Method for differentiating human embryonic stem cells into β-cells for the treatment of type I diabetes
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.
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.
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.
11. The method of claim 1 , wherein the pO 2cell of step (1) 36 mmHg.
12. The method of claim 6 , wherein the human pluripotent stem cells are human embryonic stem cells.
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.