Method for differentiating human embryonic stem cells into β-cells for the treatment of type I diabetes

• US 9,447,378 B2
• Filed: 04/29/2013
• Issued: 09/20/2016
• Est. Priority Date: 04/27/2012
• Status: Active Grant

• Current Assignee: Massachusetts Institute of Technology
• Original Assignee: Massachusetts Institute of Technology
• Inventors: Millman, Jeffrey R., Colton, Clark K., Dilenno, Amanda
• Primary Examiner(s): BERTOGLIO, VALARIE E
• Application Number:
  US13/873,020
• Publication Number:
  US 20130287743A1
• Time in Patent Office: 1,240 Days
• US Class Current: 1/1
• CPC Class Codes: C12N 2500/02 Atmosphere, e.g. low oxygen conditions
• CPC Class Codes: C12N 2506/02 from embryonic 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 invention provides inter alia methods for differentiating embryonic stem cells into insulin producing cells, as well as compositions comprising such cells, and therapeutic uses of such compositions.

Claims

1. A method for producing insulin+ and/or C-peptide+ cells, comprising

(1) differentiating human pluripotent stern cells to posterior foregut endoderm in vitro, at an oxygen partial pressure to which cells are exposed (pO_{2 cell}) that is greater than 7 mmHg and less than 142 mmHg, in the presence of a TGF-β

superfamily member and a retinoic acid receptor agonist, and

(2) differentiating posterior foregut endoderm to insulin+ and/or C-peptide+ cells at a pO_{2 cell} that is 142 mmHg or higher.

2. The method of claim 1, wherein the human pluripotent stem cells are differentiated on an oxygen permeable membrane.

3. The method of claim 2, wherein the oxygen permeable membrane is an oxygen permeable silicone rubber membrane.

4. The method of claim 2, wherein the oxygen permeable silicone rubber membrane is coated with extracellular matrix (ECM).

5. The method of claim 1, wherein the iman pluripotent stem cells are human embryonic stem cells.

11. The method of claim 1, wherein the pO_2cell of step (1) 36 mmHg.

13. The method of claim 1, wherein the pO_2cell of step (1) is 7 mmHg to 57 mmHg.

14. The method of claim 1, wherein the pO_2cell of step (1) is 21 mmHg to 57 mmHg.

15. The method of claim 1, wherein the pO_2cell of step (2) is 142 mmHg.

16. The method of claim 1, wherein the pO_2cell of step (2) is higher than 142 mmHg.

6. A method for producing insulin+ and/or C-peptide+ cells comprising

(1) differentiating human pluripotent stem cells to definitive endoderm in the presence of a TGF-β

superfamily member at a pO_2cell that is in a range of 7 mmHg to 57 mmHg,

(2) differentiating definitive endoderm to posterior foregut endoderm in the presence of a retinoic acid receptor antagonist at a pO_2cell that is in a range of 21 mmHg to 71 mmHg, and

(3) differentiating posterior foregut endoderm to insulin+ and/or C-peptide+ cells at a pO_2cell that is 142 mmHg or higher.

7. The method of claim 6, wherein the the pO_2cell of step (1) is 36 mmHg.

8. The method of claim 6, wherein the pO_2cell of step (2) is 36 mmHg.

9. The method of claim 6, wherein the the pO_2cell of step (3) is 142 mmHg.

10. The method of claim 6, wherein the the pO_2cell of step (3) is higher than 142 mmHg.

12. The method of claim 6, wherein the human pluripotent stem cells are human embryonic stem cells.