WO2011130217A1 - Cellules souches pluripotentes induites et leurs utilisations - Google Patents

Cellules souches pluripotentes induites et leurs utilisations Download PDF

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WO2011130217A1
WO2011130217A1 PCT/US2011/032044 US2011032044W WO2011130217A1 WO 2011130217 A1 WO2011130217 A1 WO 2011130217A1 US 2011032044 W US2011032044 W US 2011032044W WO 2011130217 A1 WO2011130217 A1 WO 2011130217A1
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cell
mutation
agent
stem cell
ptpnl
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Xonia Carvajal
Bruce D. Gelb
Ihor Lemischka
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Mount Sinai School Of Medicine
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0657—Cardiomyocytes; Heart cells
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/603—Oct-3/4
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/604—Klf-4
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/606—Transcription factors c-Myc
    • C—CHEMISTRY; METALLURGY
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells

Definitions

  • 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)).
  • 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.
  • 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)).
  • iPS cells 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
  • 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
  • Yu et al. Science, 318:1917-20
  • WO2008/0233610 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.
  • 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.
  • Noonan syndrome 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.
  • NS is a relatively common syndrome with an estimated incidence of 1 : 1000 to 1 :2500 live births.
  • PTPNl 1 non-receptor type 11
  • SHP2, Syp, SHPTP2, PTP2C, PTP1D or BPTP3 wild-type PTPNl 1 protein
  • PTP2C protein tyrosine phosphatase oncogenes
  • BPTP3 protein tyrosine kinase oncogenes
  • 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
  • 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).
  • PTP protein-tyrosine phosphatases
  • PTK protein-tyrosine kinases
  • 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.
  • Noonan syndrome e.g., heart defects, undescended testicles, or excessively short stature
  • LEOPARD syndrome e.g., cryptorchidism, hypospadias, or severe skeletal deformity
  • no therapeutic treatment of the underlying disorder has been determined so far.
  • 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.
  • 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.
  • PTPNl 1 protein tyrosine phosphatase non-receptor type 11
  • the PTPNl 1 gene comprises a mutation associated with a cardiac anomaly.
  • the cardiac anomaly is hypertrophic cardiomyopathy.
  • the PTPNl 1 gene comprises a mutation associated with LEOPARD Syndrome, Noonan Syndrome or leukemia.
  • 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.
  • 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 .
  • 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
  • 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.
  • the pluripotent stem cell forms a teratoma in an immunocompromised mouse.
  • 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 Phosphatase, Telomerase, or CD30, or a combination thereof.
  • the pluripotent stem cell is capable of differentiating into a cardiomyocyte.
  • stem cell culture of the stem cells described herein exemplary stem cell lines were deposited on , 2010 with American Type Culture Collection
  • 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.
  • the invention provides a cell culture of a
  • cardiomyocyte as described herein.
  • Exemplary cardiomyocyte cell lines were deposited on
  • the primate is human.
  • the somatic cell is a fibroblast.
  • 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.
  • 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.
  • 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.
  • 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.
  • one or more nucleic acid molecules that encode Oct3/4, Sox2, Klf4, and c-Myc are introduced into the somatic cell.
  • the somatic cell is a fibroblast.
  • 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).
  • BMP4 bone morphogenetic protein 4
  • bFGF basic fibroblast growth factor
  • VEGF vascular endothelial growth factor
  • DK dickkopf homo log 1
  • 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.
  • 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.
  • 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.
  • the stem cells of the invention are capable of differentiating into a hematopoietic cell.
  • 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.
  • methods of identifying an agent for use in treatment of a disease that is associated with a mutation in the PTPN11 gene 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.
  • the disease is leukemia.
  • methods of identifying an agent for use in treatment of leukemia 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.
  • 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 ⁇ -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.
  • Figs. 2a-2b demonstrate that LS-iPSC can differentiate in vitro and in vivo into all three germ layers.
