MULTIPOTENT/PLURIPOTENT CELLS AND METHODS

• US 20100120069A1
• Filed: 09/22/2009
• Published: 05/13/2010
• Est. Priority Date: 06/15/2007
• Status: Abandoned Application

• Current Assignee: Kyoto University
• Original Assignee: Kyoto University
• Inventors: Ishikawa, Tetsuya, Sakurada, Kazuhiro, Masaki, Hideki, Takahashi, Shunichi
• Application Number:
  US12/564,836
• Publication Number:
  US 20100120069A1
• Time in Patent Office: Days
• US Class Current: 435/7.210
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 2501/602 Sox-2
• CPC Class Codes: C12N 2501/603 Oct-3/4
• CPC Class Codes: C12N 2501/604 Klf-4
• CPC Class Codes: C12N 2501/606 c-Myc
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2799/027 where the vector is derived from a retrovirus
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS

Abstract

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

Claims

1. A human stem cell that is pluripotent, somatic, non-embryonic, and having the property of long-term self renewal.

2-132. -132. (canceled)

133. A method of reprogramming primate somatic cells, the method comprising the steps of:

exposing a plurality of potency-determining factors to the primate somatic cells under conditions sufficient to reprogram the cells, wherein the potency-determining factors do not comprise c-Myc and Klf4; and

culturing the exposed cells to obtain reprogrammed cells having a higher potency level than the primate somatic cells.

134. The method of claim 133, wherein the primate somatic cells are obtained from a post-natal individual.

135. The method of claim 134, wherein the reprogrammed cells are substantially genetically identical to the post-natal individual.

136. The method of claim 133, wherein the exposing step includes the step of introducing a vector encoding one or more potency-determining factors into the primate somatic cells.

137. The method of claim 136, wherein the vector is a viral-based vector.

138. The method of claim 137, wherein the viral-based vector is a retroviral vector.

139. The method of claim 138, wherein the retroviral vector is a lentiviral vector.

140. The method of claim 133, wherein the potency-determining factors are introduced to the somatic cells as a reprogramming sequence in which a nucleic acid sequence encoding the potency-determining factor is operably linked to a heterologous promoter.

141. The method of claim 133, wherein the plurality of potency-determining factors is selected from the group consisting of Oct-4, Sox2, Nanog and Lin28.

142. The method of claim 133, wherein the potency-determining factors are Oct-4, Sox2, and at least one of Nanog and Lin28.

143. The method of claim 133, wherein the potency-determining factors are Oct-4 and Sox2.

144. The method of claim 133, wherein the reprogrammed cells are pluripotent.

145. The method of claim 133, wherein the reprogrammed cells (i) express a cell marker selected from the group consisting of Oct-4, SSEA3, SSEA4, Tra-1-60 and Tra-1-81;

(ii) exhibit morphology characteristic of pluripotent cells; and

(iii) form teratomas when introduced into an immunocompromised animal.

146. An enriched population of primate pluripotent cells produced according to a method comprising the step of:

introducing a plurality of potency-determining factors into primate somatic cells under conditions sufficient to express the potency-determining factors, thereby reprogramming the somatic cells to produce euploid primate pluripotent cells, wherein the potency-determining factors do not comprise c-Myc or Klf4.

147. The enriched population of cells as claimed in claim 146, wherein the primate pluripotent cells (i) express a cell surface marker selected from the group consisting of Oct-4, SSEA3, SSEA4, Tra-1-60 and Tra-1-81;

(ii) exhibit morphology characteristic of pluripotent cells; and

(iii) form teratomas when introduced into an immunocompromised animal.

148. The enriched population of cells as claimed in claim 146, wherein the potency-determining factors are Oct-4, Sox2 and at least one of Nanog and Lin28.

149. The enriched population of cells as claimed in claim 146, wherein the potency-determining factors are Oct-4 and Sox2.

150. The enriched population of cells as claimed in claim 146, wherein the primate pluripotent cells account for at least 60% of the population.

151. The enriched population of cells as claimed in claim 146, wherein the primate pluripotent cells account for at least 80% of the population.

152. The enriched population of cells as claimed in claim 146, wherein the primate pluripotent cells account for at least 95% of the population.

153. A cell culture comprising euploid pluripotent cells having a genome of a pre-existing differentiated cell of an individual primate.

154. The cell culture of claim 153, wherein the primate is a human.

155. The cell culture of claim 153, wherein the cells further comprise in the genome a plurality of introduced polynucleotides encoding potency-determining factors, wherein the potency-determining factors do not comprise c-Myc and Klf4.

156. A method for assessing suitability of at least one putative potency-determining factor to convert primate somatic cells to pluripotent cells, the method comprising the steps of:

exposing primate somatic cells to the at least one putative potency-determining factor, the primate somatic cells being receptive to uptake of the factor and comprising a marker gene under control of a regulated promoter active in a pluripotent cell; and

evaluating whether the marker gene is expressed in the cells after exposure to the at least one factor, expression indicating suitability of the at least one factor to convert the primate somatic cells to pluripotent cells.

