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 cell population comprising one or more of differentiated epithelial cells, transit-amplifying epithelial cells, lineage-committed epithelial cells, and basal epithelial cells under expansion culture conditions, thereby generating an expanded epithelial cell population, wherein:
the expansion culture conditions comprise a transforming growth factor beta (TGF-beta) inhibitor and an agent that disrupts cytoskeletal structure; and
the expansion culture conditions are serum-free and feeder-cell free culture conditions.
2. The method of claim 1 , wherein the TGF-beta inhibitor is an ALK5 inhibitor.
3. The method of claim 2 , wherein the ALK5 inhibitor is selected from A83-01, GW788388, RepSox, and SB 431542.
4. The method of claim 1 , wherein the agent that disrupts cytoskeletal structure is chosen from a Rho-associated protein kinase inhibitor, a p21-activated kinase (PAK) inhibitor, and a myosin II inhibitor.
5. The method of claim 1 , wherein the agent that disrupts cytoskeletal structure is a Rho-associated protein kinase 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 1 , wherein the agent that disrupts cytoskeletal structure is a PAK inhibitor.
8. The method of claim 7 , wherein the PAK inhibitor is IPA3.
9. The method of claim 1 , wherein the agent that disrupts cytoskeletal structure is a myosin II inhibitor.
10. The method of claim 9 , wherein the myosin II inhibitor is blebbistatin.
11. The method of claim 1 , wherein the expansion culture conditions comprise a beta-adrenergic receptor agonist.
12. The method of claim 11 , wherein the beta-adrenergic receptor agonist is isoproterenol.
13. The method of claim 1 , wherein the expansion culture conditions comprise calcium at a concentration below 200 μM.
14. The method of claim 1 , wherein the expansion culture conditions comprise calcium at a concentration below 100 μM.
15. The method of claim 1 , wherein the expansion culture conditions comprise one or more mitogenic growth factors.
16. The method of claim 15 , wherein the one or more mitogenic growth factors comprise EGF, FGF, or EGF and FGF.
17. The method of claim 1 , wherein the originating cell population is capable of 25 population doublings or more when cultured under the expansion culture conditions; and the originating cell population is capable of no more than 20 population doublings when cultured under control culture conditions that do not include the transforming growth factor beta (TGF-beta) inhibitor and the agent that disrupts cytoskeletal structure.
18. The method of claim 17 , wherein the originating cell population is capable of 50 population doublings or more when cultured under the expansion culture conditions.
19. The method of claim 17 , wherein the originating cell population is capable of 80 population doublings or more when cultured under the expansion culture conditions.
20. The method of claim 17 , wherein the originating cell population is capable of 100 population doublings or more when cultured under the expansion culture conditions.
21. The method of claim 1 , wherein the originating cell population is derived from differentiated tissue.
22. The method of claim 1 , wherein the originating cell population is derived from embryonic stem (ES) cells.
23. The method of claim 1 , wherein the originating cell population is derived from induced pluripotent stem cells (iPSCs).
24. The method of claim 1 , wherein the originating cell population does not comprise pluripotent cells.
25. The method of claim 1 , wherein the expansion culture conditions do not comprise one or more of extracellular matrix comprising heterogeneous components, a Wnt agonist or a beta-catenin agonist.
26. The method of claim 1 , wherein the expansion culture conditions comprise inhibitors consisting of an ALK5 inhibitor and a Rho-associated protein kinase inhibitor.
27. The method of claim 26 , wherein the expansion culture conditions comprise inhibitors consisting of A83-01 and Y-27632.
28. The method of claim 1 , wherein the expansion culture conditions comprise inhibitors consisting of an ALK5 inhibitor and a p21-activated kinase (PAK) inhibitor.
29. The method of claim 28 , wherein the expansion culture conditions comprise inhibitors consisting of A83-01 and IPA3.
30. The method of claim 1 , wherein the expansion culture conditions comprise inhibitors consisting of an ALK5 inhibitor and a myosin II inhibitor.
31. The method of claim 30 , wherein the expansion culture conditions comprise inhibitors consisting of A83-01 and blebbistatin.
32. The method of claim 1 , wherein the originating cell population comprises differentiated epithelial cells.
33. The method of claim 1 , wherein the originating cell population comprises transit-amplifying epithelial cells.
34. The method of claim 1 , wherein the originating cell population comprises lineage-committed epithelial cells.
35. The method of claim 1 , wherein the originating cell population comprises basal epithelial cells.
36. A method for proliferating epithelial cells ex vivo, comprising:
expanding the number of cells in an originating cell population comprising one or more of differentiated epithelial cells, transit-amplifying epithelial cells, lineage-committed epithelial cells, and basal epithelial cells under expansion culture conditions, thereby generating an expanded epithelial cell population, wherein:
the expansion culture conditions comprise components consisting essentially of i) a transforming growth factor beta (TGF-beta) signaling inhibitor; ii) a Rho-associated protein kinase inhibitor, a p21-activated kinase (PAK) inhibitor, or a myosin II inhibitor; iii) a mitogenic growth factor; iv) a beta-adrenergic receptor agonist; v) calcium at a concentration below 100 μM; and vi) a serum-free base medium; and
the expansion culture conditions are serum-free and feeder-cell free culture conditions.
37. A population of ex vivo expanded epithelial cells produced by a method comprising:
expanding the number of cells in an originating cell population comprising one or more of differentiated epithelial cells, transit-amplifying epithelial cells, lineage-committed epithelial cells, and basal epithelial cells under expansion culture conditions, thereby generating an expanded epithelial cell population, wherein:
the expansion culture conditions comprise a transforming growth factor beta (TGF-beta) inhibitor and an agent that disrupts cytoskeletal structure; and
the expansion culture conditions are serum-free and feeder-cell free culture conditions.
38. The population of ex vivo expanded epithelial cells of claim 37 , wherein the TGF-beta inhibitor is an ALK5 inhibitor.
39. The population of ex vivo expanded epithelial cells of claim 37 , wherein the agent that disrupts cytoskeletal structure is chosen from a Rho-associated protein kinase inhibitor, a PAK inhibitor, and a myosin II inhibitor.
40. The population of ex vivo expanded epithelial cells of claim 37 , wherein the expansion culture conditions comprise calcium at a concentration below 100 μM.
41. A method for genetically modifying cells, comprising contacting cells with one or more agents that introduce a genetic modification to the cells, wherein the cells have been expanded by a method comprising:
expanding the number of cells in an originating cell population comprising one or more of differentiated epithelial cells, transit-amplifying epithelial cells, lineage-committed epithelial cells, and basal epithelial cells under expansion culture conditions, thereby generating an expanded epithelial cell population, wherein:
the expansion culture conditions comprise a transforming growth factor beta (TGF-beta) inhibitor and an agent that disrupts cytoskeletal structure; and
the expansion culture conditions are serum-free and feeder-cell free culture conditions.
42. The method of claim 41 , wherein the TGF-beta inhibitor is an ALK5 inhibitor.
43. The method of claim 41 , wherein the agent that disrupts cytoskeletal structure is chosen from a Rho-associated protein kinase inhibitor, a PAK inhibitor, and a myosin II inhibitor.
44. The method of claim 41 , wherein the expansion culture conditions comprise calcium at a concentration below 100 μM.