초록

Provided are assay systems for determining the therapeutic or toxic effect of a putative drug based on assaying its activity in cells which have been differentiated in vitro from stem cells, and induced to display a phenotype that resembles a disease to be treated.

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1. An in vitro screening method to identify a candidate drug substance capable of ameliorating hypertrophic cardiomyopathy, comprising: (i) providing a cardiomyocyte, the cardiomyocyte having been obtained by differentiating a multipotent or pluripotent stem cell in vitro;(ii) inducing a cardiac hyp

1. An in vitro screening method to identify a candidate drug substance capable of ameliorating hypertrophic cardiomyopathy, comprising: (i) providing a cardiomyocyte, the cardiomyocyte having been obtained by differentiating a multipotent or pluripotent stem cell in vitro;(ii) inducing a cardiac hypertrophic phenotype in said cardiomyocyte, wherein the cardiac hypertrophic phenotype is selected from the group consisting of: increased cell size, increased protein synthesis, increased sarcomeric organization, activation of gene expression patterns characteristic of cardiomyopathic cells, and activation of ANF and/or BNP expression;(iii) contacting the cardiomyocyte displaying the cardiac hypertrophic phenotype with a drug substance; and(iv) determining a responsive change in the cardiac hypertrophic phenotype of the cardiomyocyte;wherein the responsive change is a decrease in or loss of the cardiac hypertrophic phenotype and said decrease or loss identifies the drug substance as a candidate drug capable of ameliorating hypertrophic cardiomyopathy. 2. The method of claim 1, wherein the cardiac hypertrophic phenotype is activation of gene expression patterns characteristic of cardiomyopathic cells. 3. The method of claim 1, wherein the cardiac hypertrophic phenotype is activation of ANF and/or BNP expression. 4. The method of claim 1, wherein the inducing step comprises a nucleic acid encoding a calcineurin operably linked to a constitutive promoter, wherein said calcineurin is constitutively expressed in said cardiomyocyte. 5. The method of claim 1, wherein the pluripotent stem cell is an embryonic stem cell. 6. The method of claim 1, wherein inducing comprises contacting the cardiomyocyte with a hypertrophic agonist. 7. The method of claim 6, wherein the hypertrophic agonist is selected from the group consisting of endothelin, phenylephrine, angiotensin, and alpha-1-adrenergic agonist. 8. The method of claim 6, wherein the cardiomyocyte further comprises a selectable marker operably linked to a cell-type specific regulatory sequence. 9. The method of claim 8, wherein the selectable marker gene confers resistance to puromycin, streptomycin, neomycin, gentamycin, hygromycin, aminopterine, methotrexate, vinblastin, doxorubicin, or actinomycin D. 10. The method of claim 9, wherein the selectable marker gene confers resistance to puromycin. 11. The method of claim 10, wherein the cardiomyocyte further comprises a reporter gene operably linked to a cell type specific regulatory sequence. 12. An in vitro screening method to identify the toxicity of a candidate drug substance demonstrated to ameliorate hypertrophic cardiomyopathy, comprising: (i) providing a cardiomyocyte, the cardiomyocyte having been obtained by differentiating a multipotent or pluripotent stem cell in vitro;(ii) inducing a cardiac hypertrophic phenotype in said cardiomyocyte, wherein the cardiac hypertrophic phenotype is selected from the group consisting of: increased cell size, increased protein synthesis, increased sarcomeric organization, activation of gene expression patterns characteristic of cardiomyopathic cells, and activation of ANF and/or BNP expression;(iii) contacting the cardiomyocyte displaying the cardiac hypertrophic phenotype with a drug substance demonstrated to ameliorate hypertrophic cardiomyopathy; and(iv) determining a responsive change in the cardiac hypertrophic phenotype of the cardiomyocyte;wherein the responsive change is enhancement or onset of a cardiomyopathic phenotype and said enhancement or onset identifies the drug substance as a substance that is toxic to cardiomyocytes with hypertrophic cardiomyopathy. 13. The method of claim 12, wherein the cardiac hypertrophic phenotype is increased activation of gene expression patterns characteristic of cardiomyopathic cells. 14. The method of claim 12, wherein the cardiac hypertrophic phenotype is activation of ANF and/or BNP expression. 15. The method of claim 12, wherein the inducing step comprises a nucleic acid encoding a calcineurin operably linked to a constitutive promoter, wherein said calcineurin is constitutively expressed in said cardiomyocyte. 16. The method of claim 12, wherein the pluripotent stem cell is an embryonic stem cell. 17. The method of claim 12, wherein inducing comprises contacting the cardiomyocyte with a hypertrophic agonist. 18. The method of claim 17, wherein the hypertrophic agonist is selected from the group consisting of endothelin, phenylephrine, angiotensin, and alpha-1-adrenergic agonist. 19. The method of claim 17, wherein the cardiomyocyte further comprises a selectable marker operably linked to a cell-type specific regulatory sequence. 20. The method of claim 19, wherein the selectable marker gene confers resistance to puromycin, streptomycin, neomycin, gentamycin, hygromycin, aminopterine, methotrexate, vinblastin, doxorubicin, or actinomycin D. 21. The method of claim 20, wherein the selectable marker gene confers resistance to puromycin. 22. The method of claim 21, wherein the cardiomyocyte further comprises a reporter gene operably linked to a cell type specific regulatory sequence.