157. The method of claim 156, wherein the regulated promoter is an Oct4 promoter.

158. The method of claim 156, wherein the at least one putative potency-determining factor is Oct-4, Sox2 and at least one of Nanog and Lin28.

159. A method of inducing pluripotency of human or non-human primate somatic cells, the method comprising the steps of:

forcing expression of a plurality of factors in the human or non-human primate somatic cells, wherein the factors do not comprise c-Myc and Klf4; and

culturing the human or non-human primate somatic cells to obtain induced pluripotent stem cells.

160. The method of claim 159, wherein the human or non-human primate somatic cells are obtained from post-natal tissue.

161. The method of claim 159, wherein the induced pluripotent stem cells comprise the genome of the human or non-human primate somatic cells or are immunologically compatible with the human or non-human primate somatic cells.

162. The method of claim 159, further comprising introducing a vector encoding one or more factors into the human or non-human primate somatic cells.

163. The method of claim 162, wherein the vector is a viral-based vector.

164. The method of claim 163, wherein the viral-based vector is a retroviral vector.

165. The method of claim 164, wherein the retroviral vector is a lentiviral vector.

166. The method of claim 159, wherein each factor is introduced to the human or non-human primate somatic cells as a nucleic acid sequence encoding the factor and wherein the nucleic acid is operably linked to a promoter.

167. The method of claim 159, wherein the factors comprise Oct3/4 and c-Myc or Sox2 and Klf4.

168. The method of claim 159, wherein the factors are Oct-4, Sox2, Klf4 and at least one of Nanog and Lin28.

169. The method of claim 159, wherein the factors are Oct-4 and Sox2.

170. The method of claim 159, wherein the human or non-human primate somatic cells (i) express a cell marker selected from the group consisting of Oct-4, SSEA3, SSEA4, Tra-1-60 and Tra-1-81;

(ii) exhibit morphology characteristic of pluripotent cells; and

(iii) form teratomas when introduced into an immunocompromised animal.

171. The method of claim 159, wherein the factors do not comprise factors that might increase the risk of cell transformation.

172. The method of claim 159, wherein the factors do not comprise factors that might increase the risk of inducing cancer.

173. The method of claim 159, wherein the factors do not comprise c-Myc.

190. The method of claim 159, 174, or 181, wherein the factor is an induction factor.

174. A population of human or non-human primate pluripotent cells produced according to a method comprising the step of:

forcing the expression of factors in human or non-human primate somatic cells and inducing the human or non-human primate somatic cells to become human or non-human primate pluripotent cells with a normal diploid karyotype, wherein the factors do not comprise c-Myc.

175. The population of cells as claimed in claim 174, wherein the human or non-human primate pluripotent cells (i) express a cell surface marker selected from the group consisting of Oct-4, SSEA3, SSEA4, Tra-1-60 and Tra-1-81;

(ii) exhibit morphology characteristic of pluripotent cells; and

(iii) form teratomas when introduced into an immunocompromised animal.

176. The population of cells as claimed in claim 174, wherein the factors are Oct-4 and Sox2.

177. The population of cells as claimed in claim 174, wherein the factors are Oct-4, Sox2, and Klf4.

178. The population of cells as claimed in claim 174, wherein the factors are Oct-4 and Sox2 and one or more additional factors selected from the group consisting of:

Nanog, TERT, LIN28, CYP26A1, GDF3, FoxD3, Zfp42, Dnmt3b, Ecat1, and Tcl1.

179. The population of cells as claimed in claim 174, wherein the factors are Oct-4, Sox2 and Klf4 and one or more additional factors selected from the group consisting of:

Nanog, TERT, LIN28, CYP26A1, GDF3, FoxD3, Zfp42, Dnmt3b, Ecat1, and Tcl1.

180. The population of cells as claimed in claim 174, wherein the human or non-human primate pluripotent cells are a pure population.

181. A cell culture comprising pluripotent cells with a normal diploid karyotype having a genome of a differentiated cell of a human or non-human primate.

182. The cell culture of claim 181, wherein the human or non-human primate is a human.

183. The cell culture of claim 181, wherein the pluripotent cells further comprise a genome comprising a plurality of exogenous polynucleotides encoding factors, wherein the factors do not comprise c-Myc and Klf4.

184. The cell culture of claim 183, wherein the factors do not comprise factors that might increase the risk of cell transformation.

185. The cell culture of claim 183, wherein the factors do not comprise factors that might increase the risk of inducing cancer.

186. The cell culture of claim 183, wherein the factors do not comprise factors c-Myc.

188. The method of claim 186, wherein the promoter is an Oct4 promoter.

189. The method of claim 186, wherein the induction factors are one or more induction factors selected from the group consisting of:

Oct3/4, Sox2, Klf4 and c-Myc.

187. A method for screening a test agent for inducing human or non-human primate somatic cells to become human pluripotent stem cells comprising:

(a) contacting the human or non-human primate somatic cells with a test agent in combination with forcing expression of a set of one or more induction factors, wherein the somatic cells comprise a reporter construct containing one or more elements from an embryonic stem cell marker gene promoter; and

(2) assaying the activity of the promoter.