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J. Orasky, M.J. Young, D.S. Sakaguchi; Influence of Extracellular Matrix Molecules on the Adhesion and Differentiation of Murine Retinal and Brain Progenitor Cells in vitro . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5762.
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© ARVO (1962-2015); The Authors (2016-present)
To characterize the effects of the extracellular matrix (ECM) molecules fibronectin, laminin, and collagen–I on the adhesion and differentiation of mouse retinal and brain progenitor cells (RPCs and BPCs) in vitro.
Enhanced green fluorescent protein expressing murine RPCs and BPCs were cultured under differentiation conditions on substrates of purified fibronectin, laminin, or collagen–I coated over poly–ornithine. Control substrates consisted of only poly–ornithine. To investigate the influence of the ECM on progenitor cell differentiation, the RPCs and BPCs were plated onto the purified substrates and cultured for 7 days and then prepared for immunocytochemical analysis. The following antibodies were used to identify cell specific markers: progenitor cells (Nestin), neurons (TUJ1, MAP2ab, PKC), and glia (GFAP, GS). We also stained for the following integrin subunits with specific antibodies for the α2, 3, 5, 6, V, and ß1integrin subunits.
Our results indicate that the three ECM substrates fibronectin, laminin, and collagen, as well as poly–ornithine, all strongly support cell adhesion. We have used echistatin, an RGD–containing short monomeric disintegrin, to investigate the role of integrin–mediated adhesion. No difference was noted in the adhesion of cells in the presence of echistatin to laminin, collagen or poly–ornithine, but fewer cells were found attached to the fibronectin substrates. After 7 days of differentiation, RPCs cultured on fibronectin and laminin had a higher proportion of cells with neuronal and glial phenotypes, while more RPCs cultured on collagen remained in a progenitor, nestin positive state. BPCs cultured on fibronectin had a higher proportion of cells with neuronal and glial phenotypes when compared to BPCs cultured on laminin or collagen. Antibody labeling revealed that the RPCs and BPCs express α2, 3, 5, 6, V, and ß1 integrin subunits.
These results suggest that specific ECM substrates may enhance adhesion and influence differentiation of neural progenitor cells.
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