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Sayan Basu, Andrew Hertsenberg, Martha L Funderburgh, James L Funderburgh; Limbal Biopsy Derived Stromal Stem Cells Prevent Corneal Scarring. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5172.
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Corneal allografting for stromal opacities carries the risk of failure due to immunological rejection. This study explored the possibility of developing an autologous stem cell-based technique for the treatment of blinding corneal stromal diseases including ulceration and scarring.
Tiny clinically replicable superficial biopsies of the limbus were obtained from human cadaveric corneo-scleral rims. The cells isolated from the biopsies were cultured in media containing either fetal bovine or human serum with or without cholera toxin. The limbal stromal cells were characterized based on their potential for clonal expansion, stem cell gene expression and differentiation into functional keratocytes. The limbal biopsy derived stromal stem cells were then incorporated in fibrin gel and layered on wounded mouse corneas and compared to a control group which received fibrin gel alone in terms of: restoration of corneal transparency, maintenance of collagen architecture and expression of fibrotic markers.
Results showed that cells with mesenchymal morphology could be rapidly isolated from limbal biopsies by collagenase digestion and initial co-culture with epithelial cells in media containing human serum without cholera toxin. These limbal stromal cells were highly clonogenic and expressed stem cell genes (ABCG2, Nestin, NGFR, Oct4, PAX6, and Sox2). These cells differentiated into functional keratocytes capable of expressing specific gene markers (ALDH3A1, AQP1, Keratocan and PTGDS) and generating a multi-layered matrix of collagen I and keratan-sulfate proteoglycans in-vitro. When layered on mouse corneal wounds the limbal biopsy derived stromal stem cells prevented stromal ulceration, vascularization and scarring. As compared to the control group mouse corneas treated with stromal stem cells showed significant reduction in : (a) light scattering and scar volume as assessed by high resolution optical coherence tomography ; (b) collagen fibril derangement on transmission electron microscopy; and (c) expression of fibrotic markers on qPCR and immune-staining.
The findings suggest that limbal biopsy derived stromal stem cells isolated using potentially autologous culture conditions prevent stromal ulceration and scarring and restore corneal transparency in wounded mouse corneas. This opens up the possibility of developing an autologous non-invasive stem cell-based therapy for corneal stromal diseases.
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