WO2011130217A1

WO2011130217A1 - Induced pluripotent stem cells and uses thereof

Info

Publication number: WO2011130217A1
Application number: PCT/US2011/032044
Authority: WO
Inventors: Xonia Carvajal; Bruce D. Gelb; Ihor Lemischka
Original assignee: Mount Sinai School Of Medicine
Priority date: 2010-04-14
Filing date: 2011-04-12
Publication date: 2011-10-20

Abstract

This invention generally relates to disease-specific pluripotent stem cells that are derived from somatic cells that carry a mutation in the PTPNl 1 gene. The stem cells are capable of differentiating into cells or tissues affected by the PTPNl 1 mutation. The stem cells and differentiated cells can be used to study various diseases that are associated with mutations in the PTPNl 1 gene, and to identify novel therapeutic agents for diagnosis and treatment of diseases that are associated with mutations in PTPNl 1.

Description

INDUCED PLURIPOTENT STEM CELLS AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application relates to U.S. Provisional Application Serial

No. 61/324,150, filed April 14, 2010, which is incorporated by reference in its entirety.

GOVERNMENT SUPPORT

[0002] The subject matter of this application has been supported by a research grant from the National Institutes of Health under grant number 5R01GM078465 and a grant from NYSTEM. The United States government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terms as provided for by the terms of this grant.

BACKGROUND OF THE INVENTION

[0003] Pluripotent stem cells can grow indefinitely while maintaining pluripotency, and can differentiate into cells of all three germ layers (Evans & Kaufman, Nature 292: 154-156 (1981)). Human pluripotent stem cells have promise for treating diseases such as Parkinson's disease, spinal cord injury and diabetes (Thomson et al, Science 282:1145-1147 (1998)).

[0004] Somatic cells can be reprogrammed into pluripotent stem cells by transferring their nuclear contents into oocytes (Wilmut et al, Nature 385:810-813(1997)) or by fusion with embryonic stem (ES) cells (Cowan et al, Science 309: 1369-1373 (2005)), indicating that unfertilized eggs and ES cells contain factors that confer totipotency or pluripotency in somatic cells. For example, Yu et al. showed that cells derived by in vitro differentiation from an Oct4 knock-in human ES cell line did not express EGFP, but that EGFP expression was restored upon cell-cell fusion with human ES cells (Yu et al, Stem Cells 24: 168-176 (2006)).

[0005] Induced pluripotent stem cells, commonly abbreviated as "iPS cells" or "iPSCs," are pluripotent stem cell derived from non-pluripotent cells (e.g., adult somatic cells). iPS cells are typically produced by introducing into somatic cells certain transcription factors that are involved in maintaining ES cell pluripotency. Although the transcriptional

determination of pluripotency is not fully understood, several transcription factors, including Oct 3/4 (Nichols et al, Cell 95:379-391(1998)), Sox2 (Avilion et al, Genes Dev. 17: 126-140 (2003)) and Nanog (Chambers et al., Cell 113:643-655(2003)) were found to be involved in maintaining ES cell pluripotency.

[0006] Takahashi & Yamanaka introduced four reprogramming factors (Oct3/4, Sox2, c-Myc and Klf ) into mouse adult fibroblasts to obtain iPSCs. These iPSCs exhibited mouse ES cell morphology and growth properties, and expressed mouse ES cell marker genes (Takahashi & Yamanaka, Cell 126:663-676 (2006)). Notably, exogenous Oct-4 introduced into the mouse fibroblasts resulted in only marginal Oct-4 expression. Subcutaneous transplantation of iPS cells into nude mice resulted in tumors containing a variety of tissues from all three germ layers. Following injection into blastocysts, iPS cells contributed to mouse embryonic

development. These data demonstrate that pluripotent cells can be directly generated from mouse fibroblast cultures by adding only a few defined factors using retroviral transduction. One year later, using the same principle developed in mouse models, Takahashi et al.

successfully transformed human fibroblasts into pluripotent stem cells using the same four genes: Oct3/4, Sox2, Klf4, and c-Myc with a retroviral system (Cell, 131 :861-72, 2007).

[0007] Yu et al. (Science, 318:1917-20) and WO2008/0233610 (Thomson and Yu) disclose the reprogramming of human fibroblast cells into pluripotent stem cells using a lentiviral system and a different set of factors: OCT4, SOX2, NANOG, and LIN28.

WO2008/0233610 notes that ES cells from mice and humans require distinct sets of factors to remain undifferentiated.

[0008] LEOPARD syndrome (LS; also known as "Cardiocutaneous syndrome," "Gorlin syndrome II," "Lentiginosis profusa syndrome," "Progressive cardiomyopathic lentiginosis," "Capute-Rimoin-Konigsmark-Esterly-Richardson syndrome," or "Moynahan syndrome") is a rare autosomal dominant, multisystem disease having a complex of features, mostly involving the skin, skeletal and cardiovascular systems. The name of the condition is a mnemonic, as the condition is characterized by the following seven conditions, the first letters of which spell LEOPARD: Lentigines, Electrocardiographic abnormalities, Ocular hypertelorism, Pulmonary valve stenosis, Abnormal genitalia, Retardation of growth and Deafness. These conditions may not be present in all patients. [0009] Noonan syndrome (NS) is an autosomal dominant disorder characterized by dysmorphic facial features, proportionate short stature, and heart disease, i.e., pulmonic stenosis and hypertrophic cardiomyopathy most commonly (Noonan, Am. J. Dis. Child. 1968, 116:373- 380; Allanson, J. Med. Genet. 1987, 24:9-13). Webbed neck, chest deformity, cryptorchidism, mental retardation, and bleeding diatheses constitute other frequently associated findings. NS is a relatively common syndrome with an estimated incidence of 1 : 1000 to 1 :2500 live births.

[0010] Approximately 90% of LS cases, and 45% of NS, are caused by missense mutations in the protein tyrosine phosphatase, non-receptor type 11 (PTPNl 1) gene, which encodes a protein tyrosine phosphatase. PTPNl 1 is ubiquitously expressed and essential for normal development. Further, wild-type PTPNl 1 protein (also known as SHP2, Syp, SHPTP2, PTP2C, PTP1D or BPTP3) mediates cell signaling of many protein tyrosine kinase oncogenes, such as ErbB and Met. PTPNl 1 protein is necessary for embryonic development and for growth factor, cytokine, and extra-cellular matrix signaling, and is involved in the regulation of cell proliferation, differentiation, and migration.

[0011] Somatic mutations in the PTPNl 1 gene also contribute to leukemogenesis in children (e.g., juvenile myelomonocytic leukemia (JMML), or acute myelogenous leukemia). JMML is a progressive myelodysplastic/myeloproliferative disorder characterized by

overproduction of tissue-infiltrating myeloid cells. Somatic mutations in PTPNl 1 account for about 35% of JMML patients that do not have Ras or neurofibromin mutations (Tartaglia et al., Nature Genetics, Jun;34(2), 2003: 148-50; Kratz et al, Blood 106(6):2183-2185, 2005).

[0012] Tyrosine phosphorylation is involved in the regulation of human cellular processes from cell differentiation and growth to apoptosis. The process of tyrosine

phosphorylation is regulated by protein-tyrosine phosphatases (PTP) and protein-tyrosine kinases (PTK). When this regulation is disrupted, diseases such as cancer can arise. For example, activated PTPNl 1 protein interacts with the Gab family of docking proteins. This interaction activates a pathway leading to cell proliferation and tumorigenesis. Therefore, the PTPNl 1 protein signaling pathway can be an attractive therapeutic target for treating, for example, cancer, Noonan syndrome, and LEOPARD syndrome. [0013] A number of therapeutic or surgical treatments are available to alleviate various symptoms of Noonan syndrome (e.g., heart defects, undescended testicles, or excessively short stature) or LEOPARD syndrome (e.g., cryptorchidism, hypospadias, or severe skeletal deformity). However, no therapeutic treatment of the underlying disorder has been determined so far.

[0014] Therefore, there is a need to identify novel therapeutic agents for diagnosis and treatment of diseases that are associated with mutations in the PTPNl 1 gene and/or abnormal PTPNl 1 signaling. There is also a need to provide novel tools that would facilitate the identification of such therapeutic agents.

SUMMARY OF THE INVENTION

[0015] This invention generally relates to disease-specific pluripotent stem cells that are derived from somatic cells that carry a mutation in the PTPNl 1 gene. The stem cells are capable of differentiating into cells or tissues affected by the PTPNl 1 mutation, thereby providing a powerful tool to study various diseases that are associated with mutations in the PTPNl 1 gene, and to identify novel therapeutic agents for diagnosis and treatment of diseases that are associated with mutations in the PTPNl 1 gene.

[0016] In one aspect, the invention provides an isolated primate pluripotent stem cell derived from a somatic cell, wherein the stem cell comprises a mutation in the protein tyrosine phosphatase non-receptor type 11 (PTPNl 1) gene.

[0017] In certain embodiments, the PTPNl 1 gene comprises a mutation associated with a cardiac anomaly. In certain embodiments, the cardiac anomaly is hypertrophic cardiomyopathy.

[0018] In certain embodiments, the PTPNl 1 gene comprises a mutation associated with LEOPARD Syndrome, Noonan Syndrome or leukemia.

[0019] In certain embodiments, the mutation in the PTPNl 1 gene is a deletion mutation, a substitution mutation, an addition mutation, or a combination thereof, in the coding region of the PTPNl 1 gene. In certain embodiments, the mutation in the PTPNl 1 gene results in a mutation in the encoded PTPNl 1 protein.

[0020] In certain embodiments, the mutation in the PTPNl 1 gene results in a threonine to methionine substitution in the encoded PTPNl 1 protein at an amino acid residue position corresponding to position 468 of SEQ ID NO: l, or a tyrosine to cysteine substitution in the encoded PTPNl 1 protein at an amino acid residue position corresponding to position 279 of SEQ ID NO: l .

[0021] In certain embodiments, the mutation in the PTPNl 1 gene results in a tyrosine to cysteine substitution in the encoded PTPNl 1 protein at an amino acid residue position corresponding to position 279 of SEQ ID NO: 1, a tyrosine to serine substitution in the encoded PTPNl 1 protein at an amino acid residue position corresponding to position 279 of SEQ ID NO: 1, an alanine to threonine substitution in the encoded PTPNl 1 protein at an amino acid residue position corresponding to position 461 of SEQ ID NO: 1, a glycine to alanine substitution in the encoded PTPNl 1 protein at an amino acid residue position corresponding to position 464 of SEQ ID NO: 1, a threonine to methionine substitution in the encoded PTPNl 1 protein at an amino acid residue position corresponding to position 468 of SEQ ID NO: 1, a threonine to proline substitution in the encoded PTPNl 1 protein at an amino acid residue position corresponding to position 468 of SEQ ID NO: 1, an arginine to tryptophan substitution in the encoded PTPNl 1 protein at an amino acid residue position corresponding to position 498 of SEQ ID NO: 1, an arginine to leucine substitution in the encoded PTPNl 1 protein at an amino acid residue position corresponding to position 498 of SEQ ID NO: 1, a glutamine to proline substitution in the encoded PTPNl 1 protein at an amino acid residue position corresponding to position 506 of SEQ ID NO: 1, a glutamine to proline substitution in the encoded PTPNl 1 protein at an amino acid residue position corresponding to position 510 of SEQ ID NO: l, a glutamine to glutamic acid substitution in the encoded PTPNl 1 protein at an amino acid residue position corresponding to position 510 of SEQ ID NO : 1 , or a glutamine to glycine substitution in the encoded PTPN 11 protein at an amino acid residue position corresponding to position 510 of SEQ ID NO: 1

[0022] In certain embodiments, the PTPNl 1 gene encodes a polypeptide comprising SEQ ID NO: 1, and the mutation in the PTPNl 1 gene results in a threonine to methionine substitution at amino acid residue position 468 of SEQ ID NO: 1 , or a tyrosine to cysteine substitution at amino acid residue position 279 of SEQ ID NO: 1.

[0023] In certain embodiments, the pluripotent stem cell forms a teratoma in an immunocompromised mouse.

[0024] In certain embodiments, the pluripotent stem cell expresses SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, Nanog, OCT-4, Sox2, GDF3, DPPA4, REX1, TERT, Alkaline

Phosphatase, Telomerase, or CD30, or a combination thereof.

[0025] In certain embodiments, the pluripotent stem cell is capable of differentiating into a cardiomyocyte.

[0026] Also provided is a cell culture of the stem cells described herein. Exemplary stem cell lines were deposited on , 2010 with American Type Culture Collection

(ATCC), P.O. Box 1549, Manassas, VA 20108, USA, under ATCC Accession Nos. , and .

[0027] In another aspect, the invention provides a cardiomyocyte comprising a mutation in the PTPNl 1 gene, wherein the cardiomyocyte is produced by differentiating a disease-specific pluripotent stem cell as described herein.

[0028] In certain embodiments, the invention provides a cell culture of a

cardiomyocyte as described herein. Exemplary cardiomyocyte cell lines were deposited on

, 2010 with American Type Culture Collection (ATCC), P.O. Box 1549, Manassas, VA

20108, USA, under ATCC Accession Nos. , and .

[0029] In certain embodiments, the primate is human.

[0030] In certain embodiments, the somatic cell is a fibroblast.

[0031] In another aspect, the invention provides a method of identifying an agent for use in treatment of a disease that is associated with a mutation in the PTPNl 1 gene, comprising (1) contacting a disease-specific pluripotent stem cell as described herein with the agent; and (2) determining the effect of the agent on survival, proliferation, phenotype, or differentiation of the pluripotent stem cell.

[0032] In another aspect, the invention provides a method of identifying an agent for use in treatment of a disease that is associated with a mutation in the PTPNl 1 gene, comprising (1) contacting a disease-specific cardiomyocyte as described herein with the agent; and (2) determining the effect of the agent on survival, proliferation, or phenotype of the cardiomyocyte.

[0033] In another aspect, the invention provides a method of identifying an agent for use in treatment of the cardiac hypertrophy that is associated with a mutation in the PTPNl 1 gene, comprising: (1) contacting a cardiomyocyte as described herein with the agent; and (2) determining the effect of the agent on the hypertrophic state of the cardiomyocyte. A decrease in the hypertrophic state of the cardiomyocyte, as compared to that of a control cell, is indicative that the agent is an agent for use in treatment of a disease that is associated with a mutation in the PTPNl 1 gene.

[0034] In another aspect, the invention provides a method of producing a pluripotent stem cell as described herein, comprising: (a) obtaining a somatic cell from a donor whose genome comprises a mutation in the PTPNl 1 gene; (b) introducing into the somatic cell: (i) a nucleic acid molecule that encodes a pluripotency-inducing protein; or (ii) a polypeptide that comprises a pluripotency-inducing protein; wherein said pluripotency-inducing protein is Oct3/4, Sox2, Klf4, Nanog, Lin28, c-Myc, or a combination thereof.

[0035] In certain embodiments, one or more nucleic acid molecules that encode Oct3/4, Sox2, Klf4, and c-Myc are introduced into the somatic cell.

[0036] In certain embodiments, the somatic cell is a fibroblast.

[0037] In another aspect, the invention provides a method of differentiating a pluripotent stem cell as described herein into a cardiomyocyte, comprising: culturing a pluripotent stem cell as described herein in a serum- free medium that comprises: activin A, bone morphogenetic protein 4 (BMP4), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and dickkopf homo log 1 (DK 1). [0038] In another aspect, the invention provides a method of identifying an agent as a candidate Ras-signaling pathway inhibitor, comprising: (1) contacting a pluripotent stem cell as described herein with the agent; and (2) determining the effect of the agent on tyrosine phosphorylation of EGFR or MEK1. A decrease of tyrosine phosphorylation of EGFR or MEK1, as compared to that of a control cell, is indicative that the agent is a Ras-signaling pathway inhibitor.

