Methods of Reprogramming Animal Somatic Cells

Abstract

This invention generally relates to methods to obtain mammalian cells and tissues with patterns of gene expression similar to that of a developing mammalian embryo or fetus, and the use of such cells and tissues in the treatment of human disease and age-related conditions. More particularly, the invention relates to methods for identifying, expanding in culture, and formulating mammalian pluripotent stem cells and differentiated cells that differ from cells in the adult human in their pattern of gene expression, and therefore offer unique characteristics that provide novel therapeutic strategies in the treatment of degenerative disease.

1 . A method for reprogramming an animal differentiated somatic cell to an undifferentiated stem cell, comprising the steps of:
 (a) injecting one or more animal differentiated somatic cells into an oocyte, wherein said oocyte is from a species that is different from said somatic cell, and allowing the remodeling of the nucleus of said somatic cell, wherein said remodeling results in
 (i) the remodeling of the nuclear envelope of the nucleus of said somatic cell, wherein one or more components of the nuclear envelope of said nucleus are removed and wherein one or more components from said oocyte are added to the nuclear envelope of said nucleus of said somatic cell, and 
 (ii) the reprogramming of the chromatin of said somatic cell resulting in the expression of genes of an undifferentiated cell, wherein said reprogramming of said chromatin comprises the removal of regulatory factors from said chromatin and the addition of regulatory factors from said oocyte to said chromatin; and 
   (b) transferring or fusing the resulting remodeled nucleus from step (a) into the enucleated cytoplasm of an undifferentiated embryonic cell to form an undifferentiated stem cell, and allowing the resulting undifferentiated stem cell to grow.   
     
     
         2 . The method of  claim 1 , further comprising the step of:
 (c) analyzing the karyotype of the resulting undifferentiated stem cells from step (b), and analyzing the gene expression pattern of said resulting undifferentiated stem cells to determine if said resulting undifferentiated stem cells display a pattern of gene expression expected of embryonic stem cells.   
     
     
         3 . The method of  claim 2 , wherein if said resulting cells do not display a gene expression pattern that is expected of embryonic stem cells, steps (a)-(c) are repeated one or more times until the gene expression pattern of the resulting cells from step (b) display a pattern of gene expression that is the pattern expected of embryonic stem cells. 
     
     
         4 . The method of  claim 1 , wherein the enucleated embryonic cell of step (b) is selected from the group consisting of enucleated blastomere cell, enucleated inner cell mass cell, enucleated oocyte, enucleated morula cell, enucleated ES cell, enucleated EG cell, and enucleated EC-derived cell. 
     
     
         5 . The method of  claim 1 , wherein at least two remodeled nuclei from step (a) are transferred or are fused with at least two enucleated cytoplasms in step (b). 
     
     
         6 . The method of  claim 1 , wherein said animal differentiated somatic cell is a human cell. 
     
     
         7 . The method of  claim 1 , wherein said oocyte is from the genus  Xenopus.    
     
     
         8 . The method of  claim 1 , wherein said animal differentiated somatic cell is permeabilized prior to injection. 
     
     
         9 . The method of  claim 1 , wherein said oocyte expresses one or more factors selected from the group consisting of CROC4, DNMT3B, H2AFX, HIST1H2AB, HIST1H4J, HMGB2, LEFTB, MYBL2, MYC, MYCN, NANOG, OCT3/4 (POU5F1), OTX2, SALL4, SOX2, TERF1, TERT, ZNF206. 
     
     
         10 . A method for reprogramming an animal differentiated somatic cell to an undifferentiated stem cell, comprising the steps of:
 (a) isolating the nucleus from one or more animal differentiated somatic cells and injecting said nucleus into an oocyte, wherein said oocyte is from a species that is different from the species of said somatic cell, and allowing the remodeling of the nucleus of said somatic cell, wherein said remodeling results in
 (i) the remodeling of the nuclear envelope of said nucleus, wherein one or more components of the nuclear envelope of said nucleus are removed and wherein one or more components from said oocyte are added to the nuclear envelope of said nucleus of said somatic cell, and 
 (ii) the reprogramming of the chromatin of said somatic cell resulting in the expression of genes of an undifferentiated cell, wherein said reprogramming of said chromatin comprises the removal of regulatory factors from said chromatin and the addition of regulatory factors from said oocyte to said chromatin; and 
   (b) transferring or fusing the resulting remodeled nucleus from step (a) into the enucleated cytoplasm of an undifferentiated embryonic cell to form an undifferentiated stem cell, and allowing the resulting undifferentiated stem cell to grow.   
     
