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Jee Min, Joe Phillips, William Daly, William Murphy, David M Gamm; Evaluating the effects of ECM and elastic modulus on human pluripotent stem cell-derived photoreceptor enrichment. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1150.
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© ARVO (1962-2015); The Authors (2016-present)
Human pluripotent stem cell (hPSC)-based photoreceptor replacement strategies require efficient generation and isolation of photoreceptors, as well as the removal of proliferative retinal progenitor cells (RPCs). Extrinsic factors such as the extracellular matrix (ECM), as well as the stiffness (elastic modulus) of the extracellular environment are known to modulate cellular adherence, differentiation, and maturation. The purpose of this study was to determine the potential of these extrinsic factors to promote photoreceptor enrichment while reducing the number of RPCs.
Using our established protocols, hPSC-derived optic vesicle-like (OV) structures were generated, isolated, and differentiated to day 80. OVs were then dissociated and plated onto various substrates and maintained for one week. Two experiments were performed to evaluate the following substrates: Experiment 1) 96 well plates were pre-coated with ECMs and adhesion promoting molecules, including fibronectin, laminin, vitronectin, collagen, matrigel, poly-L-lysine (PLL), poly-D-lysine (PDL), and poly-L-ornithine (PLO). Experiment 2) Polyethylene glycol based (PEG) hydrogels were spotted onto 384 well plates. A slow degrading and a fast degrading MMP-labile crosslinker, and a non MMP-labile cross-linker were added to PEG at varying concentrations to vary stiffness and degradability of the hydrogel. Furthermore, the optimal concentration of integrin binding peptides were also probed. Quantitative immunocytochemistry analysis was performed on the Operetta High Content Screening System to determine the number of photoreceptors (RECOVERIN+) and RPCs (Ki67+) in both experiments.
Initial results suggested that PLO and PLL promoted the greatest degree of photoreceptor enrichment and RPC reduction in experiment 1. In experiment 2, medium stiffness Tryptophan crosslinked hydrogels with a high concentration of IKVAV provided the most photoreceptor enrichment and RPC reduction.
In this study, high content screening was used to identify target substrates for photoreceptor enrichment. Knowledge gained from this study can be used to improve photoreceptor production and isolation methods. Additionally, these results are also being used to inform hydrogel design, as transplanting photoreceptors in an encapsulating hydrogel may improve transplant efficiency and cell survival.
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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