[0039] In another aspect, the invention provides a method of identifying an agent as a candidate Ras-signaling pathway inhibitor, comprising: (1) contacting a cardiomyocyte as described herein with the agent; and (2) determining the effect of the agent on tyrosine phosphorylation of EGFR or MEK1. A decrease of tyrosine phosphorylation of EGFR or MEK1, as compared to that of a control cell, is indicative that the agent is a Ras-signaling pathway inhibitor.

[0040] In another aspect, the invention provides a method of identifying an agent for use in treatment of a disease that is associated with a mutation in the PTPNl 1 gene, comprising: (1) contacting a pluripotent stem cell as described herein with the agent; (2) differentiating the stem cell into a cardiomyocyte; and (3) determining the hypertrophic state of the cardiomyocyte. A decrease in the hypertrophic state of the cardiomyocyte, as compared to that of a control cell, is indicative that the agent is an agent for use in treatment of a disease that is associated with a mutation in the PTPNl 1 gene.

[0041] In another aspect of the invention, the stem cells of the invention are capable of differentiating into a hematopoietic cell. Such hematopoietic cells may comprise a mutation in the PTPNl 1 gene, wherein the hematopoietic cell is produced by differentiating the stem cells of the invention.

[0042] Cell cultures of these hematopoietic cells are also provided. Exemplary hematopoietic cell lines were deposited on , 2010 with American Type Culture

Collection (ATCC), P.O. Box 1549, Manassas, VA 20108, USA, under ATCC Accession Nos.

, and . [0043] In another aspect, methods of identifying an agent for use in treatment of a disease that is associated with a mutation in the PTPN11 gene are provided, comprising (1) contacting the pluripotent stem cells with the agent; (2) differentiating the stem cell into a hematopoietic cell; and (3) determining the effect of the agent on survival, proliferation, phenotype, or differentiation of the hematopoietic cell. In one embodiment, the disease is leukemia.

[0044] In yet another aspect, methods of identifying an agent for use in treatment of leukemia are provided, comprising contacting the pluripotent stem cell with the agent; and differentiating the stem cell into a hematopoietic cell; and determining the proliferative state of the hematopoietic cell, wherein a decrease in the proliferative state of the hematopoietic cell, as compared to that of a control cell, is indicative that the agent is an agent for use in treatment of leukemia.

BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figs, la-lc demonstrate that the gene expression profile of LS-iPSC is similar to that of HESC. la, quantitative real-time PCR assay for the expression of endogenous OCT4, NANOG and SOX2 in iPSC and parental fibroblasts (Fib). PCR reactions were normalized against β-ACTIN and plotted relative to expression levels in HES2. Error bars indicate ± s.d. of triplicates, lb, bisulfite sequencing analyses of the OCT4 and NANOG promoters. The cell line and the percentage of methylation is indicated to the left of each cluster, lc, heat map showing hierarchical clustering of 3657 genes with at least two-fold expression change between the average of the three fibroblast cell lines versus all the iPSC lines/HES samples. Expression levels are represented by color; red indicates lower and yellow higher expression.

[0046] Figs. 2a-2b demonstrate that LS-iPSC can differentiate in vitro and in vivo into all three germ layers. 2a, L2-iPS6 cells were differentiated as floating EBs for eight days and then plated onto gelatin-coated dishes and allowed to differentiate for another eight days. Immunocytochemistry showed cell types positively stained for differentiation markers including Desmin/SMA (mesoderm), AFP (endoderm), vimentin (mesoderm), and GFAP/piII-Tubulin (ectoderm). The arrow indicates a βΙΙΙ-tubulin-positive cell. Scale bar, 100 μιη. 2b, HES2, Ll- iPSC and L2-iPSC were injected subcutaneously into the right hindleg of immuno-compromised NOD-SCID mice. The resulting teratomas were stained with hematoxylin and eosin and tissues representative of all three germ layers were observed.

[0047] Figs. 3a-3d show that cardiomyocytes derived from LS-iPSC had hypertrophic features. 3a, HES2, HI, wt S3-iPS4 and three LS-iPS clones were differentiated into cardiac lineage. Cell areas of 50 random cTNT-positive cardiomyocytes of each cell line were measured using Image J. Boxes show the span from the median (50th percentile) to the first and third quartiles. The lines represent the largest/smallest sizes that are no more than 1.5 times the median to quartile distance. Additional points drawn represent extreme values. 3b, Sarcomeric organization was assessed in 50 cTNT positive (red) cardiomyocytes. Data are presented as mean ± s.d. n = 3; ** < 0.01 (Student's t-test). 3c, S3-iPS4 and L2-iPS10 cells-derived cardiomyocytes were restained with NFATc4 antibody, and the nuclear versus cytosolic expression was analyzed, n = 3; **P < 0.01 (Student's t-test). 3d, Nuclear localization of NFATc protein in a cTNT -positive cell from L2-iPS10 is shown.

[0048] Figs 4a-4c show the results of phosphoproteomic and MAPK activation analyses. 4a, Protein extracts of two iPSC from each LS patient (LI and L2), wt iPSC (BJ- iPSB5) and HES2 were hybridized to an antibody microarray. The heat map represents the most significant protein changes preserved in all the comparison groups. 4b, pMEKl and pEGFR expression was confirmed by Western blot using phospho-specific antibodies. Band density was measured (ImageJ software), and normalized to β-Actin. 4c, HES2, wt S3-iPS4, and LS-iPSC were serum- and bFGF-starved for 6 hours and then treated with bFGF (20 ng/ml) for the indicated time. Phosphorylated ERK1/2 (p-ER l/2) and total ER were assessed by

immunoblotting and quantitated. p-ERKl/2 levels were compared to the untreated p-ERKl/2 level in each sample, normalized to the total ERK1/2 and represented graphically at the right of each panel.

[0049] Figs 5a-5b illustrate the formation of LS-iPSC. 5a, schematic representation of iPSC generation. 5b, typical image of a TRA-1-81 positive colony growing in the plate three weeks after infection.

[0050] Figs. 6a-6b demonstrate that LS-iPSC lines were derived from their parental fibroblasts and maintained normal karyotypes. 6a, the inventors PCR-amp lifted across three discrete genomic loci containing highly variable numbers of tandem repeats with different primer sets (D10S1214, D17S1290, and D21S2055). The resulting amplification patterns confirmed that each iPSC line was derived from its indicated parental fibroblast. 6b, G-banding of HES2 cell line, wild-type iPS clone BJ-iPSB5, and LS-iPSC lines (one clone of each patient) demonstrates normal diploid chromosomal contents.

[0051] Figs. 7a-7b show the result οΐΡΤΡΝΙΙ T468M mutation analysis in LS-iPSC and fibroblasts. 7a, the T468M point mutation in exon 12 of one allele of PTPN11 gene was verified by DNA sequencing. 7b, the inventors amplified by RT-PCR a 1.2 Kb region containing the mutation sequence and the DNA was digested with BsmFI, an enzyme that cuts the 5'-GGGAC(N)io-3' sequence of the wild type allele but does not cut the mutant allele. In all the LS samples, an undigested 1.2 Kb band was observed.

[0052] Figs. 8a-8b show the integration of retroviral transgenes in iPSC. 8a, transgene-specific primers were used to amplify OCT4, SOX2, KLF4 and c-MYC. Three LS- derived iPSC lines from each patient were analyzed, and HES2 cells and parental fibroblasts were used as negative controls. 8b, Southern blot analyses of Bgl-II digested gDNA extracted from HES2 cells, parental fibroblasts and iPSC, using DIG-labeled DNA probes against OCT4, SOX2, KLF4 and c-MYC. Retrovirally-inserted transgenic copies of these genes are indicated by asterisks and the number of detected bands is shown at the bottom. The parental fibroblasts and HES2 cells share bands in common with all the iPSC lines (arrowheads), which reflect the endogenous loci (including potential pseudogenes).

[0053] Figs. 9a-9b demonstrate the silencing of retroviral transgenes in iPSC lines. 9a, transgene-specific primers were used to determine OCT4, SOX2, KLF4 and c-MYC expression. 9b, qPCR analysis of retroviral transgene (Tg) expression. Results are normalized against β-ACTIN and plotted relative to the expression levels in transfected GP2 cells.

[0054] Figs. 10a- 10b demonstrate that LS-iPSC expressed cell markers that are common to pluripotent cells. 10a, SSEA4 expression was determined by flow cytometry in two iPSC lines derived from two LEOPARD syndrome (LS) patients (Ll-iPSl, Ll-iPS13, L2-iPS6 and L2-iPS16), and a wt-iPSC line derived from BJ fibroblasts (BJ-iPSB5). HES2 cell line was used as positive control. 10b, HES2 and iPSC were grown on MEFs, and were fixed and stained for the following pluripotency markers: alkaline phosphatase (AP), TRA-1-81, TRA-1-60, NANOG and OCT4. Nuclei were stained with DAPI.

[0055] Figs. 1 la-1 lb show the result of gene expression analysis of the cells. 11a, qPCR was used to evaluate the expression of GDF3,

REXl and TERT in two LS- iPSC lines from each patient and compared to their parental fibroblasts. HES2 cells were used as positive control of pluripotency gene expression. PCR reactions were normalized against β- ACTIN and represented relative to the expression in HES2 cells, lib, two sample comparison plots of log 2 expression values, with the line Y=X as a basis for comparison along with Y = X +/- 1 or 2 lines, representing 2-fold and 4-fold expression changes, respectively. In each scatter- plot the position of SOX2, LIN28 and OCT4 transcription factors is indicated.

[0056] Figs. 12a-12b illustrate in vitro differentiation of iPSC lines. 12a, day 8 EBs of HES2, BJ-iPSB5, Ll-iPS13 and L2-iPS6 cells. 12b, after 16 days of differentiation, cells were stained with SMA, GFAP and Vimentin antibodies. Scale bar, 100 μιη.

[0057] Figs. 13a- 13b show that LS-iPSC differentiated into hematopoietic cells. 13a, red EBs, an indication of erythrocyte development, were observed in Ll-iPSC plates 14 days after differentiation. Scale bar, 100 μπι. 13b, CD41, CD45, CD1 lb, CD71 and CD235a hematopoietic markers were analyzed by flow cytometry in EBs derived from HES2 and Ll- iPSC, 18 days after induction of hematopoietic differentiation.

[0058] Figs. 14a- 14c show the characterization of wt S3-iPS4 and L2-iPSC. 14a, the iPSC had the four transgenes integrated in their genome. 14b, Tg silencing analysis. 14c,

Fingerprinting analysis of the iPSC lines using their parental fibroblasts and HES2 cells as controls.

[0059] Figs. 15a-15c show the pluripotency of S3-iPS4 and L2-iPSC. 15a and 15b, S3-iPS4, L2-iPS10 and L2-iPS20 expressed pluripotency markers. 15c, the iPSC differentiated into derivatives of the three germ layers.

[0060] Figs. 16a- 16c show the disruption of MAPK activation upon bFGF

stimulation in LS-iPSC. 16a, FGF receptors expression analysis by RT-qPCR in LS-iPSC and fibroblasts compared to HES2. 16b, Ll-iPSl, Ll-iPS6 and HES2 cells were serum- and bFGF- starved overnight. The following day, the cells were treated with bFGF for 10 minutes (+) or not (-). Total ER 1/2 and p-ER l/2 expression was analyzed by immunobloting of total lysates. 16c, basal p-ERKl/2 was quantified in each sample and compared to HES2C (top left panel). The relative increase of p-ERKl/2 level upon stimulation was quantified in each sample (top right and lower panels). All the p-ERKl/2 values were normalized to their corresponding total ERK1/2 values.

[0061] Fig. 17 is a table showing the primer sets that were used for PCRs.

[0062] Fig. 18 is a schematic drawing showing PTPN11 gene organization and domain structure. The numbered, filled boxes at the top indicate the coding exons; the positions of the ATG and TGA codons are shown. The functional domains of the PTPN11 protein, consisting of two tandemly arranged src-homology 2(SH2) domains at the N-terminus (N-SH2 and C-SH2) followed by a protein tyrosine phosphatase (PTP) domain, are shown below. The numbers below that cartoon indicate the amino acid boundaries of those domains.

[0063] Fig. 19 shows the distribution of PTPN11 (SHP-2) mutations and their relative prevalence in Noonan syndrome.

[0064] Fig. 20 shows the cDNA sequence of human PTPN11 (SEQ ID NO: 2).

[0065] Fig. 21 shows the amino acid sequence of human PTPN11 (SEQ ID NO: 1). DETAILED DESCRIPTION OF THE INVENTION 1. OVERVIEW

[0066] This invention generally relates to disease-specific pluripotent stem cells that are derived from somatic cells that carry a mutation in the PTPN11 gene. The stem cells are capable of differentiating into cells or tissues (e.g., cardiomyocytes) affected by the PTPN11 mutation, thereby recapitulating both normal and pathologic tissue formation in vitro. The stem cells and cardiomyocytes provided herein can facilitate disease investigation and drug development - they may be used to study various diseases that are associated with mutations in the PTPN11 gene, and to identify novel therapeutic agents for diagnosis and treatment of diseases that are associated with mutations in PTPN11. [0067] The present invention is based on the discovery that somatic cells from

LEOPARD syndrome (LS) patients, which carry a mutation in the PTPN11 gene, can be reprogrammed into pluripotent stem cells. Further, the stem cells may be cultured under appropriate differentiation conditions to generate differentiated, disease-specific cardiomyocytes. Such disease-specific stem cells and cardiomyocytes are useful for studying the genetic basis of disease progression and pathogenesis, e.g., by comparing the survival, proliferation, phenotype, or differentiation of the disease-specific cells to that of cells that do not carry the mutation in the PTPN11 gene.

[0068] For example, as described and exemplified herein, this approach is used to develop an in vitro model of LEOPARD syndrome and cardiac hypertrophy. The inventors generated LS-specific induced pluripotent stem cell (LS-iPSC) lines from fibroblasts of LS patients. The pluripotency of the induced LS-specific stem cells were confirmed by the expression of certain stem cell markers, chromatin methylation patterns, genome-wide mRNA expression profiles, teratoma formation, and differentiability. Then the LS-specific stem cells were differentiated into cardiomyocytes in vitro. Consistent with the observation that a major disease phenotype in patients with LEOPARD syndrome is hypertrophic cardiomyopathy, in vz^'tro-derived cardiomyocytes from LS-iPSC were larger, had a higher degree of sarcomeric organization and showed preferential localization of NFATc4 in the nucleus, when compared to cardiomyocytes derived from human embryonic stem cells (HESC) or iPSCs derived from an unaffected brother (i.e., not having the PTPN11 mutation) of one of the LS patients.

[0069] The disease-specific iPSCs and cardiomyocytes provided herein offer several advantages for disease study and drug screening as compared to existing cell lines. For example, most of the human cell lines in wide use today are derived either from malignant tissues or are genetically modified to drive immortal growth. On the other hand, primary human cells have a limited life span in culture, a constraint that thwarts inquiry into the regulation of tissue formation, regeneration, and repair. Therefore, the immortal pluripotent stem cells and the in vitro differentiated cardiomyocytes provided herein are particularly valuable as disease models for studying mutations in the PTPN11 gene. [0070] Also provided herein are methods of identifying an agent for use in treatment of a disease that is associated with a mutation in the PTPN11 gene; methods of identifying an agent for use in treatment of the cardiac hypertrophy that is associated with a mutation in the PTPN11 gene; methods of producing an LS-specific pluripotent stem cell; methods of differentiating an LS-specific pluripotent stem cell into a cardiomyocyte; and methods of identifying an agent as a candidate Ras-signaling pathway inhibitor.

