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 a population comprising at least 15% definitive endoderm cells, and
(c) differentiating the definitive endoderm cells of step (b) in the presence of a retinoid to obtain PDX1-positive foregut endoderm cells, wherein said PDX1-positive foregut endoderm cells are multipotent and can differentiate into cells, tissues or organs from the anterior portion of the gut tube.
2. The method of claim 1 further comprising the step of allowing sufficient time for PDX1-positive human foregut endoderm cells to form, wherein said sufficient time for PDX1-positive human foregut endoderm cells to form has been determined by detecting the presence of PDX1-positive human foregut endoderm cells in said cell population.
3. The method of claim 1 , wherein at least 2% of said human definitive endoderm cells differentiate into PDX1-positive foregut endoderm cells.
4. The method of claim 1 , wherein at least 5% of said human definitive endoderm cells differentiate into PDX1-positive foregut endoderm cells.
5. The method of claim 1 , wherein at least 10% of said human definitive endoderm cells differentiate into PDX1-positive foregut endoderm cells.
6. The method of claim 1 , wherein at least 25% of said human definitive endoderm cells differentiate into PDX1-positive foregut endoderm cells.
7. The method of claim 2 , wherein detecting the presence of PDX1-positive foregut endoderm cells in said cell population comprises detecting the expression of PDX1.
8. The method of claim 7 , wherein the expression of PDX1 is greater than the expression of a marker selected from the group consisting of alpha-fetoprotein (AFP), SOX7, SOX1, ZIC1 and NFM in said PDX1-positive foregut endoderm cells.
9. The method of claim 7 , wherein the expression of said PDX1 is determined by quantitative polymerase chain reaction (Q-PCR).
10. The method of claim 7 , wherein the expression of PDX1 is determined by immunocytochemistry.
11. The method of claim 1 , wherein said retinoid is retinoic acid (RA).
12. The method of claim 11 , wherein RA is provided in a concentration ranging from about 0.01 μM to about 50 μM.
13. The method of claim 11 , wherein RA is provided when said population is about 4-days-old.
14. The method of claim 1 , wherein said growth factor of the TGFβ superfamily is activin A.
15. The method of claim 14 , wherein said activin A is provided to said population of human pluripotent stem cells at a concentration of about 100 ng/ml.
16. The method of claim 1 , wherein said growth factor of the TGFβ superfamily is removed from said cell population prior to providing said retinoid.
17. The method of claim 1 , wherein said retinoid is removed from the cell population after 3 days.