Method for making human pluripotent suspension cultures and cells derived therefrom
First Claim
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1. A method of differentiating pluripotent cells comprising:
- a. treating pluripotent stem cells cultured in a planar adherent culture with a chelating agent or enzyme to release cell aggregates from the planar adherent culture;
b. suspending the cell aggregates from the planar adherent culture in a culture medium in the presence of a Rho-kinase inhibitor without centrifuging the cell aggregates and without dissociating the cell aggregates to single cells;
c. diluting the culture using culture media and a Rho-kinase inhibitor to a concentration of cells from about 1 to about 1.5 million cells/ml;
d. transferring the suspension of cell aggregates to a dynamic suspension culture;
e. expanding the suspension of cell aggregates in the dynamic suspension culture to generate pluripotent stem cell clusters; and
f. differentiating the pluripotent stem cell clusters in the dynamic suspension culture system, wherein the step of differentiating comprises differentiating posterior foregut (Stage
3) cells into pancreatic endocrine precursor (Stage
4) cells in a medium supplemented with 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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28 Claims
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1. A method of differentiating pluripotent cells comprising:
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a. treating pluripotent stem cells cultured in a planar adherent culture with a chelating agent or enzyme to release cell aggregates from the planar adherent culture; b. suspending the cell aggregates from the planar adherent culture in a culture medium in the presence of a Rho-kinase inhibitor without centrifuging the cell aggregates and without dissociating the cell aggregates to single cells; c. diluting the culture using culture media and a Rho-kinase inhibitor to a concentration of cells from about 1 to about 1.5 million cells/ml; d. transferring the suspension of cell aggregates to a dynamic suspension culture; e. expanding the suspension of cell aggregates in the dynamic suspension culture to generate pluripotent stem cell clusters; and f. differentiating the pluripotent stem cell clusters in the dynamic suspension culture system, wherein the step of differentiating comprises differentiating posterior foregut (Stage
3) cells into pancreatic endocrine precursor (Stage
4) cells in a medium supplemented with a Cyp26 inhibitor. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28)
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Specification