Early mesoderm cells, a stable population of mesendoderm cells that has utility for generation of endoderm and mesoderm lineages and multipotent migratory cells (MMC)
First Claim
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1. A method of differentiating primate Pluripotent Stem Cells (pPSCs) into Isl1+ mesoderm cells comprising (a) providing pPSCs;
- (b) contacting the pPSCs with an effective amount of at least one GSK inhibitor selected from the group consisting of a GSK3 inhibitor and a Wnt protein in a cell differentiation medium for a period sufficient to produce mesendoderm cells; and
(c) subsequently contacting the cells obtained from step (b) with an effective amount of a bone morphogenic protein (BMP) and optionally, a GSK inhibitor selected from the group consisting of a GSK3 inhibitor and a Wnt protein for a period sufficient to produce said mesoderm cells; and
(d) optionally, isolating said mesoderm cells.
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Abstract
The present invention relates to the production of multipotent migratory cell (MMCs) which can be differentiated into mesoderm or endoderm lineages. Multipotent Migratory Cells (MMC) are stable and robust and can be passaged at least 20 times (perhaps indefinitely), can be recovered after freezing, reamplified and differentiated into multiple lineages. They are therefore storage stable. The method of producing these cells points to a way to generate a multipotent cell type (mesendoderm) from blastocycts for the generation of therapeutically useful cell types without going through a classical hESC state. The production of multipotent migratory cells, mesendoderm cells and mesoderm cells (Isl1+) is also described.
36 Citations
40 Claims
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1. A method of differentiating primate Pluripotent Stem Cells (pPSCs) into Isl1+ mesoderm cells comprising (a) providing pPSCs;
- (b) contacting the pPSCs with an effective amount of at least one GSK inhibitor selected from the group consisting of a GSK3 inhibitor and a Wnt protein in a cell differentiation medium for a period sufficient to produce mesendoderm cells; and
(c) subsequently contacting the cells obtained from step (b) with an effective amount of a bone morphogenic protein (BMP) and optionally, a GSK inhibitor selected from the group consisting of a GSK3 inhibitor and a Wnt protein for a period sufficient to produce said mesoderm cells; and
(d) optionally, isolating said mesoderm cells. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)
- (b) contacting the pPSCs with an effective amount of at least one GSK inhibitor selected from the group consisting of a GSK3 inhibitor and a Wnt protein in a cell differentiation medium for a period sufficient to produce mesendoderm cells; and
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19. A method of differentiating primate Pluripotent Stem Cells (pPSCs) into Isl1+ mesoderm cells comprising (a) providing pPSCs;
- (b) contacting the pPSCs with an effective amount of at least one GSK inhibitor selected from the group consisting of a GSK3 inhibitor and a Wnt protein in a cell differentiation medium for a period ranging from about 18 hours to about 72 hours to produce mesendoderm cells; and
(c) subsequently contacting the cells obtained from step (b) with an effective amount of a bone morphogenic protein (BMP) and optionally, a GSK inhibitor selected from the group consisting of a GSK3 inhibitor or a Wnt protein for a period of about two days to about 9 days to produce said mesoderm cells; and
(d) optionally, isolating said mesoderm cells.
- (b) contacting the pPSCs with an effective amount of at least one GSK inhibitor selected from the group consisting of a GSK3 inhibitor and a Wnt protein in a cell differentiation medium for a period ranging from about 18 hours to about 72 hours to produce mesendoderm cells; and
- 20. A method of producing Isl1+ mesoderm cells from pPSCs comprising culturing said pPSCs in a differentiation medium with an effective amount of a GSK inhibitor in combination with an effective amount of a bone morphogenic protein and optionally, isolating said Isl1+ mesoderm cells.
- 32. A method of differentiating mesendoderm cells into Isl1+ mesoderm cells comprising contacting said mesendoderm cells with an effective amount of a bone morphogenic protein and optionally, a GSK inhibitor in a cell differentiation medium for a period ranging from about 2-9 days to produce Isl1+ mesoderm cells and optionally, isolating said mesoderm cells.
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Current AssigneeThe University of Georgia Research Foundation, Inc.
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Original AssigneeThe Theuniversity of Georgia Research Foundation Incorporated
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InventorsDalton, Stephen, Reynolds, David
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Primary Examiner(s)Ton, Thaian N
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Application NumberUS12/449,237Publication NumberTime in Patent Office2,834 DaysField of SearchUS Class Current1/1CPC Class CodesA61P 9/00 Drugs for disorders of the ...A61P 9/10 for treating ischaemic or a...C12N 2500/90 Serum-free medium, which ma...C12N 2500/99 Serum-free mediumC12N 2501/155 Bone morphogenic proteins [...C12N 2501/16 Activin; Inhibin; Mullerian...C12N 2501/195 Heregulin, neu differentiat...C12N 2501/415 Wnt; FrizzeledC12N 2501/70 EnzymesC12N 2506/02 from embryonic cellsC12N 5/0603 Embryonic cells production ...C12N 5/0606 Pluripotent embryonic cells...