2. DEFINITIONS

[0071] As used herein, the singular forms "a," "an" and "the" include plural references unless the content clearly dictates otherwise.

[0072] The term "about", as used here, refers to +/- 10% of a value.

[0073] As used herein, the term "pluripotency" refers to the nature of a cell, i.e., an ability to differentiate into a variety of tissues or organs. Typically, the pluripotency of a differentiated cell is limited.

[0074] As used herein, "reprogramming" refers to a genetic process whereby differentiated somatic cells are converted into de-differentiated, pluripotent cells, and thus having a greater pluripotency potential than the cells from which they were derived. For example, the reprogrammed cells may express at least one of the following pluripotent cell- specific markers: SSEA-3, SSEA-4, TRA-1-60 or TRA 1-81. Preferably, the reprogrammed cells express all these markers.

3. DISEASE-SPECIFIC PLURIPOTENT STEM CELLS

[0075] In one aspect, the invention provides an isolated primate pluripotent stem cell derived from a somatic cell, wherein the stem cell comprises a mutation in the protein tyrosine phosphatase non-receptor type 11 (PTPN11) gene. Mutations in the PTPN11 gene have been implicated in several diseases, such as LEOPARD syndrome, Noonan syndrome, and leukemia. Therefore, the stem cells provided herein are useful for studying the pathologic tissue formation in vitro, and for screening novel therapeutic agents for treating or diagnosing diseases that are associated with PTPN11 mutations. A. PTPN11 Gene and PTPN11 Protein

[0076] PTPN11 protein (also known as SHP2, Syp, SHPTP2, PTP2C, PTP1D or BPTP3) is a member of a small subfamily of cytoplasmic, SH2-domain-containing protein tyrosine phosphatases that control cellular proliferation and differentiation (Feng, Exp Cell Res 1999;253:47-54; Tartaglia et al, Nat genet 2001;29:465-68; Tartaglia et al, Am J Hum Genet 2002;70: 1555-63). Human PTPN11 is ubiquitously expressed in all tissues examined, with higher levels of expression in the heart and the brain (Ahmad et al, Proc Natl Acad Sci USA 1993;90:2197-2201; Bastien et al, Biochem Biophys Res Commun 1993 ; 196: 124-133; Freeman et al, Proc Natl Acad Sci USA, 1992;89: 11239-11243).

[0077] PTPN11 is a key molecule in intracellular signaling and is necessary for activation of the RAS/MAPK cascade in response to a variety of growth factors, hormones and cytokines (Maroun et al, Mol Cell Biol 2000;20:8513-25; Shi Z-Q, et al, Mol Cell Biol 2000; 20: 1526-1536; Cunnick et al, J Biol Chem 2002;277:9498-504). PTPN11 is required during embryogenesis for mesodermal patterning (Tang et al, Cell 1995;80:473-483), semilunar valvuogenesis (Chen et al, Nat Genet 2000;24:296-9) and skeletal and limb development (Qu et al, Mol Cell Biol 1998; 18:6075-82; Saxon et al, Nat Genet 2000;24:420-3). Loss of murine SHP2 severely suppresses development of erythroid/myeloid and lymphoid cell progenitors, (Qu et al, Mol Cell Biol 1998; 18:6075-82; Qu et al, Mol Cell Biol 1997; 17:5499-507; Qu et al, Blood 2001;97:911-4), suggesting that it participates in early events during hematopoetic stem/progenitor cell commitment and differentiation. PTPN11 also controls cell differentiation at later stages of hematopoiesis, and has a role in the function of differentiated erythroid, myeloid and lymphoid cells (Pazdrak et al, J Exp Med 1997; 186:561-8; Edmead et al, FEBS Lett 1999;459:27-32; Ohtani et al, Immunity 2000;12:95-105; Tamir et al, Curr Opin Immunol 2000;12:307-15; Bordin et al, Blood 2002; 100:276-82). These effects appear to be mediated through a signal relay downstream of receptors for a number of hematopoietic growth factors and cytokines, including GM-CSF (Pazdrak et al, J Exp Med 1997;186:561-8; Welham et al, J Biol Chem 1994;269:23764-8; Tauchi et al, J Biol Chem 1995;270:5631-5; Tauchi et al, J Biol Chem 1994;269:25206-1138; Ward et al, Biochem Biophys Acta 1998;1448:70-6; Lecoq-Lafon et al, Blood 1999;93:2578-85; You et al, J exp Med 2001;193: 101-10). [0078] The human PTPNl 1 gene organization and intron boundary sequence can be established using cDNA (GENBANK Accession Nos. NM_002834; amino acid and nucleotide sequences represented herein as SEQ ID Nos.: 1 and 2, respectively) and genomic sequences (GENBANK Accession Nos. NG 007459).

[0079] Figure 18 shows the organization of the PTPNl 1 gene and the functional domains of the PTPNl 1 protein. The PTPNl 1 protein comprises two SH2 (src-homology 2) domains, one from amino acid 3 to amino acid 104, the other from amino acid 112 to amino acid 216, and one PTP (protein tyrosine phosphatase) domain, from amino acid 221 to amino acid 524.

[0080] The PTPNl 1 gene of the invention also encompasses nucleic acid sequences comprising a coding sequence as set forth in SEQ ID NO:2, homo logs (including allelic variants and orthologs), sequence-conservative variants, or function-conservative variants thereof. The PTPNl 1 protein of the invention also encompasses amino acid sequences comprising the amino acid sequence as set forth in SEQ ID NO: l, homo logs (including orthologs), or function- conservative variants thereof.

[0081] As used herein, the term "homologous" in all its grammatical forms and spelling variations refers to the relationship between polynucleotides or proteins that possess a "common evolutionary origin," including polynucleotides or proteins from superfamilies and homologous polynucleotides or proteins from different species (Reeck et al, Cell 50:667, 1987). Such polynucleotides or proteins have sequence homology, as reflected by their sequence similarity, whether in terms of percent identity or the presence of specific amino acids or motifs at conserved positions.

[0082] "Sequence-conservative variants" of a polynucleotide sequence are those in which a change of one or more nucleotides in a given codon position results in no alteration in the amino acid encoded at that position.

[0083] "Function-conservative variants" are those in which one or more nucleotides of a polynucleotide sequence have been changed without altering the overall conformation and function of the polypeptide encoded by the polynucleotide; or one or more amino acid residues of a polypeptide sequence have been changed without altering the overall conformation and function of the polypeptide. Function-conservative variants includes, e.g., replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like). Amino acids with similar properties are well known in the art. For example, arginine, histidine and lysine are hydrophilic- basic amino acids and may be interchangeable. Similarly, isoleucine, a hydrophobic amino acid, may be replaced with leucine, methionine or valine. Such changes are expected to have little or no effect on the apparent molecular weight or isoelectric point of the protein or polypeptide.

[0084] An amino acid position "corresponding to" a position in another sequence is the position that lines up to the reference position when the amino acid sequence is aligned with the reference sequence. Alignments can be done by hand or by using well-known sequence alignment programs such as ClustalW2.

[0085] Other variants of PTPN11 gene may include those polynucleotide sequences comprising a coding sequence that is at least about 70%, about 75%, about 80%>, about 85%, about 90%, about 95%, or about 99% identical to SEQ ID NO:2. Preferably the variant gene encodes a protein that has the same or substantially similar properties or functions as the native or parent PTPN11 protein.

[0086] Other variants of PTPN11 protein may include those polypeptide sequences comprising an amino acid sequence that is at least about 70%>, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% identical to SEQ ID NO: 1. Preferably the variant protein has the same or substantially similar properties or functions as the native or parent PTPN11 protein.

B. Diseases Associated with Mutations in the PTPN11 Gene

[0087] A disease is "associated with" a mutation in the PTPN11 gene when the disease is directly or indirectly caused by, or correlates with a mutation in the PTPN11 gene. Such mutation of PTPN11 may lead to a non- functioning or defective PTPN11 protein, or altered expression level of the PTPN11 gene. Such mutation may be a substitution mutation (i.e., a mutation in which one or more bases within the nucleic acid sequence have been replaced by a different base), an insertion mutation (i.e, a mutation in which the total length of the gene of interest has been increased by the insertion of one or more bases), a deletion mutation (a mutation in which the total length of the gene of interest has been decreased by removal of one or more bases) or an inversion mutation (a mutation in which a region of two or more bases has been rotated 180 degrees), or a combination thereof. The mutation may be directly, or indirectly, fully or partly responsible for the disease, or alternatively, the mutation may not be responsible for the disease but correlates with the diseased state in the sense that it is diagnostic/indicative of the diseased state.

[0088] In certain embodiments, the mutation in the PTPN11 gene results in a mutation in the encoded PTPN11 protein.

[0089] In certain embodiments, the stem cells described herein comprise a mutation in the PTPN11 gene that is associated with Noonan Syndrome.

[0090] "Noonan syndrome" (NS) encompasses all forms of the disorder as described under the accession No. MIM 163950 in the Online Mendelian Inheritance in Man (OMIM) at World-Wide Web Address www.ncbi.nlm.nih.gov/Omim, (as accessed in April 2010), as well as disorders similar or related to NS. Such disorders include, but are not limited to, the Watson (MIM 193520) and LEOPARD (MIM 151100) Syndromes (Mendez and Opitz, Am J Med Genet 1985;21 :493-506); male Turner and female pseudo-Turner Syndrome, as well as Turner phenotype with normal karyotype (see MIM 163950); Noonan syndrome with multiple giant-cell lesions (MIM 163955; Tartaglia et al, Am J Hum Genet 2002;70: 1555-1563) and/or Noonan syndrome with multiple caf-au-lait spots (also known as LEOPARD syndrome, MIM 151100; Digilio et al, Am J Hum Genet 2002;71 :389-394; Legius et al, J Med Genet 2002;39:571-574); valvular sclerosis (Snellen et al, Circulation 1968;38(1 Suppl):93-101); and idiopathic short stature (Attie K M, Curr Opin Pediatr 2000;12:400-404).

[0091] NS encompasses familial or sporadic forms, including NS1, whose locus has been identified on chromosome 12. The present invention takes into consideration, however, that NS and its related disorders are genetically heterogeneous, but share phenotypical features. The features of NS have been well described and a clinical scoring system devised. See, Mendez and Opitz, Am J Med Genet 1985;21 :493-506; Noonan, Clin Pediatr (Phila) 1994;33:548-555; Sharland et al, Arch Dis Child 1992;67: 178-183; Duncan et al, Am J Med Genet 1981;10:37- 50).

[0092] Approximately 45% of NS are caused by missense mutations in the PTPN11 gene. For example, in more than 50% of patients with Noonan syndrome, Tartaglia et al.

(Nature Genet. 29: 465-468, 2001; Errata: Nature Genet. 29: 491, 2001; Nature Genet. 30: 123, 2002) identified several missense mutations in the PTPN11 gene: a G-to-T mutation at position 214 in exon 3, resulting in an Ala72-to-Ser (A72S) substitution in the N-SH2 domain; a C-to-G mutation at nucleotide 215 in exon 3, resulting in an Ala72-to-Gly (A72G) substitution; and an A-G mutation in exon 8, resulting in an Asn308-to-Asp (N308D) substitution in the

phosphotyrosine phosphatase (PTP) domain. About one-third of the patients who had mutations in the PTPN11 gene had the N308D mutation, which was by far the most common. That codon 308 is a hotspot for Noonan syndrome is further supported by the finding of an Asn308-to-Ser missense mutation in 2 families that had typical features of Noonan syndrome associated with multiple giant cell lesions in bone (Tartaglia et al., Am. J. Hum. Genet. 70: 1555-1563, 2002). All the PTPN11 missense mutations identified are clustered in the interacting portions of the amino N-SH2 domain and the phosphotyrosine phosphatase (PTP) domains, which are involved in switching the protein between its inactive and active conformations. The findings suggest that gain-of-function changes resulting in excessive SHP-2 activity underlie the pathogenesis of Noonan syndrome.

[0093] Using direct DNA sequencing, Maheshwari et al. (Hum. Mutat. 20: 298-304, 2002) surveyed 16 subjects with the clinical diagnosis of Noonan syndrome from 12 families and their relevant family members for mutations in the PTPN11/SHP2 gene, and found 3 different mutations among 5 families. Two unrelated subjects shared a Ser502-to-Thr (S502T) substitution in exon 13; 2 additional unrelated families had a Tyr63-to-Cys (Y63C) mutation in exon 3; and 1 subject had a Tyr62-to-Asp (Y62D) substitution, also in exon 3. In the mature protein model, the exon 3 mutants and the exon 13 mutant amino acids cluster at the interface between the N-terminal SH2 domain and the phosphatase catalytic domain. Six of 8 subjects with mutations had pulmonary valve stenosis. These results confirm that mutations in PTPN11 underlie a common form of Noonan syndrome, and that the disease exhibits both allelic and locus heterogeneity. The observation of recurrent mutations supports the hypothesis that a special class of gain-of-function mutations in SHP2 gives rise to Noonan syndrome.

[0094] Kosaki et al. (J. Clin. Endocr. Metab. 87: 3529-3533, 2002) analyzed the PTPN11 gene in 21 Japanese patients with Noonan syndrome. Mutation analysis of the 15 coding exons and their flanking introns by denaturing HPLC and direct sequencing revealed 6 different heterozygous missense mutations in 7 cases. The mutations clustered either in the N- Src homology 2 domain or in the protein-tyrosine phosphatase domain.

[0095] Musante et al. {Europ. J. Hum. Genet. 11 : 201-206, 2003; Erratum: Europ. J. Hum. Genet. 11 : 551, 2003) screened the PTPN11 gene for mutations in 96 familial or sporadic Noonan syndrome patients. They identified 15 mutations, all of which were missense mutations; 11 of them were located in exon 3, which encodes the N-SH2 domain.

[0096] Tartaglia et al. (Am. J. Hum. Genet. 75: 492-497, 2004) investigated the parental origin of de novo PTPN11 lesions and explored the effect of paternal age in Noonan syndrome. By analyzing intronic positions that flank the exonic PTPN11 lesions in 49 sporadic Noonan syndrome cases, they traced the parental origin of mutations in 14 families. All mutations were inherited from the father.

[0097] Yoshida et al. (J. Clin. Endocr. Metab. 89: 3359-3364, 2004) reported PTPN11 mutation analysis and clinical assessment in 45 Japanese patients with Noonan syndrome. Sequence analysis of the coding exons 1 through 15 of PTPN11 revealed a novel 3- bp deletion (181delGTG, resulting in deletion of the Gly60 codon in the N-SH2 domain of the protein) and 10 recurrent missense mutations in 18 patients. Because Gly60 is directly involved in the N-SH2/PTP interaction, loss of this residue was predicted to disrupt N-SH2/PTP binding, activating the phosphatase function.

[0098] Becker et al. (Am. J. Med. Genet. 143A: 1249-1252, 2007) reported what they stated was the first known case of compound heterozygosity for NS-causing mutations in the PTPN11 gene (compound heterozygosity for N308S and Y63C mutations), resulting in early fetal death. [0099] Other PTPNl 1 mutations that have been associated with Noonan syndrome include, e.g., A-to-G mutation at nucleotide 182 in exon 3 (Asp61-to-Gly, D61G), 218C-T mutation in exon 3 (Thr73-to-Ile, T73I), T-to-C mutation at nucleotide 854 in exon 8 (Phe285-to- Ser, F285S), 1909A-G mutation (Gln510-to-Arg); 236A-G mutation in exon 3 (Gln79-to-Arg, Q79R); and T-to-C mutation in exon 11 (Thr411 -to-Met, T411M). See, Kosaki et al. (J. Clin. Endocr. Metab. 87: 3529-3533, 2002); Bertola et al. (Am. J. Med. Genet. 136A: 242-245, 2005); Schollen et al. (Europ. J. Hum. Genet. 11 : 85-88, 2003).

