Multipotent/pluripotent cells and methods

Abstract

Described herein are multipotent stem cells, e.g., human and other mammalian pluripotent stem cells, and related methods.

1 . A human stem cell that is pluripotent, somatic, non-embryonic, and having the property of long-term self renewal. 
     
     
         2 .- 132 . (canceled) 
     
     
         133 . A purified preparation of isolated induced pluripotent somatic mammalian cells, wherein the cells: (a) express endogenous Oct4 and Nanog, (b) differentiate into tissues having the characteristics of endoderm, mesoderm, and ectoderm when injected into SCID mice; (c) have at least one exogenous gene integrated into the genome; and (d) do not express an exogenously introduced pluripotency gene. 
     
     
         134 . The purified preparation of cells of  claim 133 , wherein the cells share the same genome as the donor of said somatic cells and wherein said donor is a subject in need of cell transplantation. 
     
     
         135 . A method of deriving an induced pluripotent stem cell from a somatic cell, the method comprising steps of: (a) providing somatic cells that do not contain a selectable marker, wherein at least some of which are induced pluripotent stem cells; and (b) selecting a cell or colony of cells having a morphology characteristic of a human ES cell colony. 
     
     
         136 . The method of  claim 135 , wherein the provided somatic cells do not comprise a selectable marker construct. 
     
     
         137 . The method of  claim 135 , wherein the somatic cells contain at least one exogenously introduced induction factor. 
     
     
         138 . The method of  claim 137 , wherein said exogenously introduced induction factor is a protein introduced by protein transduction or transient transfection of a construct encoding said protein. 
     
     
         139 . The method of  claim 135 , wherein the somatic cells of step (a) contain one or more exogenously introduced pluripotency genes or proteins encoded by such genes. 
     
     
         140 . The method of  claim 135 , wherein the somatic cells contain or express at least two exogenous proteins selected from the group consisting of Oct-4, Sox-2, c-Myc, and KIf4. 
     
     
         141 . The method of  claim 135 , wherein the somatic cells of step (a) contain one or more exogenously introduced genes selected from the group consisting of: Oct-4, Sox-2, c-Myc, KIf4, and combinations thereof. 
     
     
         142 . The method of  claim 135 , wherein the selected cells display the following characteristics: (i) ability to differentiate into cells having characteristics of endoderm, mesoderm, and ectoderm when injected into SCID mice; (ii) expression of endogenous Oct4, Nanog, or both; and (iii) expression of an ES cell marker. 
     
     
         143 . The method of  claim 142 , wherein the ES cell marker is selected from the group consisting of: Alkaline Phosphatase (AP), SSEA-I, SSEA-3, SSEA-4, and TRA-1-60. 
     
     
         144 . The method of  claim 135 , further comprising subjecting the identified cells to one or more selection steps. 
     
     
         145 . The method of  claim 144 , wherein said one or more selection steps comprise (e) passaging or subcloning said colony and identifying cells or a cell colony having an ES-like morphology from among progeny of the cells of said colony. 
     
     
         146 . A method of identifying a somatic cell that is an induced pluripotent stem cell, the method comprising steps of :
 (a) providing a population of cells, at least some of which are induced pluripotent stem cells, wherein said cell does not comprise an induction factor promoter reporter construct, and   (b) identifying a cell or colony of cells having a morphology characteristic of an ES cell or ES cell colony.   
     
     
         147 . The method of  claim 146 , further comprising separating said cell or at least some cells from said colony of cells from other cells in the population lacking such morphology. 
     
     
         148 . The method of  claim 146 , wherein the cells of step (a) contain one or more exogenously introduced genes selected from the group consisting of: Oct-4, Sox-2, c-Myc, KIf4, and combinations thereof. 
     
     
         149 . A method of identifying a somatic cell that is an induced pluripotent stem cell, the method comprising steps of:
 (a) introducing one or more induction factors to cells, wherein said induction factors are sufficient to induce the cells to become pluripotent stem cells;   (b) maintaining said cells in culture for a suitable period of time to allow colonies containing ES− like cells to develop; and   (c) identifying at least one such colony of ES-like cells without employing selection.   
     
     
         150 . The method of  claim 149 , wherein step (a) comprises introducing one or more genes selected from the group consisting of: Oct-4, Sox-2, c-Myc, KIf4, and combinations thereof into the cells. 
     
     
         151 . A purified preparation of isolated induced pluripotent stem cells from somatic mammalian cells, wherein the cells (a) express endogenous Oct4 and Nanog; (b) differentiate into tissues having the characteristics of endoderm, mesoderm, and ectoderm when injected into SCID mice; and (c) do not contain a DNA encoding a selectable marker operably linked to an endogenous pluripotency gene . 
     
     
         152 . The purified preparation of cells of  claim 151 , wherein the cells comprise the genome of a donor of said somatic cells or a donor, wherein said donor is an individual in need of cell transplantation. 
     
     
         153 . An induced pluripotent stem cell, wherein the induced pluripotent stem cell does not comprise a selectable marker. 
     
