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 differentiated tissue, embryonic stem (ES) cells, or induced pluripotent stem cells (iPSCs) under expansion culture conditions, thereby generating an expanded epithelial cell population, wherein:
the expansion culture conditions comprise a first agent consisting of an ALK5 inhibitor and a second agent chosen from a Rho-associated protein kinase inhibitor, a p21-activated kinase (PAK) inhibitor, and a myosin II inhibitor;
the expansion culture conditions are serum-free and feeder-cell free culture conditions, and are defined culture conditions, xeno-free culture conditions, or defined and xeno-free culture conditions;
the originating epithelial cell population is capable of 25 population doublings or more when cultured under the expansion culture conditions; and
the originating epithelial cell population is capable of no more than 20 population doublings when cultured under control culture conditions that do not include the first agent and the second agent.
2. The method of claim 1 , wherein the ALK5 inhibitor is selected from A83-01, GW788388, RepSox, and SB 431542.
3. The method of claim 1 , wherein the second agent is a Rho-associated protein kinase inhibitor.
4. The method of claim 3 , wherein the Rho-associated protein kinase inhibitor is selected from Y-27632, SR 3677, thiazovivin, HA1100 hydrochloride, HA1077 and GSK-429286.
5. The method of claim 1 , wherein the second agent is a PAK inhibitor.
6. The method of claim 5 , wherein the PAK inhibitor is IPA3.
7. The method of claim 1 , wherein the second agent is a myosin II inhibitor.
8. The method of claim 7 , wherein the myosin II inhibitor is blebbistatin.
9. The method of claim 1 , wherein the expansion culture conditions 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 200 μM.
12. The method of claim 1 , wherein the expansion culture conditions comprise calcium at a concentration below 100 μM.
13. The method of claim 1 , wherein the expansion culture conditions comprise one or more mitogenic growth factors.
14. The method of claim 13 , wherein the one or more mitogenic growth factors comprise EGF, FGF, or EGF and FGF.
15. The method of claim 1 , wherein the originating epithelial cell population is capable of 50 population doublings or more when cultured under the expansion culture conditions.
16. The method of claim 1 , wherein the originating epithelial cell population is capable of 80 population doublings or more when cultured under the expansion culture conditions.
17. The method of claim 1 , wherein the originating epithelial cell population is capable of 100 population doublings or more when cultured under the expansion culture conditions.
18. 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.
19. The method of claim 1 , wherein the expansion culture conditions comprise inhibitors consisting of an ALK5 inhibitor and a Rho-associated protein kinase inhibitor.
20. The method of claim 19 , wherein the expansion culture conditions comprise inhibitors consisting of A83-01 and Y-27632.
21. The method of claim 1 , wherein the expansion culture conditions comprise inhibitors consisting of an ALK5 inhibitor and a p21-activated kinase (PAK) inhibitor.
22. The method of claim 21 , wherein the expansion culture conditions comprise inhibitors consisting of A83-01 and IPA3.
23. The method of claim 1 , wherein the expansion culture conditions comprise inhibitors consisting of an ALK5 inhibitor and a myosin II inhibitor.
24. The method of claim 23 , wherein the expansion culture conditions comprise inhibitors consisting of A83-01 and blebbistatin.
25. A method for proliferating epithelial cells ex vivo, comprising:
expanding the number of cells in an originating epithelial cell population derived from differentiated tissue, embryonic stem (ES) cells, or induced pluripotent stem cells (iPSCs) 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;
the expansion culture conditions are serum-free and feeder-cell free culture conditions, and are defined culture conditions, xeno-free culture conditions, or defined and xeno-free culture conditions;
the originating epithelial cell population is capable of 25 population doublings or more when cultured under the expansion culture conditions; and
the originating epithelial cell population is capable of no more than 20 population doublings when cultured under control culture conditions that do not include the first agent and the second agent.
26. The method of claim 25 , wherein the transforming growth factor beta (TGF-beta) signaling inhibitor is an ALK 5 inhibitor selected from A83-01, GW788388,RepSox, and SB 431542.
27. The method of claim 25 , wherein the Rho-associated protein kinase inhibitor is selected from Y-27632, SR 3677, thiazovivin, HA1100 hydrochloride, HA1077 and GSK-429286.
28. The method of claim 25 , wherein the PAK inhibitor is IPA3.
29. The method of claim 25 , wherein the myosin II inhibitor is blebbistatin.
30. A population of ex vivo expanded epithelial cells produced by a method comprising:
expanding the number of cells in an originating epithelial cell population derived from differentiated tissue, embryonic stem (ES) cells, or induced pluripotent stem cells (iPSCs) under expansion culture conditions, thereby generating an expanded epithelial cell population, wherein:
the expansion culture conditions comprise a first agent consisting of an ALK5 inhibitor and a second agent chosen from a Rho-associated protein kinase inhibitor, a p21-activated kinase (PAK) inhibitor, and a myosin II inhibitor;
the expansion culture conditions are serum-free and feeder-cell free culture conditions, and are defined culture conditions, xeno-free culture conditions, or defined and xeno-free culture conditions;
the originating epithelial cell population is capable of 25 population doublings or more when cultured under the expansion culture conditions; and
the originating epithelial cell population is capable of no more than 20 population doublings when cultured under control culture conditions that do not include the first agent and the second agent.
31. The population of ex vivo expanded epithelial cells of claim 30 , wherein the ALK 5 inhibitor is selected from A83-01, GW788388, RepSox, and SB 431542.
32. The population of ex vivo expanded epithelial cells of claim 30 , wherein the second agent is a Rho-associated protein kinase inhibitor selected from Y-27632, SR 3677, thiazovivin, HA1100 hydrochloride, HA1077 and GSK-429286.
33. The population of ex vivo expanded epithelial cells of claim 30 , wherein the second agent is a PAK inhibitor, and the PAK inhibitor is IPA3.
34. The population of ex vivo expanded epithelial cells of claim 30 , wherein second agent is a myosin II inhibitor, and the myosin II inhibitor is blebbistatin.
35. The population of ex vivo expanded epithelial cells of claim 30 , wherein the expansion culture conditions comprise calcium at a concentration below 100 μM.
36. 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 epithelial cell population derived from differentiated tissue, embryonic stem (ES) cells, or induced pluripotent stem cells (iPSCs) under expansion culture conditions, thereby generating an expanded epithelial cell population, wherein:
the expansion culture conditions comprise a first agent consisting of an ALK5 inhibitor and a second agent chosen from a Rho-associated protein kinase inhibitor, a p21-activated kinase (PAK) inhibitor, and a myosin II inhibitor;
the expansion culture conditions are serum-free and feeder-cell free culture conditions, and are defined culture conditions, xeno-free culture conditions, or defined and xeno-free culture conditions;
the originating epithelial cell population is capable of 25 population doublings or more when cultured under the expansion culture conditions; and
the originating epithelial cell population is capable of no more than 20 population doublings when cultured under control culture conditions that do not include the first agent and the second agent.
37. The method of claim 36 , wherein the ALK5 inhibitor is selected from A83-01,GW788388, RepSox, and SB 431542.
38. The method of claim 36 , wherein the second agent is a Rho-associated protein kinase inhibitor selected from Y-27632, SR 3677, thiazovivin, HA1100 hydrochloride, HA1077 and GSK-429286.
39. The method of claim 36 , wherein the second agent is a PAK inhibitor, and the PAK inhibitor is IPA3.
40. The method of claim 36 , wherein second agent is a myosin II inhibitor, and the myosin II inhibitor is blebbistatin.
41. The method of claim 36 , wherein the expansion culture conditions comprise calcium at a concentration below 100 μM.