[00100] Fig. 19 shows the distribution of PTPNl 1 (SHP-2) mutations and their relative prevalence in Noonan syndrome

[00101] In certain embodiments, the stem cells described herein comprise a mutation in the PTPNl 1 gene that is associated with LEOPARD Syndrome.

[00102] LEOPARD syndrome is an autosomal dominant disorder characterized by lentigines and cafe-au-lait spots, facial anomalies, and cardiac defects, sharing several clinical features with Noonan syndrome. Digilio et al. (Am. J. Hum. Genet. 71 : 389-394, 2002) screened 9 patients with LEOPARD syndrome (including a mother-daughter pair), and 2 children with Noonan syndrome who had multiple cafe-au-lait spots, for mutations in the PTPNl 1 gene. They found two missense mutations: an A-to-G mutation at nucleotide 836 in exon 7, resulting in a Tyr279-to-Cys (Y279C) mutation; and a Thr468-to-Met (T468M) mutation resulting from a C- to-T mutation at nucleotide 1403 in exon 12, respectively. Both mutations affected the PTPNl 1 phosphotyrosine phosphatase domain, which is involved in less than 30% of the Noonan syndrome PTPNl 1 mutations. This study demonstrated that LEOPARD syndrome and Noonan syndrome are allelic disorders. The detected mutations suggested that distinct molecular and pathogenetic mechanisms cause the peculiar cutaneous manifestations of the LEOPARD syndrome subtype of Noonan syndrome.

[00103] In 4 of 6 Japanese patients with LEOPARD syndrome, Yoshida et al. (Am. J. Med. Genet. 130A: 432-434, 2004) identified 3 heterozygous missense mutations: Tyr279 to Cys (Y279C), Ala461 to Thr (A461T, resulting from a 1381G-A mutation in exon 12), or Gly464 to Ala (G464A, resulting from a 1391G-C mutation in exon 12). [00104] Kontaridis et al. (J. Biol. Chem. 281 : 6785-6792, 2006) examined the enzymatic properties of mutations in PTPNl 1 causing LEOPARD syndrome and found that, in contrast to the activating mutations that cause Noonan syndrome and neoplasia, LEOPARD syndrome mutants are catalytically defective and act as dominant-negative mutations that interfere with growth factor/ERK-MAPK-mediated signaling. Molecular modeling and biochemical studies suggested that LEOPARD syndrome mutations control the SHP2 catalytic domain and result in open, inactive forms of SHP2. Kontaridis et al. concluded that the pathogenesis of LEOPARD syndrome is distinct from that of Noonan syndrome and suggested that these disorders should be distinguished by mutation analysis rather than clinical

presentation.

[00105] Other PTPN 11 mutations that have been associated with LEOPARD syndrome include, e.g., 1529A-C mutation in exon 13 (Gln510-to-Pro, Q510P) (Kalidas et al. (J. Hum. Genet. 50: 21-25, 2005) and the following missense PTPNl I mutations, in exon 7, 12 and 13: Tyr279Cys/Ser, Ala461Thr, Gly464Ala, Thr468Met/Pro, Arg498Trp/Leu, Gin506Pro, and GlnS lOGlu/Giy (Sarkozy A., et al, Orphanet J Rare Dis 3, 13 (2008)).

[00106] Other diseases that are known to be associated with a mutation in the PTPNl 1 gene include, e.g., cardiofaciocutaneous syndrome (MIM 163955), juvenile myelomonocytic leukemia (NIM 607785), and certain other malignancies.

[00107] In certain embodiments, the stem cells described herein comprise a mutation in the PTPNl 1 gene that is associated with leukemia.

[00108] Juvenile myelomonocytic leukemia (JMML), a disorder with excessive proliferation of myelomonocytic cells, constitutes approximately 30% of childhood cases of myelodysplasia syndrome (MDS) and 2% of leukemia. JMML is observed occasionally in patients with Noonan syndrome. In 5 unrelated children with Noonan syndrome and JMML, Tartaglia et al. (Nature Genet. 34: 148-150, 2003) found heterozygosity with respect to a mutation in exon 3 of PTPNl 1. Four of the children shared the same mutation (218C-T). In 2 unrelated individuals with growth retardation, pulmonic stenosis, and JMML, they found missense defects in PTPNl 1 : the 218C-T transition, and a defect in exon 13 affecting the protein tyrosine phosphatase domain. [00109] Tartaglia et al. also identified missense mutations in PTPNl 1 in 21 of 62 individuals with JMML but without Noonan syndrome, with 9 different molecular defects in exon 3 and 1 in exon 13. Nonhemato logic DNAs were available for 9 individuals with a mutation in PTPNl 1 in their leukemic cells, and none harbored the defect.

[00110] Other PTPNl 1 mutations that have been associated with JMML include, e.g., 226G-A mutation (Glu76-to-Lys, E76K), Glu76-to-Val, Glu76-to-Gly, Glu76-to-Ala (Tartaglia et al).

[00111] Tartaglia et al. also investigated the prevalence of somatic mutations in PTPNl 1 among 50 children with myelodysplasia syndrome. They identified missense mutations in exon 3 in 5 of 27 children with an excess of blasts. Three of these mutations were also associated with JMML in other patients. Among 24 children with de novo AML (MIM 601626), they identified a novel trinucleotide substitution in an infant with acute monoblastic leukemia.

[00112] In certain embodiments, the stem cells described herein comprise a mutation in the PTPNl 1 gene that is associated with a cardiac anomaly.

[00113] A cardiac anomaly refers to any structural or functional abnormality or defect of the heart or great vessels. The general effects of cardiac malformations on cardiovascular functioning are increased cardiac workload, increased pulmonary vascular resistance, inadequate cardiac output, and, in the case of cyanotic anomalies, decreased oxygen saturation. The general physical symptoms of these pathophysiologic alterations are growth retardation, decreased exercise tolerance, recurrent respiratory infections, dyspnea, tachypnea, tachycardia, cyanosis, tissue hypoxia, and murmurs, all of which vary in severity, depending on the type and degree of the defect.

[00114] Patients affected with cardiofaciocutaneous syndrome (CFC) present with symptoms that some considered to represent a more severe expression of Noonan syndrome, namely, congenital heart defects, cutaneous abnormalities, Noonan-like facial features, and severe psychomotor developmental delay. Bertola et al. {Am. J. Med. Genet. 130A: 378-383, 2004) identified a T-to-C mutation in exon 11 of the PTPNl 1 gene, resulting in a Thr411 -to-Met (T411M) substitution. [00115] In certain embodiments, the stem cells described herein comprise a mutation in the PTPNl 1 gene that is associated with cardiac hypertrophy (also known as hypertrophic cardiomyopathy) .

[00116] Cardiac hypertrophy refers to the process in which cardiac myocytes respond to stress through hypertrophic growth. Such growth is characterized by cell size increases without cell division, assembling of additional sarcomeres within the cell to maximize force generation, and an activation of a fetal cardiac gene program. Pathologic cardiac hypertrophy is often associated with increased risk of morbidity and mortality, and thus studies aimed at understanding the molecular mechanisms of cardiac hypertrophy could have a significant impact on human health.

[00117] In certain embodiments, the stem cells described herein comprise a threonine to methionine substitution in PTPNl 1 protein at an amino acid residue position corresponding to position 468 of SEQ ID NO: 1 , or a tyrosine to cysteine substitution in PTPNl 1 protein at an amino acid residue position corresponding to position 279 of SEQ ID NO: 1. In certain embodiments, the PTPNl 1 protein comprises SEQ ID NO: 1, and the mutation is a threonine to methionine substitution at amino acid residue position 468 of SEQ ID NO: 1 , or a tyrosine to cysteine substitution at amino acid residue position 279 of SEQ ID NO: 1.

C. Characterization of the Diseases-Specific Pluripotent Stem Cells

[00118] The disease-specific pluripotent stem cells provided herein show two important characteristics that distinguish them from other types of cells. First, they are non- lineage committed cells that are capable of maintaining their pluripotent state and of renewing themselves for long periods through cell division. Second, under appropriate conditions, they can be induced to differentiate into cells with specialized functions.

[00119] The disease-specific pluripotent stem cells may be characterized by any of several criteria of pluripotency known in the art. For example, the stem cells are capable of continuous indefinite replication in vitro. Continued proliferation for a long period of time (e.g., at least about 1 month, at least about 2 months, at least about 4 months, at least about 6 months, at least about 9 months, at least about one year) of a culture is sufficient evidence of immortality, as primary cell cultures without this property fail to continuously divide for such a length of time (Freshney, Culture of animal cells. New York: Wiley-Liss, 1994).

[00120] Additionally or alternatively, the disease-specific pluripotent stem cells may be characterized by the expression of certain markers, including but not limited to cell surface markers. Stem cells from different species may exhibit species-specific markers on their cell surfaces. For example, Thomson (U.S. Pat. Nos. 5,843,780 and 6,200,806) discloses certain cell surface markers that may be used to identify pluripotent stem cells derived from primates.

Furthermore, Stage Specific Embryonic Antigens (SSEAs) are unique carbohydrate epitopes that may be used to characterize pluripotent stem cells. Stem cells derived from different species exhibit different patterns of SSEAs. For example, undifferentiated primate pluripotent stem cells (including human pluripotent stem cells) express SSEA-3 and SSEA-4, but not SSEA-1.

Undifferentiated mouse pluripotent stem cells express SSEA-1, but not SSEA-3 or SSEA-4.

[00121] Additionally or alternatively, markers that are not exhibited on the surface of a cell may be used to characterize a pluripotent stem cell. For example, the homeodomain transcription factor Oct 4 (also termed Oct-3 or Oct3/4) is frequently used as a marker for pluripotent stem cells.

[00122] In certain embodiments, the pluripotent stem cells provided herein express SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, Nanog, OCT-4, Sox2, GDF3, DPPA4, REX1, TERT, Alkaline Phosphatase, Telomerase, or CD30, or a combination thereof.

[00123] The disease-specific pluripotent stem cells described herein are capable of differentiating into all three embryonic germ layers (endoderm, mesoderm, and ectoderm) in a fashion similar to embryonic stem cells. Such potency and differentiability can be demonstrated by, e.g., teratoma formation (e.g., injecting the stem cells into an immunodeficient mouse such as a SCID mouse, and then histologically examining the resulting tumors). Additionally or alternatively, the disease-specific pluripotent stem cells may be characterized by the capacity to develop into fully differentiated somatic cell lineages (e.g., neurons, cardiomyocytes, etc) and/or the germ line. Additionally or alternatively, the disease-specific pluripotent stem cells may be characterized by the capacity to participate in normal development when transplanted into a preimplantation embryo to generate a chimeric embryo. [00124] Additionally or alternatively, the disease-specific pluripotent stem cells may be characterized by their morphology (a stem cell has round shape, large nucleolus and scant cytoplasm; human iPSCs form sharp-edged, flat, tightly-packed colonies similar to human embryonic stem cells), telomerase activity (pluripotent stem cells express high telomerase activity to sustain self-renewal and proliferation), promoter demethylation (promoters of pluripotency-inducing genes, such as Oct-3/4, Rexl, and Nanog, are generally demethylated in iPSCs), or histone demethylation (H3 histones associated with Oct-3/4, Sox2, and Nanog genes are generally demethylated).

[00125] Exemplary stem cell lines were deposited on , 2010 with American

Type Culture Collection (ATCC), P.O. Box 1549, Manassas, VA 20108, USA, under ATCC Accession No. and .

[00126] In certain embodiments, the pluripotent stem cell of the present invention is a primate stem cell. In certain embodiments, the primate is human. In other embodiments, the pluripotent stem cell is a non-human stem cell (e.g., a mouse stem cell, a rat stem cell, a pig stem cell, a sheep stem cell, a goat stem cell, or a non-human primate stem cell).

[00127] The capacity of pluripotent stem cells to self renew in culture, while retaining their pluripotent potential, provides the opportunity to produce virtually unlimited numbers of undifferentiated and differentiated cell types for in vitro and in vivo investigation of disease mechanisms (Lerou, P. H. & Daley, G. Q. Therapeutic potential of embryonic stem cells. Blood Rev 19, 321-31, 2005; Ben-Nun, I. F. & Benvenisty, N. Human embryonic stem cells as a cellular model for human disorders. Mol Cell Endocrinol 252, 154-9, 2006). Pluripotent stem cells carrying the genes responsible for a particular disease (e.g., a mutation in the PTPN11 gene) can be induced to differentiate into the cell types affected in that disease. Studies of the differentiated cells in culture could provide important information regarding the molecular and cellular nature of events leading to pathology.

[00128] In certain embodiments, this approach is used to develop an in vitro model of LEOPARD syndrome and cardiac hypertrophy. As described more fully below in the Examples section, induced pluripotent stem cell lines were derived from two LEOPARD syndrome patients, and an unaffected brother not bearing the T468M mutation of one of the LS patients. These three pluripotent stem cell lines were differentiated into cardiomyocytes in culture. These in vitro differentiated cardiomyocytes could be maintained in culture, providing the opportunity to detect differences between the cardiomyocytes derived from stem cells carrying a mutant PTPN11 and those derived from stem cells that do not carry the mutation.

D. Differentiation of the Disease-Specific Pluripotent Stem Cells

[00129] The disease-specific pluripotent stem cells described herein may be induced in vitro to differentiate into a variety of somatic cell lineages (e.g., neurons, cardiomyocytes, etc). In one aspect, the invention provides cardiomyocytes comprising a mutation in the PTPN11 gene, wherein the cardiomyocyte is produced by differentiating the disease-specific pluripotent stem cell described herein.

[00130] The disease-specific pluripotent stem cells may be subjected to any conditions that induce the stem cells to differentiate into cardiomyocytes. For example, the disease-specific pluripotent stem cells may be suspended into a single-cell suspension, allowed to spontaneously aggregate into embryoid bodies over a first period of time (e.g. 48 hours, although such a period of time may be increased or decreased depending on other conditions to which the embryonic stem cells are subjected), and then treated with a suitable differentiation factor or factors for a second period of time such that the stem cells differentiate into cardiomyocytes. By way of example, such differentiation factors may include activin A, bone morphogenetic protein 4 (BMP4), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) dickkopf homo log 1 (DK 1), or a combination thereof.

[00131] In certain embodiments, the invention provides a method of differentiating a pluripotent stem cell as described herein into a cardiomyocyte, comprising culturing the stem cell in a serum- free medium that comprises: activin A, bone morphogenetic protein 4 (BMP4), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and dickkopf homolog 1 (DKK1).

[00132] Alternatively, the stem cells may be induced to differentiate into

cardiomyocytes in the presence of a prostaglandin, analogue or functional equivalent thereof, alone or in combination with other factors (such as minerals (e.g., selenium), small molecules (e.g., a p38 MAPK inhibitor such as SB203580), transferring, protein growth factors of the FGF, IGF and BMP families (e.g., IGF1, FGF2, BMP2, BMP4, BMP6, or a homologue or functional equivalent thereof), β-ΜΕ, non-essential amino acids, L-glutamine, etc.), as described in U.S. Application Publication No. 20070204351. The prostaglandin, analogue or functional equivalent thereof may be prostacyclin (PGI2), an analogue or functional equivalent thereof, including its naturally breakdown form of 6-keto-Prostaglandin Fla (6k-PGFla) and 2,3-dinor-6-keto- Prostaglandin Fla (2,3d-6k-PGFla); its synthetic analogs, such as iloprost, cicaprost, and carbarprostacyclin (cPGI) and stable chemical structures (Whittle, 1980, Town, 1982,

Sturzebecher, 1986); and its derivatives, as described in U.S. Application Publication No.

