Methods of Reprogramming Animal Somatic Cells

Abstract

This invention generally relates to methods to obtain mammalian cells and tissues with patterns of gene expression similar to that of a developing mammalian embryo or fetus, and the use of such cells and tissues in the treatment of human disease and age-related conditions. More particularly, the invention relates to methods for identifying, expanding in culture, and formulating mammalian pluripotent stem cells and differentiated cells that differ from cells in the adult human in their pattern of gene expression, and therefore offer unique characteristics that provide novel therapeutic strategies in the treatment of degenerative disease.

1 . A method for reprogramming an animal differentiated somatic cell to an undifferentiated stem cell, comprising the steps of:
 (a) injecting one or more animal differentiated somatic cells into an oocyte, wherein said oocyte is from a species that is different from said somatic cell, and allowing the remodeling of the nucleus of said somatic cell, wherein said remodeling results in
 (i) the remodeling of the nuclear envelope of the nucleus of said somatic cell, wherein one or more components of the nuclear envelope of said nucleus are removed and wherein one or more components from said oocyte are added to the nuclear envelope of said nucleus of said somatic cell, and 
 (ii) the reprogramming of the chromatin of said somatic cell resulting in the expression of genes of an undifferentiated cell, wherein said reprogramming of said chromatin comprises the removal of regulatory factors from said chromatin and the addition of regulatory factors from said oocyte to said chromatin; and 
   (b) transferring or fusing the resulting remodeled nucleus from step (a) into the enucleated cytoplasm of an undifferentiated embryonic cell to form an undifferentiated stem cell, and allowing the resulting undifferentiated stem cell to grow.   
     
     
         2 - 51 . (canceled) 
     
     
         52 . A method for in vitro reprogramming a differentiated somatic cell to an undifferentiated stem cell, comprising contacting the differentiated somatic cell in culture with OCT3/4 (POU5F1) protein under conditions in which chromatin remodeling and telomere elongation occurs such that the somatic cell becomes an undifferentiated stem cell. 
     
     
         53 . The method of  claim 52 , wherein the differentiated somatic cell is selected from the group consisting of fibroblasts, keratinocytes, lymphocytes, monocytes, epithelial cells and hematopoietic cells. 
     
     
         54 . The method of  claim 52 , wherein the differentiated somatic cell is a fibroblast. 
     
     
         55 . The method of  claim 52 , wherein the differentiated somatic cell is permeabilized before being contacted with the OCT3/4 protein in culture. 
     
     
         56 . The method of  claim 52 , wherein the differentiated somatic cell is further contacted in culture with one or more proteins selected from the group consisting of SOX2, NANOG, cMYC, DNMT3B, and embryonic histones. 
     
     
         57 . The method of  claim 52 , wherein the differentiated somatic cell is further contacted in culture with one or more proteins selected from the group consisting of SOX2, NANOG, DNMT3B, CROC4, H2AFX, HIST1H2AB, HIST1H4J, HMGB2, LEFTB, MYBL2, MYC, MYCN, NANOG, OTX2, SALL4, TERF1, TERT and ZNF206. 
     
     
         58 . The method of  claim 52 , wherein said undifferentiated stem cell is a pluripotent cell. 
     
     
         59 . The method of  claim 58 , further comprising differentiating said pluripotent cell. 
     
     
         60 . The method of  claim 59 , wherein said pluripotent cell is differentiated into a cell type selected from the group consisting of: cartilage, bone, skeletal muscle, cardiac muscle, renal, hepatic, blood, blood forming, vascular precursor, vascular endothelial, pancreatic beta, neuron, glia, retinal, inner ear follicle, intestinal, and lung.