Multipotent/pluripotent cells and methods

• US 20090191159A1
• Filed: 06/13/2008
• Published: 07/30/2009
• Est. Priority Date: 06/15/2007
• Status: Active Grant

• Current Assignee: Kyoto University
• Original Assignee: Kyoto University
• Inventors: Ishikawa, Tetsuya, Takahashi, Shunichi, Sakurada, Kazuhiro, Masaki, Hideki
• Granted Patent: US 8,211,697 B2
• Time in Patent Office: Days
• US Class Current: 424/93.7
• 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

1-132. -132. (canceled)

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

134. The human stem cell of claim 133, wherein the human stem cell comprises exogenous genes encoding two or more of the following:

an Oct 3/4 polypeptide, a Sox2 polypeptide, a Klf4 polypeptide, and a c-Myc polypeptide

135. The human stem cell of claim 134, wherein the human stem cell comprises exogenous genes encoding three or more of the following:

an Oct3/4 polypeptide, a Sox2 polypeptide, a Klf4 polypeptide, and a c-Myc polypeptide.

136. The human stem cell of claim 133, comprising endogenous and exogenous genes, wherein the exogenous genes comprise a first exogenous gene encoding an Oct3/4 polypeptide, a second exogenous gene encoding a Sox2 polypeptide, and a third exogenous gene encoding a Klf4 polypeptide.

137. The human stem cell of claim 136, wherein the exogenous genes consist essentially of the first exogenous gene, the second exogenous gene, and the third exogenous gene.

138. The human stem cell of claim 135, wherein the human stem cell does not comprise an exogenous gene capable of inducing cancer.

139. The human stem cell of claim 138, wherein the human stem cell does not comprise an exogenous gene encoding a c-Myc polypeptide.

140. The human stem cell of claim 136, wherein the human stem cell further comprises an exogenous gene encoding a c-Myc polypeptide

141. The human stem cell of claim 140, wherein the exogenous genes consist essentially of a first exogenous gene encoding an Oct3/4 polypeptide, a second exogenous gene encoding a Sox2 polypeptide, a third exogenous gene encoding a Klf4 polypeptide, and a fourth exogenous gene encoding a c-Myc polypeptide.

142. The human stem cell of claim 133, wherein the cell was induced from a population comprising adult human skin fibroblasts, adult peripheral blood mononuclear cells, adult human bone marrow-derived mononuclear cells, neonatal human skin fibroblasts, human umbilical vein endothelial cells, human umbilical artery smooth muscle cells, human postnatal skeletal muscle cells, human postnatal adipose cells, human postnatal peripheral blood mononuclear cells, or human cord blood mononuclear cells.

143. A differentiated cell obtained by differentiating the human stem cell of claim 133.

144. The differentiated cell of claim 143, wherein the differentiated cell is a pancreatic beta cell, a neural stem cell, a dopaminergic neuron, an oligodendrocyte, a hepatocyte, or a cardiac muscle cell.

145. A human stem cell that is somatic, non-embryonic, alkaline phosphatase positive, and expresses two or more genes selected from the group consisting of TDGF 1, Dnmt3b, FoxD3, GDF3, Cyp26a1, TERT, zfp42, Sox2, Oct3/4, and Nanog.

146. The human stem cell of claim 145, wherein the human stem cell is pluripotent.

147. The human stem cell of claim 145, wherein the telomeres in the human stem cell are at least 0.25 kB shorter than telomeres in embryonic stem cell lines.

148. The human stem cell of claim 145, wherein the human stem cell comprises exogenous genes encoding two or more of the following:

an Oct 3/4 polypeptide, a Sox2 polypeptide, a Klf4 polypeptide, and a c-Myc polypeptide.

149. The human stem cell of claim 145, wherein the human stem cell comprises exogenous genes encoding three or more of the following:

an Oct3/4 polypeptide, a Sox2 polypeptide, a Klf4 polypeptide, and a c-Myc polypeptide.

150. The human stem cell of claim 149, wherein the human stem cell comprises only three exogenous genes encoding induction factors and wherein the genes encode an Oct3/4 polypeptide, a Sox2 polypeptide, and a Klf4 polypeptide.

151. The human stem cell of claim 145, wherein the human cell comprises four exogenous genes encoding induction factors and wherein the four exogenous genes encode the following:

an Oct3/4 polypeptide, a Sox2 polypeptide, a Klf4 polypeptide, and a c-Myc polypeptide

152. The human stem cell of claim 145, wherein the cell was induced from a population comprising adult human skin fibroblasts, adult peripheral blood mononuclear cells, adult human bone marrow-derived mononuclear cells, neonatal human skin fibroblasts, human umbilical vein endothelial cells, human umbilical artery smooth muscle cells, human postnatal skeletal muscle cells, human postnatal adipose cells, human postnatal peripheral blood mononuclear cells, or human cord blood mononuclear cells.

