PDX1 expressing endoderm
Disclosed herein are cell cultures comprising PDX1-positive endoderm cells and methods of producing the same. Also disclosed herein are cell populations comprising substantially purified PDX1-positive endoderm cells as well as methods for enriching, isolating and purifying PDX1-positive endoderm cells from other cell types. Methods of identifying differentiation factors capable of promoting the differentiation of endoderm cells, such as PDX1-positive foregut endoderm cells and PDX1-negative definitive endoderm cells, are also disclosed.
1. An in vitro method of producing human PDX1-positive foregut endoderm cells, said method comprising:
(a) culturing a population of human pluripotent stem cells,
(b) differentiating the human pluripotent stem cells in the presence of an exogenous TGFβ superfamily growth factor to obtain definitive endoderm cells,
(c) removing the exogenous TGFβ superfamily growth factor from the definitive endoderm cells, and
(d) differentiating the definitive endoderm cells of step (c) in the presence of a retinoid to obtain PDX1-positive foregut endoderm cells.
2. The in vitro method of claim 1 , wherein step (d) further comprises providing an FGF-family growth factor.
3. The in vitro method of claim 2 , wherein the FGF-family growth factor is provided at approximately the same time as said retinoid.
4. The in vitro method of claim 2 , wherein said FGF-family growth factor comprises FGF-10.
5. An in vitro method of producing human PDX1-positive endoderm cells, said method comprising:
(a) culturing a population of pluripotent human stem cells,
(b) differentiating the human pluripotent stem cells in the presence of an exogenous TGFβ superfamily growth factor to obtain definitive endoderm cells,
(c) removing the exogenous TGFβ superfamily growth factor from the definitive endoderm cells, and
(d) differentiating the definitive endoderm cells of step (c) in the presence of a retinoid to obtain a population of PDX1-positive endoderm cells, wherein at least 25% of said cell population expresses PDX1.
6. An in vitro method of producing human PDX1-positive endoderm cells, said method comprising:
(a) culturing a population of pluripotent human stem cells,
(b) differentiating the population of pluripotent human cells in the presence of an exogenous TGFβ superfamily growth factor to obtain definitive endoderm cells,
(c) removing the exogenous TGFβ superfamily growth factor from the definitive endoderm cells, and
(d) differentiating the definitive endoderm cells of step (c) in the presence of a retinoid to obtain a population of PDX1-positive endoderm cells, wherein the expression of PDX1 is greater than the expression of a marker selected from the group consisting of α-fetoprotein, SOX7, SOX1, ZIC1 and NFM in said population of PDX1-positive endoderm cells as determined by quantitative polymerase chain reaction.
7. The in vitro method of claim 1 , wherein the expression of PDX1 is determined by immunocytochemistry.
8. The in vitro method of claim 1 , wherein said retinoid is retinoic acid (RA).
9. The in vitro method of claim 8 , wherein RA is provided in a concentration ranging from 0.01 μM to 50 μM.
10. The in vitro method of claim 8 , wherein RA is provided in a concentration ranging from 0.1 μM to 10 μM.
11. The in vitro method of claim 1 , wherein the exogenous TGFβ superfamily growth factor comprises activin A.
12. The in vitro method of claim 5 , wherein the expression of PDX1 is determined by immunocytochemistry.
13. The in vitro method of claim 5 , wherein said retinoid is RA.
14. The in vitro method of claim 13 , wherein RA is provided in a concentration ranging from 0.01 μM to 50 μM.
15. The in vitro method of claim 13 , wherein RA is provided in a concentration ranging from 0.1 μM to 10 μM.
16. The in vitro method of claim 5 , wherein the exogenous TGFβ superfamily growth factor comprises activin A.
17. The in vitro method of claim 6 , wherein said retinoid is RA.
18. The in vitro method of claim 17 , wherein RA is provided in a concentration ranging from 0.01 μM to 50 μM.
19. The in vitro method of claim 17 , wherein RA is provided in a concentration ranging from 0.1 μM to 10 μM.
20. The in vitro method of claim 6 , wherein the exogenous TGFβ superfamily growth factor comprises activin A.