Gynogenetic or androgenetic production of pluripotent cells and cell lines, and use thereof to produce differentiated cells and tissues

Abstract

Methods for obtaining pluripotent (embryonic stem) cells from parthenogenetic embryos, especially primates, are provided. These cells are useful for producing differentiated cells, tissues and organs, especially human and non-human primate cells, tissues and organs.

What is claimed is: 
     
         1 . A method for producing pluripotent (ES) cells that can be used to produce differentiated cells and tissues comprising:
 (a) obtaining a haploid cell in metaphase II that comprises DNA derived from a single individual male or female, which optionally may be genetically modified;   (b) activating said haploid cell by a method selected from the group consisting of:
 (1) conditions that do not result in second polar body extrusion; 
 (2) conditions that provide for polar body extrusion but in the presence of an agent that inhibits polar body extrusion; and 
 (3) conditions that prevent the initial cleavage, and culturing said activated cell to produce a gynogenetic or androgenetic embryo comprising a discernible trophectoderm and an inner cell mass; 
   (c) isolating said inner cell mass or cells therefrom and transferring said inner cell mass or cells to an in vitro media that inhibits differentiation of said inner cell mass derived therefrom; and   (d) culturing said inner cell mass cells or cells derived therefrom to maintain said cells in an undifferentiated pluripotent state.   
     
     
         2 . The method of  claim 1 , wherein the metaphase II cell is an oocyte or blastomere. 
     
     
         3 . The method of  claim 2 , wherein the haploid cell is a human, non-human primate, bovine, porcine, or ovine oocyte or blastomere. 
     
     
         4 . The method of  claim 3 , wherein the haploid DNA derived from a single individual is human, bovine, primate, ovine, or porcine. 
     
     
         5 . The method of  claim 4 , wherein the cell is a human or bovine oocyte and the haploid DNA is human DNA. 
     
     
         6 . The method of  claim 1 , where said activation conditions include the use of DMAP (phosphorylation inhibitor) or other compound that inhibits second polar body extrusion. 
     
     
         7 . The method of  claim 1 , wherein activation conditions include use of a compound that inhibits microfilament or protein production. 
     
     
         8 . The method of  claim 7 , wherein said compound is cycloheximide or cytochalasin B. 
     
     
         9 . The method of  claim 1 , wherein the haploid DNA is of a female origin. 
     
     
         10 . The method of  claim 1 , wherein haploid DNA is of male origin. 
     
     
         11 . The method of  claim 1 , wherein the haploid cells are human oocytes containing human male or female DNA. 
     
     
         12 . The method of  claim 1 , wherein said cultured cells of (d) are allowed to differentiate. 
     
     
         13 . The method of  claim 1 , wherein said cells are implanted at a desired site in vivo that is to be engrafted with cells or tissue. 
     
     
         14 . The method of  claim 13  wherein said cells are implanted in an immunocompromised non-human animal. 
     
     
         15 . The method of  claim 13 , wherein said site is a wound, a joint, muscle, bone, or the central nervous system. 
     
     
         16 . The method of  claim 1 , wherein the cell obtained by (d) is genetically modified.