Primate totipotent and pluripotent stem cells produced by somatic cell nuclear transfer

Abstract

Purified totipotent stem cells and pluripotent stems cells derived by somatic cell nuclear transfer are disclosed herein, as well as cell lines, multipotent cells and differentiated cells produced from these stem cells. The stem cells are produced from an enucleated host cell from a first donor and nuclear genetic material from a somatic cell of a second donor. Methods for making and using such compositions of such stem cells are also provided.

1 . A method for producing a reconstituted primate embryo or a totipotent stem cell, comprising:
 removing the nuclear genetic material from a recipient primate host oocyte cell from a first primate using a real-time non-UV-based spindle polarized light imaging system in the absence of staining and in a manner that does not lower levels of maturation promoting factor (MPF) to form an enucleated oocyte containing the oocyte cell's mitochondria and mitochondrial DNA (mtDNA);   introducing a nucleus of a donor primate somatic cell from a second primate into the enucleated oocyte in Ca 2+  free medium under conditions that reduce or eliminate calcium oscillations, wherein the nucleus comprises nuclear genetic material and the oocyte comprises mtDNA and wherein the first primate and the second primate are from the same primate species, to produce a reconstituted primate embryo or a totipotent stem cell,   wherein the reconstituted primate embryo or the totipotent stem cell (i) is capable of four or more cell divisions; (ii) maintains a normal karyotype while in culture; (iii) is capable of differentiating into ectoderm, mesoderm, and endoderm layers; and (iv) comprises mtDNA derived from the oocyte of the first primate recipient and nuclear genetic material from the donor primate somatic cell of the second primate.   
     
     
         2 . The method of  claim 1 , wherein a Major Histocompatibility (MHC) locus of the donor primate somatic cell matches at least one MHC locus of an individual in need of stem cell therapy. 
     
     
         3 . The method of  claim 1 , wherein the first primate recipient and the second primate donor are a human. 
     
     
         4 . The method of  claim 1 , wherein the somatic cell is a skin cell, a fibroblast, a nucleated hematopoietic cell, a muscle cell, a hair follicle cell, a adult stem cell or an adipose cell. 
     
     
         5 . The method of  claim 1 , further comprising one or more of detecting chromosome condensation, detecting nuclear envelope breakdown, and detecting disappearance of laminin A/C. 
     
     
         6 . The method of  claim 1 , wherein the method produces the reconstituted primate embryo or the totipotent stem cell with an efficiency of greater than about 10%. 
     
     
         7 . The method of  claim 1 , wherein the reconstituted primate embryo or the totipotent stem cell expresses an imprinted gene. 
     
     
         8 . The method of  claim 7 , wherein the imprinted gene is H19, CDKNIC, PHLDA2, DLX5, ATP10A, SLC22A18, TP73, IGF2, NDN, SNRPN, MEST, MAGEL2 or PEG3. 
     
     
         9 . The method of  claim 1 , wherein telomeres of the reconstituted primate embryo or the totipotent stem cell are elongated in comparison to telomeres of a fibroblast from the second primate donor. 
     
     
         10 . The method of  claim 1 , wherein the reconstituted primate embryo or the totipotent stem cell expresses an increased amount of telomerase as compared to a fibroblast from the second primate donor. 
     
     
         11 . The method of  claim 1 , wherein the reconstituted primate embryo or the totipotent stem cell comprises two X chromosomes. 
     
     
         12 . The method of  claim 11 , wherein the reconstituted primate embryo or the totipotent stem cell expresses XIST. 
     
     
         13 . The method of  claim 1 , wherein the reconstituted primate embryo or the totipotent stem cell comprises an X and a Y chromosome. 
     
     
         14 . The method of  claim 1 , further comprising activating the reconstituted primate embryo or the totipotent stem cell. 
     
     
         15 . The method of  claim 1 , further comprising producing a pluripotent primate stem cell, where in the method further comprises,
 culturing the reconstituted primate embryo to the blastocyst stage;   isolating an inner cell mass cell from the blastocyst; and   culturing the inner cell mass cell on a fibroblast feeder layer so that the cells divide, thereby producing the pluripotent primate stem cell.   
     
     
         16 . The method of  claim 15 , wherein said isolating the inner cell mass cell does not comprise immunosurgery. 
     
     
         17 . A totipotent primate stem cell, wherein the totipotent stem cell:
 is capable of four or more cell divisions;   maintains a normal karyotype while in culture;   is capable of differentiating into ectoderm, mesoderm, and endoderm layers; and   comprises mitochondrial DNA derived from the oocyte of a first primate recipient and nuclear genetic material from a somatic cell of a second primate donor, wherein the first primate recipient and the second primate donor are from the same species.   
     
     
         18 . The totipotent primate stem cell of  claim 17 , wherein the totipotent primate stem cell is totipotent and can differentiate into germ cells, trophectoderm, ectoderm, mesoderm, and endoderm cells in vitro. 
     
     
         19 . The totipotent stem cell of  claim 17 , wherein the first primate recipient is a human and the second primate donor is a human. 
     
     
         20 . The totipotent stem cell of  claim 17 , wherein the first primate recipient and the second primate donor are non-human primates. 
     
     
         21 . The totipotent stem cell of  claim 17 , wherein the somatic cell is a skin cell, a fibroblast, a nucleated hematopoietic cell, a muscle cell, a hair follicle cell, an adult stem cell or an adipose cell. 
     
     
         22 . The totipotent stem cell of  claim 17 , wherein the totipotent stem cell is a clonal match to the second primate donor Major Histocompatibility Complex (MHC) locus. 
     
     
         23 . The totipotent stem cell of  claim 17 , wherein the totipotent stem cell expresses an imprinted gene. 
     
     
         24 . The totipotent stem cell of  claim 23 , wherein the imprinted gene is H19, CDKNIC, PHLDA2, DLX5, ATP10A, SLC22A18, TP73, IGF2, NDN, SNRPN, MEST, MAGEL2 or PEG3. 
     
     
         25 . The totipotent stem cell of  claim 17 , wherein telomeres of the totipotent stem cell are elongated in comparison to telomeres of a fibroblast from the second primate donor. 
     
     
         26 . The totipotent stem cell of  claim 17 , wherein the totipotent stem cell expresses an increased amount of telomerase as compared to a somatic cell from the second primate donor. 
     
     
         27 . The totipotent stem cell of  claim 17 , wherein the totipotent stem cell comprises two X chromosomes. 
     
     
         28 . The totipotent stem cell of  claim 17 , wherein the totipotent stem cell comprises an X and a Y chromosome.