153. The human stem cell of claim 145, wherein the human stem cell does not comprise exogenous genes encoding one or more of the following:

a TERT polypeptide, a SV40 Large T antigen polypeptide, HPV16 E6 polypeptide, a HPV16 E7 polypeptide or a Bmi1 polypeptide.

154. The human stem cell of claim 145, wherein, compared to a human embryonic stem cell, the human stem cell has a higher level of gene expression in 1 to 1000 genes.

155. The human stem cell of claim 154, wherein, compared to a human embryonic stem cell, the human stem cell has a higher level of gene expression in 1 to 300 genes.

156. A differentiated cell obtained by differentiating the human stem cell of claim 145.

157. The differentiated cell of claim 156, wherein the differentiated cell is a pancreatic beta cell, a neural stem cell, a dopaminergic neuron, an oligodendrocyte, a hepatocyte, or a cardiac muscle cell.

158. A human stem cell generated by a method comprising forcing the expression of an Oct3/4 polypeptide, a Sox2 polypeptide, and a Klf4 polypeptide in human postnatal cells to obtain one or more colonies of cells that have a high nucleus to cytoplasm ratio and are smaller in size than cells surrounding the one or more colonies, and isolating at least one of the one or more colonies.

159. The human stem cell of claim 158, wherein the human stem cell is pluripotent.

160. The human stem cell of claim 158, wherein the Oct3/4 polypeptide comprises an amino acid sequence at least 70% identical to SEQ ID NO:

7, the Sox2 polypeptide comprises an amino acid sequence at least 70% identical to SEQ ID NO;

9, and the Klf4 polypeptide comprises an amino acid sequence at least 70% identical to SEQ ID NO;

11.

161. The human stem cell of claim 160, wherein the Oct3/4 polypeptide comprises an amino acid sequence at least 85% identical to SEQ ID NO:

7, the Sox2 polypeptide comprises an amino acid sequence at least 85% identical to SEQ ID NO;

9, and the Klf4 polypeptide comprises an amino acid sequence at least 85% identical to SEQ ID NO;

11.

162. The human stem cell of claim 158, wherein the human stem cell does not comprise an exogenous gene capable of inducing cancer.

165. The human stem cell of claim 158, wherein the method further comprises forcing expression of a c-Myc polypeptide.

166. A purified population of human stem cells comprising the human stem cell of claim 158.

167. A composition comprising the human stem cell of claim 158 and a cryopreservation medium

168. The human stem cell of claim 158, wherein the population cells was prepared from a composition of cells that had been stored frozen and then thawed before the preparation.

164. The human stem cell of claim 164, wherein the method does not comprise forcing expression of a c-Myc polypeptide.

163. The human stem cell of claim 164, wherein the method does not comprise forcing expression of an exogenous gene encoding a c-Myc polypeptide.

169. A method for generating a human stem cell, comprising forcing expression of polypeptides in human postnatal cells, wherein the polypeptides comprise two or more of an Oct3/4 polypeptide, a Sox2 polypeptide, a Klf4 polypeptide, and a c-Myc polypeptide.

170. The method of claim 169, wherein the method does not comprise forcing expression of a c-Myc polypeptide.

171. The method of claim 169, wherein the method comprises forcing expression of a c-Myc polypeptide.

172. The method of claim 169, comprising forcing expression of three or more of the polypeptides in human postnatal cells.

173. The method of claim 172, comprising forcing expression of an Oct 3/4 polypeptide, a Sox2 polypeptide, and a Klf4 polypeptide.

174. The method of claim 172, further comprising contacting the human postnatal cells with a histone deacetylase inhibitor.

175. The method of claims 172, wherein the human postnatal cells are from an adult human.

176. The method of claims 172, wherein the human postnatal cells comprise adult human bone marrow-derived mononuclear cells, neonatal human skin fibroblasts, umbilical vein endothelial cells, umbilical artery smooth muscle cells, postnatal skeletal muscle cells, postnatal adipose cells, postnatal peripheral blood mononuclear cells, cord blood mononuclear cells, or placental cells.

177. The method of claims 172, wherein forcing the expression of the Oct3/4, Sox2, and Klf4 polypeptides, comprises introducing into the human postnatal cells one or more expression vectors encoding the Oct3/4, Sox2, and Klf4 polypeptides.

178. The method of claim 177, wherein the one or more expression vectors comprise recombinant retroviruses, lentiviruses, or adenoviruses.

