Differentiation of human embryonic stem cells
View Patent ↗The present invention provides methods to promote the differentiation of pluripotent stem cells into cells expressing markers characteristic of the pancreatic endocrine lineage that co-express PDX1, NKX6.1, but do not express CDX2 and NGN3.
1. A method to differentiate a population of human pluripotent stem cells into a population of pancreatic endoderm cells that co-express PDX-1 and NKX-6.1, but do not express CDX-2 and NGN-3 comprising the steps of:
(a) culturing human pluripotent stem cells,
(b) differentiating the human pluripotent stem cells into definitive endoderm cells by treating the human pluripotent stem cells with a medium supplemented with a TGF-β receptor agonist,
(c) differentiating the definitive endoderm cells into a population of pancreatic endoderm cells that co-express PDX-1 and NKX6.1, but do not express CDX2 and NGN3 by;
i) culturing the definitive endoderm cells in a first medium supplemented with FGF7, followed by
ii) culturing the cells from step i) in a second medium supplemented with FGF7, a factor capable of inhibiting BMP, activin A, retinoic acid, and a hedgehog signaling pathway inhibitor, and
(d) selecting cells which co-express PDX-1 and NKX6.1 and do not express CDX2 and NGN3 to obtain a purified population of pancreatic endoderm cells that co-express PDX-1 and NKX-6.1, but do not express CDX-2 and NGN-3.
2. The method of claim 1 , wherein the factor capable of inhibiting BMP is noggin.
3. The method of claim 1 , wherein the hedgehog signaling pathway inhibitor is cyclopamine-KAAD.
4. A method of differentiating definitive endoderm cells into a purified population of pancreatic endoderm cells that co-express PDX-1 and NKX6.1, but do not express CDX2 and NGN3 by:
(a) treating definitive endoderm cells with a first medium supplemented with FGF7; and
(b) culturing the treated definitive endoderm cells in a second medium supplemented with FGF7, a factor capable of inhibiting BMP, activin A, retinoic acid, and a hedgehog signaling pathway inhibitor to obtain a population of pancreatic endoderm cells that co-express PDX- 1 and NKX-6.1, but do not express CDX-2 and NGN-3, and
(c) selecting cells which co-express PDX-1 and NKX6.1 and do not express CDX2 and NGN3 to obtain a purified population of pancreatic endoderm cells that co-express PDX-1 and NKX-6.1, but do not express CDX-2 and NGN-3.