  • 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 ⁇ -tubulin-positive cell. Scale bar, 100 ⁇ .
  • HES2 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.
  • 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).
  • 3d Nuclear localization of NFATc protein in a cTNT -positive cell from L2-iPS10 is shown.
  • 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 ⁇ -Actin.
  • 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
  • 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.
  • 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.
  • 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.
  • Figs. 7a-7b show the result ⁇ 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.
  • 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).
  • 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 ⁇ -ACTIN and plotted relative to the expression levels in transfected GP2 cells.
  • 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.
  • 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.
  • 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.
  • the position of SOX2, LIN28 and OCT4 transcription factors is indicated.
  • 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 ⁇ .
  • 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 ⁇ . 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.
  • 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,
  • 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.
  • Figs. 16a- 16c show the disruption of MAPK activation upon bFGF
  • 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.
  • Fig. 17 is a table showing the primer sets that were used for PCRs.
  • 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.
  • Fig. 19 shows the distribution of PTPN11 (SHP-2) mutations and their relative prevalence in Noonan syndrome.
  • Fig. 20 shows the cDNA sequence of human PTPN11 (SEQ ID NO: 2).
  • Fig. 21 shows the amino acid sequence of human PTPN11 (SEQ ID NO: 1). DETAILED DESCRIPTION OF THE INVENTION 1. OVERVIEW
  • 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.
  • 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.
  • LS-iPSC LS-specific induced pluripotent stem cell
  • 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.
  • HESC human embryonic stem cells
  • iPSCs derived from an unaffected brother i.e., not having the PTPN11 mutation
  • 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.
  • 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.
  • 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.
  • 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.
  • the reprogrammed cells express all these markers.
  • 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.
  • PTPN11 protein also known as SHP2, Syp, SHPTP2, PTP2C, PTP1D or BPTP3
  • SHP2, Syp SHPTP2, PTP2C, PTP1D or BPTP3
  • SH2-domain-containing protein tyrosine phosphatases that control cellular proliferation and differentiation
  • 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).
  • 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).
  • 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • “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.
  • arginine, histidine and lysine are hydrophilic- basic amino acids and may be interchangeable.
  • 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.
  • 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.
  • 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.
  • the variant gene encodes a protein that has the same or substantially similar properties or functions as the native or parent PTPN11 protein.
  • 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.
  • the variant protein has the same or substantially similar properties or functions as the native or parent PTPN11 protein.
  • 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.
  • the mutation in the PTPN11 gene results in a mutation in the encoded PTPN11 protein.
  • the stem cells described herein comprise a mutation in the PTPN11 gene that is associated with Noonan Syndrome.
  • NS Noonan syndrome
  • 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
  • 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).
  • 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).
  • 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.
  • S502T Ser502-to-Thr
  • Y63C Tyr63-to-Cys
  • Y62D Tyr62-to-Asp
  • Tartaglia et al. 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.
  • A-to-G mutation at nucleotide 182 in exon 3 Asp61-to-Gly, D61G
  • 218C-T mutation in exon 3 Thr73-to-Ile, T73I
  • Fig. 19 shows the distribution of PTPNl 1 (SHP-2) mutations and their relative prevalence in Noonan syndrome
  • the stem cells described herein comprise a mutation in the PTPNl 1 gene that is associated with LEOPARD Syndrome.
  • 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.
  • 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
  • PTPNl 1 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.
  • MIM 163955 cardiofaciocutaneous syndrome
  • NIM 607785 juvenile myelomonocytic leukemia
  • certain other malignancies include, e.g., cardiofaciocutaneous syndrome (MIM 163955), juvenile myelomonocytic leukemia (NIM 607785), and certain other malignancies.
  • the stem cells described herein comprise a mutation in the PTPNl 1 gene that is associated with leukemia.
  • JMML Juvenile myelomonocytic leukemia
  • MDS myelodysplasia syndrome
  • JMML is observed occasionally in patients with Noonan syndrome.
