Ex vivo proliferation of epithelial cells
The technology relates in part to methods and compositions for ex vivo proliferation and expansion of epithelial cells.
1. A method for proliferating epithelial cells ex vivo, comprising:
expanding the number of cells in an originating epithelial cell population derived from induced pluripotent stem cells (iPSCs) under serum-free and feeder-cell free expansion culture conditions, thereby generating an expanded epithelial cell population, wherein the expansion culture conditions comprise one or more agents that inhibit transforming growth factor beta (TGF-beta) signaling in the population.
2. The method of claim 1 , the one or more agents that inhibit transforming growth factor beta (TGF-beta) signaling in the population comprise one or more inhibitors of ALK5, ALK4, and/or ALK7.
3. The method of claim 2 , wherein the one or more inhibitors of ALK5, ALK4, and/or ALK7 are selected from A83-01, GW788388, RepSox, and SB 431542.
4. The method of claim 1 , wherein the expansion culture conditions comprise a second agent that modulates cytoskeletal structure in the population.
5. The method of claim 4 , wherein the second agent comprises an inhibitor selected from a Rho-associated protein kinase inhibitor, a p21-activated kinase (PAK) inhibitor, and a myosin II inhibitor.
6. The method of claim 5 , wherein the Rho-associated protein kinase inhibitor is selected from Y-27632, SR 3677, thiazovivin, HA1100 hydrochloride, HA1077 and GSK-429286.
7. The method of claim 5 , wherein the PAK inhibitor is IPA3.
8. The method of claim 5 , wherein the myosin II inhibitor is blebbistatin.
9. The method of claim 1 , wherein the expansion culture conditions further comprise a beta-adrenergic receptor agonist.
10. The method of claim 9 , wherein the beta-adrenergic receptor agonist is isoproterenol.
11. The method of claim 1 , wherein the expansion culture conditions comprise calcium at a concentration below 100 μM.
12. The method of claim 1 , wherein the expansion culture conditions comprise one or more mitogenic growth factors.
13. The method of claim 12 , wherein the one or more mitogenic growth factors comprise EGF, FGF, or EGF and FGF.
14. The method of claim 1 , wherein the originating epithelial cell population comprises transit-amplifying epithelial cells.
15. The method of claim 1 , wherein the originating epithelial cell population comprises epithelial progenitor cells.
16. The method of claim 1 , wherein the originating epithelial cell population comprises lineage-committed epithelial cells.
17. The method of claim 1 , wherein the expansion culture conditions comprise inhibitors consisting of an ALK5 inhibitor and a Rho-associated protein kinase inhibitor; an ALK5 inhibitor and a p21-activated kinase (PAK) inhibitor; or an ALK5 inhibitor and a myosin II inhibitor.
18. A method for proliferating epithelial cells ex vivo, comprising:
expanding the number of cells in an originating epithelial cell population derived from induced pluripotent stem cells (iPSCs) under serum-free and feeder-cell free expansion culture conditions, thereby generating an expanded epithelial cell population, wherein the expansion culture conditions comprise
a) one or more transforming growth factor beta (TGF-beta) inhibitors;
b) one or more agents that modulate cytoskeletal structure;
c) a beta-adrenergic receptor agonist;
d) one or more mitogenic growth factors; and
e) calcium at a concentration below 100 μM.
19. The method of claim 18 , wherein the one or more transforming growth factor beta (TGF-beta) inhibitors comprise one or more inhibitors of ALK5, ALK4, and/or ALK7; and the one or more agents that modulate cytoskeletal structure comprise one or more inhibitors selected from a Rho-associated protein kinase inhibitor, a p21-activated kinase (PAK) inhibitor, and a myosin II inhibitor.
20. The method of claim 19 , wherein the one or more inhibitors of ALK5, ALK4, and/or ALK7 are selected from A83-01, GW788388, RepSox, and SB 431542; the Rho-associated protein kinase inhibitor is selected from Y-27632, SR 3677, thiazovivin, HA1100 hydrochloride, HA1077 and GSK-429286; the PAK inhibitor is IPA3; and the myosin II inhibitor is blebbistatin.