Pluripotent stem cell culture on micro-carriers

• US 9,969,972 B2
• Filed: 02/25/2015
• Issued: 05/15/2018
• Est. Priority Date: 11/20/2008
• Status: Active Grant

• Current Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Inventors: Nelson, Shelley
• Primary Examiner(s): Ton, Thaian N
• Assistant Examiner(s): Montanari, David A.
• Application Number:
  US14/631,019
• Publication Number:
  US 20150166950A1
• Time in Patent Office: 1,175 Days
• CPC Class Codes: C12N 2501/70 Enzymes
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2509/00 Methods for the dissociation of cells, e.g. specific use of enzymes
• CPC Class Codes: C12N 2531/00 Microcarriers
• CPC Class Codes: C12N 5/0075 using microcarriers
• CPC Class Codes: C12N 5/0602 Vertebrate cells
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0676 Pancreatic cells

Abstract

The present invention is directed to methods for the growth, expansion and differentiation of pluripotent stem cells on micro-carriers.

Claims

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 μ

M to about 10 μ

M.

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 μ

M to about 10 μ

M 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 μ

M to about 5 μ

M 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×

10⁴ cells/cm².

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 μ

M to about 10 μ

M.

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.