     
         154 . A method of generating a stem cell, comprising the steps of:
 (a) providing a differentiated somatic cell that contains at least one exogenously introduced induction factor that contributes to inducing said cell to become an induced pluripotent stem cell;   (b) maintaining said cell under conditions appropriate for proliferation of said cell and for activity of said at least one exogenously introduced induction factor for a period of time sufficient to activate at least one endogenous pluripotency gene; and   (c) inactivating said at least one exogenously-introduced induction factor.   
     
     
         155 . The method of  claim 154 , further comprising identifying cells that express one or more markers of pluripotency. 
     
     
         156 . The method of  claim 154 , wherein said differentiated somatic cell is terminally differentiated. 
     
     
         157 . The method of  claim 154 , wherein said differentiated somatic cell is a blood cell. 
     
     
         158 . The method of  claim 154 , wherein said differentiated somatic cell is obtained from peripheral blood. 
     
     
         159 . The method of  claim 154 , wherein said at least one induction factor is a polynucleotide. 
     
     
         160 . The method of  claim 154 , wherein said at least one induction factor is a polypeptide. 
     
     
         161 . The method of  claim 154 , wherein said at least one induction factor is selected from the group consisting of Oct4, Sox2, Nanog, Lin28, c-Myc and combinations thereof. 
     
     
         162 . The method of  claim 154 , wherein said differentiated somatic cell contains exogenously introduced Oct4, Sox2, and KIf-4. 
     
     
         163 . The method of  claim 154 , wherein said differentiated somatic cell contains exogenously introduced Oct4, Sox2, KIf-4 and c-Myc. 
     
     
         164 . The method of  claim 154 , wherein said differentiated somatic cell further contains exogenously introduced genes Oct4. Sox2 and KIf-4 and further contains at least one exogenously introduced induction factor. 
     
     
         165 . The method of  claim 164 , wherein said at least one exogenously introduced induction factor is a polynucleotide. 
     
     
         166 . The method of  claim 164 , wherein said at least one exogenously introduced induction factor is a polypeptide. 
     
     
         167 . The method of  claim 164 , wherein said at least one exogenously introduced induction factor is a polypeptide. 
     
     
         168 . The method of  claim 154 , wherein said at least one exogenously introduced factor is introduced using a vector. 
     
     
         169 . The method of  claim 154 , wherein said at least one exogenously introduced factor is introduced using an inducible vector. 
     
     
         170 . The method of  claim 154 , wherein said at least one exogenously introduced factor is introduced using a vector which is not subject to methylation-mediated silencing. 
     
     
         171 . The method of  claim 154 , wherein said at least one exogenously introduced factor is introduced using a viral vector. 
     
     
         172 . The method of  claim 154 , wherein said at least one exogenously introduced factor is introduced using a retroviral vector. 
     
     
         173 . The method of  claim 154 , wherein said at least one exogenously introduced factor is introduced using a lentiviral vector. 
     
     
         174 . The method of  claim 154 , wherein said differentiated somatic cell is maintained in the presence of a growth factor. 
     
     
         175 . The method of  claim 154 , wherein said endogenous pluripotency gene is selected from the group consisting of Nanog, Oct4, Sox2 and combinations thereof. 
     
     
         176 . An isolated pluripotent cell produced by a method comprising:
 (a) providing a differentiated somatic cell that contains at least one exogenously introduced induction factor that contributes to inducing said cell to a pluripotent state;   (b) maintaining said cell under conditions appropriate for proliferation of said cell and for activity of said at least one exogenously introduced factor for a period of time sufficient to activate at least one endogenous pluripotency gene;   (c) functionally inactivating said at least one exogenously introduced factor; and   (d) differentiating cells which display one or more markers of pluripotency from cells which do not.   
     
     
         177 . A purified population of somatic cells produced by a method comprising:
 (a) providing a differentiated somatic cell that contains at least one exogenously introduced induction factor;   (b) maintaining said cell under conditions appropriate for proliferation of said cell and for activity of said at least one exogenously introduced factor for a period of time sufficient to activate at least one endogenous pluripotency gene;   (c) functionally inactivating said at least one exogenously introduced induction factor; and   (d) differentiating cells which display one or more markers of pluripotency from cells which do not.   
     
     
         178 . A purified preparation of isolated pluripotent reprogrammed somatic mammalian cells, wherein the cells (a) express endogenous Oct4 and Nanog, (b) differentiate into tissues having the characteristics of endoderm, mesoderm, and ectoderm when injected into SCID mice; (c) have at least one genetic modification; and (d) do not express an exogenously introduced pluripotency gene or selectable marker operably linked to an endogenous pluripotency gene. 
     
     
         179 . The purified preparation of cells of  claim 178 , wherein the cells are genetically matched to a donor of said somatic cells or a donor of a precursor cell of said somatic cells, wherein said donor is an individual in need of cell therapy. 
     
     
         180 . A method of deriving a somatic cell that has an increased likelihood of having been reprogrammed to an ES-like state, the method comprising steps of:
 (a) providing somatic cells that do not contain a genetic modification usable for chemical selection of said cells, wherein at least some of the cells have been at least in part reprogrammed to an ES− like state; and   (b) selecting a cell or colony of cells having a morphology characteristic of an ES cell or ES cell colony.   
     
