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Sayan Basu, Martha Funderburgh, James Funderburgh; Limbal Biopsy As a Source of Stem Cells for Autologous Stromal Cell-based Therapy. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4557.
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Mesenchymal stem cells have been identified in the superficial stromal layers of the peripheral cornea and limbus. This study addresses the hypothesis that human stromal stem cells can be isolated from clinically replicable limbal biopsies and cultivated in conditions suitable for potentially autologous cell-based therapy.
One clock hour wide superficial stromal biopsies were obtained from the limbal region of human corneo-scleral rims. Stromal mesenchymal cells were isolated by collagenase digestion of the limbal fragment, with or without epithelial removal by dispase, and cultured in media containing 2% bovine or human serum. At the third passage stromal cells were evaluated for: a) stem cell gene expression; b) clonal growth; c) sphere formation; d) expression of keratocytes marker genes in differentiation conditions; and e) organization of collagen lamellae on aligned nanofiber substratum.
Collagenase digestion without epithelial removal was the most efficient method of isolating stromal cells, which readily expanded in media containing human serum. Epithelial cells were lost from the cultures by serial passage. P3 stromal cells had high clonogenecity compared to human fibroblasts, readily formed spheres and expressed stem cell gene markers (ABCG2, BMI1, CXCR4, Nestin, NOTCH1, Oct4 and SSEA4). The stromal cells cultured using both bovine and human serum demonstrated similar ability to differentiate into functional keratocytes, characterized by upregulation of keratocyte gene markers (ALDH, AQPR1, B3GnT7, CD34, CHST6, Keratocan and PTGDS) and generation of a multi-layered matrix of aligned collagen fibers.
Mesenchymal stem cells can be isolated from tiny superficial biopsies of the human limbal stroma and expanded in vitro using potentially autologous culture conditions. The ability of these stromal stem cells to differentiate into functional keratocytes opens the possibility of therapeutic strategies to cure blindness resulting from corneal stromal diseases with patient derived-cells isolated using autologous, xenobiotic-free conditions.
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