MULTIPOTENT/PLURIPOTENT CELLS AND METHODS
First Claim
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1. A human stem cell that is pluripotent, somatic, non-embryonic, and having the property of long-term self renewal.
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Abstract
Described herein are multipotent stem cells, e.g., human and other mammalian pluripotent stem cells, and related methods.
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Citations
202 Claims
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1. A human stem cell that is pluripotent, somatic, non-embryonic, and having the property of long-term self renewal.
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2-132. -132. (canceled)
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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. - View Dependent Claims (134)
- (a) express endogenous Oct4 and Nanog, (b) differentiate into tissues having the characteristics of endoderm, mesoderm, and ectoderm when injected into SCID mice;
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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. - View Dependent Claims (136, 137, 138, 139, 140, 141, 142, 143, 144, 145)
- (a) providing somatic cells that do not contain a selectable marker, wherein at least some of which are induced pluripotent stem cells; and
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146. A method of identifying a somatic cell that is an induced pluripotent stem cell, the method comprising steps of :
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(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. - View Dependent Claims (147, 148)
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149. A method of identifying a somatic cell that is an induced pluripotent stem cell, the method comprising steps of:
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(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. - View Dependent Claims (150)
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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 . - View Dependent Claims (152)
- (b) differentiate into tissues having the characteristics of endoderm, mesoderm, and ectoderm when injected into SCID mice; and
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153. An induced pluripotent stem cell, wherein the induced pluripotent stem cell does not comprise a selectable marker.
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154. A method of generating a stem cell, comprising the steps of:
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(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. - View Dependent Claims (155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175)
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176. An isolated pluripotent cell produced by a method comprising:
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(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.
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177. A purified population of somatic cells produced by a method comprising:
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(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.
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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. - View Dependent Claims (179)
- (c) have at least one genetic modification; and
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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:
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(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.
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181. A method of selecting induced pluripotent stem cells, the method comprising:
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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. - View Dependent Claims (182, 183, 184, 185, 186, 187, 188, 189, 190, 191)
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192. A method of selecting induced pluripotent stem cells, the method comprising:
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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. - View Dependent Claims (193, 194, 195, 196, 197, 198, 199, 200, 201, 202)
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Specification