Generation of anterior foregut endoderm from pluripotent cells
The invention is directed to in vitro methods of inducing differentiation of anterior foregut endoderm and the enriched populations of anterior foregut endoderm produced by such methods. Such enriched populations are useful for studies of the molecular events that occur during differentiation and for generating cells for cell replacement therapy.
1. A method of preparing an enriched population of anterior foregut endoderm cells from pluripotent cells, the method comprising:
(i) culturing isolated mammalian pluripotent cells under conditions that induce the pluripotent cells to form definitive endoderm cells; and
(ii) culturing the definitive endoderm cells in the presence of an inhibitor of a bone morphogenetic protein (“BMP”) and an inhibitor of a transforming growth factor beta (“TGF-β”) such that definitive endoderm cells form anterior foregut endoderm cells.
2. The method of claim 1 , wherein the pluripotent cells are cultured in the presence of basic fibroblast growth factor (“bFGF”), bone morphogenetic protein (“BMP”) and Activin A to induce said pluripotent cells to form said definitive endoderm cells.
3. The method of claim 1 , wherein the definitive endoderm cells are cultured in the absence of Activin A.
4. The method of claim 1 , wherein said inhibitor of bone morphogenetic protein (“BMP”) is Noggin or Chordin.
5. The method of claim 4 , wherein said inhibitor of bone morphogenetic protein (“BMP”) is Noggin.
6. The method of claim 1 , wherein the inhibitor of transforming growth factor beta (“TGF-beta”) signaling is SB-431542.
7. The method of claim 1 , wherein the pluripotent cells are cultured for 1-4 days.
8. The method of claim 1 , wherein the definitive endoderm cells are cells are cultured for 2-6 days.
9. The method of claim 1 , further comprising isolating said anterior foregut endoderm cells.
10. The method of claim 1 , wherein the pluripotent cells are ES cells.
11. The method of claim 1 , wherein the pluripotent cells are iPS cells.
12. The method of claim 11 , wherein the pluripotent cells are human cells.
13. The method of claim 1 , wherein the definitive endoderm cell is an embryoid body cell.