu Ser Val Tyr Thr Pro Asp Ser Asp Val Ala Ala Pro Gln 260 265 270Ile Asn Arg Asn Leu Ile Gln Lys Ala Gly Tyr Leu Asn Leu Arg Asn 275 280 285Lys Thr Gly Leu Val Thr Thr Thr Trp Glu Arg Leu Tyr Phe Phe Thr 290 295 300Gln Gly Gly Asn Leu Met Cys Gln Pro Arg Gly Ala Val Ala Gly Gly305 310 315 320Leu Ile Gln Asp Leu Asp Asn Cys Ser Val Met Ala Val Asp Cys Glu 325 330 335Asp Arg Arg Tyr Cys Phe Gln Ile Thr Thr Pro Asn Gly Lys Ser Gly 340 345 350Ile Ile Leu Gln Ala Glu Ser Arg Lys Glu Asn Glu Glu Trp Ile Cys 355 360 365Ala Ile Asn Asn Ile Ser Arg Gln Ile Tyr Leu Thr Asp Asn Pro Glu 370 375 380Ala Val Ala Ile Lys Leu Asn Gln Thr Ala Leu Gln Ala Val Thr Pro385 390 395 400Ile Thr Ser Phe Gly Lys Lys Gln Glu Ser Ser Cys Pro Ser Gln Asn 405 410 415Leu Lys Asn Ser Glu Met Glu Asn Glu Asn Asp Lys Ile Val Pro Lys 420 425 430Ala Thr Ala Ser Leu Pro Glu Ala Glu Glu Leu Ile Ala Pro Gly Thr 435 440 445Pro Ile Gln Phe Asp Ile Val Leu Pro Ala Thr Glu Phe Leu Asp Gln 450 455 460Asn Arg Gly Ser Arg Arg Thr Asn Pro Phe Gly Glu Thr Glu Asp Glu465 470 475 480Ser Phe Pro Glu Ala Glu Asp Ser Leu Leu Gln Gln Met Phe Ile Val 485 490 495Arg Phe Leu Gly Ser Met Ala Val Lys Thr Asp Ser Thr Thr Glu Val 500 505 510Ile Tyr Glu Ala Met Arg Gln Val Leu Ala Ala Arg Ala Ile His Asn 515 520 525Ile Phe Arg Met Thr Glu Ser His Leu Met Val Thr Ser Gln Ser Leu 530 535 540Arg Leu Ile Asp Pro Gln Thr Gln Val Ser Arg Ala Asn Phe Glu Leu545 550 555 560Thr Ser Val Thr Gln Phe Ala Ala His Gln Glu Asn Lys Arg Leu Val 565 570 575Gly Phe Val Ile Arg Val Pro Glu Ser Thr Gly Glu Glu Ser Leu Ser 580 585 590Thr Tyr Ile Phe Glu Ser Asn Ser Glu Gly Glu Lys Ile Cys Tyr Ala 595 600 605Ile Asn Leu Gly Lys Glu Ile Ile Glu Val Gln Lys Asp Pro Glu Ala 610 615 620Leu Ala Gln Leu Met Leu Ser Ile Pro Leu Thr Asn Asp Gly Lys Tyr625 630 635 640Val Leu Leu Asn Asp Gln Pro Asp Asp Asp Asp Gly Asn Pro Asn Glu 645 650 655His Arg Gly Ala Glu Ser Glu Ala 660131067PRTHomo sapiens 13Met Ala Ser Pro Thr Ser Thr Asn Pro Ala His Ala His Phe Glu Ser1 5 10 15Phe Leu Gln Ala Gln Leu Cys Gln Asp Val Leu Ser Ser Phe Gln Glu 20 25 30Leu Cys Gly Ala Leu Gly Leu Glu Pro Gly Gly Gly Leu Pro Gln Tyr 35 40 45His Lys Ile Lys Asp Gln Leu Asn Tyr Trp Ser Ala Lys Ser Leu Trp 50 55 60Thr Lys Leu Asp Lys Arg Ala Gly Gln Pro Val Tyr Gln Gln Gly Arg65 70 75 80Ala Cys Thr Ser Thr Lys Cys Leu Val Val Gly Ala Gly Pro Cys Gly 85 90 95Leu Arg Val Ala Val Glu Leu Ala Leu Leu Gly Ala Arg Val Val Leu 100 105 110Val Glu Lys Arg Thr Lys Phe Ser Arg His Asn Val Leu His Leu Trp 115 120 125Pro Phe Thr Ile His Asp Leu Arg Ala Leu Gly Ala Lys Lys Phe Tyr 130 135 140Gly Arg Phe Cys Thr Gly Thr Leu Asp His Ile Ser Ile Arg Gln Leu145 150 155 