  • Tartaglia et al. found heterozygosity with respect to a mutation in exon 3 of PTPNl 1.
  • Four of the children shared the same mutation (218C-T).
  • missense defects in PTPNl 1 the 218C-T transition, and a defect in exon 13 affecting the protein tyrosine phosphatase domain.
  • 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.
  • 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).
  • 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.
  • the stem cells described herein comprise a mutation in the PTPNl 1 gene that is associated with a cardiac anomaly.
  • 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.
  • CFC cardiofaciocutaneous syndrome
  • Noonan syndrome namely, congenital heart defects, cutaneous abnormalities, Noonan-like facial features, and severe psychomotor developmental delay.
  • T411M Thr411 -to-Met
  • the stem cells described herein comprise a mutation in the PTPNl 1 gene that is associated with cardiac hypertrophy (also known as hypertrophic cardiomyopathy) .
  • 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.
  • 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.
  • 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.
  • 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.
  • the disease-specific pluripotent stem cells may be characterized by any of several criteria of pluripotency known in the art.
  • 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).
  • 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.
  • 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.
  • Stage Specific Embryonic Antigens 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.
  • markers that are not exhibited on the surface of a cell may be used to characterize a pluripotent stem cell.
  • the homeodomain transcription factor Oct 4 also termed Oct-3 or Oct3/4 is frequently used as a marker for pluripotent stem cells.
  • 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.
  • 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.
  • somatic cell lineages e.g., neurons, cardiomyocytes, etc
  • 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.
  • 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).
  • ATCC Type Culture Collection
  • P.O. Box 1549 Manassas, VA 20108, USA, under ATCC Accession No. and .
  • the pluripotent stem cell of the present invention is a primate stem cell.
  • the primate is human.
  • 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).
  • 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
  • Studies of the differentiated cells in culture could provide important information regarding the molecular and cellular nature of events leading to pathology.
  • this approach is used to develop an in vitro model of LEOPARD syndrome and cardiac hypertrophy.
  • 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.
  • 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).
  • somatic cell lineages e.g., neurons, cardiomyocytes, etc.
  • 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.
  • the disease-specific pluripotent stem cells may be subjected to any conditions that induce the stem cells to differentiate into cardiomyocytes.
  • 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.
  • 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.
  • BMP4 bone morphogenetic protein 4
  • bFGF basic fibroblast growth factor
  • VEGF vascular endothelial growth factor
  • DK 1 dickkopf homo log 1
  • 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).
  • BMP4 bone morphogenetic protein 4
  • bFGF basic fibroblast growth factor
  • VEGF vascular endothelial growth factor
  • DKK1 dickkopf homolog 1
  • 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), ⁇ - ⁇ , non-essential amino acids, L-glutamine, etc.), as described in U.S. Application Publication No. 20070204351.
  • 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), ⁇ - ⁇ , non-essential amino acids, L-
  • 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,
  • 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.
  • 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.
  • a cardiomyocyte differentiated from a stem cell comprises a mutation in the PTPN11 gene.
  • the mutation is associated with a cardiac anomaly (such as hypertrophic cardiomyopathy).
  • the mutation is associated with LEOPARD Syndrome, Noonan Syndrome or leukemia.
  • 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.
  • the invention provides a cell culture of the cardiomyocytes described herein. Exemplary cardiomyocyte cell lines were deposited on
  • the invention provides methods of producing the disease-specific pluripotent stem cells as described herein.
  • 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.
  • 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.
  • 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).
  • 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
  • the pluripotency-inducing proteins are referred to as "nuclear reprogramming factors" by Yamanaka, and “potency-determining factors” by Thomson.
  • 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.
  • 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.
  • a set of two or more pluripotency-inducing proteins are introduced into the somatic cells.
  • the set include Oct-4 and Sox2.
