Differentiation of human embryonic stem cells into pancreatic endocrine cells using thyroid hormone and/or alk5, an inhibitor of tgf-beta type 1 receptor
The present invention provides methods to promote differentiation of pluripotent stem cells to pancreatic endoderm cells expressing PDX1, NKX6.1, and HB9. In particular, the methods encompass culturing Stage 4 to Stage 6 cells with a thyroid hormone (e.g. T3), an ALK5 inhibitor, or both.
1. An in vitro cell culture for differentiating cells derived from human pluripotent stem cells comprising:
a) a culture vessel;
b) a volume of differentiation medium; and
c) a population of differentiated cells derived from human pluripotent stem cells wherein at least ten percent of said differentiated cells co-express PDX1, NKX6.1 and HB9, wherein said differentiation medium comprises a growth medium supplemented with a thyroid hormone selected from the group consisting of triiodothyronine, thyroxine, analogues of triiodothyronine, analogues of thyroxine and mixtures thereof, or
both the thyroid hormone and ALK5 inhibitor.
2. The cell culture of claim 1 , wherein said ALK5 inhibitor is SD208, ALK5 inhibitor II, or ALX-270-445.
3. The cell culture of claim 2 , wherein said growth medium is MCDB131.
4. The cell culture of claim 1 , wherein said differentiated cells comprise cells expressing pancreatic endocrine cell markers.
5. The cell culture of claim 2 , wherein said growth medium is further supplemented with one or more of:
a) a smoothened receptor inhibitor selected from MRT10 or cyclopamine;
b) a SHH signaling pathway antagonist selected from SANT-1 or HPI-1;
c) a BMP Receptor Inhibitor selected from LDN-193189, Noggin or Chordin;
d) a PKC activator selected from TPB, PDBu, PMA, and ILV;
e) a fibroblast growth factor selected from FGF7 or FGF10;
f) retinoic acid;
g) ascorbic acid;
h) heparin; and
i) zinc sulfate.
6. The cell culture of claim 5 , wherein said growth medium is further supplemented with SANT-1, retinoic acid and ascorbic acid.
7. An in vitro cell culture comprising a population of differentiated human pluripotent stem cells expressing pancreatic endocrine cell markers wherein at least ten percent of said cells express HB9, PDX1 and NKX6.1.
8. The cell culture of claim 7 , wherein at least thirty percent of the cells that express both NKX6.1 and PDX1 also express HB9.
9. The cell culture of claim 7 , wherein at least fifty percent of the cells that express both NKX6.1 and PDX1 also express HB9.
10. The cell culture of claim 7 , wherein at least eighty percent of the cells that express both NKX6.1 and PDX1 also express HB9.
11. The cell culture of claim 4 , wherein said differentiated cells comprise cells expressing β cell markers.
12. The cell culture of claim 11 , wherein said differentiated cells produce insulin.