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