Pluripotent stem cell culture on micro-carriers

1. A method to differentiate human pluripotent stem cells to insulin-expressing cells on micro-carriers comprising the steps of:

a. attaching the human pluripotent stem cells to a volume of dextran micro-carriers;

b. differentiating the human pluripotent stem cells on the dextran microcarriers to definitive endoderm cells using Activin A;

c. differentiating the definitive endoderm cells on the dextran microcarriers into pancreatic endoderm cells;

d. differentiating the pancreatic endoderm cells on the dextran microcarriers into pancreatic endocrine cells; and

e. differentiating the pancreatic endocrine cells on the dextran microcarriers into insulin-expressing cells, wherein the step of attaching comprises culturing the human pluripotent stem cells in medium containing a Rho kinase inhibitor at a concentration of between about 0.25 μM to about 10 μM.

2. The method of claim 1 , wherein the micro-carriers are coated with denatured collagen.

3. The method of claim 1 , wherein the dextran micro-carriers are agitated.

4. The method of claim 1 , wherein the Rho kinase inhibitor is Y27632 or Glycyl-H-1152 dihydrochloride.

5. The method of claim 1 , wherein the step of attaching comprises culturing the human pluripotent stem cells in medium containing from about 1 μM to about 10 μM of the Rho kinase inhibitor Y27632.

6. The method of claim 1 , wherein the attaching comprises culturing the human pluripotent stem cells in medium containing from about 0.25 μM to about 5 μM of the Rho kinase Glycyl-H-1152 dihydrochloride.

7. The method of claim 1 , wherein the step of attaching comprises seeding at least 3.0×10 4 cells/cm 2 .

8. The method of claim 1 , wherein the method further comprises releasing the pancreatic endocrine cells by treatment with an enzyme.

9. A method to differentiate human pluripotent stem cells to pancreatic endoderm cells on micro-carriers comprising the steps of:

a. differentiating human pluripotent stem cells attached to dextran microcarriers to definitive endoderm cells using Activin A; and

b. differentiating the definitive endoderm cells on the dextran microcarriers into pancreatic endoderm cells, wherein the human pluripotent stem cells were attached to the dextran microcarriers using a medium containing a Rho kinase inhibitor at a concentration of between about 0.25 μM to about 10 μM.

10. The method of claim 9 , wherein the method further comprises differentiating the pancreatic endoderm cells on the dextran microcarriers into pancreatic endocrine cells.

11. The method of claim 10 , further comprising differentiating the pancreatic endocrine cells on the dextran microcarriers into insulin-expressing cells.

12. The method of claim 9 , wherein the dextran micro-carriers are coated with denatured collagen.

13. The method of claim 9 , wherein the micro-carriers are agitated.

14. The method of claim 9 , wherein the Rho kinase inhibitor is Y27632 or Glycyl-H-1152 dihydrochloride.

15. A method to differentiate human pluripotent stem cells to pancreatic endoderm cells on micro-carriers comprising the steps of:

a. differentiating human pluripotent stem cells attached to dextran microcarriers to definitive endoderm cells using Activin A; and

b. differentiating the definitive endoderm cells on the dextran microcarriers into pancreatic endoderm cells.