Compositions and Methods Useful for Culturing Differentiable Cells

Abstract

The present invention relates to cell culture methods and compositions that are essentially serum-free and comprise a basal salt nutrient solution and an ErbB3 ligand.

1 - 34 . (canceled) 
     
     
         35 . A method for passaging single pluripotent cells said method comprising:
 a) obtaining a population of pluripotent cells; and   b) disassociating the pluripotent cells to single cells in a medium comprising at least one growth factor and an agent capable of disassociating pluripotent cells to single cells.   
     
     
         36 . The method of  claim 35 , wherein the at least one growth factor is selected from the group consisting of an ErbB3 ligand or a functional fragment thereof; a TGF-β family member or a functional fragment thereof; an activator of insulin-like growth factor receptor (IGF-1R) or a functional fragment thereof; an activator of a fibroblast growth factor (FGF) receptor or a functional fragment thereof; and combinations thereof. 
     
     
         37 . The method of  claim 36 , wherein the ErbB3 ligand is selected from the group consisting of Neuregulin-β, Heregulin-β(HRG-β), Heregulin-α (HRG-α), Neu differentiation factor (NDF), acetylcholine receptor-inducing activity (ARIA), glial growth factor 2 (GGF2), motor-neuron derived factor (SMDF), Neuregulin-2, Neuregulin-2β (NRG2-β), Epiregulin, Biregulin and functional fragments thereof. 
     
     
         38 . The method of  claim 37 , wherein the ErbB3 ligand is HRG-β or a functional fragment thereof. 
     
     
         39 . The method of  claim 35 , wherein the medium is free of exogenous insulin and insulin substitutes. 
     
     
         40 . The method of  claim 35 , wherein the medium is free of exogenous fibroblast growth factor. 
     
     
         41 . The method of  claim 36 , wherein the FGF receptor is selected from the group consisting of FGF-2, FGF-7, FGF-10, FGF-22 and variants and functional fragments thereof. 
     
     
         42 . The method of  claim 35 , wherein the pluripotent cells are selected from the group consisting of ES cells, EPL cells, ICM/epiblast cells, EG cells, and pluripotent cells derived by dedifferentiation or nuclear transfer. 
     
     
         43 . The method of  claim 35 , wherein the pluripotent cells are human embryonic stem cells. 
     
     
         44 . The method of  claim 36 , wherein the TGF-β family member is selected from the group consisting of Nodal, Activin A, Activin B, bone morphogenic protein-2 (BMP2), bone morphogenic protein-4 (BMP4), and functional fragments thereof. 
     
     
         45 . The method of  claim 44 , wherein said TGF-β family member is Activin A. 
     
     
         46 . The method of  claim 35 , wherein the agent capable of disassociating the pluripotent cells to single cells is selected from the group consisting of ACCUTASE™, Trypsin, TrypLE, and VERSENE. 
     
     
         47 . The method of  claim 35 , wherein the agent capable of disassociating the pluripotent cells to single cells is a mixture of enzymes with proteolytic, collagenolytic, and DNAse activities. 
     
     
         48 . The method of  claim 35 , wherein the single pluripotent cells are karyotypically normal. 
     
     
         49 . The method of  claim 35 , wherein the pluripotent cells can be passaged at least 2 times. 
     
     
         50 . The method of  claim 35 , wherein the pluripotent cells retain expression of pluripotency markers upon passaging. 
     
     
         51 . The method of  claim 35 , wherein the medium is serum free. 
     
     
         52 . The method of  claim 35 , wherein the medium is activin A free. 
     
     
         53 . A method for passaging single pluripotent cells said method comprising:
 a) obtaining a population of pluripotent cells; and   b) disassociating the pluripotent cells to single cells in a medium comprising a basal salt nutrient solution, an ErbB3 ligand or a functional fragment thereof and an agent capable of disassociating pluripotent cells to single cells.   
     
     
         54 . A method of identifying a compound that modulates proliferation and/or differentiation of pluripotent stem cells, the method comprising:
 culturing the pluripotent stem cells in a cell medium comprising an ErbB3 ligand and a candidate compound;   determining whether an increase or decrease in proliferation and/or differentiation occurs in the pluripotent stem cells cultured with the compound relative to a control, wherein the increase or decrease indicates that the compound modulates proliferation and/or differentiation of the pluripotent stem cells.