RNAi methods and compositions for stimulating proliferation of cells with adherent junctions
Described herein are methods and compositions for stimulating proliferation of cells that express adherent junctions and cease proliferation, for example, human corneal endothelial cells, by downregulation of certain cell-cell junctions. In one embodiment, downregulation is achieved using RNA interference, and contacting the cells with mitogenic growth factors and an agent that elevates intracytoplasmic cAMP. Furthermore, described herein are methods of isolating human corneal endothelial cells from keratocytes, and methods of preserving and maintaining viability of human corneal endothelial cell aggregates. Also described are surgical grafts comprising human corneal endothelial cells that have been isolated, optionally stored, and transiently contacted with an agent that downregulates expression of p 120, and a biocompatible support. The methods and compositions described herein can be used in novel therapies to help expand human corneal endothelial cells during in vitro tissue engineering and for in vivo treatment of corneal endothelial dysfunction.
1. A method of stimulating the proliferation of a plurality of endothelial cells or epithelial cells with adherent junctions in vivo, comprising transiently contacting a plurality of endothelial cells or epithelial cells with adherent junctions in an eye of a mammal with an agent that downregulates expression of E-cadherin, VE-cadherin, P-cadherin, N-cadherin, α-catenin, β-catenin, p120 catenin, p190, or any combination thereof; wherein the agent is an agent that induces RNAi, thereby stimulating the proliferation of the plurality of cells.
2. The method of claim 1 , comprising downregulating the expression of p120 catenin.
3. The method of claim 1 , wherein the contacting occurs in pulses that are followed by withdrawal of the agent that induces RNAi.
4. The method of claim 1 , wherein the pulse of the agent that induces RNAi is administered for at least 12 hours.
5. The method of claim 1 , wherein the mammal has a corneal endothelial dysfunction.
6. The method of claim 1 , comprising administering the agent to an anterior chamber of the eye of the mammal.
7. The method of claim 1 , wherein the plurality of endothelial cells or epithelial cells with adherent junctions are human corneal epithelial cells.
8. A method of generating an expanding monolayer of human corneal endothelial cells in culture, comprising: (a) transiently contacting an aggregate or monolayer of human corneal endothelial cells with an RNAi-inducing agent that downregulates expression of N-cadherin, α-catenin, β-catenin, p120 catenin, p190, or any combination thereof, to generate expandable human corneal epithelial cells; and (b) culturing the expandable human corneal endothelial cells in media free of Cholera toxin and bFGF, such that they form an expanding monolayer of human corneal endothelial cells.
9. The method of claim 8 , comprising downregulating the expression of p120 catenin.
10. The method of claim 8 , wherein the contacting occurs in pulses that are followed by withdrawal of the RNAi-inducing agent.
11. The method of claim 10 , wherein the pulse of the RNAi-inducing agent is administered for at least 12 hours.
12. The method of claim 8 , wherein the human corneal endothelial cells are in early or late confluence.
13. The method of claim 4 , wherein the pulse of the agent that induces RNAi is administered daily, three times a week, twice a week, or weekly.
14. The method of claim 8 , further comprising contacting the cells with a mitogenic growth factor, an agent that elevates intracytoplasmic cAMP, or a combination thereof.
15. The method of claim 8 , wherein a pulse of an agent that induces RNAi is administered daily, three times a week, twice a week, or weekly.
16. The method of claim 8 , wherein the expanded aggregate or monolayer of human corneal endothelial cells is used to make a surgical graft.
17. The method of claim 1 , wherein the plurality of endothelial cells or epithelial cells with adherent junctions are human corneal endothelial cells.
18. The method of claim 1 , further comprising contacting the cells with a mitogenic growth factor, an agent that elevates intracytoplasmic cAMP, or a combination thereof.
19. The method of claim 18 , wherein the agent that elevates intracytoplasmic cAMP is selected from 8-bromo-cAMP, dibutyryl cAMP, isobutylmethylxanthine, Pentoxifylline, forskolin, cholera toxin, prostaglandin E2 (PGE2), phenylbutyrate, Butaprost, or Iloprost.
20. The method of claim 14 , wherein the agent that elevates intracytoplasmic cAMP is selected from 8-bromo-cAMP, dibutyryl cAMP, isobutylmethylxanthine, Pentoxifylline, forskolin, cholera toxin, prostaglandin E2 (PGE2), phenylbutyrate, Butaprost, or Iloprost.