160Gln Leu Leu Leu Leu Lys Val Ala Leu Leu Leu Gly Val Glu Ile His 165 170 175Trp Gly Val Thr Phe Thr Gly Leu Gln Pro Pro Pro Arg Lys Gly Ser 180 185 190Gly Trp Arg Ala Gln Leu Gln Pro Asn Pro Pro Ala Gln Leu Ala Asn 195 200 205Tyr Glu Phe Asp Val Leu Ile Ser Ala Ala Gly Gly Lys Phe Val Pro 210 215 220Glu Gly Phe Lys Val Arg Glu Met Arg Gly Lys Leu Ala Ile Gly Ile225 230 235 240Thr Ala Asn Phe Val Asn Gly Arg Thr Val Glu Glu Thr Gln Val Pro 245 250 255Glu Ile Ser Gly Val Ala Arg Ile Tyr Asn Gln Ser Phe Phe Gln Ser 260 265 270Leu Leu Lys Ala Thr Gly Ile Asp Leu Glu Asn Ile Val Tyr Tyr Lys 275 280 285Asp Asp Thr His Tyr Phe Val Met Thr Ala Lys Lys Gln Cys Leu Leu 290 295 300Arg Leu Gly Val Leu Arg Gln Asp Trp Pro Asp Thr Asn Arg Leu Leu305 310 315 320Gly Ser Ala Asn Val Val Pro Glu Ala Leu Gln Arg Phe Thr Arg Ala 325 330 335Ala Ala Asp Phe Ala Thr His Gly Lys Leu Gly Lys Leu Glu Phe Ala 340 345 350Gln Asp Ala His Gly Gln Pro Asp Val Ser Ala Phe Asp Phe Thr Ser 355 360 365Met Met Arg Ala Glu Ser Ser Ala Arg Val Gln Glu Lys His Gly Ala 370 375 380Arg Leu Leu Leu Gly Leu Val Gly Asp Cys Leu Val Glu Pro Phe Trp385 390 395 400Pro Leu Gly Thr Gly Val Ala Arg Gly Phe Leu Ala Ala Phe Asp Ala 405 410 415Ala Trp Met Val Lys Arg Trp Ala Glu Gly Ala Glu Ser Leu Glu Val 420 425 430Leu Ala Glu Arg Glu Ser Leu Tyr Gln Leu Leu Ser Gln Thr Ser Pro 435 440 445Glu Asn Met His Arg Asn Val Ala Gln Tyr Gly Leu Asp Pro Ala Thr 450 455 460Arg Tyr Pro Asn Leu Asn Leu Arg Ala Val Thr Pro Asn Gln Val Arg465 470 475 480Asp Leu Tyr Asp Val Leu Ala Lys Glu Pro Val Gln Arg Asn Asn Asp 485 490 495Lys Thr Asp Thr Gly Met Pro Ala Thr Gly Ser Ala Gly Thr Gln Glu 500 505 510Glu Leu Leu Arg Trp Cys Gln Glu Gln Thr Ala Gly Tyr Pro Gly Val 515 520 525His Val Ser Asp Leu Ser Ser Ser Trp Ala Asp Gly Leu Ala Leu Cys 530 535 540Ala Leu Val Tyr Arg Leu Gln Pro Gly Leu Leu Glu Pro Ser Glu Leu545 550 555 560Gln Gly Leu Gly Ala Leu Glu Ala Thr Ala Trp Ala Leu Lys Val Ala 565 570 575Glu Asn Glu Leu Gly Ile Thr Pro Val Val Ser Ala Gln Ala Val Val 580 585 590Ala Gly Ser Asp Pro Leu Gly Leu Ile Ala Tyr Leu Ser His Phe His 595 600 605Ser Ala Phe Lys Ser Met Ala His Ser Pro Gly Pro Val Ser Gln Ala 610 615 620Ser Pro Gly Thr Ser Ser Ala Val Leu Phe Leu Ser Lys Leu Gln Arg625 630 635 640Thr Leu Gln Arg Ser Arg Ala Lys Glu Asn Ala Glu Asp Ala Gly Gly 645 650 655Lys Lys Leu Arg Leu Glu Met Glu Ala Glu Thr Pro Ser Thr Glu Val 660 665 670Pro Pro Asp Pro Glu Pro Gly Val Pro Leu Thr Pro Pro Ser Gln His 675 680 685Gln Glu Ala Gly Ala Gly Asp Leu Cys Ala Leu Cys Gly Glu His Leu 690 695 700Tyr Val Leu Glu Arg Leu Cys Val Asn Gly His Phe Phe His Arg Ser705 710 715 720Cys Phe Arg Cys His Thr Cys