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Abstract
Purpose: Transplantation of autologous corneal stem cells in not possible in cases of bilateral limbal stem cell deficiency (LSCD). To restore the ocular surface in these patients, an autologous extraocular source of stem cells is desirable to avoid dependence on deceased donor tissue and host immunosuppression of allogenic transplants. While bone marrow-derived mesenchymal stem cells (MSCs) can acquire certain characteristics of corneal epithelial cells, subcutaneous adipose tissue (AT) is more readily available and accessible. The aim of this study was to determine if extraocular human AT-derived MSCs (hAT-MSCs) can acquire in vitro some features of corneal epithelial-like cells.
Methods: hAT-MSCs were isolated from human lipoaspirates and expanded up to 3–4 passages. We studied the immunophenotype of MSCs and demonstrated its multipotent capacity to differentiate toward osteoblasts, adipocytes and chondrocytes. To test the capacity of differentiation of hAT-MSCs toward corneal epithelial-like cells, hAT-MSCs were cultured on substrata of plastic or collagen IV. We used basal culture medium (BM), BM conditioned with human corneal epithelial cells (HCEcBM) and BM conditioned with limbal fibroblasts (LFcBM).
Results: The hAT-MSCs incubated for 15 days with HCEcBM acquired more polygonal and complex morphology as evaluated by phase-contrast microscopy and flow cytometry. Additionally, the expression of transforming growth factor-β receptor CD105 and corneal epithelial marker CK12 got increased as evaluated by flow cytometry, real-time reverse-transcription polymerase chain reaction, western blot and immunostaining. These changes were absent in hAT-MSCs incubated with unconditioned BM or with LFcBM.
Conclusions: Corneal epithelial-like cells can be induced from extraocular hAT-MSCs by subjecting them to an in vitro microenvironment containing conditioning signals derived from differentiated human corneal epithelial cells. Our results suggest that hAT-MSCs could provide a novel source of stem cells that hold the potential to restore sight lost in patients suffering from bilateral ocular surface failure due to LSCD.
Acknowledgements
The authors thank M. J. Villanueva (Europa Clinic, Valladolid, Spain) and M. F. de la Paz (Barraquer Eye Bank, Barcelona, Spain) for their support in providing human liposuctions and human corneoscleral tissues, respectively. The authors thank R. M. Corrales (Department of Ophthalmology, Baylor College of Medicine, Houston, TX) for the initial advice, M.T. García-Montes (Department of Hematology, Salamanca University Hospital, Salamanca, Spain) for her initial technical assistance, I. Fernández (IOBA, University of Valladolid, Valladolid, Spain) for statistical assistance, and B. Bromberg (Certified Editor in Life Science of Xenofile Editing) for his assistance in the editing of this article.