September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Evaluating the effects of ECM and elastic modulus on human pluripotent stem cell-derived photoreceptor enrichment
Author Affiliations & Notes
  • Jee Min
    Waisman Center, University of Wisconsin - Madison, Madison, Wisconsin, United States
  • Joe Phillips
    Waisman Center, University of Wisconsin - Madison, Madison, Wisconsin, United States
    McPherson Eye Research Institute, Madison, Wisconsin, United States
  • William Daly
    Department of Materials Science and Engineering, University of Wisconsin Madison, Madison, Wisconsin, United States
    Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • William Murphy
    Department of Materials Science and Engineering, University of Wisconsin Madison, Madison, Wisconsin, United States
    Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • David M Gamm
    Department of Ophthalmology and Visual Sciences, University of Wisconsin - Madison, Madison, Wisconsin, United States
    McPherson Eye Research Institute, Madison, Wisconsin, United States
  • Footnotes
    Commercial Relationships   Jee Min, None; Joe Phillips, None; William Daly, None; William Murphy, None; David Gamm, None
  • Footnotes
    Support  Foundation Fighting Blindness Wynn-Gund TRAP Award, NIH RO1 EY21218, Retinal Research Foundation Emmett A. Humble Distinguished Directorship, McPherson Eye Research Institute (Sandra Lemke Trout Chair), Research to Prevent Blindness, Carl and Mildred Reeves Foundation, NIH P30HD03352, Muskingum County Community Foundation, Choroideremia Research Foundation
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 1150. doi:
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    • Get Citation

      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)

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Abstract

Purpose : 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.

Methods : 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.

Results : 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.

Conclusions : 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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