Glu Ala Thr Leu Trp Pro Gly Gly Tyr 725 730 735Glu Gln His Pro Gly Asp Gly His Phe Tyr Cys Leu Gln His Leu Pro 740 745 750Gln Thr Asp His Lys Ala Glu Gly Ser Asp Arg Gly Pro Glu Ser Pro 755 760 765Glu Leu Pro Thr Pro Ser Glu Asn Ser Met Pro Pro Gly Leu Ser Thr 770 775 780Pro Thr Ala Ser Gln Glu Gly Ala Gly Pro Val Pro Asp Pro Ser Gln785 790 795 800Pro Thr Arg Arg Gln Ile Arg Leu Ser Ser Pro Glu Arg Gln Arg Leu 805 810 815Ser Ser Leu Asn Leu Thr Pro Asp Pro Glu Met Glu Pro Pro Pro Lys 820 825 830Pro Pro Arg Ser Cys Ser Ala Leu Ala Arg His Ala Leu Glu Ser Ser 835 840 845Phe Val Gly Trp Gly Leu Pro Val Gln Ser Pro Gln Ala Leu Val Ala 850 855 860Met Glu Lys Glu Glu Lys Glu Ser Pro Phe Ser Ser Glu Glu Glu Glu865 870 875 880Glu Asp Val Pro Leu Asp Ser Asp Val Glu Gln Ala Leu Gln Thr Phe 885 890 895Ala Lys Thr Ser Gly Thr Met Asn Asn Tyr Pro Thr Trp Arg Arg Thr 900 905 910Leu Leu Arg Arg Ala Lys Glu Glu Glu Met Lys Arg Phe Cys Lys Ala 915 920 925Gln Thr Ile Gln Arg Arg Leu Asn Glu Ile Glu Ala Ala Leu Arg Glu 930 935 940Leu Glu Ala Glu Gly Val Lys Leu Glu Leu Ala Leu Arg Arg Gln Ser945 950 955 960Ser Ser Pro Glu Gln Gln Lys Lys Leu Trp Val Gly Gln Leu Leu Gln 965 970 975Leu Val Asp Lys Lys Asn Ser Leu Val Ala Glu Glu Ala Glu Leu Met 980 985 990Ile Thr Val Gln Glu Leu Asn Leu Glu Glu Lys Gln Trp Gln Leu Asp 995 1000 1005Gln Glu Leu Arg Gly Tyr Met Asn Arg Glu Glu Asn Leu Lys Thr 1010 1015 1020Ala Ala Asp Arg Gln Ala Glu Asp Gln Val Leu Arg Lys Leu Val 1025 1030 1035Asp Leu Val Asn Gln Arg Asp Ala Leu Ile Arg Phe Gln Glu Glu 1040 1045 1050Arg Arg Leu Ser Glu Leu Ala Leu Gly Thr Gly Ala Gln Gly 1055 1060 106514125PRTHomo sapiens 14Met Pro His Ser Ser Asp Ser Ser Asp Ser Ser Phe Ser Arg Ser Pro1 5 10 15Pro Pro Gly Lys Gln Asp Ser Ser Asp Asp Val Arg Arg Val Gln Arg 20 25 30Arg Glu Lys Asn Arg Ile Ala Ala Gln Lys Ser Arg Gln Arg Gln Thr 35 40 45Gln Lys Ala Asp Thr Leu His Leu Glu Ser Glu Asp Leu Glu Lys Gln 50 55 60Asn Ala Ala Leu Arg Lys Glu Ile Lys Gln Leu Thr Glu Glu Leu Lys65 70 75 80Tyr Phe Thr Ser Val Leu Asn Ser His Glu Pro Leu Cys Ser Val Leu 85 90 95Ala Ala Ser Thr Pro Ser Pro Pro Glu Val Val Tyr Ser Ala His Ala 100 105 110Phe His Gln Pro His Val Ser Ser Pro Arg Phe Gln Pro 115 120 12515660PRTHomo sapiens 15Met Leu Val Cys Tyr Ser Val Leu Ala Cys Glu Ile Leu Trp Asp Leu1 5 10 15Pro Cys Ser Ile Met Gly Ser Pro Leu Gly His Phe Thr Trp Asp Lys 20 25 30Tyr Leu Lys Glu Thr Cys Ser Val Pro Ala Pro Val His Cys Phe Lys 35 40 45Gln Ser Tyr Thr Pro Pro Ser Asn Glu Phe Lys Ile Ser Met Lys Leu 50 55 60Glu Ala Gln Asp Pro Arg Asn Thr Thr Ser Thr Cys Ile Ala Thr Val65 70 75 80Val Gly Leu Thr Gly Ala Arg Leu Arg Leu Arg Leu Asp Gly Ser Asp 85 90

