Methods and compositions for generating or maintaining pluripotent cells
AssigneeRegeneron Pharmaceuticals, Inc.
InventorsJunko Kuno, Wojtek Auerbach, David M. Valenzuela
Life of this patent
Estimating the term…
Abstract
Methods and compositions are provided for generating or maintaining human iPS cells in culture. Methods include the use of a low osmolality medium to make human iPS cells, or use of a low osmolality medium to maintain human iPS cells. Methods for making targeted genetic modification to human iPS cells cultured in low osmolality medium are also included. Compositions include human iPS cells cultured and maintained using the low osmolality medium defined herein.
Claims56
An in vitro culture comprising:
- (a) a population of hiPSCs;
- and
- (b) a low osmolality medium comprising a base medium and supplements, wherein the low osmolality medium comprises:
- (i) a leukemia inhibitory factor (LIF) polypeptide;
- (ii) a glycogen synthase kinase 3 (GSK3) inhibitor;
- and
- (iii) a MEK inhibitor;
- wherein the base medium has an osmolality of about 180 mOsm/kg to about 250 mOsm/kg.
A population of hiPSCs made or maintained in a low osmolality medium comprising a base medium and supplements, wherein the low osmolality medium comprises:
- (a) a leukemia inhibitory factor (LIF) polypeptide;
- (b) a glycogen synthase kinase 3 (GSK3) inhibitor;
- and
- (c) a MEK inhibitor;
- wherein the base medium has an osmolality of about 180 mOsm/kg to about 250 mOsm/kg.
A method for making a population of human induced pluripotent stem cells (hiPSCs), the method comprising culturing in vitro a population of non-pluripotent cells, transformed to express a pluripotent state, in a low osmolality medium comprising a base medium and supplements, wherein the low osmolality medium comprises:
- (a) a leukemia inhibitory factor (LIF) polypeptide;
- (b) a glycogen synthase kinase 3 (GSK3) inhibitor;
- and
- (c) a MEK inhibitor;
- wherein the base medium has an osmolality of about 180 mOsm/kg to about 250 mOsm/kg.
-72. (canceled)
A method for maintaining a population of hiPSCs in an in vitro culture, the method comprising culturing the population of hiPSCs in a low osmolality medium comprising a base medium and supplements, wherein the low osmolality medium comprises:
- (a) a leukemia inhibitory factor (LIF) polypeptide;
- (b) a glycogen synthase kinase 3 (GSK3) inhibitor;
- and
- (c) a MEK inhibitor;
- wherein the base medium has an osmolality of about 180 mOsm/kg to about 250 mOsm/kg.
-96. (canceled)
A method for modifying a target genomic locus in a hiPSC, comprising:
- (a) introducing into the hiPSC a targeting vector comprising an insert nucleic acid flanked by 5′ and 3′ homology arms corresponding to 5′ and 3′ target sites at the target genomic locus;
- and
- (b) identifying a genetically modified hiPSC comprising in its genome the insert nucleic acid integrated at the target genomic locus;
- wherein the hiPSC is cultured in a low osmolality medium comprising a base medium and supplements, wherein the low osmolality medium comprises:
- (i) a leukemia inhibitory factor (LIF) polypeptide;
- (ii) a glycogen synthase kinase 3 (GSK3) inhibitor;
- and
- (iii) a MEK inhibitor;
- wherein the base medium has an osmolality of about 180 mOsm/kg to about 250 mOsm/kg.
-127. (canceled)
A method for modifying a target genomic locus in a hiPSC, comprising:
- (a) introducing into the hiPSC one or more nuclease agents that induces one or more nicks or double-strand breaks at a recognition site at the target genomic locus;
- and
- (b) identifying at least one cell comprising in its genome a modification at the target genomic locus;
- wherein the hiPSC is cultured in a low osmolality medium comprising a base medium and supplements, wherein the low osmolality medium comprises:
- (i) a leukemia inhibitory factor (LIF) polypeptide;
- (ii) a glycogen synthase kinase 3 (GSK3) inhibitor;
- and
- (iii) a MEK inhibitor;
- wherein the base medium has an osmolality of about 180 mOsm/kg to about 250 mOsm/kg.
-154. (canceled)
Dependencies are inferred from the claim text.