Abstract
Purpose:
To establish an in vitro model of proliferative vitreoretinopathy (PVR) using retinal pigment epithelial cells derived from induced pluripotent stem cells (iPS-RPE).
Methods:
RPE was spontaneously derived from iPS. Colonies exhibiting RPE characteristics were selected and enriched. iPS-RPE was analyzed for expression of RPE genes RPE65, LRAT, and CRALBP. iPS-RPE was seeded onto fibronectin-coated transwells. At days 1 through 35 after seeding, the iPS-RPE was processed for immunofluorescence analysis of markers of epithelial-mesenchymal transition (EMT) including smooth muscle actin, vimentin, S100A4, cytokeratin, β-catenin and MITF. In a separate assay,IPS-RPE was grown to confluency on fibronectin-coated transwells unitl the entire cell monolayer was pigmented and hexagonal. A wound healing assay was performed by scratching the iPS-RPE monolayer. Cells were treated with TGF-β2 to induce EMT. The monolayer was observed for migration of cells into the wound area, and phenotypic changes of cells in response to wounding.
Results:
After trypsinization and seeding onto fibronectin, the iPS-RPE lost pigmentation and became fibroblastic in appearance. The cells expressed markers of EMT including vimentin, S100A4, and smooth muscle actin. However, within 14 days after seeding, the cells started to regain pigmentation and become hexagonal. The expression of EMT markers was reduced. By 35 days after seeding, the cells were highly pigmented, hexagonal, and expression of EMT markers was very low. In the wound healing assay, both TGF-β2 treated and untreated iPS-RPE migrated into the wound area and became fibroblastic. Within 60 days after wounding, most cells in the wound area had regained hexagonal , pigmented morphology, although TGF-β2 treated iPS-RPE migrated and proliferated more quickly than the untreated control iPS-RPE.
Conclusions:
EMT is required for the migration of epithelial cells during the wound healing process. Abnormal EMT contributes to the development of PVR after rhegmatogenous retinal detachment. After trypsinization and seeding onto fibronectin, iPS-RPE expresses markers of EMT but regains the RPE phenotype. Both TGF-β2 treated and untreated iPS-RPE regained RPE phenotype in the wound healing assay.Once the in vitro model of PVR has been established, iPS-RPE will provide a useful tool to screen small molecules and pharmaceuticals for the prevention and treatment of PVR.
Keywords: 655 proliferative vitreoretinopathy •
701 retinal pigment epithelium •
512 EMT (epithelial mesenchymal transition)