High-throughput image-based chemical screening in zebrafish blastomere cell culture

Abstract

Disclosed are methods of inducing differentiation of stem into myogenic cells without gene manipulation and for inducing proliferation of satellite cells. The cells can be used as a source of cells for transplantation in a subject in need thereof. Also disclosed is a screening assay for screening test compounds using blastomere cultures.

What is claimed is: 
     
         1 . A composition comprising: at least two of: (i) a GSK3 pathway inhibitor; (ii) a compound that increases intracellular levels of 3′,5′-cyclic adenosine monophosphate (cAMP); and (iii) a FGF pathway activator. 
     
     
         2 . The composition of  claim 1 , wherein the composition further comprises a stem cell. 
     
     
         3 . The composition of  claim 1 , wherein the composition is a cell culture media. 
     
     
         4 . A reagent kit for inducing differentiation from a pluripotent cell to a skeletal muscle progenitor cell, wherein the kit comprises: at least two of: (i) a GSK3 pathway inhibitor; (ii) a compound that increases intracellular levels of 3′,5′-cyclic adenosine monophosphate (cAMP); and (iii) a FGF pathway activator. 
     
     
         5 . The kit of  claim 4 , wherein the kit further comprises a stem cell. 
     
     
         6 . A method of increasing satellite cell proliferation, the method comprising: culturing a satellite cell in the presence of a compound that increases intracellular levels of 3′,5′-cyclic adenosine monophosphate. 
     
     
         7 . The method of  claim 6 , wherein the compound is an activator of adenylyl cyclase. 
     
     
         8 . A method of treating a subject for damaged muscle tissue or for increasing muscle mass, the method comprising co-administering to the subject in need thereof at least two of: (i) a GSK3 pathway inhibitor; (ii) a compound that increases intracellular levels of 3′,5′-cyclic adenosine monophosphate (cAMP); and (iii) a FGF pathway activator. 
     
     
         9 . A screening assay comprising culturing a blastomere in presence of a test compound, wherein the blastomere comprises a reporter gene, wherein the reporter gene encodes a cell lineage specific marker and produces a detectable signal when expressed; and measuring/detecting the detectable signal, wherein a change in level or amount of the detectable signal indicating that the test compound modulates the development or function of cell lineage that express the cell lineage specific marker.