Engineered Cellular Pathways for Programmed Autoregulation of Differentiation

Abstract

The present invention provides compositions and methods for programming mammalian cells to perform desired functions. In particular, the present invention provides compositions and methods for programming stem cells to differentiate into a desired cell type. A quorum sensing systems that regulates the expression of cell fate regulators is introduced into mammalian host cells, such as stem cells. The quorum sensing systems generally comprises vectors that express the components of a bacterial quorum sensing pathway, including proteins which catalyze the synthesis of an autoinducer and a gene encoding a regulatory partner of the autoinducer, and vectors in which genes encoding cell fate regulators are operably linked to a promoter induced by the autoinducer/regulatory partner complex. The system can also comprise vectors in which genes encoding additional cell fate regulators are operably linked to a promoter that is induced by a factor synthesized in response to a first stage of differentiation, so that a second stage of differentiation is triggered.

1 - 77 . (canceled) 
     
     
         78 . A composition comprising one or more mammalian vectors that comprise:
 a) a first nucleic acid sequence capable of producing a first cell fate regulator protein that is capable of inducing differentiation of a first cell type into a second cell type that expresses a protein marker, and   b) a second nucleic acid sequence capable of producing a second cell fate regulator protein that is operably linked to a cell type specific promoter of said second cell type, and that is capable of inducing differentiation of said second cell type into a third cell type   
     
     
         79 . The composition of  claim 78 , wherein said one or more vectors further comprises
 c) a third nucleic acid sequence capable of producing a third cell fate regulator protein that is operably linked to a cell type specific promoter of said second cell type, and that is capable of inducing differentiation of said second cell type into said third cell type.   
     
     
         80 . The composition of  claim 78 , wherein said composition is comprised in a mammalian cell, wherein said vectors are exogenous to said mammalian cell. 
     
     
         81 . The composition of  claim 78 , wherein said first cell fate regulator protein is selected from the group consisting of Sox17, Gata4, Gata6, Pdx1, Ngn3, Nkx6.1, Nkx2.2, Fgf4, BRA, Wnt9, NCAD, CER, FoxA2, CxcR4, Hnf1B, Hnf4A, Hnf6, HlxB9, Pax4, Cgc, GHRL, SST, PPY, Activin, Fgf10, Cyc, RA, Ex4, DAPT, HGF and Igf1. 
     
     
         82 . The composition of  claim 79 , wherein one or more of said second cell fate regulator protein and said third cell fate regulator protein is selected from the group consisting of Pdx1, Ngn3, Nkx6.1, Nkx2.2, Fgf4, BRA, Wnt9, NCAD, CER, FoxA2, CxcR4, Hnf1B, Hnf4A, Hnf6, HlxB9, Pax4, Cgc, GHRL, SST, PPY, Activin, Fgf10, Cyc, RA, Ex4, DAPT, HGF and Igf1. 
     
     
         83 . The composition of  claim 78 , wherein one or more of said first cell type and said second cell type is an epiblast-like stem cell (ELSC) and said protein marker is selected from the group consisting of stage-specific embryonic antigen-4, stage-specific embryonic antigen-1, stage-specific embryonic antigen-3, and carcinoembryonic antigen cell adhesion molecule-1. 
     
     
         84 . The composition of  claim 83 , wherein said second cell type is a myoblast cell, and said protein marker is dystrophin. 
     
     
         85 . The composition of  claim 83 , wherein said second cell type is an adipocyte cell, and said protein marker is PPAR(. 
     
     
         86 . The composition of  claim 83 , wherein said second cell type is an endoderm cell, and said protein marker is selected from the group consisting of Hnf3∃, lamininB1, and ∀-fetoprotein (AFP). 
     
     
         87 . The composition of  claim 83 , wherein said second cell type is an ectoderm cell and said protein marker is nestin. 
     
     
         88 . The composition of  claim 78 , wherein said first cell fate regulator protein comprises Gata4. 
     
     
         89 . The composition of  claim 78 , wherein said second cell fate regulator protein comprises Pdx1. 
     
     
         90 . The composition of  claim 78 , wherein said second cell fate regulator protein comprises Ngn3 and Pdx1. 
     
     
         91 - 125 . (canceled)