Method for making human pluripotent suspension cultures and cells derived therefrom

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.