20070204351.

[00133] Also encompassed within the invention are cardiomyocyte progenitors that are differentiated from the pluripotent stem cells described herein. A cardiomyocyte progenitor, or cardiac progenitor cell, refers to a cell that is resident in the heart, or that comes into the heart from elsewhere after acute ischemia, is smaller than a mature cardiomyocyte, expresses a- sarcomeric actin but is negative for troponin, is normally quiescent but can be induced to go into the cell cycle as defined by positive Ki67 staining.

[00134] The cardiomyocytes and cardiomyocyte progenitors described herein may be beating. The cardiomyocytes may be fixed and stained with a-actinin antibodies to confirm muscle phenotype. a-troponin, a-tropomysin and a-MHC antibodies also give characteristic muscle staining.

[00135] In certain embodiments, a cardiomyocyte differentiated from a stem cell provided herein comprises a mutation in the PTPN11 gene. In certain embodiments, the mutation is associated with a cardiac anomaly (such as hypertrophic cardiomyopathy). In certain embodiments, the mutation is associated with LEOPARD Syndrome, Noonan Syndrome or leukemia. For example, in humans, a threonine to methionine substitution at amino acid residue position 468 of PTPN11 protein (SEQ ID NO: 1), or a tyrosine to cysteine substitution at amino acid residue position 279 of PTPN11 protein (SEQ ID NO: 1) is associated with LEOPARD syndrome/ hypertrophic cardiomyopathy. [00136] In certain embodiments, the invention provides a cell culture of the cardiomyocytes described herein. Exemplary cardiomyocyte cell lines were deposited on

20108, USA, under ATCC Accession No. and .

4. METHODS OF MAKING DISEASE-SPECIFIC PLURIPOTENT STEM CELLS

[00137] In another aspect, the invention provides methods of producing the disease- specific pluripotent stem cells as described herein.

[00138] In certain embodiment, the invention provides methods of producing the disease-specific pluripotent stem cells comprising (1) obtaining a somatic cell from a donor whose genome comprises a mutation in the PTPN11 gene, and (2) reprogramming the somatic cell into a pluripotent stem cell. In certain embodiments, the reprogramming comprises introducing into the somatic cell: (i) a nucleic acid molecule that encodes a pluripotency- inducing protein; or (ii) a polypeptide that comprises a pluripotency-inducing protein.

[00139] Methods for nuclear reprogramming of a somatic cell nucleus have been reported. One technique for nuclear reprogramming involves nuclear transfer into oocytes (Wakayama et al. Nature 394:369-374, 1998; Wilmut et al. Nature 385:810-813, 1997). Another method of reprogramming a somatic cell nucleus involves fusing a somatic cell and an ES cell (Tada et al. Curr. Biol. 11 : 1553-1558, 2001; Cowan et al. Science 309: 1369-73, 2005).

Alternatively, a somatic cell nucleus can be reprogrammed by treating a differentiated cell with an undifferentiated human carcinoma cell extract (Taranger et al. Mol. Biol. Cell 16:5719-35, 2005).

[00140] Induced pluripotent stem cells typically can be produced by introducing into somatic cells certain proteins (in particular, transcription factors) that are involved in maintaining ES cell pluripotency. These proteins are referred herein as "pluripotency-inducing proteins." Exemplary pluripotency-inducing proteins and genes encoding pluripotency-inducing proteins have been disclosed by Yamanaka et al. (EP Application Publication No. 1970446; US

Application Publication Nos. 2009/0068742 and 2009/0047263), and Thomson et al. (US Application Publication No. 2008/0233610). The pluripotency-inducing proteins are referred to as "nuclear reprogramming factors" by Yamanaka, and "potency-determining factors" by Thomson.

[00141] Exemplary pluripotency-inducing protein include, but are not limited to, Oct- 4, Sox2, FoxD3, UTF1, Stella, Rexl, ZNF206, Soxl5, Mybl2, Lin28, Nanog, DPPA2, ESG1, Otx2, Klf4, c-Myc, or combinations thereof.

[00142] In certain embodiments, the pluripotency-inducing protein is a protein from the Oct family (e.g., Oct3/4), Klf family (e.g., Klf4), Sox family (e.g., Sox2), or a combination thereof.

[00143] In certain embodiments, a set of two or more pluripotency-inducing proteins are introduced into the somatic cells. In embodiments, the set include Oct-4 and Sox2. In certain embodiments, the set include Oct3/4, Sox2, Klf4, and c-Myc. In other embodiments, the set include Oct3/4, Klf and c-Myc. Yet in another embodiment, the set include OCT4, SOX2, NANOG, and LIN28.

[00144] It is specifically envisioned that the set of pluripotency-inducing proteins sufficient to reprogram somatic cells can vary with the cell type of the somatic cells. One can identify a set of pluripotency-inducing proteins sufficient to reprogram other cells using different combinations of pluripotency-inducing proteins.

[00145] Suitable somatic cells can be any somatic cell, although higher

reprogramming frequencies are observed when the starting somatic cells have a doubling time of about twenty-four hours. Somatic cells useful in the invention may be non-embryonic cells obtained from a fetal, newborn, juvenile or adult primate, including a human. Examples of somatic cells that can be used with the methods described herein include, but are not limited to, bone marrow cells, epithelial cells, fibroblast cells, hematopoietic cells, hepatic cells, intestinal cells, mesenchymal cells, myeloid precursor cells and spleen cells. Another type of somatic cell is a mesenchymal cell that attaches to a substrate. Alternatively, the somatic cells can be cells that can themselves proliferate and differentiate into other types of cells, including blood stem cells (multipotent hematopoietic cells), muscle/bone stem cells, brain stem cells, liver stem cells, etc. [00146] In certain embodiments, the somatic cell is a fibroblast.

[00147] Suitable somatic cells are receptive, or can be made receptive using methods generally known in the art, to uptake pluripotency-inducing proteins or nucleic acid sequences that encode the pluripotency-inducing proteins. Uptake-enhancing methods can vary depending on the cell type and expression system. Exemplary conditions used to prepare receptive somatic cells having suitable transduction efficiency are known in the art (such as electroporation).

[00148] A nucleic acid encoding a pluripotency-inducing protein can be introduced by transfection or transduction into the somatic cells using a vector, such as an integrating- or non- integrating vector (e.g., a retroviral vector, an adenoviral vector). For example, a retroviral vectors (e.g., a lentiviral vector) may be transduced by packaging the vector into virions prior to contact with a cell. After introduction, the DNA segment(s) encoding the pluripotency-inducing protein can be located extra-chromosomally (e.g., on an episomal plasmid) or stably integrated into cellular chromosome(s).

[00149] A viral-based gene transfer and expression vector is a genetic construct that enables efficient and robust delivery of genetic material to most cell types, including non- dividing and hard-to-transfect cells (primary, blood, stem cells) in vitro or in vivo. Often, viral- based constructs that are integrated into genomic DNA result in high expression levels. In addition to a DNA segment that encodes a pluripotency-inducing protein of interest, the vectors may include a transcription promoter and a polyadenylation signal operatively linked, upstream and downstream, respectively, to the DNA segment. For example, nucleic acid encoding a pluripotency-inducing protein may be operably linked to a heterologous promoter, which may become inactive after somatic cells are reprogrammed. The heterologous promoter is any promoter sequence that can drive expression of a polynucleotide sequence encoding the pluripotency-inducing protein in the somatic cell, such as an Oct4 promoter.

[00150] The vector can include a single DNA segment encoding a single pluripotency- inducing protein factor or encoding a plurality of pluripotency-inducing proteins. A plurality of vectors can be introduced into a single somatic cell. The vector can optionally encode a selectable marker to identify cells that have taken up and express the vector. As an example, when the vector confers antibiotic resistance on the cells, antibiotic can be added to the culture medium to identify successful introduction of the vector into the cells. Either integrating vectors or non-integrating vectors may be used. Suitable integrating vectors include retroviral (e.g., lentiviral) vectors. Suitable non-integrating vectors include Epstein-Barr virus (EBV) vectors (Ren C, et al, Acta. Biochim. Biophys. Sin. 37:68-73 (2005); and Ren C, et al, Stem Cells 24: 1338-1347 (2006)). Adenoviral vectors may also be used to transport the nucleic acid into the somatic cell. Since the adenovirus does not integrated into cellular chromosome(s), the risk of creating tumors is reduced. Stadtfeld et al, Science, 332: 945 - 949 (2008). If desired, non- integrating vectors can be lost from cells by dilution after reprogramming.

[00151] To facilitate the identification of induced pluripotent stem cells, a non-lethal marker, such as Green Fluorescent Protein (GFP), Enhanced Green Fluorescent Protein (EGFP) or luciferase, under the control of a promoter active only after the somatic cell has been converted to a pluripotent state, may be used. A selectable marker gene may be used to identify the reprogrammed cells expressing the marker through visible cell selection techniques, such as fluorescent cell sorting techniques. Alternatively, the reprogrammed cells can be produced without a selectable marker. For example, a marker may be integrated into the genome of the somatic cells downstream of the promoter that regulates Oct4 expression. The endogenous Oct4 promoter is active in undifferentiated, pluripotent stem cells.

[00152] The non-lethal marker can be constructed to enable its subsequent removal using any of a variety of art-recognized techniques, such as removal via Cre -mediated, site- specific gene excision. For example, it may become desirable to delete the marker gene after the pluripotent cell population is obtained, to avoid interference by the marker gene product in the experiment or process to be performed with the cells. Targeted deletions can be accomplished by providing structure(s) near the marker gene that permits its ready excision. For example, a Cre/Lox genetic element can be used. The Lox sites can be built into the cells. If it is desirable to remove the marker from the pluripotent cells, the Cre agent can be added to the cells. Other similar systems also can be used. Because Cre/Lox excision can introduce chromosomal rearrangements and can leave residual genetic material after excision, it may also be desirable to introduce the pluripotency-inducing proteins into the somatic cells using non-integrating, episomal vectors and obtaining cells from which the episomal vectors are lost (e.g., at a rate of about 5% per generation) by subsequently withdrawing the drug selection used to maintain the vectors during the reprogramming step.

[00153] The vectors described herein can be constructed and engineered using art- recognized techniques. Standard techniques for the construction of expression vectors suitable for use are well-known in the art and can be found in publications such as Sambrook J, et al., "Molecular cloning: a laboratory manual," (3rd ed. Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001).

[00154] It is also possible to generate iPS cells by direct delivery of pluripotency- inducing proteins, thus eliminating the need for viruses or genetic modification of the somatic cells. For example, it has been reported that repeated treatment of the cells with certain proteins channeled into the cells via poly-arginine anchors was sufficient to induce pluripotency (Zhou et al, Cell Stem Cell, Volume 4, 381-384 (2009)). The expression of pluripotency-inducing genes can also be increased by treating somatic cells with FGF2 under low oxygen conditions.

[00155] The relative ratio of pluripotency-inducing proteins may be adjusted to increase reprogramming efficiency. For example, it has been reported that linking Oct-4 and Sox2 in a 1 : 1 ratio on a single vector increased reprogramming efficiency in cells by a factor of four, compared to reprogramming efficiency wherein the pluripotency-inducing genes were provided to cells in separate constructs and vectors, where the uptake ratio of the respective pluripotency-inducing genes into single cells was uncontrolled. Although the ratio of pluripotency-inducing proteins may differ depending upon the set of pluripotency-inducing proteins used, one of ordinary skill can readily determine optimal ratios of pluripotency-inducing proteins.

[00156] Pluripotent stem cells can be cultured in any medium used to support growth of pluripotent cells. Typical culture medium includes, but is not limited to, a defined medium, such as TeSR™ (StemCell Technologies, Inc.; Vancouver, Canada), mTeSR™ (StemCell Technologies, Inc.) and StemLine® serum-free medium (Sigma; St. Louis, Mo.), as well as a conditioned medium, such as mouse embryonic fibroblast (MEF)-conditioned medium.

Alternatively, cells can be maintained on MEFs in culture medium. 5. DISEASE MODELING AND DRUG SCREENING

[00157] In another aspect, the invention provides a method of identifying an agent for use in treatment of a disease that is associated with a mutation in the PTPN11 gene, comprising (1) contacting a disease-specific pluripotent stem cell as described herein with the agent; and (2) determining the effect of the agent on survival, proliferation, phenotype, or differentiation of the pluripotent stem cell.

[00158] In another aspect, the invention provides a method of identifying an agent for use in treatment of a disease that is associated with a mutation in the PTPN11 gene, comprising (1) contacting a cardiomyocyte as described herein with the agent; and (2) determining the effect of the agent on survival, proliferation, or phenotype of the cardiomyocyte.

[00159] In another aspect, the invention provides a method of identifying an agent for use in treatment of the cardiac hypertrophy that is associated with a mutation in the PTPN11 gene, comprising: (1) contacting a cardiomyocyte as described herein with the agent; and (2) determining the effect of the agent on the hypertrophic state of the cardiomyocyte, wherein a decrease in the hypertrophic state of the cardiomyocyte, as compared to that of a control cell, is indicative that the agent is an agent for use in treatment of a disease that is associated with a mutation in the PTPN11 gene.

[00160] The disease-specific pluripotent stem cells and cardiomyocytes described herein may be used to screen for test agents that affect survival, proliferation, phenotype, or differentiation of the cells. For example, the disease-specific stem cells may be induced to differentiate into cardiomyocytes by placing it under appropriate differentiation conditions. Before, during and/or after differentiation, the cell may be subjected to a test agent in order to determine whether that agent has an effect on survival, proliferation, phenotype, or

differentiation of the cells. The agents can be used for treatment, preventing or ameliorating the symptoms of the diseases.

[00161] The disease-specific stem cells and cardiomyocytes described herein may also be used to develop personalized treatment regimens. For example, certain patients may respond better to a given therapy or drug regimen than other patients. Additionally or alternatively, certain patients may experience fewer and/or less severe side effects after being administered a given therapy or drug regimen than other patients. By utilizing stem cells that contain the genetic complement of a patient suffering from and/or predicted to suffer from a disease of interest, and permitting such cells to differentiate into a cell type associated with that disease, it will be possible to better predict which therapy or drug regimen will be most beneficial and/or result in the least detrimental side effects.

[00162] A control cell may be a parallel sample cell that has not been treated with the agent (e.g., recipient cells mock-treated with buffers), or a cell that has been treated with an agent having a known effect (e.g., a positive effect, a negative effect, or no effect).

Alternatively, a control cell may be a cell having known or pre-determined properties (e.g., a characterized cell line from a database or a cell not carrying the PTPNl 1 mutation of the disease- specific cell).

[00163] In certain embodiments, a disease of interest may be modeled and/or studied by creating a disease-specific pluripotent stem cell line, and inducing the cell line to differentiate under appropriate differentiation conditions. For example, a disease-specific pluripotent stem cell line may be generated in which the stem cells carry a mutation in the PTPNl 1 gene that is associated with LEOPARD syndrome, or a cardiac anomaly. By observing survival,

proliferation, phenotype, or differentiation of the stem cells and the in vitro differentiated cardiomyocytes, and comparing them to stem cells or cardiomyocytes that do not carry the mutation, one can better understand the genetic basis of disease progression and pathogenesis. The cells are particularly useful in studying and/or modeling diseases that have not been amenable to such study and/or modeling.

