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Maryam Ghiassi-Nejad, Deepti Singh, SHAO-BIN WANG, Laurel T Tainsh, Bo Chen, Ron A Adelman, Lawrence J Rizzolo; Scaffold Composition Affects the Proliferation and Differentiation of Retinal Progenitor Cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1149. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal progenitor cells (RPC) can form neurospheres that resemble retinal structure, but their small spherical geometry limits their utility for experimentation and implantation into diseased retinas. We examined different scaffolds for biocompatibility, and ability to foster stem cell growth and differentiation.
Scaffolds were fabricated from electrospun polycaprolactone (PCL) or from alginate or gelatin that was decorated with hyaluronic acid and chondroitin sulfate. The later scaffolds were freeze-dried to create large pores, rehydrated, and sectioned into discs varying from 60-120 microns thick. Pore size was evaluated by scanning electron microscopy, and potential toxicity was tested by implanting the scaffolds into mouse retinas. Human embryonic stem cells (WA09) were differentiated, and in some experiments, co-cultured with human fetal (hfRPE). The cell differentiation was monitored using qRT-PCR and immunofluorescence every two weeks for two months. The cultures were followed up to three months and monitored by quantitative RT-PCR (qPCR) or immunofluorescence.
Scaffolds had interconnected pores 50-100 micron in diameter, but for PCL pores were flattened in the XZ plane. Stem cells penetrated PCL only 20-30 micron, and the cultures were not uniform. RPC were able to differentiate on PCL and gelatin scaffolds, but growth and differentiation was impaired on alginate scaffolds. We proceeded with gelatin-based scaffolds. Scaffolds degraded slowly in culture, but were degraded within three weeks following implantation into the subretinal space. There was no evidence of overt inflammation, retinal edema, or degeneration. When plated at low density and co-cultured with hfRPE, RPC transformed from spherical cells to broad, flattened cells that extended neurite-like projections. When plated at high density, cells uniformly populated the full thickness of scaffolds as thick as 120 microns and expressed retinal markers.
Scaffolds based on freeze-dried gelatin were most successful at satisfying 3 criteria: full penetration of the scaffolds, uniform cell density in the xy-plane, and no cytotoxicity upon implantation. Retinal lamina began to form, but further experimentation is required to achieve the structures found in neurospheres.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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