April 2011
Volume 52, Issue 14
Free
ARVO Annual Meeting Abstract  |   April 2011
Extracellular Matrix Promotes Embryonic Stem Cell Differentiation Towards a Retinal Progenitor Fate
Author Affiliations & Notes
  • Jie Gong
    Ophthalmology, Columbia University, New York, New York
  • Mark A. Fields
    Ophthalmology, Columbia University, New York, New York
  • Hui Cai
    Ophthalmology, Columbia University, New York, New York
  • Stephen H. Tsang
    Ophthalmology, Columbia University, New York, New York
  • Lucian V. Del Priore
    Ophthalmology, Columbia University, New York, New York
  • Footnotes
    Commercial Relationships  Jie Gong, None; Mark A. Fields, None; Hui Cai, None; Stephen H. Tsang, None; Lucian V. Del Priore, None
  • Footnotes
    Support  Research to Prevent Blindness, Robert L. Burch III Fund, Retina Society, Hickey’s Family Foundation and the Foundation Fighting Blindness.
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 2255. doi:https://doi.org/
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      Jie Gong, Mark A. Fields, Hui Cai, Stephen H. Tsang, Lucian V. Del Priore; Extracellular Matrix Promotes Embryonic Stem Cell Differentiation Towards a Retinal Progenitor Fate. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2255. doi: https://doi.org/.

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

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Abstract

Purpose: : Embryonic stem cell (ESC) transplantation is a promising therapeutic approach for the replacement of degenerated neural cells and retinal pigment epithelium (RPE) cells in patients with age-related macular degeneration (AMD), retinitis pigmentosa (RP), and other disorders. We used extracellular matrix environs to induce ESC differentiation toward neural progenitors.

Methods: : Mouse ESCs from American Type Culture Collection, Manassas, VA were cultured in serum-free floating culture with Dickkopf-Related Protein 1 (DKK1), a Wnt pathway inhibitor and Left-Right Determination Factor A (Lefty A), a Nodal pathway inhibitor and serum (Osakada F, Nature Biotechnology 2008) for 9 days. Then the cells were treated with N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), a Notch signal inhibitor, in retinal differentiation medium containing retinoic acid, taurine, basic fibroblast growth factor (FGF), and acid FGF and cultured up to 28 days on dishes coated with: (1) poly-D-lysine plus laminin (330 µg/mL); (2) poly-D-lysine plus laminin (330 µg/mL), vitronectin (33 µg/mL), and fibronectin (250 µg/mL); or (3) Matrigel (12 µl/mL Becton, Dickinson and Co. hESC-qualified Matrix). We performed immunofluorescence microscopy on the differentiated cells using retinal progenitor markers [beta-tubulin, nestin, neural retina leucine zipper (Nrl), and neurofilament 200 (NF200)], retinal photoreceptor markers [rhodopsin, cone opsin, and cone-rod homeobox (CRX)], and RPE markers [zonula occludens protein-1 (ZO-1) and Bestrophin].

Results: : After 24 hour treatment with Wnt and Nodal inhibitors, mouse ESCs in a suspension culture formed embryoid body aggregates. Within 10 days, monolayers formed on all 3 types of coated dishes. At 28 days in retinal differentiation medium, these cells developed a neuronal phenotype and were positive for beta-tubulin, NF200, Nrl and nestin and negative for cone opsin, CRX or rhodopsin. In dishes with the poly-D-lysine plus laminin, vitronectin, and fibronectin, the ESCs formed more neuronal structures compared to culturing on poly-D-lysine plus laminin. Few cells cultured on Matrigel expressed beta-tubulin, but most of the cells showed pigmentation and expressed RPE markers, ZO-1 and bestrophin.

Conclusions: : Modulation of the extracellular matrix can modulate the expression of neural markers in ESCs and differentiate them towards either a neural or RPE fate. Additional studies are required to achieve the goal of stem cell differentiation within a complex 3-dimensional environment required for retinal reconstruction.

Keywords: extracellular matrix • photoreceptors • retinal pigment epithelium 
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