Cell culture platform for single cell sorting and enhanced reprogramming of ipscs
AssigneeFate Therapeutics, Inc.
InventorsBahram Valamehr, Ramzey Abujarour, Peter Flynn
Life of this patent
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Abstract
The invention provides cell culture conditions for culturing stem cells, including feeder-free conditions for generating and culturing human induced pluripotent stem cells (iPSCs). More particularly, the invention provides a culture platform that allows long-term culture of pluripotent cells in a feeder-free environment; reprogramming of cells in a feeder-free environment; single-cell dissociation of pluripotent cells; cell sorting of pluripotent cells; maintenance of an undifferentiated status; improved efficiency of reprogramming; and generation of a naïve pluripotent cell.
Claims33
-126. (canceled)
An induced pluripotent cell, a cell line thereof, or a population thereof, wherein the cell has improved genomic stability, and wherein the cell is obtained by a method comprising the steps of:
- (a) introducing into cells without ground state pluripotency one or more polynucleotides encoding proteins comprising Oct4, and optionally, one or more of Sox2 and Klf4, wherein the cells without ground state pluripotency are somatic cells, progenitor cells, or multipotent cells;
- and
- (b) culturing the cells resulting from step (a) in a culture medium composition comprising a Rock inhibitor, and one or more of a GSK3 inhibitor, a MEK inhibitor and a TGFβ inhibitor for a sufficient amount of time to reprogram the cells without ground state pluripotency into pluripotent cells, wherein the pluripotent cells have improved genomic stability, as indicated by a lower propensity for genomic abnormalities than pluripotent cells obtained by step (a) but not cultured in the culture medium composition of (b).
A single dissociated induced pluripotent cell, a cell line thereof, or a population thereof, wherein the cell has improved genomic stability, and wherein the cell is obtained by a method comprising the steps of:
- (a) introducing into cells without ground state pluripotency one or more polynucleotides encoding proteins comprising Oct4, and optionally, one or more of Sox2 and Klf4, wherein the cells without ground state pluripotency are somatic cells, progenitor cells, or multipotent cells;
- (b) culturing the cells resulting from step (a) in a culture medium composition comprising a Rock inhibitor, and one or more of a GSK3 inhibitor, a MEK inhibitor and a TGFβ inhibitor for a sufficient amount of time to reprogram the cells without ground state pluripotency into pluripotent cells, wherein the pluripotent cells have improved genomic stability, as indicated by a lower propensity for genomic abnormalities than pluripotent cells obtained by step (a) but not cultured in the culture medium composition of (b);
- (c) contacting the cells resulting from step (b) with one or more dissociating enzyme to obtain single cell dissociated pluripotent cells;
- and
- (d) sorting the single cell dissociated pluripotent cells by selecting and isolating one or more single dissociated pluripotent cells expressing one or more markers of pluripotency to obtain a single dissociated induced pluripotent cell or a population thereof.
An induced pluripotent cell, a cell line thereof, or a population thereof, wherein the cell has improved genomic stability, and wherein the cell is obtained by a method comprising the steps of:
- culturing an induced pluripotent cell or population thereof in a culture medium composition comprising a Rock inhibitor, and one or more of a GSK3 inhibitor, a MEK inhibitor and a TGFβ inhibitor for a sufficient amount of time, thereby obtaining induced pluripotent cells having improved genomic stability, as indicated by a lower propensity for genomic abnormalities than the pluripotent cells not cultured in said culture medium composition;
- wherein the induced pluripotent cells for culturing are obtained by introducing into somatic cells one or more polynucleotides encoding proteins comprising Oct4, and optionally, one or more of Sox2 and Klf4, thereby generating induced pluripotent cells.
A method of manufacturing therapeutic cells comprising an induced pluripotent cell, a cell line thereof, or a population thereof, wherein the method comprises:
- (a) introducing into cells without ground state pluripotency one or more polynucleotides encoding proteins comprising Oct4, and optionally, one or more of Sox2 and Klf4, wherein the cells without ground state pluripotency are somatic cells, progenitor cells, or multipotent cells;
- and
- (b) culturing the cells resulting from step (a) in a culture medium composition comprising a Rock inhibitor, and one or more of a GSK3 inhibitor, a MEK inhibitor and a TGFβ inhibitor for a sufficient amount of time to reprogram the cells without ground state pluripotency into pluripotent cells, wherein the pluripotent cells have improved genomic stability, as indicated by a lower propensity for genomic abnormalities than pluripotent cells obtained by step (a) but not cultured in the culture medium composition of (b).
A method of manufacturing therapeutic cells comprising a single dissociated induced pluripotent cell, a cell line thereof, or a population thereof, wherein the method comprises:
- (a) introducing into cells without ground state pluripotency one or more polynucleotides encoding proteins comprising Oct4, and optionally, one or more of Sox2 and Klf4, wherein the cells without ground state pluripotency are somatic cells, progenitor cells, or multipotent cells;
- (b) culturing the cells resulting from step (a) in a culture medium composition comprising a Rock inhibitor, and one or more of a GSK3 inhibitor, a MEK inhibitor and a TGFβ inhibitor for a sufficient amount of time to reprogram the cells without ground state pluripotency into pluripotent cells, wherein the pluripotent cells have improved genomic stability, as indicated by a lower propensity for genomic abnormalities than pluripotent cells obtained by step (a) but not cultured in the culture medium composition of (b);
- (c) contacting the cells resulting from step (b) with one or more dissociating enzyme to obtain single cell dissociated pluripotent cells;
- and
- (d) sorting the single cell dissociated pluripotent cells by selecting and isolating one or more single dissociated pluripotent cells expressing one or more markers of pluripotency to obtain a single dissociated induced pluripotent cell or a population thereof.
A method of manufacturing therapeutic cells comprising a genomically stable induced pluripotent cell, a cell line thereof, or a population thereof, wherein the method comprises:
- culturing an induced pluripotent cell or a population thereof in a culture medium composition comprising a Rock inhibitor, and one or more of a GSK3 inhibitor, a MEK inhibitor and a TGFβ inhibitor for a sufficient amount of time, thereby obtaining induced pluripotent cells having improved genomic stability, as indicated by a lower propensity for genomic abnormalities than the pluripotent cells without being cultured in said culture medium composition;
- wherein the induced pluripotent cells for culturing are obtained by introducing into somatic cells one or more polynucleotides encoding proteins comprising Oct4, and optionally, one or more of Sox2 and Klf4, thereby generating induced pluripotent cells.
Dependencies are inferred from the claim text.