Methods for producing insulin-secreting beta cells from human pluripotent stem cells

1. A method of culturing posterior foregut endoderm cells to produce cells of the pancreatic lineage, the method comprising the step of:

(a) culturing the posterior foregut endoderm cells for about 7 days in the presence of a serum free chemically defined medium (ITSFINE) comprising an effective amount of;

i) insulin, transferrin, selenium, FGF7, islet neogenesis associated peptide (INGAP), nicotinamide, and exendin-4, and

ii) Noggin,

wherein the cells are cultured on an extracellular matrix coated onto a membrane, and

wherein pancreatic lineage cells are produced, wherein the pancreatic lineage cells are insulin + cells.

2. A method of culturing human pluripotent stem cells to produce cells of the pancreatic lineage, the method comprising the steps of:

(a) culturing human pluripotent stem cells for about 3 days in the presence of a chemically defined medium under conditions that induce formation of mesendoderm/primitive streak and definitive endoderm cells, wherein the medium comprises an effective amount of;

i) fibroblast growth factor (FGF),

ii) Activin A, and

iii) bone morphogenetic protein (BMP);

(b) culturing the cells from step (a) for about 3 days in the presence of a chemically defined medium comprising an effective amount of;

i) insulin, transferrin, and selenium (ITS), and

ii) fibroblast growth factor (FGF);

(c) culturing the cells from step (b) for about 4 days in the presence of a chemically defined medium under conditions that induce formation of posterior foregut cells, wherein the medium comprises an effective amount of;

i) insulin, transferrin, and selenium (ITS), and

ii) Noggin-Nicotinamide-Retinoic acid (NNR); and

(d) culturing the cells from step (c) for about 7 days in the presence of a serum free chemically defined medium (ITSFINE) comprising an effective amount of;

i) insulin, transferrin, selenium, FGF7, islet neogenesis associated peptide (INGAP), nicotinamide, and exendin-4, and

ii) Noggin,

wherein the cells are cultured on an extracellular matrix coated onto a membrane, and

wherein pancreatic lineage cells are produced, wherein the pancreatic lineage cells are insulin + cells.

3. The method of claim 2 , wherein the stem cells are selected from the group consisting of human embryonic stem cells and human induced pluripotent stem cells.

4. The method of claim 2 , wherein the extracellular matrix is selected from the group consisting of Matrigel™ and Laminin511.

5. The method of claim 4 , wherein the extracellular matrix is Matrigel™.

6. The method of claim 2 , wherein the membrane is a polycarbonate or polyester membrane.

7. The method of claim 6 , wherein the polycarbonate or polyester membrane is Transwell™.

8. The method of claim 2 , wherein in step (a) the effective amount of:

i) FGF ranges from about 10 ng/ml to about 200 ng/ml,

ii) Activin A ranges from about 10 ng/ml to about 200 ng/ml, and

iii) BMP ranges from about 5 ng/ml to about 50 ng/ml.

9. The method of claim 8 , wherein the FGF is selected from the group consisting of FGF2, FGF4, FGF7, FGF10 and the mixtures thereof.

10. The method of claim 8 , wherein the BMP is selected from the group consisting of BMP2, BMP4, BMP7 and the mixtures thereof.

11. The method of claim 2 , wherein in step (b) the FGF is selected from the group consisting of FGF2, FGF7 and the mixtures thereof.

12. The method of claim 2 , wherein in step (b) the effective amount of FGF ranges from about 10-200 ng/ml.

13. The method of claim 2 , wherein in step (c) the effective amount of NNR comprises;

i) Nicotinamide at about 10 mM,

ii) Noggin at from about 10 ng/ml to about 1000 ng/ml, and

iii) Retinoic acid at about 100 nM to about 10 μM.

14. The method of claim 2 , wherein in step (d) the effective amount of:

i) FGF7 ranges from about 10 ng/ml to about 200 ng/ml,

ii) Nicotinamide is about 10 mM,

iii) Exendin-4 ranges from about 1 nM to about 100 nM, and

iv) Noggin ranges from about 10 ng/ml to about 300 ng/ml.

15. The method of claim 2 , wherein the pancreatic lineage cells co-express PDX-1, Nkx6.1, Ngn3, Sox9, and FoxA2.

16. The method of claim 2 , wherein the pancreatic lineage insulin + cells comprise PDX1 + insulin + cells.

17. The method of claim 2 , wherein the pancreatic lineage cells produce and process insulin endogenously and secrete C-peptide.

18. The method of claim 1 , wherein the extracellular matrix is selected from the group consisting of Matrigel™ and Laminin511.

19. The method of claim 18 , wherein the extracellular matrix is Matrigel™.

20. The method of claim 1 , wherein the membrane is a polycarbonate or polyester membrane.

21. The method of claim 20 , wherein the polycarbonate or polyester membrane is Transwell™.