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Y. Du, M.L. Funderburgh, M.M. Mann, N. SundarRaj, J.L. Funderburgh; Multipotent Stem Cells in Human Corneal Stroma . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3830.
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Purpose: Keratocytes are important for corneal integrity and transparency. Keratocyte proliferation and maintenance of a differentiated phenotype in vitro will be essential for corneal tissue engineering, but in culture human keratocytes change into fibroblasts losing their unique phenotype. Side population (SP) cells are defined by their active efflux of Hoechst 33342 dye, via a transporter protein known as ABCG2. SP cells from several tissues are enriched in adult stem cells. In this study, we investigated the presence of SP cells in the human corneal stroma and whether these cells can generate multiple, differentiated daughter populations. Methods: Human corneal stromal cells were cultured and passaged in medium containing 2% fetal bovine serum (FBS) with growth factors. SP cells were isolated by cell sorting. The sorted SP cells were cloned and passaged and then induced to differentiate into different cell types. Neural cells were induced by FGF2, endothelial cells by VEGF, keratocytes by low mitogen horse serum or Advanced–MEM (Invitrogen). The expression of ABCG2, nestin, keratocan, keratan sulfate, ALDH, collagen IV, NG2, neurofilament protein, and von Willebrand Factor (vWF) were detected by immunoblotting, RT–PCR, or Real–Time PCR in cells cultured in different media. Results: We identified SP cells in early passage human stroma cells but the proportion of SP cells decreased gradually and disappeared eventually when cells were passaged. Purified SP cells could be cloned and expressed ABCG2 and nestin but were negative for keratocan. SP cells in differentiation media changed their phenotype and gene expression patterns. In media with horse serum or in Advanced–MEM, the stromal SP cell expression of keratocan and keratan sulfate was up–regulated. Cells in FGF2 expressed neural cell markers (neurofilament and NG2). Cells in VEGF expressed vWF. Conclusions: Viable SP cells can be isolated from human corneal stroma; they are clonogenic and express stem cell markers. These can be induced to express markers of differentiated keratocytes, neural cells, and endothelial cells. Horse serum and Advanced–MEM both initiate differentiated keratocyte phenotype. Ability to expand cultures of human cells that can give rise to functional keratocytes will be important for development of cell based therapies and assembly of bioprosthetic corneas.
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