Methods of identifying small molecules for renewal, survival and migration of cardiac progenitors

Abstract

The present invention relates to a small molecule high-throughput screening assay consisting of detectably labeled cardiac progenitor cells. The invention also describes a method of identifying small molecules from the high-throughput assay affecting cardiogenesis and/or modulating cardiac progenitor cell development. Also described are methods of stimulating maturation of cardiac progenitor cells using a GSK-3β inhibitor.

1 . A high-throughput screening assay comprising:
 (a) injecting tamoxifen into a transgenic non-human animal having a tamoxifen-dependent Cre-recombinase in the isl1 locus (isl1-mER-Cre-mER) and an Cre reporter; and   (b) isolating at least one cell fraction from the animal, wherein the cells express a detectably labeled marker.   
     
     
         2 . The assay of  claim 1 , wherein the labeled marker is β-galactosidase. 
     
     
         3 . The assay of  claim 1 , wherein the labeled marker is LacZ. 
     
     
         4 . The assay of  claim 1 , wherein the non-human animal is a rat or mouse. 
     
     
         5 . A method of identifying a small molecule affecting cardiogenesis comprising:
 (a) labeling a cell fraction isolated from a transgenic non-human animal having a tamoxifen-dependent Cre-recombinase in the isl1 locus (isl1-mER-Cre-mER) and an Cre reporter (R16R) with a fluorescent label, wherein the cell fraction contains detectably labeled cardiac progenitor cells;   (b) identifying a test molecule from the cell fraction having increased fluorescence as compared to a control molecule;   (c) contacting the test molecule with the detectably labeled cardiac progenitor cells; and   (d) determining the expression of the isl1+ transcription factor,   thereby identifying a small molecule affecting cardiogenesis.   
     
     
         6 . The method of  claim 5 , wherein the non-human animal is a rat or mouse. 
     
     
         7 . The method of  claim 5 , further comprising detecting expression of one or more growth factors selected from the group consisting of BMP4, BMP7, and FGF10. 
     
     
         8 . A method of identifying a small molecule which regulates cardiogenesis comprising:
 (a) labeling a cell fraction isolated from a transgenic non-human animal having a tamoxifen-dependent Cre-recombinase in the isl1 locus (isl1-mER-Cre-mER) and an Cre reporter with a fluorescent label, wherein the cell fraction contains detectably labeled cardiac progenitor cells;   (b) identifying a test molecule from the cell fraction having increased fluorescence as compared to a control molecule;   (c) contacting the test molecule with the detectably labeled cardiac progenitor cells;   (d) determining the expression of the isl1+ transcription factor; and   (e) identifying a factor which is modulates isl1+ expression,   thereby identifying a small molecule which regulates cardiogenesis.   
     
     
         9 . The method of  claim 8 , wherein the non-human animal is a rat or mouse. 
     
     
         10 . The method of  claim 8 , further comprising detecting expression of one or more growth factors selected from the group consisting of BMP4, BMP7, and FGF10. 
     
     
         11 . A method for stimulating maturation of cardiac progenitor cells comprising contacting the cells with an effective amount of a GSK-3β inhibitor, thereby stimulating maturation of cardiac progenitor cells. 
     
     
         12 . The method of  claim 11 , wherein the inhibitor is 6-bromoindirubin-3′-oxime (BIO). 
     
     
         13 . A method for generating an Isl1 lineage-traced cell comprising contacting an undifferentiated progenitor cell that expresses Isl1 with a GSK-3 inhibitor that activates or enhances expression of Isl1 in the cell, thereby generating a Isl1 lineage-traced cell. 
     
     
         14 . The method of  claim 13 , wherein the inhibitor is 6-bromoindirubin-3′-oxime (BIO). 
     
     
         15 . The method of  claim 13 , wherein the activation or enhancement of Isl1 expression causes the cell to differentiate into a cell selected from the group consisting of cardiomyocyte, endothelial cell, and smooth muscle cell. 
     
     
         16 . The method of  claim 13 , wherein the undifferentiated progenitor cell is selected from the group consisting of embryonic heart muscle cell, post-natal heart muscle cells, embryonic stem cells, progenitors from cord blood, and adult progenitor cells. 
     
     
         17 . The method of  claim 16 , wherein the adult progenitor cells are bone marrow cells or adipose tissue cells. 
     
     
         18 . The method of  claim 16 , wherein the cardiac cell is derived from a rat, mouse or human.