Reprogramming of cells to a new fate

Abstract

The present invention generally provides methods and compositions for transdifferentiation of an animal cell from a first non-pluripotent cell fate to a second non-pluripotent cell fate. Also provided are methods and compositions for the transdifferentiation of an animal cell from a non-pluripotent mesodermal, endodermal, or ectodermal cell fate to a different non-pluripotent mesodermal, endodermal, or ectodermal cell fate.

1 - 48 . (canceled) 
     
     
         49 . A composition for efficiently transdifferentiating an animal cell from a first non-pluripotent cell fate to a second non-pluripotent cell fate, the composition comprising:
 (a) a non-pluripotent intermediate cell obtained by a method of
 (i) introducing a polynucleotide encoding an Oct4 polypeptide, and optionally one or more reprogramming factors comprising polynucleotides encoding a Klf polypeptide, a Sox2 polypeptide, or a c-Myc polypeptide; or 
 contacting cells having a first non-pluripotent cell fate with an Oct4 polypeptide, and optionally one or more reprogramming factors comprising a Klf polypeptide, a Sox2 polypeptide, or a c-Myc polypeptide; and 
 (ii) limiting the expression of endogenous Nanog in the cells from step (i) to a level substantially lower than the level of expression of endogenous Nanog in an induced pluripotent cell (iPSC), thereby generating a non-pluripotent intermediate cell; and 
   (b) one or more of a growth factor, a cytokine, and/or a small molecule, wherein the one or more of a growth factor, a cytokine, and/or a small molecular are present in amounts effective to induce differentiation of the non-pluripotent intermediate cell into a cell having a second non-pluripotent cell fate.   
     
     
         50 . The composition of  claim 49 , wherein the composition further comprises a chemically defined medium. 
     
     
         51 . The composition of  claim 50 , wherein the chemically defined medium is serum-free. 
     
     
         52 . The composition of  claim 49 , wherein step (a)(ii) comprises eliminating exogenous LIF, a JAK inhibitor, a PI3K inhibitor, a CDK2 inhibitor, a CDK4 inhibitor, a glycolysis inhibitor, a Wnt inhibitor, or a combination thereof. 
     
     
         53 . The composition of  claim 49 , wherein the first non-pluripotent cell is a fibroblast or a neural precursor cell. 
     
     
         54 . The composition of  claim 49 , wherein the second non-pluripotent cell fate is a cardiomyocyte. 
     
     
         55 . The composition of  claim 54 , wherein the one or more of a growth factor, a cytokine, and/or a small molecule that induces differentiation comprises a GSK-3 inhibitor, a calcium channel agonist, a Gas activating agent, a cAMP analog, and/or a BMP protein. 
     
     
         56 . The composition of  claim 55 , wherein the non-pluripotent intermediate cell from step (a)(ii) is contacted with a JAK inhibitor. 
     
     
         57 . The composition of  claim 49 , wherein the second non-pluripotent cell fate is a neural progenitor cell. 
     
     
         58 . The composition of  claim 57 , wherein the one or more of a growth factor, a cytokine, and/or a small molecule that induces differentiation comprises FGF2, FGF4, and EGF. 
     
     
         59 . The composition of  claim 58 , wherein the non-pluripotent intermediate cell from step (a)(ii) is contacted with a Wnt inhibitor, a JAK inhibitor, or both a Wnt inhibitor and a JAK inhibitor. 
     
     
         60 . The composition of  claim 49 , wherein the second non-pluripotent cell fate is an induced definitive endoderm (iDE) cell. 
     
     
         61 . The composition of  claim 60 , wherein the one or more of a growth factor, a cytokine, and/or a small molecule that induces differentiation comprises a GSK-3 inhibitor, an HDAC inhibitor, and/or a TGFβ/Activin/Nodal family member. 
     
     
         62 . The composition of  claim 49 , wherein the second non-pluripotent cell fate is a pancreatic beta cell. 
     
     
         63 . The composition of  claim 62 , wherein the one or more of a growth factor, a cytokine, and/or a small molecule that induces differentiation comprises:
 (a) GSK-3 inhibitor, an HDAC inhibitor, and/or a TGFβ/Activin/Nodal family member;   (b) FGF7, retinoic acid (RA), a Hedgehog pathway inhibitor, a BMP inhibitor, and/or a TGFβ/ALK5 inhibitor;   (c) EGF and/or a Notch inhibitor; and   (d) bFGF, nicotinamide, and/or extendin-4.