179. The method of claim 177, wherein the one or more expression vectors are nucleic acid expression vectors.

180. The method of claim 169, wherein forcing the expression of the Oct3/4, Sox2 polypeptide, and Klf4 polypeptides comprises contacting a human postnatal cell population with at least one of:

(i) a purified polypeptide comprising the amino acid sequence of a protein transduction domain and an Oct3/4 polypeptide;

(ii) a carrier reagent and a purified Oct3/4 polypeptide;

(iii) a purified polypeptide comprising the amino acid sequence of a protein transduction and a Sox2 polypeptide;

(iv) a carrier reagent and a purified Sox2 polypeptide;

(v) a purified polypeptide comprising the amino acid sequence of a protein transduction domain and a Klf4 polypeptide;

(vi) a carrier reagent and a purified Klf4 polypeptide;

(vii) a purified polypeptide comprising the amino acid sequence of a protein transduction domain and a c-Myc polypeptide;

or

(viii) any combination of (i) to (vii).

181. A method for generating an autologous stem cell from a human subject, comprising forcing expression of an Oct3/4 polypeptide, a Sox2 polypeptide, and a Klf4 polypeptide in a cultured population of non-embryonic postnatal cells from the human subject.

182. The method of claim 181, wherein the stem cell is capable of forming a teratoma.

183. The method of claim 181, wherein the stem cell is pluripotent.

184. The method of claims 181, wherein the human postnatal cells have been passaged four or fewer times after preparation from a biological sample.

185. The method of claim 181, wherein the human postnatal cells are cultured in the presence of a serum concentration of 5% or less prior to the forced expression

186. The method of claim 181, wherein the human postnatal cells are cultured in the presence of bFGF, FGF-2, PDGF, EGF, IGF, insulin, TGFb-1, activin A, Noggin, BDNF, NGF, NT-1, NT-2, NT-3 or FGF-growth factor family member prior to the forced expression.

187. The method of claim 181, further comprising isolating, after the forced expression step, one or more colonies of cells smaller in size than surrounding cells, and identifying at least one of the one or more colonies that expresses alkaline phosphatase, nanog, TDGF1, Dnmt3b, FoxD3, GDF3, CYP26A1, TERT, and zfp4.

188. A method for performing cell transplantation in a subject in need thereof, comprising:

(i) identifying a donor that is immunocompatible with the subject;

(ii) generating an induced pluripotent stem cell line from postnatal cells of the healthy donor; and

(iii) transplanting one or more cells differentiated from the induced pluripotent stem cell line into the subject.

189. The method of claim 188, wherein the induced pluripotent stem cell line is induced from a mononuclear blood cell.

190. A method for identifying an agent that stimulates pluripotency or multipotency in a human somatic cell comprising:

(i) providing first and second cultured human somatic cells;

(ii) contacting the first cultured human somatic cell with a test agent;

(iii) contacting the second cultured human somatic cell with a negative control agent;

(iv) determining expression levels of an embryonic stem cell marker gene in the contacted first and second cultured cells; and

comparing the expression levels determined in step (iii) and indicating that the test agent stimulates pluripotency or multipotency if the embryonic stem cell marker gene expression level in the contacted first cultured cell is greater than that determined in the contacted second cultured cell, and indicating that the test agent fails to stimulate multipotency or pluripotency if the expression level of the embryonic stem cell marker gene in the contacted first cultured cell is the same or less than that determined in the contacted second cultured cell, wherein determining the expression levels of the embryonic stem cell marker gene comprises determining the expression levels of Tert or Cyp26A1.

191. The method of claim 190, wherein the test agent comprises one or more nucleic acids.

192. The method of claim 191, wherein the one or more nucleic acids comprises an RNAi, an RNAa, or an antisense oligonucleotide.

193. The method of claim 190, wherein the test agent comprises a small molecule compound.

194. A composition comprising at least one of:

(i) a purified polypeptide comprising the amino acid sequence of a protein transduction domain and an Oct3/4 polypeptide;

(ii) a carrier reagent and a purified Oct3/4 polypeptide;

(iii) a purified polypeptide comprising the amino acid sequence of a protein transduction and a Sox2 polypeptide;

(iv) a carrier reagent and a purified Sox2 polypeptide;

(v) a purified polypeptide comprising the amino acid sequence of a protein transduction domain and a Klf4 polypeptide;

(vi) a carrier reagent and a purified Klf4 polypeptide;

(vii) a purified polypeptide comprising the amino acid sequence of a protein transduction domain and a c-Myc polypeptide;

(viii) a carrier reagent and a purified c-Myc;

or

(ix) any combination of (i) to (viii)

195. The composition of claim 194, comprising at least two of:

(i) to (viii).