Cytoplasmic transfer to de-differentiate recipient cells

Abstract

Methods for de-differentiating or altering the life-span of desired “recipient” cells, e.g., human somatic cells, by the introduction of cytoplasm from a more primitive, less differentiated cell type, e.g., oocyte or blastomere are provided. These methods can be used to produce embryonic stem cells and to increase the efficiency of gene therapy by allowing for desired cells to be subjected to multiple genetic modifications without becoming senescent. Such cytoplasm may be fractionated and/or subjected to subtractive hybridization and the active materials (sufficient for de-differentiation) identified and produced by recombinant methods.

What is claimed is: 
     
         1 . A method for producing a totipotent cell comprising:
 a) transferring all or part of the cytoplasm of a donor cell into an isolated recipient cell; and   b) transferring a telomerase or a DNA construct that provides for the expression of telomerase into the recipient cell or recipient cell nucleus;   
       wherein the donor cell is at or near senescence. 
     
     
         2 . The method of  claim 1 , wherein the donor cell is less differentiated than the recipient cell or is an undifferentiated cell. 
     
     
         3 . The method of  claim 1 , wherein said donor cell is an oocyte or an embryonic cell. 
     
     
         4 . The method of  claim 1 , wherein the telomerase DNA under the control of a regulatable promoter. 
     
     
         5 . The method of  claim 1 , wherein said recipient cell is a mammalian cell. 
     
     
         6 . The method of  claim 5 , wherein said mammalian cell is derived from a mammal selected from the group consisting of non-human primate, human, rat, guinea pig, mouse, rabbit, dog, cat, hamster, goat, cattle, sheep, horse, bison and buffalo. 
     
     
         7 . The method of  claim 6 , wherein said mammalian cell is selected from the group consisting of cardiac, lung, skin, liver, stomach, intestine, neural, muscle, bone, cartilage, immune, pancreatic, spleen, esophageal, and corneal cells. 
     
     
         8 . The method of  claim 1 , wherein said recipient cell or recipient cell nucleus is genetically modified prior, concurrent or subsequent to the introduction of said cytoplasm. 
     
     
         9 . The method of  claim 10 , wherein (a) said genetically modified cells comprise several genetic modifications; or (b) said genetically modified recipient cell or recipient cell nucleus comprises a recombinant DNA that encodes a desired polypeptide. 
     
     
         10 . The method of  claim 1 , which results in the increased life-span of a mammalian recipient cell or recipient cell nucleus. 
     
     
         11 . The method of  claim 1 , wherein said donor cell is of a different species than the recipient cell. 
     
     
         12 . The method of  claim 11 , wherein said donor cell is a non-human primate oocyte or embryonic cell and the recipient cell is a human somatic cell. 
     
     
         13 . An in vitro method for producing an embryonic stem cell, the method comprising:
 a) providing a nucleus isolated from a cell;   b) transferring part of the cytoplasm of a cytoplasm donor cell into said nucleus;   c) introducing a telomerase or a DNA construct that provides for the expression of telomerase into said nucleus or the cell from which said nucleus is isolated; and   d) introducing said nucleus into a cytoplast.   
     
     
         14 . The method of  claim 13 , wherein the embryonic stem cell has an increased life-span relative to the cell from which said nucleus is isolated, and wherein the donor cell is at or near senescence. 
     
     
         15 . The method of  claim 13 , wherein the cytoplasm is derived from an oocyte or embryonic cell. 
     
     
         16 . The method of  claim 13 , wherein the telomerase DNA under the control of a regulatable promoter. 
     
     
         17 . The method of  claim 13 , wherein the cell from which said nucleus is isolated is a mammalian cell. 
     
     
         18 . The method of  claim 13 , wherein said recipient cell or recipient cell nucleus is genetically modified prior, concurrent or subsequent to the introduction of said cytoplasm. 
     
     
         19 . The method of  claim 13 , wherein said donor cell is of a different species than the recipient cell. 
     
     
         20 . The method of  claim 19 , wherein said donor cell is a non-human primate oocyte or embryonic cell and the recipient cell is a human somatic cell.