     
         181 . A method of selecting induced pluripotent stem cells, the method comprising:
 a) re-programming a differentiated primary cell to a pluripotent phenotype, wherein the differentiated primary cell does not express Nanog mRNA when measured by RT-PCR;   b) culturing the cell re-programmed in step (a) in the absence of a selection agent after re-programming;   c) microscopically observing the culture of step (b), and isolating a clone of cells in the culture which have become smooth and rounded in appearance; and   d) testing cells of the clone for the expression of a stem cell marker; wherein the detection of stem cell marker expression is indicative that the cells are induced pluripotent stem cells.   
     
     
         182 . The method of  claim 181  wherein said re-programming comprises one of: introducing nucleic acid sequences encoding the transcription factors Oct4, Sox2, c-Myc and KIf4 to said differentiated somatic cell, the sequences operably linked to regulatory elements for the expression of the factors; introducing one or more protein factors that re-program the cell's differentiation state; and contacting said cell with a small molecule that induces a re-programming of the cell's differentiated state. 
     
     
         183 . The method of  claim 181  further comprising the step of introducing cells of a said clone that express a stem cell marker into nude mice and performing histology on a tumor arising from the cells, wherein the growth of a tumor comprising cells from all three germ layers further indicates that the cells are pluripotent stem cells. 
     
     
         184 . The method of  claim 181  wherein the step of culturing further comprises passaging said cells. 
     
     
         185 . The method of  claim 181  wherein said differentiated somatic cell has a morphology distinctly different from that of an ES cell. 
     
     
         186 . The method of  claim 181  wherein the differentiated primary cell is a fibroblast, and wherein said fibroblast is flattened and irregularly shaped prior to said re-programming. 
     
     
         187 . The method of  claim 181  wherein the stem cell marker is selected from the group consisting of SSEA1, CD9, Nanog, Fbx15, Ecat1, Esg1, Eras, GdO, Fgf4, Cripto, Dax1, Zpf296, Slc2a3, Rex1, Utf1, and Oct4. 
     
     
         188 . The method of  claim 182  wherein said nucleic acid sequences are comprised in a viral vector or a plasmid. 
     
     
         189 . The method of  claim 188  wherein said viral vector is a retroviral vector, a lentiviral vector or an adenoviral vector. 
     
     
         190 . The method of  claim 181  further comprising the step of testing cells of said clone for the expression of exogenous Oct4, Sox2, c-Myc and/or Klf4. 
     
     
         191 . The method of  claim 181 , wherein said cell comprises a human cell. 
     
     
         192 . A method of selecting induced pluripotent stem cells, the method comprising:
 a) inducing a differentiated primary cell to a pluripotent phenotype, wherein the differentiated primary cell does not express Nanog mRNA when measured by RT-PCR;   b) culturing the cell induced in step (a) in the absence of a selection agent after inducing pluripotency;   c) microscopically observing the culture of step (b), and isolating a clone of cells in the culture which have become smooth and rounded in appearance; and   d) testing cells of the clone for the expression of a stem cell marker; wherein the detection of stem cell marker expression is indicative that the cells are induced pluripotent stem cells.   
     
     
         193 . The method of  claim 192  wherein said inducing comprises one of: introducing nucleic acid sequences encoding the transcription factors Oct4, Sox2, c-Myc and Klf4 to said differentiated somatic cell, the sequences operably linked to regulatory elements for the expression of the factors; introducing one or more protein factors that induce pluripotency of said differentiated somatic cell; and contacting said differentiated somatic cell with a small molecule that induces the cell to become pluripotent. 
     
     
         194 . The method of  claim 192  further comprising the step of introducing cells of said clone that express a stem cell marker into nude mice and performing histology on a teratoma arising from the cells, wherein the growth of a teratoma comprising cells from all three germ layers further indicates that the cells are pluripotent stem cells. 
     
     
         195 . The method of  claim 192  wherein the step of culturing further comprises passaging said cells. 
     
     
         196 . The method of  claim 192  wherein said differentiated somatic cell has a morphology distinctly different from that of an ES cell. 
     
     
         197 . The method of  claim 192  wherein the differentiated primary cell is a fibroblast, and wherein said fibroblast is flattened and irregularly shaped prior to said inducing. 
     
     
         198 . The method of  claim 192  wherein the stem cell marker is selected from the group consisting of SSEA1, CD9, Nanog, Fbx15, Ecat1, Esg1, Eras, GdO, Fgf4, Cripto, Dax1, Zpf296, Slc2a3, Rex1, Utf1, and Oct4. 
     
     
         199 . The method of  claim 193  wherein said nucleic acid sequences are comprised in a viral vector or a plasmid. 
     
     
         200 . The method of  claim 199  wherein said viral vector is a retroviral vector, a lentiviral vector or an adenoviral vector. 
     
     
         201 . The method of  claim 192  further comprising the step of testing cells of said clone for the expression of exogenous Oct4, Sox2, c-Myc or Klf4. 
     
     
         202 . The method of  claim 192 , wherein said cell comprises a human cell.