  • the set include Oct3/4, Sox2, Klf4, and c-Myc.
  • the set include Oct3/4, Klf and c-Myc.
  • the set include OCT4, SOX2, NANOG, and LIN28.
  • the set of pluripotency-inducing proteins sufficient to reprogram somatic cells can vary with the cell type of the somatic cells.
  • Suitable somatic cells can be any somatic cell, although higher
  • Somatic cells useful in the invention may be non-embryonic cells obtained from a fetal, newborn, juvenile or adult primate, including a human.
  • 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.
  • 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.
  • blood stem cells multipotent hematopoietic cells
  • muscle/bone stem cells muscle/bone stem cells
  • brain stem cells brain stem cells
  • liver stem cells etc.
  • the somatic cell is a fibroblast.
  • 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).
  • 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).
  • a retroviral vectors e.g., a lentiviral vector
  • 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).
  • 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.
  • the vectors may include a transcription promoter and a polyadenylation signal operatively linked, upstream and downstream, respectively, to the DNA segment.
  • 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.
  • 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)).
  • EBV Epstein-Barr virus
  • 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.
  • a non-lethal marker such as Green Fluorescent Protein (GFP), Enhanced Green Fluorescent Protein (EGFP) or luciferase
  • GFP Green Fluorescent Protein
  • EGFP Enhanced Green Fluorescent Protein
  • luciferase luciferase
  • 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.
  • the reprogrammed cells can be produced without a selectable marker.
  • 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.
  • 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.
  • 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.
  • 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).
  • pluripotency-inducing proteins 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.
  • 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.
  • 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 TeSRTM (StemCell Technologies, Inc.; Vancouver, Canada), mTeSRTM (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.
  • cells can be maintained on MEFs in culture medium. 5. DISEASE MODELING AND DRUG SCREENING
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • the agents can be used for treatment, preventing or ameliorating the symptoms of the diseases.
  • 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.
  • 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.
  • 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).
  • the agent e.g., recipient cells mock-treated with buffers
  • an agent having a known effect e.g., a positive effect, a negative effect, or no effect.
  • 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).
  • 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.
  • 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.
  • the cells are particularly useful in studying and/or modeling diseases that have not been amenable to such study and/or modeling.
  • the test agent may be any molecule that increases or decreases the expression level or the activity level of PTPNl 1 protein.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • iPSC reprogrammed induced pluripotent stem cells
  • 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
  • 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.
  • HESC human embryonic stem cells
  • wt wild type iPSC derived from an unaffected, non-PTPNl lbrother of one of the LS patients.
  • DMEM Dulbecco's modified Eagle medium
  • 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 ⁇ -mercaptoethanol ( ⁇ - ⁇ , Sigma-Aldrich).
  • MEFs irradiated Swiss Webster mouse embryonic feeder cells
  • 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.
  • 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.
  • pMXs-EGFP vector was used to estimate the infection efficiency (data not shown).
  • GP-2 cells were plated at 8xl0 6 cells per 10-cm dish and transfected with pMXs, VSV-G and Gag-Pol vectors using SuperFect transfection reagent on the following day.
  • human fibroblasts were seeded at 8xl0 5 cells per 10-cm dish.
  • the four retroviruses hOCT4, hSOX2, hKLF-4 and hC- MYC
  • 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.
  • Genomic DNA was isolated with Easy-DNATM kit (Invitrogen). Fifty nanograms of genomic DNA was used per reaction. Primers are summarized in Figure 17 (Table 1).
  • HES2 and iPSC were grown on Matrigel-coated glass coverslip dishes (MatTek, Ma). The day of culture harvest, 20 ⁇ of colcemid (5 ⁇ g/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.
  • 2cc of hypotonic solution sodium citrate solution 0.86%
  • fixative methanol: glacial acetic acid; 3: 1
  • 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).
  • qPCR and transgenes integration were performed 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
  • 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), ⁇ -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.
  • 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.
