PDX1 positive foregut endoderm cells and methods of production
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 cell culture comprising:
human pancreatic and duodenal homeobox 1 (PDX1)-negative definitive endoderm cells;
a medium comprising an effective amount of B27, a retinoid and a fibroblast growth factor (FGF), to differentiate the PDX1-negative definitive endoderm cells to PDX1-positive foregut endoderm cells; and
PDX1-positive foregut endoderm cells,
such that the amount of PDX1 expressed by the PDX1-positive foregut endoderm cells is increased in the in vitro cell culture as compared to a second in vitro cell culture comprising human PDX1-negative definitive endoderm cells, PDX1-positive foregut endoderm cells, and the medium without B27.
2. The in vitro cell culture of claim 1 , wherein B27 is present in the in vitro cell culture at a concentration of about 0.1% v/v to about 20% v/v.
3. The in vitro cell culture of claim 1 , wherein the retinoid is retinoic acid (RA).
4. The in vitro cell culture of claim 3 , wherein RA is present in the in vitro cell culture and the second in vitro cell culture at a concentration of about 0.01 μM to about 50 μM.
5. The in vitro cell culture of claim 1 , wherein the FGF is FGF-10 or FGF-4.
6. The in vitro cell culture of claim 1 , wherein the in vitro cell culture and the second in vitro cell culture further comprise a transforming growth factor beta (TGFβ) family member.
7. The in vitro cell culture of claim 6 , wherein the TGFβ family member is Activin A.
8. An in vitro method of producing human PDX1-positive foregut endoderm cells, comprising:
(a) culturing a population of human pluripotent stem cells in vitro,
(b) differentiating the human pluripotent stem cells in vitro in the presence of an exogenous transforming growth factor beta (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 in vitro to obtain PDX1-positive foregut endoderm cells, wherein the PDX1-positive foregut endoderm cells are multipotent and can differentiate into cells, tissues or organs from the posterior portion of the gut tube.