ASSAY FOR DRUG DISCOVERY BASED ON IN VITRO DIFFERENTIATED CELLS

1 - 60 . (canceled)

61 . An in vitro method for screening, identifying, or determining the toxicity of, a candidate drug substance capable of ameliorating hypertrophic cardiomyopathy, comprising:

(i) contacting a cardiomyocyte with a drug substance to be screened; and

(ii) determining a responsive change in the cardiac hypertrophic phenotype of the cardiomyocyte;

wherein the cardiomyocyte was derived from a pluripotent stem cell in vitro and displays a cardiac hypertrophic phenotype selected from the group consisting of cell size, cell shape, protein synthesis, organization of actin/myosin filament, activation of gene expression pattern characteristic of cardiomyopathic cells, and activation of genes expressed during early embryonic development; and

wherein a responsive change preventing or delaying the onset or the progression of the cardiac hypertrophic phenotype is indicative of a useful drug substance, or a responsive change enhancing the onset or progression of the cardiac hypertrophic phenotype is indicative of the toxicity of the drug substance.

62 . The method of claim 61 , wherein the cardiac hypertrophic phenotype is selected from the group consisting of cell size, cell shape, gene activation, and protein synthesis.

63 . The method of claim 61 , wherein the cardiac hypertrophic phenotype is selected from the group consisting of cell size, cell shape, protein synthesis, and organization of actin/myosin filament.

64 . The method of claim 61 , wherein the displayed cardiac hypertrophic phenotype relates to the expression of at least one gene selected from the group consisting of atrial natriuretic factor (ANF), b-type natriuretic peptide (BNP), β-myosin heavy chain gene, an α-skeletal actin gene, cardiac troponin T (cTnT), Nkx-2.5, c-FOS, e-JUN, c-MYC, early growth response genes, heat shock protein 70, alpha-myosin heavy chain, collagen III, preproendothelin-1, myosin light chain 2, Na + /H + exchanger, cardiac alpha-actin, Na + /Ca 2+ exchanger, phosphatidylinositol-3 receptor, angiotensin-converting enzyme, collagen I, collagen XV, sarcoplasmic reticulum Ca-ATPase-2 alpha, beta-adrenoreceptor, protein kinase C, and phospholamban.

65 . The method of claim 64 , wherein said at least one gene is mutated.

66 . The method of claim 64 , wherein said at least one gene is overexpressed.

67 . The method of claim 64 , wherein said one or more genes is up-regulated inducing the cardiomyocyte to display the cardiac hypertrophic phenotype.

68 . The method of claim 64 , wherein said one or more genes is down-regulated inducing the cardiomyocyte to display the cardiac hypertrophic phenotype.

69 . The method of claim 61 , wherein the displayed cardiac hypertrophic phenotype relates to the activity of at least one gene product selected from the group consisting of atrial natriuretic factor (ANF), b-type natriuretic peptide (BNP), β-myosin heavy chain gene, an α-skeletal actin gene, cardiac troponin T (cTnT), Nkx-2.5, c-FOS, e-JUN, c-MYC, early growth response genes, heat shock protein 70, alpha-myosin heavy chain, collagen III, preproendothelin-1, myosin light chain 2, Na + /H + exchanger, cardiac alpha-actin, Na + /Ca 2+ exchanger, phosphatidylinositol-3 receptor, angiotensin-converting enzyme, collagen I, collagen XV, sarcoplasmic reticulum Ca-ATPase-2 alpha, beta-adrenoreceptor, protein kinase C, and phospholamban.

70 . The method of claim 61 , wherein the pluripotent stem cell is an embryonic stem cell.

71 . The method of claim 61 , wherein obtaining the cardiomyocyte to display a cardiac hypertrophic phenotype comprises differentiating and/or inducing the pluripotent stem cell in vitro.

72 . The method of claim 61 , wherein inducing comprises contacting the pluripotent stem cell with a hypertrophic agonist.

73 . The method of claim 72 , wherein the hypertrophic agonist is selected from the group consisting of endothelin, phenylephrine, and alpha-1-adrenergic agonist.

74 . The method of claim 72 , wherein the hypertrophic agonist comprises a nucleotide sequence comprising a regulatory sequence of a gene expressed in cardiac tissue.

75 . The method of claim 72 , wherein the hypertrophic agonist comprises a nucleotide sequence comprising a coding sequence of a gene expressed in cardiac tissue.

76 . The method of claim 75 , wherein the nucleotide sequence encodes a mutant or wild type protein selected from the group consisting of troponin, myosin heavy chain, calcineurin, calmodulin, protein kinase C, phospholamban, beta-myosin heavy chain, regulatory myosin light chain, alpha-tropomyosin, and calcium calmodulin dependent kinase IV (CaMKIV).

77 . The method of claim 71 , wherein inducing the cardiomyocyte to display a cardiac hypertrophic phenotype comprises the expression of a nucleotide sequence encoding a mutant or wild type protein selected from the group consisting of troponin, heavy myosin chain, calcineurin, calmodulin, protein kinase C, phospholamban, and calcium calmodulin dependent kinase IV (CaMKIV).

78 . The method of claim 71 , wherein the cardiomyocyte further comprises a selectable marker operably linked to a cell type specific regulatory sequence.

79 . The method of claim 78 , wherein the selectable marker gene confers resistance to puromycin, streptomycin, neomycin, gentamycin, hygromycin, aminopterine, methotrexate, vinbiastin, doxorubicin, or actinomycin D.

80 . The method of claim 79 , wherein the selectable marker gene confers resistance to puromycin.

81 . The method of claim 80 , wherein the cardiomyocyte further comprises a reporter gene operably linked to a cell-type specific regulatory sequence.

82 . The method of claim 81 , wherein said cell-type specific regulatory sequence is atrial and/or ventricular specific.

83 . The method of claim 71 , wherein the pluripotent stem cell is derived from a patient suffering from cardiac hypertrophy disease.