     
         11 . The method of  claim 10 , further comprising the step of (c) analyzing the karyotype of the resulting undifferentiated stem cells from step (b), and analyzing the gene expression pattern of said resulting undifferentiated stem cells to determine if said resulting undifferentiated stem cells display a pattern of gene expression expected of embryonic stem cells. 
     
     
         12 . The method of  claim 11 , wherein if said resulting cells do not display a gene expression pattern that is expected of embryonic stem cells, steps (a)-(c) are repeated one or more times until the gene expression pattern of the resulting cells from step (b) display a pattern of gene expression that is the pattern expected of embryonic stem cells. 
     
     
         13 . The method of  claim 10 , wherein at least two somatic cell nuclei are remodeled within one oocyte in step (a). 
     
     
         14 . The method of  claim 10 , wherein the enucleated embryonic cell of step (b) is selected from the group consisting of enucleated blastomere cell, enucleated inner cell mass cell, enucleated oocyte, enucleated morula cell, enucleated ES cell, enucleated EG cell, and enucleated EC-derived cell. 
     
     
         15 . The method of  claim 16 , wherein at least two remodeled nuclei from step (a) are transferred or are fused with at least two enucleated cytoplasms in step (b). 
     
     
         16 . The method of  claim 10 , wherein the differentiated somatic cell is human. 
     
     
         17 . The method of  claim 10  wherein the oocyte is from the genus  Xenopus.    
     
     
         18 . The method of  claim 10  wherein said oocyte expresses one or more factors selected from the group consisting of CROC4, DNMT3B, H2AFX, HIST1H2AB, HIST1H4J, HMGB2, LEFTB, MYBL2, MYC, MYCN, NANOG, OCT3/4 (POU5F1), OTX2, SALL4, SOX2, TERF1, TERT, ZNF206. 
     
     
         19 . The method of  claim 10 , wherein the method further comprises the step of replacing one or more genes in the somatic cell genome by homologous recombination, wherein said homologous recombination comprises the addition of one or more DNA-targeting constructs and a protein or extract preparation proficient in homologous recombination, and wherein said step is performed before step (b). 
     
     
         20 . The method of  claim 19 , wherein said extract proficient in homologous recombination is an extract from the chicken pre-B cell line DT40 or the human lymphoid cell line DG75. 
     
     
         21 . A method for reprogramming an animal differentiated somatic cell to an undifferentiated stem cell, comprising the steps of:
 (a) isolating the nucleus, DNA, or chromatin from said differentiated somatic cell and encapsulating said nucleus, DNA, or chromatin in a new nuclear envelope in vitro using extract from undifferentiated cells to form a remodeled nucleus; and   (b) transferring or fusing the resulting remodeled nucleus from step (a) into the enucleated cytoplasm of an undifferentiated embryonic cell to form an undifferentiated stem cell, and allowing the resulting undifferentiated stem cell to grow.   
     
     
         22 . The method of  claim 21 , further comprising the step of:
 (c) analyzing the karyotype of the resulting undifferentiated stem cells from step (b), and analyzing the gene expression pattern of said resulting undifferentiated stem cells to determine if said resulting undifferentiated stem cells display a pattern of gene expression expected of embryonic stem cells.   
     
     
         23 . The method of  claim 22 , wherein if said resulting cells do not display a gene expression pattern that is expected of embryonic stem cells, steps (a)-(c) are repeated one or more times until the gene expression pattern of the resulting cells from step (b) display a pattern of gene expression that is the pattern expected of embryonic stem cells. 
     
