Wnt pathway stimulation in reprogramming somatic cells with nuclear reprogramming factors

Abstract

The invention provides compositions and methods of use in reprogramming somatic cells. Compositions and methods of the invention are of use, e.g., for generating or modulating (e.g., enhancing) generation of induced pluripotent stem cells by reprogramming somatic cells. The reprogrammed somatic cells are useful for a number of purposes, including treating or preventing a medical condition in an individual. The invention further provides methods for identifying an agent that reprograms somatic cells to a pluripotent state and/or enhances the speed and/or efficiency of reprogramming. Certain of the compositions and methods relate to modulating the Wnt pathway.

1 - 65 . (canceled) 
     
     
         66 . A method of reprogramming a mammalian somatic cell, comprising:
 (a) contacting a somatic cell with a Wnt-conditioned medium or an agent that modulates the Wnt pathway; and   (b) culturing the somatic cell under conditions suitable for reprogramming the somatic cell to a pluripotent state;   wherein the somatic cell over-expresses Oct4, and optionally over-expresses one or more reprogramming factors selected from the group consisting of: Klf4 and Sox2; and   wherein the agent that modulates the Wnt pathway is a recombinant, exogenous, soluble, and biologically active Wnt protein or a GSK-3 inhibitor.   
     
     
         67 . The method of  claim 66 , wherein the somatic cell is not genetically modified to express c-Myc at levels greater than normally present in somatic cells of that type. 
     
     
         68 . The method of  claim 66 , wherein the method comprises: (a) culturing the cell in culture medium containing the agent; (b) culturing the cell in culture medium comprising the Wnt-3a conditioned medium or the agent for at least 10 days; (c) contacting the cell with the Wnt-3a conditioned medium or the agent that modulates the Wnt pathway, thereby enhancing the number of ES-like cell colonies by at least 5-fold; (d) contacting the cell with the Wnt-3a conditioned medium or the agent that modulates the Wnt pathway, thereby enhancing the number of ES-like cell colonies by at least 10-fold; (e) culturing the cell in the Wnt3a-conditioned medium; (f) contacting the cell with a second agent that modulates the Wnt pathway; or (g) culturing the cell in medium containing the recombinant, exogenous, soluble, and biologically active Wnt protein. 
     
     
         69 . The method of  claim 68 , wherein the recombinant, exogenous, soluble, and biologically active Wnt protein is Wnt3a. 
     
     
         70 . The method of  claim 66 , wherein the cell: (a) is a human cell; (b) is a terminally differentiated cell; (c) is a fibroblast; or (d) is modified to express or contain at least one reprogramming factor at levels greater than normally present in cells of that type. 
     
     
         71 . The method of  claim 66 , further comprising: (a) confirming that the reprogrammed cell is pluripotent; (b) administering the reprogrammed cell to a subject; or (c) differentiating the cell to a desired cell type in vitro after reprogramming the cell. 
     
     
         72 . The method of  claim 66 , wherein the method is practiced on: (a) a population of cells and the method further comprises identifying ES-like cells by morphological criteria; (b) a population of cells and the method does not comprise imposing chemical selection to select reprogrammed cells; or (c) a population of cells and the method further comprises separating cells that are reprogrammed to a pluripotent state from cells that are not reprogrammed to a pluripotent state. 
     
     
         73 . A composition comprising:
 (a) a somatic cell over-expressing Oct4, and optionally over-expressing one or more reprogramming factors selected from the group consisting of: Klf4, and Sox2; and   (b) a Wnt-3a conditioned medium; or an agent that increases activity of a Wnt pathway selected from the group consisting of a recombinant, exogenous, soluble, and biologically active Wnt protein, and a GSK-3 antagonist.   
     
     
         74 . The composition of  claim 73 , wherein the somatic cell is not genetically modified to express c-Myc at levels greater than normally present in somatic cells of that type. 
     
     
         75 . The composition of  claim 73 , wherein the recombinant, exogenous, soluble, and biologically active Wnt protein is Wnt3a. 
     
     
         76 . A composition comprising differentiated cells of a desired cell type derived from iPS cells, wherein the iPS cells are comprised in a composition comprising (i) a Wnt-3a conditioned medium; or (ii) an agent that increases activity of a Wnt pathway selected from the group consisting of a recombinant, exogenous, soluble, and biologically active Wnt protein, and a GSK-3 antagonist. 
     
     
         77 . A therapeutic composition comprising differentiated cells according to  claim 76 . 
     
     
         78 . A therapeutic composition comprising iPS cells, and
 (i) a Wnt-3a conditioned medium; or   (ii) an agent that increases activity of a Wnt pathway selected from the group consisting of a recombinant, exogenous, soluble, and biologically active Wnt protein, and a GSK-3 antagonist.   
     
     
         79 . Therapeutic use of the therapeutic composition of  claim 78  by administering the composition to a subject suitable for such treatment. 
     
     
         80 . The therapeutic use of  claim 79 , wherein the cells comprised in the therapeutic composition are allogenic or autogenic. 
     
     
         81 . The therapeutic use of  claim 79 , wherein the subject is in need of bone marrow transplantation, or has neurological, endocrine, structural, skeletal, vascular, urinary, digestive, integumentary, blood, immune, auto-immune, inflammatory, endocrine, kidney, bladder, cardiovascular, cancer, circulatory, digestive, hematopoietic, or muscular disease, disorder, and condition. 
     
     
         82 . A method of manufacturing therapeutic cells, wherein the method comprises:
 (a) contacting a somatic cell with a Wnt-conditioned medium or an agent that modulates the Wnt pathway; and   (b) culturing the somatic cell under conditions suitable for reprogramming the somatic cell to a pluripotent state; and optionally,   (c) differentiating the cell at the pluripotent state from step (b) to a desired differentiated cell type;   wherein the somatic cell of step (a) over-expresses Oct4, and optionally over-expresses one or more reprogramming factor selected from the group consisting of: Klf4 and Sox2; and   wherein the agent that modulates the Wnt pathway is a recombinant, exogenous, soluble, and biologically active Wnt protein or a GSK-3 inhibitor.   
     
     
         83 . The method of  claim 82 , wherein the somatic cell of step (a) is not genetically modified to express c-Myc at levels greater than normally present in somatic cells of that type. 
     
     
         84 . The method of  claim 82 , wherein the somatic cell of step (a): (i) is a human cell; (ii) is a terminally differentiated cell; (iii) is a fibroblast; or (iv) is modified to express or contain at least one reprogramming factor at levels greater than normally present in cells of that type. 
     
     
         85 . The method of  claim 82 , wherein the recombinant, exogenous, soluble, and biologically active Wnt protein is Wnt3a.