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M.L. Funderburgh, M.M. Mann, J.L. Funderburgh; Keratocyte Differentiation in vitro: It's About Matrix Interaction . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2708.
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Keratocytes produce the unique extracellular matrix required for the strength and transparency of the corneal stroma. The phenotype of a differentiated keratocyte is not immutable: it changes, for example, in a healing wound to make long–lasting, non–transparent scar tissue. Similar changes are observed in cultured keratocytes. Our current study tests the hypothesis that interaction with substratum influences keratocyte matrix production.
Primary keratocytes were isolated by collagenase digestion from fresh bovine corneas. The cells were maintained in serum–free media in conditions promoting either cell attachment to a substratum or aggregation into non–attached cell spheroids. Markers of keratocyte differentiation were assayed by Real–Time qPCR for mRNA levels and secreted matrix proteoglycans were analyzed by Western blotting and fluorophore–assisted carbohydrate electrophoresis (FACE). Cell viability was determined by calcein AM staining.
Virtually all primary keratocytes were able to form aggregates, or spheroids. Cells in these balls remained viable, but did not increase in cell numbers, even under conditions stimulating growth in attached cells. Spheroid cells secreted high levels of keratocyte matrix components keratan sulfate and keratocan compared to attached cells. Cells in spheroids differed from substrate–attached cells in their response to TGF beta: stimulation of smooth muscle actin and EDA–fibronectin was decreased in the spheroids, while collagen types I and III, biglycan and hyaluronan responses were similar in the two growth conditions.
Keratocytes forming spheroids in culture remain viable even in the absence of attachment to substratum, and they are able to maintain much of their characteristic differentiated phenotype. On the other hand, attachment as a monolayer reduces expression of keratocytic markers and promotes the transition to myofibroblast. Cellular response to TGF beta is dependent on environment, but not all genes respond the same. These results imply that the transition from cell–cell interactions to cell–matrix interactions plays a role in the functional response of keratocytes in a wound–healing environment.
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