95Asn Lys Asn Asp Phe Trp Arg Leu Val Asp Ser Ala Glu Ile Gln Pro 100 105 110Ile Gly Asn Cys Glu Lys Asn Gly Gly Met Leu Gln Pro Pro Leu Gly 115 120 125Phe Arg Leu Asn Ala Ser Ser Trp Pro Met Phe Leu Leu Lys Thr Leu 130 135 140Asn Gly Ala Glu Met Ala Pro Ile Arg Ile Phe His Lys Glu Pro Pro145 150 155 160Ser Pro Ser His Asn Phe Phe Lys Met Gly Met Lys Leu Glu Ala Val 165 170 175Asp Arg Lys Asn Pro His Phe Ile Cys Pro Ala Thr Ile Gly Glu Val 180 185 190Arg Gly Ser Glu Val Leu Val Thr Phe Asp Gly Trp Arg Gly Ala Phe 195 200 205Asp Tyr Trp Cys Arg Phe Asp Ser Arg Asp Ile Phe Pro Val Gly Trp 210 215 220Cys Ser Leu Thr Gly Asp Asn Leu Gln Pro Pro Gly Thr Lys Val Val225 230 235 240Ile Pro Lys Asn Pro Tyr Pro Ala Ser Asp Val Asn Thr Glu Lys Pro 245 250 255Ser Ile His Ser Ser Thr Lys Thr Val Leu Glu His Gln Pro Gly Gln 260 265 270Arg Gly Arg Lys Pro Gly Lys Lys Arg Gly Arg Thr Pro Lys Thr Leu 275 280 285Ile Ser His Pro Ile Ser Ala Pro Ser Lys Thr Ala Glu Pro Leu Lys 290 295 300Phe Pro Lys Lys Arg Gly Pro Lys Pro Gly Ser Lys Arg Lys Pro Arg305 310 315 320Thr Leu Leu Asn Pro Pro Pro Ala Ser Pro Thr Thr Ser Thr Pro Glu 325 330 335Pro Asp Thr Ser Thr Val Pro Gln Asp Ala Ala Thr Ile Pro Ser Ser 340 345 350Ala Met Gln Ala Pro Thr Val Cys Ile Tyr Leu Asn Lys Asn Gly Ser 355 360 365Thr Gly Pro His Leu Asp Lys Lys Lys Val Gln Gln Leu Pro Asp His 370 375 380Phe Gly Pro Ala Arg Ala Ser Val Val Leu Gln Gln Ala Val Gln Ala385 390 395 400Cys Ile Asp Cys Ala Tyr His Gln Lys Thr Val Phe Ser Phe Leu Lys 405 410 415Gln Gly His Gly Gly Glu Val Ile Ser Ala Val Phe Asp Arg Glu Gln 420 425 430His Thr Leu Asn Leu Pro Ala Val Asn Ser Ile Thr Tyr Val Leu Arg 435 440 445Phe Leu Glu Lys Leu Cys His Asn Leu Arg Ser Asp Asn Leu Phe Gly 450 455 460Asn Gln Pro Phe Thr Gln Thr His Leu Ser Leu Thr Ala Ile Glu