Methods of modifying transcriptional regulatory networks in stem cells

Abstract

The vast differentiation potential of human embryonic and induced pluripotent stem cells, including their potential to cascade through all of the somatic cell lineages and to display the complete transcriptional regulatory network of human biology, has generated interest in deriving scalable, purified, and identified cell types and methods of discovering the precise structure of the human regulatory network. However, the innate capacity of pluripotent cells to display all these lineages is not necessarily reflected during their culture in vitro. The clonal isolation and propagation of progenitors greatly facilitates the generation of highly purified and indentified formulations for research and therapeutic purposes. Nevertheless, other cell types have yet to be isolated and propagated from normal cells and methods of isolating said novel cell types as well as methods for introducing perturbations into the transcriptional regulatory network in order to construct a computer model of the entire human transcriptional regulatory network would greatly benefit basic research as well as manufacturing technology for cell-based therapies.

What is claimed is: 
     
         1 . A method of generating an isolated progenitor cell line comprising:
 modulating the activity of a transcriptional regulator in a pluripotent stem cell; and   inducing the differentiation of said pluripotent stem cell in vitro to generate a progenitor cell line.   
     
     
         2 . The method of  claim 1 , wherein said pluripotent stem cell is human. 
     
     
         3 . The method of  claim 2 , wherein said human pluripotent stem cell is not derived from a human embryo. 
     
     
         4 . The method of  claim 1 , wherein said modulating comprises increasing the activity of said transcriptional regulator in said pluripotent stem cell. 
     
     
         5 . The method of  claim 1 , wherein said pluripotent stem cell result from the reprogramming of a somatic cell that is genetically modified to constitutively overexpress a transcription factor. 
     
     
         6 . The method of  claim 5 , wherein said transcriptional regulator is OCT4. 
     
     
         7 . The method of  claim 1 , wherein said modulating comprises introducing an expression vector encoding said transcriptional regulator into said pluripotent stem cell. 
     
     
         8 . The method of  claim 7 , wherein said expression vector is selected from the group consisting of: constitutive expression vector, inducible expression vector, retroviral vector, lentiviral vector, transient expression vector, and combinations thereof. 
     
     
         9 . The method of  claim 1 , wherein said modulating comprises introducing said transcriptional regulator directly into said pluripotent stem cell. 
     
     
         10 . The method of  claim 9 , wherein said transcriptional regulator is a protein. 
     
     
         11 . The method of  claim 1 , further comprising increasing cell division in derivatives of said pluripotent stem cell. 
     
     
         12 . The method of  claim 11 , wherein said increasing cell division comprises providing a cell cycle regulator that overcomes cell cycle inhibition in said pluripotent stem cell. 
     
     
         13 . The method of  claim 12 , wherein said cell cycle regulator is selected from the group consisting of: p53, SV40 T antigen, adenovirus proteins E1A and E1B, papillomavirus proteins E6 and/or E7, CDK4, and combinations thereof. 
     
     
         14 . The method of  claim 1 , wherein said isolated progenitor cell line constitutively expresses a transcriptional regulator. 
     
     
         15 . The method of  claim 14 , wherein said transcriptional regulator is selected from the group consisting of: OCT4, SIX1, FOXA1, SOX17, SIX2, SOX2, SOX21, PAX6, MYOD1, MYOG, NEUROG1, NKX2.5, and LHX8. 
     
     
         16 . The method of  claim 1 , wherein said pluripotent stem cell is selected from the group consisting of: ES cell, somatic cell reprogrammed to pluripotent cell including an iPS cell, ED cell, EG cell, and EC cell. 
     
     
         17 . The method of  claim 1 , wherein said progenitor cell line is selected from the group consisting of: endodermal cells, mesodermal cells, ectodermal cells, neuroglial precursor cells, hepatic cells or hepatic precursor cells, chondrocyte or chondrocyte precursor cells, myocardial or myocardial precursor cells, gingival fibroblast or gingival fibroblast precursor cells, pancreatic beta cells or pancreatic beta precursor cells, retinal precursor cells, hemangioblasts, dermal fibroblasts, 
     
     
         18 . An isolated progenitor cell line, wherein said isolated progenitor cell line constitutively expresses a transcription factor selected from the group consisting of: OCT4, SIX1, FOXA1, SOX17, SIX2, SOX2, SOX21, PAX6, MYOD1, MYOG, NEUROG1, NKX2.5, LHX8, and combinations thereof. 
     
     
         19 . The isolated progenitor cell line of  claim 18 , wherein said progenitor cell line is 14SKEL12Z. 
     
     
         20 . An isolated progenitor cell line, wherein said progenitor cell line is selected from the group consisting of: 14SKEL7X, 14SKEL18X, 14SKEL12Z, 14SKEL14Z, 14SKEL15Z, 14SKEL20Z, 14SKEL24Z, 14PEND2X, 14PEND11X, 14PEND12X, 14PEND14X, 14PEND20X, 14PEND23X, 14PEND24X, 14SMOO2X, 14SMOO8X, and 14PEND17Z.