Therapeutic potential of human hair follicle‐derived stem cells for ocular surface reconstruction using a rabbit stem cell deficiency model

Abstract

Abstract Purpose To examine the therapeutic potential of human hair follicle (HF)‐derived stem cells (SC) for ocular surface reconstruction. Methods HF SC were isolated enzymatically and enriched on a 3T3 layer by clonal growth assay. To support differentiation into a corneal cell phenotype, clones were transferred onto a fibrin carrier and cultivated in a conditioned medium (CM) derived from limbal stromal keratocytes. Following generation of a limbal stem cell deficiency a multilayered cell sheet grown on a fibrin carrier was transplanted onto a rabbit eye (n=3). Cell phenotype was examined by means of real time RT‐PCR, immunohistochemistry, and Western‐blotting using antibodies against corneal markers, K12 and Pax6, as well as the epidermal marker K10. To identify secreted factors in the limbal CM a Human Growth Factor Antibody Array was performed. Results Cultivation of HF cells in limbal keratatocyte–derived CM induced the expression of K12 and Pax6 on the mRNA and protein level, whereas the expression of K10 was strongly downregulated, in comparison to an unconditioned control. Several potential candidates (FGF‐4, IGFBP‐2, 4,6 and IGF‐II) involved in the corneal differentiation process could be identified. Following transplantation onto a limbal SC deficient rabbit eye, HF‐derived SC could regenerate the corneal epithelium and express the corneal differentiation marker K12 while suppressing K10 expression. Conclusion These findings suggest that human HF‐derived SC are capable of being reprogrammed into corneal cell phenotype when exposed to factors of the limbal niche in vitro and maintain the reprogrammed phenotype in vivo following transplantation onto an animal model.