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Lauren M. James, Phillippa B. Cottrill, Hari Jayaram, Hannah Levis, Karl Matter, G. Astrid Limb; Development Of Cellular Scaffolds For Neural Retinal Cell Replacement Using Adult Human Müller Stem Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2246.
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Stem cell transplantation studies to repair degenerated retina have not yet achieved adequate cellular distribution across the neural retina. This is partly attributed to the anatomical features of the retina and surrounding tissues that prevent optimal migration and integration of the injected cells. This study aimed to develop cellular scaffolds to facilitate transplantation of human Müller stem cells onto degenerated retina.
Acidic solutions of rat tail type-I Collagen (1ml vol.) were polymerized using 5M sodium hydroxide, and subsequently submitted to plastic compression for five minutes under 150g weights. Electron microscopy was used to examine the microstructure of the collagen mats. Human Müller stem cells were cultured on these mats in the presence or absence of FGF2 and the Notch1 inhibitor DAPT (N-[N-(3, 5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester) for 5days to induce retinal ganglion cell differentiation. Phenotypic differentiation of the cells was examined by confocal microscopy. Cellular scaffolds were placed onto the inner surface of explants of porcine and human retina and cultured for up to 7 days in neurobasal medium supplemented with B-27 and N-2. Cell migration and integration into the explanted retina were analyzed by immunohistochemistry.
Human Müller stem cells (hMSC) transfected with a lentivirus GFP readily adhered to collagen substrates in the presence or absence of DAPT and FGF2. Scanning electron and confocal microscopy showed that cells developed long axonal processes and firmly attached to the collagen mats. Differentiation of hMSC into RGC was confirmed by their co-expression of GFP, mature RGC markers HUD and ISLET-1 and the RGC precursor marker BRN3b. Immunostaining of sections obtained from the explant retinal tissue transplanted in vitro with the cellular scaffolds showed that cells detached from the scaffolds and migrated into the retinal explants. Cellular viability was not affected by culturing the cells on collagen mats when compared to Matrigel alone.
Compressed collagen mats support Müller stem cell adhesion, growth and differentiation. Cellular scaffolds using these constructs may constitute a viable strategy for retinal stem cell transplantation in order to provide a platform to aid uniform cellular distribution of transplanted cells into the retina.
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