Adipose-derived stem cells differentiate to keratocytes in vitro

PURPOSE Adipose-derived stem cells (ADSC) are an abundant population of adult stem cells with the potential to differentiate into several specialized tissue types, including neural and neural crest-derived cells. This study sought to determine if ADSC express keratocyte-specific phenotypic markers when cultured under conditions inducing differentiation of corneal stromal stem cells to keratocytes. METHODS Human subcutaneous adipose tissue was obtained by lipoaspiration. ADSC were isolated by collagenase digestion and differential centrifugation. Side population cells in ADSC were demonstrated using fluorescence-activated cell sorting after staining with Hoechst 33342. Differentiation to keratocyte phenotype was induced in fibrin gels or as pellet cultures with serum-free or reduced-serum media containing ascorbate. Keratocyte-specific gene expression was characterized using western blotting, quantitative RT-PCR, and immunostaining. RESULTS ADSC contained a side population and exhibited differentiation to adipocytes and chondrocytes indicating adult stem-cell potential. Culture of ADSC in fibrin gels or as pellets in reduced-serum medium with ascorbate and insulin induced expression of keratocan, keratan sulfate, and aldehyde dehydrogenase 3 family, member A1 (ALDH3A1), products highly expressed by differentiated keratocytes. Expression of differentiation markers was quantitatively similar to corneal stromal stem cells and occurred in both serum-free and serum containing media. CONCLUSIONS ADSC cultured under keratocyte-differentiation conditions express corneal-specific matrix components. Expression of these unique keratocyte products suggests that ADSC can adopt a keratocyte phenotype and therefore have potential for use in corneal cell therapy and tissue engineering.