[00164] The test agent may be any molecule that increases or decreases the expression level or the activity level of PTPNl 1 protein. For example, the agent may be a small molecule compound, an antibody, a hormone, a vitamin, a nucleic acid molecule, an enzyme, an amino acid, or a virus.

[00165] The invention also demonstrates for the first time that mutations in the PTPNl 1 gene perturbs RAS-MAPK signal transduction as early as the pluripotent stem cell stage. RAS is a family of genes encoding small GTPases that are involved in cellular signal transduction. Activation of Ras signalling causes cell growth, differentiation and survival.

Transmission of stimulatory signals from Ras to nuclear targets involves regulation of the family of kinases known as MAPKs ("mitogen-activated protein kinases") or ER s ("extracellular signal regulated kinases"). This pathway includes, but is not limited to, components such as RAFl (V-Raf-1 Murine Leukemia Viral Oncogene Homo log 1), SOS1 (Son of Sevenless Homolog 1 (Drosophila)), ERK2, KRAS (V-KI-RAS2 Kirsten Rat Sarcoma 2 Viral Oncogene Homo log), PTPN11, BRAF (B-Raf proto-oncogene serine/threonine -protein kinase), MEK1 (mitogen-activated protein kinase kinase 1), MEK2, and HRAS. Additional components of this pathway have been identified and described (see, e.g., Lee and McCubrey, Leukemia 16:486- 507, 2002).

[00166] In another aspect, the invention provides a method of identifying an agent as a candidate Ras-signaling pathway inhibitor, comprising: (1) contacting a pluripotent stem cell as described herein with the agent; and (2) determining the effect of the agent on tyrosine phosphorylation of EGFR (Epidermal growth factor receptor) or MEK1. A decrease of tyrosine phosphorylation of EGFR or MEK1, as compared to that of a control cell, is indicative that the agent is a Ras-signaling pathway inhibitor.

[00167] In another aspect, the invention provides a method of identifying an agent as a candidate Ras-signaling pathway inhibitor, comprising: (1) contacting a cardiomyocyte as described herein with the agent; and (2) determining the effect of the agent on tyrosine phosphorylation of EGFR or MEK1. A decrease of tyrosine phosphorylation of EGFR or MEK1, as compared to that of a control cell, is indicative that the agent is a Ras-signaling pathway inhibitor.

[00168] In another aspect, the invention provides a method of identifying an agent for use in treatment of a disease that is associated with a mutation in the PTPN11 gene, comprising: (1) contacting a pluripotent stem cell as described herein with the agent; (2) differentiating the stem cell into a cardiomyocyte; and (3) determining the hypertrophic state of the cardiomyocyte. A decrease in the hypertrophic state of the cardiomyocyte, as compared to that of a control cell, is indicative that the agent is an agent for use in treatment of a disease that is associated with a mutation in the PTPN11 gene. [00169] The pluripotent stem cells and cardiomyocytes described herein may also be used to test or validate the therapeutic activities of known Ras kinase inhibitors or PTPN11 inhibitors. For example, the pluripotent stem cells or cardiomyocytes described herein may be cultured in the presence of a Ras inhibitor or PTPN11 inhibitor, and effect of the inhibitor on survival, proliferation, phenotype, or differentiation of the cells may be determined. Currently, there are a few known PTPN11 inhibitors, such as CDL 4340-0580 (Hellmuth et al, P roc Natl Acad Sci USA 105,7275-7280, 2008) and NAT6-297775 (Noren-Muller et al, Proc Natl Acad Sci USA . , 103 : 10606- 11 2006).

EXAMPLES

[0170] The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.

[0171] The generation of reprogrammed induced pluripotent stem cells (iPSC) from patients with defined genetic disorders promises important avenues to understand the etiologies of complex diseases, and the development of novel therapeutic interventions. As shown by the Examples described below, the inventors have generated iPSCs from patients with LEOPARD syndrome (LS; acronym of its main features: Lentigines, Electrocardiographic abnormalities, Ocular hypertelorism, Pulmonary valve stenosis, Abnormal genitalia, Retardation of growth and Deafness), an autosomal dominant developmental disorder belonging to a relatively prevalent class of inherited RAS-MAPK signaling diseases, which also includes Noonan syndrome (NS), with pleomorphic effects on several tissues and organ systems (Gorlin, R.J., et al, Birth Defects Orig Artie Ser 07, 110-115 (1971); Sarkozy, A., et al, Orphanet J Rare Dis 3, 13 (2008)). The patient-derived cells have a mutation in the PTPN11 gene, which encodes the SHP2 phosphatase The iPSC have been extensively characterized and produce multiple differentiated cell lineages. A major disease phenotype in patients with LEOPARD syndrome is hypertrophic

cardiomyopathy. The inventors show that in vzYro-derived cardiomyocytes from LS-iPSC are larger, have a higher degree of sarcomeric organization and preferential localization of NFATc4 in the nucleus when compared to cardiomyocytes derived from human embryonic stem cells (HESC) or wild type (wt) iPSC derived from an unaffected, non-PTPNl lbrother of one of the LS patients. These features indicate a hypertrophic state. The inventors also provide molecular insights into signaling pathways that may promote the disease phenotype.

EXAMPLE 1: MATERIALS AND METHODS

[00172] Cell culture. Dermal fibroblast lines were obtained from skin biopsies, collected under an Institutional Review Board-approved protocol and with informed consent. LEOPARD syndrome patients derived fibroblasts, BJ fibroblasts (American Type Culture Collection) and GP2 cells were maintained in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum and penicillin/streptomycin (Invitrogen). HES2 and iPSC were maintained on irradiated Swiss Webster mouse embryonic feeder cells (MEFs), in a serum free HESC medium composed of DMEM/F12 (Cellgro, Mediatech) containing 20 ng/ml basic fibroblast growth factor (bFGF, R&D systems), 20% (vol/vol) KSR (Invitrogen), 5% (vol/vol) MEF-conditioned medium, Penicillin/Streptomycin, L-glutamine (L-Gln), non-essential amino acids (Invitrogen), and β-mercaptoethanol (β-ΜΕ, Sigma-Aldrich).

[00173] Plasmid construction. Full-length sequences of human OCT4, SOX2, KLF4 and c-MYC transcription factors were obtained from Open Biosystems. The coding sequences were PCR amplified using Pfu Turbo (Stratagene) and cloned into pMXs vector and verified by sequencing. pMXs-EGFP vector was constructed by introducing the BamHI/NotI EGFP fragment from FUGW (kindly provided by Dr. Lois, MIT, Massachusetts) into pMXs vector. The latter vector was used to monitor the transfection and infection efficiency.

[00174] Retroviral infection and LEOPARD syndrome iPSC generation. OCT4, SOX2, KLF4 and c-MYC transcription factors were introduced in dermal fibroblasts derived from two patients with LEOPARD syndrome via the pMXs retroviral vector. In parallel, pMXs-EGFP vector was used to estimate the infection efficiency (data not shown). Six days after infection, fibroblasts were seeded onto MEFs. The following day the medium was replaced with the HESC medium and bFGF.

[00175] Specifically, GP-2 cells were plated at 8xl0⁶ cells per 10-cm dish and transfected with pMXs, VSV-G and Gag-Pol vectors using SuperFect transfection reagent on the following day. The same day, human fibroblasts were seeded at 8xl0⁵ cells per 10-cm dish. Twenty-four hours after transfection, the four retroviruses (hOCT4, hSOX2, hKLF-4 and hC- MYC) containing supernatants were collected, and equal amounts of each were mixed and filtered through a 0.45 μιη pore-size filter, and supplemented with 4 μg/mL polybrene.

Retroviruses containing medium was added to the fibroblasts plates. The following day, forty- eight hours post-transfection, the fibroblasts were reinfected following the same procedure as the day before. Six days after transduction, fibroblasts were transferred into four dishes coated with MEFs, at 50,000 fibroblasts per plate.

[00176] DNA Fingerprinting analysis. In order to verify the genetic relatedness of the iPSC to their parental fibroblasts, the inventors PCR amplified across three discrete genomic loci containing highly variable numbers of tandem repeats. Genomic DNA (gDNA) was isolated with Easy-DNA™ kit (Invitrogen). Fifty nanograms of genomic DNA was used per reaction. Primers are summarized in Figure 17 (Table 1).

[00177] Karyotype analysis. HES2 and iPSC were grown on Matrigel-coated glass coverslip dishes (MatTek, Ma). The day of culture harvest, 20 μΐ of colcemid (5 μg/ml) was added to the in situ ESC1 culture which was 30-50% confluent. The culture was re-incubated for 15 min at 37°C. A robotic harvester (Tecan) was utilized, which included automatic addition of 2cc of hypotonic solution (sodium citrate solution 0.8%) with incubation for 20 min at room temperature, prefixation with addition of 2cc of fixative (methanol: glacial acetic acid; 3: 1), followed by addition of 4cc of fixative, twice. The coverslip was dried completely at 37 °C with 45-50%) humidity and mounted on a microscope slide and GTG-banded according to standard protocols. Metaphases were captured and karyotypes were prepared using the Cyto Vision software program (Version 3.92 Build 7, Applied Imaging).

[00178] qPCR and transgenes integration. For quantitative real-time PCR (qPCR) analyses, total RNA was extracted from cells using Trizol® Reagent (Invitrogen) and subsequently column-purified with RNeasy kit (Qiagen) and treated with RNase-free DNase (Qiagen). One microgram of total RNA was reverse transcribed into cDNA using random primers and Superscript II Reverse Transcriptase (Invitrogen). PCR for transgene silencing was performed with Expand High Fidelity Enzyme Tag Polymerase (Roche). Real-time qPCR was performed on a StepOne Plus Real-Time PCR System (Applied Biosystems) with Fast

SYBR® Green Master Mix (Applied Biosystems). The results were analyzed with the StepOne Software v2.0, normalized to β-Actin gene expression, and compared to HES2 cell expression levels. To examine the presence of transgenes in the iPSC lines, gDNA was isolated with Easy- DNA Kit (Invitrogen). PCR reactions were carried out with the Expand High Fidelity Enzyme Taq Polymerase (Roche). Primer sequences are described in Table 1 (Figure 17). Primers for FGF receptors expression analysis have been previously described (Dvorak, P., et al, Stem Cells 23, 1200-1211 (2005)).

[00179] Southern blot analyses. gDNA (2μ ) was completely digested with Bgl II, separated on a 0.8% agarose gel, transferred to a positively charged nylon membrane, and hybridized with DIG-labeled hOCT4, hSOX2, hKLF-4 and hC-MYC cDNA probes. After hybridization, membranes were washed, blocked with DIG blocking solution, and incubated with anti-DIG-AP Fab fragments (Roche). Probe-target hybrids were then incubated with

chemiluminescent CDF-Star substrates (Roche) and detected via exposure to X-ray film.

[00180] Bisulfite sequencing. 500 ng of purified gDNA were treated with sodium bisulfite using the Zymo EZ-DNA Methylation Kit, following the manufacturer's instructions. The sequences of primers used for amplification of genomic fragments were previously published (Freberg, C.T., et al, Mol Biol Cell 18, 1543-1553 (2007)). PCR products were then size fractionated in 1% TAE-agarose, extracted using the Qiaquick gel extraction kit (Qiagen) and cloned into the pGEM-T Easy Vector system (Promega). Blue-white selection was applied to eliminate false positives, and twelve random clones were picked and sequenced. Bisulfite conversion efficiency of non-CpG cytosines was >90% for all individual clones for each sample.

[00181] Immunocytochemistry, AP staining and FACS analysis. For in vivo immunostaining, HES2 and iPSC were washed once with DMEM medium supplemented with 10% FBS and antibiotics (DMEM 10%), and incubated with biotin-TRA-1-81 antibody (1 : 100, eBiosciences) for 2 h. Cells were washed three times with DMEM 10% and they were incubated with the secondary antibody streptavidin-FITC (1 : 100, eBiosciences) and the phycoeritrin TRA- 1-60 (PE-TRA-1-60) antibody (1 : 100, eBiosciences) where indicated. All the incubations were performed in a humidified incubator at 37 °C with 5% C0₂. For intracellular staining, cells were fixed in 2% paraformaldehyde for 30 min, and blocked and permeabilized in PBS containing 10% donkey serum, 1% BSA and 0.1% Triton X-100 for 45 min. Cells were incubated with primary antibody in blocking solution overnight at 4 °C, washed and incubated with the corresponding Alexa donkey secondary antibody for 1 h at room temperature (RT). Then cells were washed and stained with DAPI (1 μg/ml) for 20 min. The primary antibodies used for intracellular immunostaining were OCT4 (1 : 100, Bio Vision), NANOG (1 : 100, R&D systems), desmin (1 : 100, Lab Vision), a-SMA (pre-diluted, DAKO), vimentin (1 : 100, Chemicon), AFP (1 :500, DAKO), GFAP (1 :1000, DAKO), βΙΙΙ-Tubulin (1 : 100, Chemicon), or NFATc4 (1 : 100, Santa Cruz Biotechnology). All the secondary antibodies Alexa 488 Donkey anti-Rabbit (1 : 100), Alexa 546 donkey anti-goat (1 : 100) and Alexa 546 donkey anti-mouse (1 : 100) were obtained from Invitrogen. Alkaline phosphatase staining was detected following manufacturer's recommendations (Millipore). SSEA-4, troponin T, and hematopoietic markers expression were evaluated on a BD Biosciences LSRII FACS machine analyzer. Primary antibodies SSEA-4-PE (R&D systems), cardiac troponin T (Lab Vision), CD1 lb-APC (Caltag), and CD45-APC, CD45- PE, CD71-PE, CD41-PE and CD235a-APC were purchased from BD Biosciences.

[00182] Teratoma formation. All animal procedures were performed in accordance with the Mount Sinai Medical Center's Institutional Animal Care and Use Committee.

Approximately l-2xl0⁶ cells were injected subcutaneously into the right hindleg of immunocompromised NOD-SCID mice (The Jackson Laboratory). Teratomas were excised 6-10 weeks post-injection, fixed overnight in formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin by the Morphology and Assessment Core of the Department of Gene and Cell Medicine. Histological evaluation was performed using a Nikon TE2000-U microscope and ACT-1 software.

[00183] In vitro differentiation. For non-lineage specific and hematopoietic differentiation the inventors used previously described protocols, with certain modifications (Takahashi, K., et al, Cell 131, 861-872 (2007); Dimos, J.T., et al, Science 321, 1218-1221 (2008), unpublished data, Kennedy M. and Keller G. et al). For cardiomyocytes induction, the inventors used a well-established assay (Yang, L., et al, Nature 453, 524-528 (2008)). For embryoid body (EB) formation, HES2 and iPSC were treated with collagenase B (Roche) for 10 min, and collected by scraping. After centrifuging, cell pellets were resuspended in basic differentiation media, StemPro 34 (Invitrogen) containing 2 mM L-Gln, 4x10^"

monothioglycerol (MTG), 50 μg/ml ascorbic acid (Sigma) and 150 μg/ml transferrin (Sigma). EBs were grown in ultra low-binding plates (Costar) and medium was changed every three days. After eight days of differentiation, EBs were collected, resuspended in DMEM 10% and transferred to gelatin-coated dishes to allow them to attach and differentiate for eight additional days before processing for immunocytochemistry analyses. For hematopoietic differentiation the inventors used a described protocol (Kennedy, M., et al, Blood 109, 2679-2687 (2007)) with certain modifications (unpublished data, Kennedy M. and Keller G. et al.). For cardiomyocyte induction, the inventors used a well-established protocol (Yang, L., et al, Nature 453, 524-528 (2008)).

