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E. A. Blazejewska, U. Schloetzer-Schrehardt, K. Bitterer, P. Stockmann, F. E. Kruse; Replication of the Limbal Stem Cell Niche Allows for Differentiation of Human Hair Follicle Stem Cells Into Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2047.
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© ARVO (1962-2015); The Authors (2016-present)
Previously we demonstrated that murine hair follicle (HF) stem cells (SC) can differentiate into corneal epithelial-like cells in the presence of factors which replicate corneal-limbal SC niche in vitro. The aim of this study was to evaluate the potential of human HF as an autologous source of SC for ocular surface reconstruction and to gain further insight in the regulatory process of corneal differentiation.
Human scalp HF SC were isolated by enzymatic dissociation and clonally enriched on a 3T3 feeder cell layer. After 2-3 weeks of culture, clonal cells were further subcultivated on fibrin gels as a carrier, coated with various matrix components, such as laminin-5, type IV and type V collagen. Various conditioned media (CM) derived from epithelial cells or stromal fibroblasts either of central or peripheral cornea, or the limbus were applied for cell cultivation. Cellular phenotype and differentiation state were evaluated by light and electron microscopy, real time RT-PCR, immunohistochemistry, and Western-blotting. Antibodies against differentiation markers cytokeratin12 (K12), the paired box gene 6 (Pax6), or cytokeratin10 (K10) were used to evaluate the phenotype.
We could successfully isolate human HF bulge stem cells and expand them by clonal growth on a feeder layer. Subcultivation on fibrin gels coated with laminin-5, type IV and type V collagen, which are abundantly expressed in the corneal-limbal basement membrane zone, promoted cell adhesion, growth and formation of multilayered cell sheets. CM derived from both limbal fibroblasts and epithelial cells were found to be the most effective in inducing a corneal epithelial-like phenotype. They markedly upregulated the expression of K12, a corneal differentiation marker and its transcription factor Pax6 (about 6-fold and 3-fold, respectively) on the mRNA and protein level, whereas the expression of K10, an epidermal differentiation marker was strongly downregulated.
These findings suggest that human HF epithelial SC are capable of differentiating into corneal epithelial-like cells ex vivo when exposed to limbus-specific microenvironment. Identification of factors derived from limbal epithelial cells and fibroblasts may shed light on the regulatory process involved in the differentiation of limbal stem cells.
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