September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Integrin and extracellular matrix (ECM) protein expression in differentiating neural retina generated from human ES cell (hESC) cultures
Author Affiliations & Notes
  • Debjani Phillips
    Waisman Center, University of Wisconsin Madison, Madison, Wisconsin, United States
    McPherson Eye Research Institute, Madison, Wisconsin, United States
  • Douglas Streeten
    University of Wisconsin Madison, Madison, Wisconsin, United States
  • Isabel Pinilla
    Ophthalmology, University Hospital Lozano Blesa, Zaragoza, Spain
    University of Wisconsin Madison, Madison, Wisconsin, United States
  • William J Brunken
    Ophthalmology, Upstate Medical University, Syracuse, New York, United States
    SUNY Eye Institute, Syracuse, New York, 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   Debjani Phillips, None; Douglas Streeten, None; Isabel Pinilla, None; William Brunken, None; David Gamm, None
  • Footnotes
    Support  Retinal Research Foundation Emmet A. Humble Distinguished Directorship, McPherson Eye Research Institute (Sandra Lemke Trout Chair), Carl and Mildred Reeves Foundation, NIH P30HD03352, Muskingum County Community Foundation, Choroideremia Research Foundation
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 1148. doi:
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    • Get Citation

      Debjani Phillips, Douglas Streeten, Isabel Pinilla, William J Brunken, David M Gamm; Integrin and extracellular matrix (ECM) protein expression in differentiating neural retina generated from human ES cell (hESC) cultures. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1148.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : During retinal development, integrin heterodimers associate with ECM proteins to regulate axon guidance, spatial organization and ECM assembly, thus directing cell fate. The β1 integrin subunit has been implicated in the expansion of neural progenitor cells and is integral for maintaining the collective integrity of the developing retina. Using our established hESC model of retinal development, we sought to profile the expression of β1 integrin and its partners, α1-6 integrins and αv integrin, and the ECM proteins with which they associate at varying stages of human neural retinal differentiation.

Methods : Optic vesicles (OVs) generated from H9CRX17tdTomato hESCs were differentiated to day (D) 45, D67, D92 and D100. Expression of β1 integrin subunit, its associated α integrin subunits, and the ECM proteins fibronectin, collagen IV and laminin were observed using immunocytochemistry (ICC). Levels of integrin expression on developing CRX+ cells were analyzed by flow cytometry. The CRX+ population was gated according to tdTomato reporter expression levels.

Results : The β1 integrin subunit levels remained elevated at D45 and D100 of differentiation. In contrast, expression levels of α integrin subunits were low or undetectable at D45 but increased at D100. Fibronectin, collagen IV, and laminin expression were prevalent during differentiation of hESCs into neural retina and integrins appeared clustered alongside tdTomato-expressing CRX+ cells.

Conclusions : Our hESC-OV neural retina model is a useful tool for identifying integrins and ECM proteins that have the capacity to contribute to development and perhaps fate determination of neural retinal cells. Positive identification of the β1 integrin subunit along with all of its associated α integrin subunits in these 3-D cultures provides further evidence of their utility as models of human retinal development. Knowledge of the expression profiles of integrins and their ECM binding partners during different stages of development improves our understanding of the developing human retina and provides guidance for the manipulation of culture techniques in order to direct cells to a desired fate.

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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