Assay for drug discovery based on in vitro differentiated cells

Abstract

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 - 60 . (canceled) 
     
     
         61 . An in vitro method for screening and identifying a candidate substance capable of ameliorating hypertrophic cardiomyopathy comprising:
 (a) differentiating a pluripotent stem cell to a cardiomyocyte in vitro;   (b) inducing the cardiomyocyte to display a first cardiac hypertrophic phenotype;
 (i) wherein the inducing results in the cardiomyocyte having an increased size, protein production and/or sarcomeric assembly relative to a normal cardiomyocyte; and/or 
 (ii) wherein the inducing results in the cardiomyocyte expressing atrial natriuretic factor (ANF) and/or brain natriuretic protein (BNP) genes or their products in an amount greater than an normal cardiomyocyte; 
   (c) contacting a test sample comprising the in vitro differentiated cardiomyocyte with a substance to be tested prior, during or after said cardiomyocyte is induced to display the cardiac hypertrophic phenotype;   (d) measuring a second cardiac hypertrophic phenotype in the cardiomyocyte of step (c);   (e) comparing the measurement obtained in step (d) to that of a cardiomyocyte not subjected to the substance to be tested;   wherein the candidate substance is identified on the basis of whether the measurement of the second cardiac hypertrophic phenotype in the cardiomyocyte of step (d) is consistent with a reduction in hypertrophic cardiomyopathy; and   wherein the inducing in step (b) is the result of the expression of a gene which is present in the pluripotent stem cell.   
     
     
         62 . The method of  claim 61  wherein the expression of the gene is altered by the differentiation in step (a). 
     
     
         63 . The method of  claim 62  wherein the gene is up-regulated by the differentiation in step (a). 
     
     
         64 . The method of  claim 62  wherein the gene is down-regulated by the differentiation in step (a). 
     
     
         65 . The method of  claim 61  wherein, the gene is a transgene. 
     
     
         66 . The method of  claim 65  wherein the transgene encodes a constitutively active protein. 
     
     
         67 . The method of  claim 66  wherein the constitutively active protein is a constitutively active calcineurin. 
     
     
         68 . The method of  claim 61  wherein the gene encodes a wild type protein. 
     
     
         69 . The method of  claim 61  wherein the gene encodes a mutant protein. 
     
     
         70 . The method of  claim 61  wherein the first cardiac hypertrophic phenotype and the second cardiac hypertrophic phenotype are the same. 
     
     
         71 . The method of  claim 61  wherein the first cardiac hypertrophic phenotype and the second cardiac hypertrophic phenotype are different. 
     
     
         72 . An in vitro method for screening and identifying a candidate substance capable of ameliorating hypertrophic cardiomyopathy comprising:
 (a) obtaining a pluripotent stern cell comprising a gene capable of inducing a cardiomyocyte to display a first cardiac hypertrophic phenotype;   (b) differentiating the pluripotent stem cell in vitro to obtain an in vitro differentiated cardiomyocyte capable of displaying the first cardiac hypertrophic phenotype;
 (i) wherein the in vitro differentiated cardiomyocyte has an increased size, protein production and/or sarcomeric assembly relative to the same cardiomyocyte lacking the gene; and/or 
 (ii) wherein the in vitro differentiated cardiomyocyte expresses atrial natriuretic factor (ANF) and/or brain natriuretic protein (BNP) genes or their products in an amount greater than the same cardiomyocyte lacking the gene; 
   (c) contacting a test sample comprising the in vitro differentiated cardiomyocyte with a substance to be tested prior, during or after said in vitro differentiated cardiomyocyte displays the cardiac hypertrophic phenotype;   (d) measuring a second cardiac hypertrophic phenotype in the in vitro differentiated cardiomyocyte of step (c);   (e) comparing the measurement obtained in step (d) to that of a cardiomyocyte not subjected to the substance to be tested;   wherein the candidate substance is identified on the basis of whether the measurement of the second cardiac hypertrophic phenotype in the in vitro differentiated cardiomyocyte of step (d) is consistent with a reduction in hypertrophic cardiomyopathy.   
     
     
         73 . The method of  claim 72  wherein the gene is up-regulated by the differentiation in step (b). 
     
     
         74 . The method of  claim 72  wherein the gene is down-regulated by the differentiation in step (b). 
     
     
         75 . The method of  claim 72  wherein, the gene is a transgene. 
     
     
         76 . The method of  claim 75  wherein the transgene encodes a constitutively active protein. 
     
     
         77 . The method of  claim 76  wherein the constitutively active protein is a constitutively active calcineurin. 
     
     
         78 . The method of  claim 75  wherein the transgene encodes a wild type. 
     
     
         79 . The method of  claim 72  wherein the gene encodes a mutant protein. 
     
     
         80 . The method of  claim 72  wherein the gene encodes a wild type protein. 
     
     
         81 . The method of  claim 72  wherein the first cardiac hypertrophic phenotype and the second cardiac hypertrophic phenotype are the same. 
     
     
         82 . The method of  claim 72  wherein the first cardiac hypertrophic phenotype and the second cardiac hypertrophic phenotype are different