SUSPENSION AND CLUSTERING OF HUMAN PLURIPOTENT STEM CELLS FOR DIFFERENTIATION INTO PANCREATIC ENDOCRINE CELLS
First Claim
1. A method of expanding and differentiating pluripotent cells in a dynamically agitated suspension culture system comprising culturing pluripotent cells to aggregated cell clusters in a planar adherent culture and differentiating the pluripotent cell clusters in a dynamic agitated suspension culture system, wherein the step of differentiating comprises use of a Cyp26 inhibitor.
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Abstract
The present invention provides methods of preparing aggregated pluripotent stem cell clusters for differentiation. Specifically, the invention discloses methods of differentiating pluripotent cells into beta cell, cardiac cell and neuronal cell lineages using suspension clustering. The methods involve preparing the aggregated cell clusters followed by differentiation of these clusters.
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Citations
21 Claims
- 1. A method of expanding and differentiating pluripotent cells in a dynamically agitated suspension culture system comprising culturing pluripotent cells to aggregated cell clusters in a planar adherent culture and differentiating the pluripotent cell clusters in a dynamic agitated suspension culture system, wherein the step of differentiating comprises use of a Cyp26 inhibitor.
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6. A method of increasing the percentage of cells in G0/G1 phase of the cell cycle comprising expanding pluripotent to aggregated cell clusters in a planar adherent culture in an environment that includes from about 0.1% to about 2% of bovine serum albumin, transferring the clusters of pluripotent stem cells from the planar adherent culture to a dynamic suspension and culturing the cells in the dynamic suspension in a media supplemented with small molecule and optionally a TGFβ
- family member.
- View Dependent Claims (7, 8)
- 9. A method of differentiating pluripotent cells to definitive endoderm comprising expanding pluripotent to aggregated cell clusters in a planar adherent culture in an environment that includes from about 0.1% to about 2% of bovine serum albumin, transferring the clusters of pluripotent stem cells from the planar adherent culture to a dynamic suspension and culturing the cells in the dynamic suspension in a media supplemented with MCX and GDF8 or WNT3A and activin A.
- 11. A method of expanding and differentiating pluripotent stem cells in a dynamically agitated suspension culture system comprising expanding pluripotent stems to aggregated cell clusters in a planar adherent culture, transferring the clusters of pluripotent stem cells from the planar adherent culture to a dynamic suspension and differentiating the pluripotent cell clusters in a dynamic agitated suspension culture system, wherein the cells are expanded to aggregated cell clusters in an environment that includes from about 0.1% to about 2% of bovine serum albumin.
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15. A method of expanding and differentiating pluripotent cells in a dynamically agitated suspension culture system comprising:
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a. expanding the pluripotent cells to aggregated cell clusters in planar adherent cultures in an environment that includes from about 0.1% to about 2% of bovine serum albumin, b. transferring the clusters of pluripotent cells from the planar adherent culture to a dynamic suspension culture using an enzyme or chelating agent, c. maintaining the cell clusters in a dynamically agitated suspension culture system, and d. differentiating the pluripotent cell clusters in a dynamic agitated suspension culture system to generate a pancreatic precursor cell population, a neural precursor cell population or a cardiomyocte precursor population, wherein the differentiation environment includes an oxygen range from about hypoxia to about 30% of ambient, a lipid in the range of 0.1% to about 2%, or the combination thereof. - View Dependent Claims (16, 17, 18, 19, 20, 21)
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Specification