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Tanja Ilmarinen, Pyry Grönroos, Heidi Hongisto, Anni Sorkio, Soile Nymark, Heli Skottman; Influence of basement membrane components on the in vitro maturation of hESC-RPE cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4559.
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© ARVO (1962-2015); The Authors (2016-present)
Human pluripotent stem cell derived retinal pigment epithelial cell (hPSC-RPE) transplantation is currently under clinical evaluation as treatment for macular degeneration. For cell therapy, RPE of uniform quality and consistent level of maturation is required with potential implications to functional cell integration and survival after transplantation. We have previously shown, that the culture substrate dramatically affects human embryonic stem cell derived RPE (hESC-RPE) basal lamina production and maturation, including barrier properties. In the present study, we aimed to further identify the role of abundant RPE basement membrane (BM) proteins, type IV collagen, laminin, and nidogen-1, to the maturation and quality of hPSC-RPE.
Human ESC-RPE were seeded (2 x 105 cells/cm2) on 1 µm PET culture inserts coated with collagen IV or laminin alone, or in combination with and without nidogen-1. Both freshly differentiated and cryopreserved cells were used. The cells were matured for eight to ten weeks in serum-free culture conditions and analyzed for transepithelial electrical resistance (TER), phagocytosis activity, ion transport and markers for tight junctions and visual cycle.
The presence of laminin improved the overall consistency of the epithelium producing higher TER. Especially after cryopreservation, collagen IV coating alone resulted in a poor quality epithelium on PET inserts compared to the combination of collagen IV and laminin. Addition of nidogen did not influence the expression or polarization of Na/K-ATPase and ZO-1, but increased the expression of visual cycle proteins RPE65 and CRALBP, as well as tight junction protein claudin-19.
Collagen IV and laminin are typically used individually for coating cell culture surfaces for hPSC-RPE culture. Our results indicate, that it would be advantageous to combine these BM proteins and use extracellular matrix linker molecules such as nidogen to obtain high quality hPSC-RPE. After cryopreservation of hPSC-RPE, the culture substrate is even more relevant, with possible implications to clinical use of these cells after cell banking.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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