Tyr465 470 475 480Ser His Ser His Asp Arg Tyr Leu Pro Gly Glu Thr Phe Val Leu Gly 485 490 495Asn Ser Leu Ala Arg Ser Leu Glu Pro His Ser Asp Ser Met Asp Ser 500 505 510Ala Ser Asn Pro Thr Asn Leu Val Ser Thr Ser Gln Arg His Arg Pro 515 520 525Leu Leu Ser Ser Cys Gly Leu Pro Pro Ser Thr Ala Ser Ala Val Arg 530 535 540Arg Leu Cys Ser Arg Gly Val Leu Lys Gly Ser Asn Glu Arg Arg Asp545 550 555 560Met Glu Ser Phe Trp Lys Leu Asn Arg Ser Pro Gly Ser Asp Arg Tyr 565 570 575Leu Glu Ser Arg Asp Ala Ser Arg Leu Ser Gly Arg Asp Pro Ser Ser 580 585 590Trp Thr Val Glu Asp Val Met Gln Phe Val Arg Glu Ala Asp Pro Gln 595 600 605Leu Gly Pro His Ala Asp Leu Phe Arg Lys His Glu Ile Asp Gly Lys 610 615 620Ala Leu Leu Leu Leu Arg Ser Asp Met Met Met Lys Tyr Met Gly Leu625 630 635 640Lys Leu Gly Pro Ala Leu Lys Leu Ser Tyr His Ile Asp Arg Leu Lys 645 650 655Gln Gly Lys Phe 66016197PRTHomo sapiens 16Met Ala Ser Gln Lys Arg Pro Ser Gln Arg His Gly Ser Lys Tyr Leu1 5 10 15Ala Thr Ala Ser Thr Met Asp His Ala Arg His Gly Phe Leu Pro Arg 20 25 30His Arg Asp Thr Gly Ile Leu Asp Ser Ile Gly Arg Phe Phe Gly Gly 35 40 45Asp Arg Gly Ala Pro Lys Arg Gly Ser Gly Lys Val Pro Trp Leu Lys 50 55 60Pro Gly Arg Ser Pro Leu Pro Ser His Ala Arg Ser Gln Pro Gly Leu65 70 75 80Cys Asn Met Tyr Lys Asp Ser His His Pro Ala Arg Thr Ala His Tyr 85 90 95Gly Ser Leu Pro Gln Lys Ser His Gly Arg Thr Gln Asp Glu Asn Pro 100 105 110Val Val His Phe Phe Lys Asn Ile Val Thr Pro Arg Thr Pro Pro Pro 115 120 125Ser Gln Gly Lys Gly Arg Gly Leu Ser Leu Ser Arg Phe Ser Trp Gly 130 135 140Ala Glu Gly Gln Arg Pro Gly Phe Gly Tyr Gly Gly Arg Ala Ser Asp145 150 155 160Tyr Lys Ser Ala His Lys Gly Phe Lys Gly Val Asp Ala Gln Gly Thr 165 170 175Leu Ser Lys Ile Phe Lys Leu Gly Gly Arg Asp Ser Arg Ser Gly Ser 180 185 190Pro Met Ala Arg Arg 195