     
         24 . The method of  claim 21 , wherein the enucleated embryonic cell of step (b) is selected from the group consisting of enucleated blastomere cell, enucleated inner cell mass cell, enucleated oocyte, enucleated morula cell, enucleated ES cell, enucleated EG cell, and enucleated EC-derived cell. 
     
     
         25 . The method of  claim 21 , wherein at least two remodeled nuclei from step (a) are transferred or are fused with at least two enucleated cytoplasms in step (b). 
     
     
         26 . The method of  claim 21 , wherein said animal differentiated somatic cell is a human cell. 
     
     
         27 . The method of  claim 21 , wherein the species from which the differentiated somatic cell and the undifferentiated cell extract originate are different. 
     
     
         28 . The method of  claim 21 , wherein the undifferentiated cell extract comprises one or more factors selected from the group consisting of CROC4, DNMT3B, H2AFX, HIST1H2AB, HIST1H4J, HMGB2, LEFTB, MYBL2, MYC, MYCN, NANOG, OCT3/4 (POU5F1), OTX2, SALL4, SOX2, TERF1, TERT, ZNF206. 
     
     
         29 . The method of  claim 21 , wherein the undifferentiated cell extract is from embryonic carcinoma cells. 
     
     
         30 . The method of  claim 21 , wherein the undifferentiated cell extract is an oocyte extract. 
     
     
         31 . The method of  claim 30 , wherein the oocyte extract is from oocytes of the genus  Xenopus.    
     
     
         32 . The method of  claim 30 , wherein the oocyte extract is from oocytes of the genus  Bos  or  Mus.    
     
     
         33 . The method of  claim 21 , wherein the method further comprises a step wherein the genome of the remodeled nucleus is modified by homologous recombination, wherein homologous recombination comprises the addition of one or more DNA-targeting constructs and a protein or extract preparation proficient in homologous recombination. 
     
     
         34 . The method of  claim 33 , wherein said homologous recombination results in the deletion or replacement of one or more genes. 
     
     
         35 . The method of any of  claims 33  and  34 , wherein said protein or extract preparation proficient in homologous recombination is an extract from the chicken pre-B cell line DT40 or the human lymphoid cell line DG75. 
     
     
         36 . The method of  claim 21 , wherein step (a) further comprises condensing the somatic cell genome to a chromosome clump through the exposure of said somatic cell nuclei to an extract from mitotic cells. 
     
     
         37 . The method of  claim 36 , wherein said mitotic cells are selected from the group consisting of metaphase II oocytes and metaphase germ-line cells. 
     
     
         38 . The method of  claim 36 , wherein said mitotic cells are cells from a different species than the species of the differentiated somatic cell. 
     
     
         39 . The method of  claim 36 , wherein said extract from mitotic cells comprises one or more factors selected from the group consisting of CROC4, DNMT3B, H2AFX, HIST1H2AB, HIST1H4J, HMGB2, LEFTB, MYBL2, MYC, MYCN, NANOG, OCT3/4 (POU5F1), OTX2, SALL4, SOX2, TERF1, TERT, ZNF206. 
     
     
         40 . A method for reprogramming an animal differentiated somatic cell to an undifferentiated stem cell, comprising the steps of:
 (a) isolating the nucleus from said differentiated somatic cell and condensing the chromatin to a chromosome clump through the exposure of said isolated nucleus to an extract from mitotic cells, thereby remodeling the somatic cell genome; and   (b) transferring or fusing the resulting remodeled genome from step (a) into the enucleated cytoplasm of an undifferentiated embryonic cell to form an undifferentiated stem cell, and allowing the resulting undifferentiated stem cell to grow.   
     
     
         41 . The method of  claim 40 , further comprising the step of:
 (c) analyzing the karyotype of the resulting undifferentiated stem cells from step (b), and analyzing the gene expression pattern of said resulting undifferentiated stem cells to determine if said resulting undifferentiated stem cells display a pattern of gene expression expected of embryonic stem cells.   
     
     
         42 . The method of  claim 41 , wherein if said resulting cells do not display a gene expression pattern that is expected of embryonic stem cells, steps (a)-(c) are repeated one or more times until the gene expression pattern of the resulting cells from step (b) display a pattern of gene expression that is the pattern expected of embryonic stem cells. 
     