[00184] Microarray analysis. Gene level mRNA abundance measures were extracted using the Affymetrix GeneChip Exon 1.0 ST array according to the manufacturer's protocols by the Genomics Core Lab in The Institute for Personalized Medicine at Mount Sinai Medical Center. Microarrays were scanned and data were Robust Multi- Array (RMA)-normalized using the Affymetrix Expression Console software. Subsequently, these genes were clustered and a heat map was generated against a background subset of genes showing at least two-fold change between sample averages of iPSC/HES2 cells and fibroblast samples.

[00185] Cytology/cardiomyocyte size determination. On D18 of differentiation, beating EBs were plated on gelatin coated dishes. Three days after plating, EBs outgrowths were trypsinized, filtered through a 40 μιη size pore-size filter, and single cells were replated at low density on gelatin coated dishes. The following day, cells were fixed with 4% paraformaldehyde, permeablized, blocked in PBS/1% BSA/0.1%> Triton/10%) donkey serum, and Stained for cardiac Troponin T (1 :200, Lab Vision), overnight at 4°C. Stained cells were washed three times with PBS, and then incubated with the Alexa Fluor 547 donkey-anti-mouse antibody (Invitrogen) for 1 h. The areas of HESC- and LS-iPSC-derived cardiomyocytes were analyzed using ImageJ software (NIH).

[00186] Phosphoproteomics and Western blotting. HES2 and iPSC were incubated overnight in HESC culture medium deprived of bFGF and knockout serum replacement (KSR). Protein lysates were quantified by Bradford assay and sent to Kinexus Bioinformatics Corporation (Vancouver, Canada) for antibody microarray screening. The proteins with at least 1.5 fold change between the LS-iPSC samples and control sample (either HES2 or wt-iPSC), and conserved in the majority of the comparison groups were represented in a heat map.

[00187] Specifically, the inventors prepared a lysis buffer (pH 7.2) containing 20 mM MOPS pH 7.0, 2 mM EGTA, 5 mM EDTA, 30 mM sodium fluoride, 60 mM β- glycerophosphate, 20 mM sodium phyrophospate and 1% Triton X-100. Protease and phosphatase inhibitors (1 mM phenylmethylsulfonyl fluoride, 3 mM benzamidine, 10 μg/ml aprotinin, 10 μΜ leupeptin, 5 μΜ pepstatin, ImM dithiothreitol and 1 mM sodium

ortho vanadate) were added to the lysis buffer immediately before use. Protein extracts were sent to Kinexus Bioinformatics Corporation (Vancouver, Canada). The antibody microarray results were processed following the company recommendations. Western blot was carried out as previously described (Carvajal-Vergara, X., et al, Blood 105, 4492-4499 (2005)). The primary antibodies used were: pS6 S235/236 (1 : 1000, Cell Signaling), pEGFR Y1086 (1 :1000, Cell Signaling), pMEKl S298 (1 : 1000, Cell Signaling), β-Actin (1 :5000, Abeam), p-ER l/2

T202/Y204 (1 :2000 Cell Signaling) and ER 1 (1 :2500, Santa Cruz Biotechnology).

EXAMPLE 2: PATIENT-SPECIFIC INDUCED PLURIPOTENT STEM CELL DERIVED FROM MODELS OF LEOPARD SYNDROME

[00188] Approximately 90% of LS cases, and 45% of NS, are caused by missense mutations in the PTPN11 gene that encodes the protein tyrosine phosphatase SHP2. PTPN11 is ubiquitously expressed, essential for normal development, and somatic mutations in this gene contribute to leukemogenesis in children (Loh, M.L., et al, Blood 103, 2325-2331 (2004), Tartaglia, M., et al, Blood 104, 307-313 (2004)). For LS, two mutations, T468M and Y279C, are most recurrent (Tartaglia, M., et al., Am J Hum Genet 78, 279-290 (2006)). Hypertrophic cardiomyopathy is the most common life-threatening cardiac anomaly in LS (Sarkozy, A., et al., Orphanet J Rare Dis 3, 13 (2008)). Animal models of LS have been generated in Drosophila and zebrafish (Jopling, C, et al, PLoS Genet 3, e225 (2007); Oishi, K., et al, Hum Mol Genet (2008)), but the molecular pathogenesis of LS remains obscure.

[00189] Ectopic expression of four transcription factors (OCT4, SOX2, KLF4 and c- MYC) in adult human dermal fibroblasts can generate pluripotent iPSC (Lowry, W.E., et al., Proc Natl Acad Sci U S A 105, 2883-2888 (2008), Park, I.H., et al, Cell 134, 877-886 (2008), Takahashi, K., et al., Cell 131, 861-872 (2007)). The inventors have established iPSC lines from two LS patients, a 25-year-old female (LI), and a 34-year-old male (L2). A heterozygous T468M substitution mutation in PTPN11 is present in both.

[00190] Patient-derived fibroblasts were transduced with OCT4-, SOX2-, KLF4- and c-MYC-encoding VSV-pseudotyped Moloney-based retroviral vectors. Granular morphology and compact ESC-like colonies emerged during the ensuing two weeks, distinguishable by TRA- 1-81 staining (Fig. 5), as described previously (Lowry, W.E., et al, Proc Natl Acad Sci U S A 105, 2883-2888 (2008)). TRA- 1-81 -positive colonies were clonally expanded to create stable LS-iPSC lines. Three iPSC lines per patient were used for preliminary characterization: Ll- iPSl, Ll-iPS6, Ll-iPS13, L2-iPS6, L2-iPS16 and L2-iPS18.

[00191] To verify that the iPSC originated from patient-derived fibroblasts, the inventors performed DNA fingerprinting analysis (Fig. 6a). All iPSC had normal karyotypes of 46,XX (LI) and 46,XY (L2) (Fig. 6b and data not shown). In addition, they carried the expected T468M mutation (Fig. 7a). Restriction fragment length polymorphism analysis of an RT-PCR amplimer containing the mutation with BsmFI showed biallelic expression of PTPN11 (Fig. 7b). PCR and Southern blots indicated the presence of all four transgene proviruses in the LS-iPSC (Fig. 8) and quantitative RT-PCR (qRT-PCR) results confirmed efficient transgene silencing (Fig. 9).

[00192] To further characterize the LS-iPSC clones, expression of several HESC markers in two LS-iPSC lines from each patient (Ll-iPSl, Ll-iPS13, L2-iPS6 and L2-iPS16) was analyzed and compared to the HES2 HESC and a wt-iPSC line, BJ-iPSB5, derived in the lab from a normal human fibroblast line (BJ). The BJ-iPSB5 cell line was also karyotypically normal (46,XY), contained all four transgene proviruses, which were silenced (Fig. 6b, 8b and 9b). All LS and control iPSC lines exhibited high alkaline phosphatase activity, and expressed

pluripotency markers, including surface antigens TRA- 1-81, TRA- 1-60, and SSEA-4, as well as the nuclear transcription factors OCT4 and NANOG (Fig. 10). Quantitative RT-PCR (RT-qPCR) confirmed the activation of a series of endogenous sternness genes (OCT4, NANOG, SOX2, GDF3, DPPA4, REXl and TERT) in iPSC (Fig. la and Fig. 11a). Moreover, bisulfite sequencing analyses determined that the great majority of the CpG dinucleotides analyzed in the OCT4 and NANOG promoters were demethylated in iPSC when compared to their parental fibroblasts (Fig. lb).

[00193] The inventors next examined genome-wide mR A expression profiles of two LS-iPSC lines from each patient, the BJ-iPSB5 cell line, parental fibroblasts and HES2 cells. The resulting heat map and scatter-plot analyses indicated that iPSC lines shared a higher degree of similarity with HES2 cells than with their parental fibroblast cell lines (Fig. lc and Fig. 1 lb).

[00194] Pluripotent HESC can differentiate into cell types representative of all three germ layers. The inventors tested the differentiation abilities of the iPSC using an in vitro 8-day floating embryoid body (EBs) system, followed by replating on gelatin-coated dishes for another 8 days (Takahashi, K., et al, Cell 131, 861-872 (2007); Dimos, J.T., et al, Science 321, 1218- 1221 (2008)). Immunocytochemistry analyses detected expression of a-smooth muscle actin (a- SMA, mesoderm), desmin (mesoderm), a-fetoprotein (AFP, endoderm), vimentin (mesoderm), glial fibrillary acidic protein (GFAP, ectoderm) and βΙΙΙ-tubulin (ectoderm) markers (Fig. 2a and Fig. 12). In order to determine pluripotency in vivo, the inventors injected LS-iPS, BJ-iPSB5 and HES2 cells into immune-compromised NOD-SCID mice. Histological analyses of the resulting teratomas showed cell types representative of the three germ layers, including pigmented cells (ectoderm), lung, respiratory and gut-like epithelia (endoderm), and

mesenchyme, adipose tissue and cartilage (mesoderm) (Fig. 2b and data not shown).

[00195] As mentioned previously, hypertrophic cardiomyopathy is one of the major features of LS, affecting 80% of the patients. In addition, affected individuals occasionally manifest hematologic complications such as myelodysplasia and leukemia (Laux, D. et al., J Pediatr Hematol Oncol 30, 602-604 (2008); Ucar, C, et al, J Pediatr Hematol Oncol 28, 123-125 (2006)). Therefore, the inventors decided to explore if LS-iPSC were able to differentiate into hematopoietic and cardiac lineages. LS-iPSC from both patients differentiated into a variety of hematopoietic cell types including early hematopoietic progenitors (CD41⁺) (Mikkola, H.K., et al, Blood 101, 508-516 (2003)), early erythroblasts (CD71⁺/CD235a⁺) (Wu, C.J., et al, Blood 106, 3639-3645 (2005)), and macrophages (CD1 lb⁺) (Fan, S.T., et al, J Clin Invest 87, 50-57 (1991)) (Fig. 13 and data not shown). The cardiac hypertrophic response includes induction of immediate-early genes (such as c-jun, c-fos and c-myc), an increase in cell size, and organization of contractile proteins into sarcomeric units (Aoki, H., et al, Nat Med 6, 183-188 (2000);

Buitrago, M., et al., Nat Med 11, 837-844 (2005)). To have an appropriate control cell line to analyze some of these parameters, besides HESC, the inventors generated a wt-iPSC line (S3- iPS4) from fibroblasts obtained from an unaffected brother of LI without the T468M mutation (Fig. 14 and Fig. 15). Using a well-established cardiac differentiation protocol (Yang, L., et al, Nature 453, 524-528 (2008)), the inventors observed contracting embryoid bodies (EBs) emerging around day 11 of differentiation. In order to monitor cardiac development, the inventors analyzed cardiac troponin T (cTNT^ expression on day 18 of differentiation by flow cytometry (data not shown). Replated cells from beating EBs were processed as described in Material and Methods. Briefly, cells were fixed, immunostained for cTNT (Fig. 3b), and 50 cardiomyocytes were randomly chosen from each sample for surface area measurement using a computerized morphometric system (ImageJ software, NIH). Cardiomyocytes derived from LS- iPSC lines: Ll-iPS13, Ll-iPS6 and L2-iPS10 (Fig. 14 and Fig. 15), had a significantly increased median surface area compared to wt-iPSC cardiomyocytes; 1.8 times, 2.5 times and 4.8 times larger, respectively, whereas the area median of the cardiomyocytes obtained from HESC was similar to wt-iPSC cardiomyocytes (Fig. 3a). The inventors also observed increased sarcomere assembly in Ll-iPS6 and L2-iPS10 cells when compared to wt S3-iPS4 cells (Fig. 3b). Recently, the calcineurin-NFAT pathway has been shown to be an important regulator of cardiac hypertrophy. Active calcineurin dephosphorylates NFAT transcription factors, resulting in their nuclear translocation (Buitrago, M., et al, Nat Med 11, 837-844 (2005); Molkentin, J.D.,

Cardiovasc Res 63, 467-475 (2004)). The inventors analyzed the localization of NFAT c4 using immunocytochemistry in 50 cTNT -positive cardiomyocytes derived from the L2-iPS10 cell line, which produced the largest cardiomyocytes, and wt S3-iPS4. The inventors observed a significantly higher proportion of LS cardiomyocytes with nuclear NFATc4, (-80% versus -30%, respectively) (Fig. 3c and 3d).

EXAMPLE 3: MUTATIONS IN THE PTPN11 GENE PERTURBS RAS-MAPK SIGNAL

TRANSDUCTION IN PLURIPOTENT STEM CELLS.

[00196] In order to identify potential molecular targets that could be affected by the T468M PTPN11 mutation, protein extracts from LS-iPS, wt BJ-iPSB5 and HES2 cells were analyzed using a phosphoproteomic microarray chip containing approximately 600 pan and phospho-specific antibodies (Kinexus Bioinformatics Corporation). The inventors established eight groups for comparison, each of the LS-iPSC lines versus one control cell line, either HES2 or wt-iPSC. Proteins with a 1.5 fold change were filtered, and those that were conserved in most of the groups were represented in a heat map (Fig. 4a). Some of the proteins were more abundantly present in LS-iPS when compared to either HES2 (Tyro 10, Tyk2 and Haspin) or wt- iPSC (p-MARCKs, p-Synapsinl, p-NMDAR2B, p-MSKl, p-RSKl/3 and p-p53). The phosphorylation of other proteins was increased (p-Caveolin2, p-MEKl, p-EGFR and p-FAK) or decreased (p-Vinculin, p-S6 and p-Lck) in LS-iPSC when compared to control cell lines. In order to eliminate false positives, the inventors verified the phosphoproteomic results by Western blot (WB) for three of the most altered proteins (p-S6, p-EGFR and p-MEKl) in four LS-iPSC lines, in comparison to wt-iPSC. While the inventors did not confirm a major change in the phosphorylation status of S6 protein (data not shown), WB confirmed that the phosphorylation of EGFR and MEK1 proteins was considerably increased in the LS-iPSC samples (Fig. 4b).

[00197] RAS-MAPK represents the major signaling pathway deregulated by SHP2 mutants. NS mutants increase basal and stimulated phosphatase activity, whereas LS mutants are catalytically impaired and have dominant-negative effects, inhibiting growth factor-evoked ERK1/2 activation (Kontaridis, M.I., J Biol Chem 281, 6785-6792 (2006)). The inventors analyzed the ability of LS-iPSC to respond to external growth factors. The inventors used bFGF (basic Fibroblast Growth Factor), the main growth factor in the maintenance of HESC, to induce the stimulation of the MAPK signaling pathway. bFGF treatment increased the phosphorylation of ERK1/2 (p-ERK) levels over time in HES2 and wt S3-iPS4 cells (Fig. 4c). Although the LS- iPSC expressed the four FGF receptor (FGFR) family members (Fig. 16a), bFGF stimulation did not cause any substantial change in p-ERK levels (Fig. 4c and Figs. 16b- 16c). However, the LS- iPSC lines had higher basal p-ERK levels compared to HES2 and S3-iPS4 cells (Figs. 16b- 16c), in accordance with the increased pMEKl, ERK upstream kinase, levels found in LS-iPSC samples in phosphoproteomic array results.