Claims

1-41. (canceled)

42. A method for producing platelets, the method comprising: (i) introducing into an isolated population of pluripotent stem cells (PSCs): (a) a first transgene comprising an inducible promoter and nucleotide sequences encoding GATA binding protein 1 (GATA1), Friend leukemia virus integration 1 (FLI1) and T cell acute lymphocytic leukemia protein 1 (TAL1) proteins for their concurrent transcription under the control of the inducible promoter; and (b) a second transgene comprising a constitutive promoter and a nucleotide sequence encoding a transcription factor for the constitutive expression of the transcription factor under the control of the constitutive promoter, the transcription factor specific for binding to the inducible promoter in the first transgene thereby concurrently inducing the transcription of the GATA1, FLI1 and TAL1 proteins; and (ii) culturing the pluripotent stem cells in ultralow adherent culture conditions in a chemically defined medium (CMD) comprising at least a chemical agent, BMP4, and FGF2 to produce megakaryocyte progenitor cells for a duration wherein the megakaryocyte progenitor cells produce platelets through pro-platelet formation.

43. The method of claim 42 wherein the BMP4 and FGF2 are recombinant human proteins.

44. The method of claim 42 wherein the inducible promoter is a tetracycline responsive promoter.

45. The method of claim 44 wherein the GATA1, FLI1 and TAL1 are concurrently transcribed under the influence of the tetracycline responsive promoter.

46. The method of claim 42 wherein the constitutively expressed transcription factor is a transactivator protein.

47. The method of claim 46 wherein the transactivator protein is a reverse tetracycline-controlled transactivator protein that binds to the tetracycline responsive promoter.

48. The method of claim 42 wherein the chemical agent is tetracycline or a derivative thereof.

49. The method of claim 42, wherein the first and the second transgenes integrate at different sites in a chromosome of the pluripotent stem cells.

50. The method of claim 42, wherein the pluripotent stem cells are selected from the group consisting of embryonic stem (ES) cells, non-embryonic stem cells, fetal or adult somatic stem cells and stem cells derived from non-pluripotent cells.

51. The method of claim 42 wherein said platelets are used as a therapeutic agent in platelet-associated conditions.

52. The method of claim 42 wherein the CDM further comprises thrombopoietin protein (TPO), and one or more of Stem Cell Factor (SCF) or IL1B.

53. The method of claim 52 wherein the TPO, SCF and IL1B are recombinant human proteins.

54. The method of claim 42 wherein the induced stem cells are cultured for a period of 18 to 22 days to produce the platelets from the megakaryocyte cells.

55. The method of claim 42 wherein the induced stem cells are cultured for a period of about 20 days to produce the platelets from the megakaryocyte cells.

56. The method of claim 54 wherein at least 95% of the platelet producing megakaryocyte cells express CD41a.

57. The method of claim 54 wherein at least 50% of the platelet producing megakaryocyte cells express CD42a.

58. The method of claim 54 wherein the platelet producing megakaryocyte cells optionally produce platelet like particles (PLPs).

59. The method of claim 54 wherein the pluripotent stem cells are induced pluripotent cells (iPS).

60. The method of claim 59 wherein the iPS cells are human iPS cells.

61. The method of claim 42 wherein the chemical agent is added transiently or constitutively to the culture conditions.