  • 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.
  • MMG monothioglycerol
  • DMEM dimethyl methacrylate
  • 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.).
  • cardiomyocyte induction the inventors used a well-established protocol (Yang, L., et al, Nature 453, 524-528 (2008)).
  • 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.
  • RMA Robust Multi- Array
  • 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 ⁇ 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.
  • 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.
  • 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 ⁇ - glycerophosphate, 20 mM sodium phyrophospate and 1% Triton X-100.
  • Protease and phosphatase inhibitors (1 mM phenylmethylsulfonyl fluoride, 3 mM benzamidine, 10 ⁇ g/ml aprotinin, 10 ⁇ leupeptin, 5 ⁇ pepstatin, ImM dithiothreitol and 1 mM sodium
  • PTPN11 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)).
  • 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.
  • 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.
  • 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).
  • 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 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).
  • 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).
  • 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.
  • 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)).
  • EBs floating embryoid body
  • a-smooth muscle actin a- SMA, mesoderm), desmin (mesoderm), a-fetoprotein (AFP, endoderm), vimentin (mesoderm), glial fibrillary acidic protein (GFAP, ectoderm) and ⁇ -tubulin (ectoderm) markers (Fig. 2a and Fig. 12).
  • a- SMA smooth muscle actin
  • desmin mesoderm
  • AFP a-fetoprotein
  • vimentin mesoderm
  • GFAP glial fibrillary acidic protein
  • ectoderm glial fibrillary acidic protein
  • ⁇ -tubulin ectoderm
  • mesenchyme mesenchyme, adipose tissue and cartilage (mesoderm) (Fig. 2b and data not shown).
  • hypertrophic cardiomyopathy is one of the major features of LS, affecting 80% of the patients.
  • 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).
  • CD41 + early hematopoietic progenitors
  • CD71 + /CD235a + early erythroblasts
  • macrophages CD1 lb +
  • 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);
  • 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.,
  • 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).
  • 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).
  • HES2 Tyro 10, Tyk2 and Haspin
  • 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.
  • WB Western blot
  • 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 basic Fibroblast Growth Factor
  • bFGF treatment increased the phosphorylation of ERK1/2 (p-ERK) levels over time in HES2 and wt S3-iPS4 cells (Fig. 4c).
  • p-ERK ERK1/2
  • FGFR FGF receptor family members
  • bFGF stimulation did not cause any substantial change in p-ERK levels (Fig. 4c and Figs. 16b- 16c).
  • 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.
  • 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.
  • 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.
  • 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.
  • CD 1 lb/CD 18 to functional enhancement of effector macrophage tissue factor response. J Clin Invest 87, 50-57 (1991).

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Abstract

Cette invention concerne d'une manière générale des cellules souches pluripotentes spécifiques d'une maladie qui sont dérivées à partir de cellules somatiques qui portent une mutation dans le gène PTPNl 1. Les cellules souches sont capables de se différencier en cellules ou tissus affectés par la mutation PTPNl 1. Les cellules souches et les cellules différenciées peuvent être utilisées pour étudier diverses maladies qui sont associées à des mutations dans le gène PTPNl 1 et pour identifier de nouveaux agents thérapeutiques pour le diagnostic et le traitement de maladies qui sont associées à des mutations dans PTPNl 1.
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WO2015115690A1 (fr) * 2014-01-28 2015-08-06 한국과학기술원 Cellules souches pluripotentes induites constituant un modèle du syndrome de noonan et leur utilisation
WO2021213479A1 (fr) * 2020-04-22 2021-10-28 复星凯特生物科技有限公司 Protéine mutante inactivatrice spécifique de shp2 et son application dans la thérapie par car-t

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WO2021213479A1 (fr) * 2020-04-22 2021-10-28 复星凯特生物科技有限公司 Protéine mutante inactivatrice spécifique de shp2 et son application dans la thérapie par car-t

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