Assay for drug discovery based on in vitro differentiated cells
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.
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.