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.

1 - 19 . (canceled) 
     
     
         20 . A biologically pure culture comprising at least one mammalian cell that has been “reprogrammed” and/or had its life-span altered (increased) by the introduction of cytoplasm from an oocyte or embryonic cell of the same or different species. 
     
     
         21 . The culture of  claim 20 , wherein said mammalian cell is selected from the group consisting of human, non-human primate, mouse, rat, guinea pig, rabbit, hamster, goat, bovine, equine, ovine, canine and feline cells. 
     
     
         22 . The culture of  claim 20 , wherein said mammalian cell is a human cell. 
     
     
         23 . The culture of  claim 20 , wherein said mammalian cell comprises one or multiple genetic modifications. 
     
     
         24 - 35 . (canceled) 
     
     
         36 . A method for reprogramming or de-differentiating a recipient cell by introducing into said recipient cell all or part of cytoplasm containing active materials derived from a donor cell, the method comprising the steps of:
 a. removing cell cytoplasm containing active materials from said donor cell using a micropipette; and   b. introducing said cell cytoplasm into said recipient cell using a method selected from the group consisting of microinjection of said cytoplasm into the cytoplasm of said recipient cell, liposomal delivery of said cytoplasm into the cytoplasm of said recipient cell, and using cytoplasm blebs to introduce said cell cytoplasm into said recipient cell.   
     
     
         37 . The method of  claim 36 , wherein said donor cell is an oocyte. 
     
     
         38 . The method of  claim 36  wherein said recipient cell is a mammalian cell. 
     
     
         39 . The method of  claim 38  wherein said mammalian cell is selected from the group consisting of non-human primate, rat, guinea pig, mouse, rabbit, dog, cat, hamster, goat, cattle, horse, bison and buffalo. 
     
     
         40 . The method of  claim 38  wherein said mammalian cell is a human somatic cell. 
     
     
         41 . The method of  claim 40  wherein said human somatic 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. 
     
     
         42 . The method of  claim 36 , wherein said recipient cells are genetically modified. 
     
     
         43 . The method of  claim 42 , wherein said genetically modified cells compromise more than one genetic modification. 
     
     
         44 . The method of  claim 36  wherein said active materials are selected from the group consisting of OCT-4 and REX. 
     
     
         45 . A method for producing an embryonic stem cell by introducing into a mammalian recipient cell all or part of cytoplasm containing active materials derived from a donor cell, the method comprising the steps of:
 a. removing cell cytoplasm containing active materials from said donor cell using a micropipette; and   b. introducing said cell cytoplasm into said recipient cell using a method selected from the group consisting of microinjection of said cytoplasm into the cytoplasm of said recipient cell, liposomal delivery of said cytoplasm into the cytoplasm of said recipient cell, and using cytoplasm blebs to introduce said cell cytoplasm into said recipient cell.   
     
     
         46 . The method of  claim 45 , additionally comprising using said embryonic stem cell to treat an individual in need of alleviation of an effect selected from the group comprising diseases of immune origin, cancer, and aging. 
     
     
         47 . The method of  claim 42 , further comprising using said reprogrammed genetically modified cell for gene therapy. 
     
     
         48 . The method of  claim 42 , further comprising using said reprogrammed genetically modified cells as donor cells for nuclear transfer procedures for the production of a chimeric animal. 
     
     
         49 . The method of  claim 48 , wherein said chimeric animal is used as a research model for the study of a human disease. 
     
     
         50 . The method of  claim 45 , wherein said embryonic stem cell is used as a research tool.