[00198] In summary, the inventors have generated and characterized LS patient- specific iPSC, providing a new system for the study of disease pathogenesis. The inventors observed increases in cell size, sarcomeric organization and nuclear NFATc4 localization in LS- iPSC-derived cardiomyocytes, when compared to HESC and wt-iPSC-derived cardiomyocytes. These results are consistent with cardiac hypertrophy, a condition commonly found in LS patients (Sarkozy, A. et al., Orphanet J Rare Dis 3, 13 (2008)), and indicate that this abnormality occurs through a cell autonomous mechanism due to the PTPN11 mutation. Since many human cell types, such as cardiomyocytes, cannot be propagated readily in cell culture, iPS-derived cells exhibiting disease-relevant phenotypes provide the requisite resource for precisely elucidating pathogenesis and pursuing novel therapeutic strategies.

[00199] In the studies described herein, the inventors provided insights into the molecular events that are affected by the PTPN11 mutation in the pluripotent iPSC using antibody microarrays. The inventors found that the phosphorylation of certain proteins was increased in LS-iPSC when compared to wild-type HESC and iPSC. Interestingly, one of the more upregulated phosphoproteins was MEK1, the upstream kinase of ERK1/2, whose gene is sometimes mutated in the related disorder, cardiofaciocutaneous syndrome. PTPN11 mutations underlie 45% and 90% of NS and LS, respectively. It is not well understood how mutations that provoke opposite effects on SHP2 phosphatase activity cause syndromes with similar features (Edouard, T., et al, Cell Mol Life Sci 64, 1585-1590 (2007)). In concordance with observations in the Drosophila LS model (Oishi, K., et al, Hum Mol Genet (2008)), basal p-ERK levels were increased in LS-iPSC. Of note, receptor tyrosine kinase stimulation with bFGF in LS-iPSC failed to elicit further activation of ERK, as previously observed in a different cellular model

(Kontaridis, ML, et al, J Biol Chem 281, 6785-6792 (2006)). Interestingly, this result demonstrates for the first time that RAS-MAPK signal transduction is perturbed in LS as early as the pluripotent stem cell stage.

[00200] Taken together, this is the first described human model of an inherited RAS pathway disorder.

REFERENCES

[00201] 1. Gorlin, R. J., Anderson, R.C. & Moller, J.H. The Leopard (multiple lentigines) syndrome revisited. Birth Defects Orig Artie Ser 07, 110-115 (1971). [00202] 2. Sarkozy, A., Digilio, M.C. & Dallapiccola, B. Leopard syndrome.

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[00204] 4. Tartaglia, M., et al. Genetic evidence for lineage-related and

differentiation stage-related contribution of somatic PTPNl 1 mutations to leukemogenesis in childhood acute leukemia. Blood 104, 307-313 (2004).

[00205] 5. Tartaglia, M., et al. Diversity and functional consequences of germline and somatic PTPNl 1 mutations in human disease. Am J Hum Genet 78, 279-290 (2006).

[00206] 6. Jopling, C, van Geemen, D. & den Hertog, J. Shp2 knockdown and Noonan/LEOPARD mutant Shp2-induced gastrulation defects. PLoS Genet 3, e225 (2007).

[00207] 7. Oishi, K., et al. Phosphatase-defective LEOPARD syndrome mutations in PTPNl 1 have gain-of- function effects during Drosophila development. Hum Mol Genet (2008).

[00208] 8. Lowry, W.E., et al. Generation of human induced pluripotent stem cells from dermal fibroblasts. Proc Natl Acad Sci USA 105, 2883-2888 (2008).

[00209] 9. Park, I.H., et al. Disease-specific induced pluripotent stem cells. Cell 134, 877-886 (2008).

[00210] 10. Takahashi, K., et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861-872 (2007).

[00211] 11. Ebert, A.D., et al. Induced pluripotent stem cells from a spinal muscular atrophy patient. Nature (2008).

[00212] 12. Lee, G., et al. Modelling pathogenesis and treatment of familial dysautonomia using patient-specific iPSCs. Nature 461, 402-406 (2009).

[00213] 13. Raya, A et al. Disease-corrected haematopoietic progenitors from Fanconi anaemia induced pluripotent stem cells. Nature 460, 53-59 (2009). [00214] 14. Ye, Z et al. Human induced pluripotent stem cells from blood cells of healthy donors and patients with acquired blood disorders. Blood (2009).

[00215] 15. Dimos, J.T., et al. Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science 321, 1218-1221 (2008).

[00216] 16. Laux, D., Kratz, C. & Sauerbrey, A. Common acute lymphoblastic leukemia in a girl with genetically confirmed LEOPARD syndrome. J Pediatr Hematol Oncol 30, 602-604 (2008).

[00217] 17. Ucar, C, Calyskan, U., Martini, S. & Heinritz, W. Acute myelomonocytic leukemia in a boy with LEOPARD syndrome (PTPN11 gene mutation positive). J Pediatr Hematol Oncol 28, 123-125 (2006).

[00218] 18. Mikkola, H.K., Fujiwara, Y., Schlaeger, T.M., Traver, D. & Orkin, S.H. Expression of CD41 marks the initiation of definitive hematopoiesis in the mouse embryo. Blood 101, 508-516 (2003).

[00219] 19. Wu, C.J., et al. Evidence for ineffective erythropoiesis in severe sickle cell disease. Blood 106, 3639-3645 (2005).

[00220] 20. Fan, S.T. & Edgington, T.S. Coupling of the adhesive receptor

CD 1 lb/CD 18 to functional enhancement of effector macrophage tissue factor response. J Clin Invest 87, 50-57 (1991).

[00221] 21. Aoki, FL, Sadoshima, J. & Izumo, S. Myosin light chain kinase mediates sarcomere organization during cardiac hypertrophy in vitro. Nat Med 6, 183-188 (2000).

[00222] 22. Buitrago, M., et al. The transcriptional repressor Nabl is a specific regulator of pathological cardiac hypertrophy. Nat Med 11, 837-844 (2005).

[00223] 23. Yang, L., et al. Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population. Nature 453, 524-528 (2008). [00224] 24. Molkentin, J.D. Calcineurin-NFAT signaling regulates the cardiac hypertrophic response in coordination with the MAPKs. Cardiovasc Res 63, 467-475 (2004).

[00225] 25. Kontaridis, ML, Swanson, K.D., David, F.S., Barford, D. & Neel, B.G. PTPN11 (Shp2) mutations in LEOPARD syndrome have dominant negative, not activating, effects. J Biol Chem 281, 6785-6792 (2006).

[00226] 26. Edouard, T., et al. How do Shp2 mutations that oppositely influence its biochemical activity result in syndromes with overlapping symptoms? Cell Mol Life Sci 64, 1585-1590 (2007).

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[00233] The specification is most thoroughly understood in light of the teachings of the references cited within the specification. The embodiments within the specification provide an illustration of embodiments of the invention and should not be construed to limit the scope of the invention. The skilled artisan readily recognizes that many other embodiments are encompassed by the invention. All publications and patents cited in this disclosure are incorporated by reference in their entirety. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material. The citation of any references herein is not an admission that such references are prior art to the present invention.

[00234] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following embodiments.

Claims

An isolated primate pluripotent stem cell derived from a somatic cell, wherein said stem cell comprises a mutation in the protein tyrosine phosphatase non-receptor type 11 (PTPNl 1) gene.

The stem cell of claim 1, wherein said PTPNl 1 gene comprises a mutation associated with a cardiac anomaly.

The stem cell of claim 2, wherein said cardiac anomaly is hypertrophic cardiomyopathy.

The stem cell of claim 1, wherein said PTPNl 1 gene comprises a mutation associated with LEOPARD Syndrome, Noonan Syndrome or leukemia.

The stem cell of any one of claims 1-4, wherein the mutation is a deletion mutation, a substitution mutation, an addition mutation, or a combination thereof, in the coding region of said PTPNl 1 gene.

The stem cell of any one of claims 1-5, wherein the mutation in said PTPNl 1 gene results in a mutation in the encoded PTPNl 1 protein.

The stem cell of claim 6, wherein the mutation is a threonine to methionine substitution in the encoded PTPNl 1 protein at an amino acid residue position corresponding to position 468 of SEQ ID NO: 1, or a tyrosine to cysteine substitution in the encoded PTPNl 1 protein at an amino acid residue position corresponding to position 279 of SEQ ID NO: 1.

The stem cell of claim 7, wherein the encoded protein comprises SEQ ID NO: 1 and the mutation is a threonine to methionine substitution at amino acid residue position 468 of SEQ ID NO: l, or a tyrosine to cysteine substitution at amino acid residue position 279 of SEQ ID NO: l .

The stem cell of claim 6, wherein the mutation is selected from the group consisting of a tyrosine to cysteine substitution in the encoded PTPNl 1 protein at an amino acid residue position corresponding to position 279 of SEQ ID NO: 1, a tyrosine to serine substitution in the encoded PTPNl 1 protein at an amino acid residue position corresponding to position 279 of SEQ ID NO: 1, an alanine to threonine substitution in the encoded PTPNl 1 protein at an amino acid residue position corresponding to position 461 of SEQ ID NO: 1, a glycine to alanine substitution in the encoded PTPNl 1 protein at an amino acid residue position corresponding to position 464 of SEQ ID NO: 1, a threonine to methionine substitution in the encoded PTPNl 1 protein at an amino acid residue position corresponding to position 468 of SEQ ID NO: 1, a threonine to proline substitution in the encoded PTPNl 1 protein at an amino acid residue position corresponding to position 468 of SEQ ID NO: 1, an arginine to tryptophan substitution in the encoded PTPNl 1 protein at an amino acid residue position corresponding to position 498 of SEQ ID NO: 1, an arginine to leucine substitution in the encoded PTPNl 1 protein at an amino acid residue position corresponding to position 498 of SEQ ID NO: 1, a glutamine to proline substitution in the encoded PTPNl 1 protein at an amino acid residue position

corresponding to position 506 of SEQ ID NO: 1, a glutamine to proline substitution in the encoded PTPNl 1 protein at an amino acid residue position corresponding to position 510 of SEQ ID NO:l, a glutamine to glutamic acid substitution in the encoded PTPNl 1 protein at an amino acid residue position corresponding to position 510 of SEQ ID NO: l, and a glutamine to glycine substitution in the encoded PTPNl 1 protein at an amino acid residue position corresponding to position 510 of SEQ ID NO: 1.

10. The stem cell of any one of claims 1-9, wherein the cell forms a teratoma in an

immunocompromised mouse.

11. The stem cell of any one of claims 1-10, wherein the cell expresses SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, Nanog, OCT-4, Sox2, GDF3, DPPA4, REX1, TERT, Alkaline Phosphatase, Telomerase, or CD30, or a combination thereof.

12. The stem cell of any one of claims 1-11, wherein the stem cell is capable of

differentiating into a cardiomyocyte.

13. A cell culture of the stem cell of any one of claims 1-12.

14. The stem cell of claim 1 deposited at the American Type Culture Collection having ATCC Accession No. .

15. A cardiomyocyte comprising a mutation in the PTPN11 gene, wherein the cardiomyocyte is produced by differentiating the stem cell of any one of claims 1-12.

16. A cell culture of the cardiomyocyte of claim 15.

17. The cardiomyocyte of claim 15 deposited at the American Type Culture Collection

having ATCC Accession No. .

18. The stem cell of any one of claims 1-12, wherein the primate is human.

19. The stem cell of any one of claims 1-12, wherein the somatic cell is a fibroblast.

20. A method of identifying an agent for use in treatment of a disease that is associated with a mutation in the PTPN11 gene, comprising: contacting the pluripotent stem cell of any one of claims 1-12 with the agent; and determining the effect of the agent on survival, proliferation, phenotype, or differentiation of the pluripotent stem cell.

21. A method of identifying an agent for use in treatment of a disease that is associated with a mutation in the PTPN11 gene, comprising: contacting the cardiomyocyte of claim 15 with the agent; and determining the effect of the agent on survival, proliferation, or phenotype of the cardiomyocyte.

22. A method of identifying an agent for use in treatment of the cardiac hypertrophy that is associated with a mutation in the PTPN11 gene, comprising: contacting the cardiomyocyte of claim 15 with the agent; and determining the effect of the agent on the hypertrophic state of the cardiomyocyte, wherein a decrease in the hypertrophic state of the cardiomyocyte, as compared to that of a control cell, is indicative that the agent is an agent for use in treatment of a disease that is associated with a mutation in the PTPN11 gene.

23. A method of producing a pluripotent stem cell of any one of claims 1-12, comprising:

(a) obtaining a somatic cell from a donor whose genome comprises a mutation in the PTPN11 gene;

(b) introducing into the somatic cell: (i) a nucleic acid molecule that encodes a pluripotency-inducing protein; or (ii) a polypeptide that comprises a pluripotency- inducing protein; wherein said pluripotency-inducing protein is Oct3/4, Sox2, Klf4, Nanog, Lin28, c-Myc, or a combination thereof.

24. The method of claim 23, wherein one or more nucleic acid molecules that encode Oct3/4, Sox2, Klf4, and c-Myc are introduced into the somatic cell.

25. The methods of claims 23 or 24, wherein the somatic cell is a fibroblast.

26. A method of differentiating a pluripotent stem cell of any one of claims 1-12 into a

cardiomyocyte, comprising: culturing the stem cell in a serum- free medium that comprises: activin A, bone morphogenetic protein 4 (BMP4), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and dickkopf homo log 1 (DK 1).

27. A method of identifying an agent as a candidate Ras-signaling pathway inhibitor,

comprising: contacting the pluripotent stem cell of any one of claims 1-12 with the agent; and determining the effect of the agent on tyrosine phosphorylation of EGFR or MEK1; wherein a decrease of tyrosine phosphorylation of EGFR or MEK1, as compared to that of a control cell, is indicative that the agent is a Ras-signaling pathway inhibitor.

28. A method of identifying an agent as a candidate Ras-signaling pathway inhibitor, comprising: contacting the cardiomyocyte of claim 15 with the agent; and determining the effect of the agent on tyrosine phosphorylation of EGFR or MEKl; wherein a decrease of tyrosine phosphorylation of EGFR or MEKl, as compared to that of a control cell, is indicative that the agent is a Ras-signaling pathway inhibitor.

29. A method of identifying an agent for use in treatment of a disease that is associated with a mutation in the PTPN11 gene, comprising: contacting the pluripotent stem cell of any one of claims 1-12 with the agent; differentiating the stem cell into a cardiomyocyte; and determining the hypertrophic state of the cardiomyocyte, wherein a decrease in the hypertrophic state of the cardiomyocyte, as compared to that of a control cell, is indicative that the agent is an agent for use in treatment of a disease that is associated with a mutation in the PTPN11 gene.

30. The stem cell of any one of claims 1-11, wherein the stem cell is capable of

differentiating into a hematopoietic cell.

31. A hematopoietic cell comprising a mutation in the PTPN11 gene, wherein the

hematopoietic cell is produced by differentiating the stem cell of any one of claims 1-11 or 30.

32. A cell culture of the hematopoietic cell of claim 31.

33. The hematopoietic cell of claim 31 deposited at the American Type Culture Collection having ATCC Accession No. .

34. A method of identifying an agent for use in treatment of a disease that is associated with a mutation in the PTPN11 gene, comprising: contacting the pluripotent stem cell of any one of claims 1-11 or 30 with the agent; differentiating the stem cell into a hematopoietic cell; and determining the effect of the agent on survival, proliferation, phenotype, or differentiation of the hematopoietic cell.

35. The method of claim 34, wherein the disease is leukemia.

36. A method of identifying an agent for use in treatment of leukemia, comprising: contacting the pluripotent stem cell of any one of claims 1-11 or 30 with the agent; differentiating the stem cell into a hematopoietic cell; and determining the proliferative state of the hematopoietic cell, wherein a decrease in the proliferative state of the hematopoietic cell, as compared to that of a control cell, is indicative that the agent is an agent for use in treatment of leukemia.