     
         43 . The method of  claim 40 , wherein said mitotic cells are cells from a different species than the species of the differentiated somatic cell. 
     
     
         44 . The method of  claim 40 , wherein said extract from mitotic cells comprises one or more factors selected from the group consisting of CROC4, DNMT3B, H2AFX, HIST1H2AB, HIST1H4J, HMGB2, LEFTB, MYBL2, MYC, MYCN, NANOG, OCT3/4 (POU5F1), OTX2, SALL4, SOX2, TERF1, TERT, ZNF206. 
     
     
         45 . A method for creating an undifferentiated animal cell of specific genetic identity, the method comprising the steps of:
 (a) providing compositions of:
 (i) a differentiated animal somatic cell, or nucleus or chromatin isolated therefrom, said differentiated animal somatic cell bearing a genetic identity desired for the undifferentiated animal cell to be created; and 
 (ii) an undifferentiated embryonic cell; 
   (b) performing chromatin or nuclear transfer with the compositions of step (a), wherein the composition of (a) (i) comprises the donor material and the composition of (a) (ii) comprises the recipient cell;   (c) isolating the nucleus resulting from the chromatin or nuclear transfer of step (b) from within the undifferentiated embryonic cell; and   (d) fusing the nucleus of step (c) with an enucleated cytoplasm of an embryonic stem cell to yield an undifferentiated animal cell of specific genetic identity.   
     
     
         46 . A method for reprogramming multiple animal somatic cells, the method comprising the steps of:
 (a) providing:
 (i) at least two differentiated animal somatic cells that have their nuclear envelope remodeled to that of an undifferentiated cell; and 
 (ii) at least two undifferentiated enucleated embryonic cell cytoplasts; 
   (b) performing nuclear transfer with the cells of step (a), wherein the remodeled animal somatic cell nuclei are transferred to the cytoplasts in (ii);   (c) isolating the nuclei resulting from the nuclear transfer of step (b) from within the Undifferentiated embryonic cell; and   (d) repeating steps (a)-(c) to yield multiple reprogrammed animal pluripotent stem cells.   
     
     
         47 . An assay to identify molecules useful to include or remove from a nucleus in the process of reprogramming to an undifferentiated state comprising the steps of:
 (a) providing:
 (i) an extract prepared from undifferentiated or germ-line cells, wherein said extract is capable of remodeling the nucleus of a differentiated cell and wherein said extract is modified by the addition or deletion a particular molecular component; 
 (ii) at least one differentiated animal somatic cell; and 
 (iii) at least one undifferentiated enucleated embryonic cell cytoplast; 
   (b) remodeling the nucleus of said somatic cell of (ii) with the extract of (i);   (c) performing nuclear transfer with the remodeled somatic cell nucleus of step (b), wherein the remodeled animal somatic cell nucleus is transferred to a recipient cytoplast;   (d) culturing cells resulting from step (c); and   (e) comparing the gene expression of said resulting cells of step (c) with normal embryonic cells of the same type as the cytoplast to determine whether the added or deleted molecular component plays a role in cellular reprogramming.   
     
     
         48 . A method of deriving differentiated cell types from the undifferentiated reprogrammed cells of any of  claims 1 - 41 , wherein said differentiated cells are obtained without the generation of an ES cell line, and wherein said method comprises the steps of:
 (a) culturing said undifferentiated reprogrammed cells in the presence of at least one differentiation factor; and   (b) selecting differentiated cells from the culture of step (a), wherein said selection of differentiated cells comprises selecting cells that express one or more markers of a differentiated cell type.   
     
     
         49 . The method of  claim 48  wherein said differentiated cells are pancreatic beta cells. 
     
     
         50 . The method of  claim 48  wherein said differentiated cells are pancreatic precursor cells. 
     
     
         51 . The method of  claim 48 , wherein the method further comprises the step of permeabilizing said undifferentiated reprogrammed cells, wherein said permeabilization is performed prior to step (a).