Corneal Limbal Microenvironment Can Induce Transdifferentiation of Hair Follicle Stem Cells into Corneal Epithelial-like Cells

Abstract

The aim of this study was to investigate the transdifferentiation potential of murine vibrissa hair follicle (HF) stem cells into corneal epithelial-like cells through modulation by corneal- or limbus-specific microenvironmental factors. Adult epithelial stem cells were isolated from the HF bulge region by mechanical dissection or fluorescence-activated cell sorting using antibodies to α6 integrin, enriched by clonal expansion, and subcultivated on various extracellular matrices (type IV collagen, laminin-1, laminin-5, fibronectin) and in different conditioned media derived from central and peripheral corneal fibroblasts, limbal stromal fibroblasts, and 3T3 fibroblasts. Cellular phenotype and differentiation were evaluated by light and electron microscopy, real-time reverse transcription-polymerase chain reaction, immunocytochemistry, and Western blotting, using antibodies against putative stem cell markers (K15, α6 integrin) and differentiation markers characteristic for corneal epithelium (K12, Pax6) or epidermis (K10). Using laminin-5, a major component of the corneo-limbal basement membrane zone, and conditioned medium from limbal stromal fibroblasts, clonally enriched HF stem and progenitor cells adhered rapidly and formed regularly arranged stratified cell sheets. Conditioned medium derived from limbal fibroblasts markedly upregulated expression of cornea-specific K12 and Pax6 on the mRNA and protein level, whereas expression of the epidermal keratinocyte marker K10 was strongly downregulated. These findings suggest that adult HF epithelial stem cells are capable of differentiating into corneal epithelial-like cells in vitro when exposed to a limbus-specific microenvironment. Therefore, the HF may be an easily accessible alternative therapeutic source of autologous adult stem cells for replacement of the corneal epithelium and restoration of visual function in patients with ocular surface disorders.

Human-Derived Feeder Fibroblasts for the Culture of Epithelial Cells For Clinical use

“…In these studies CK12 [27,28] and Pax6 [28] were upregulated. Since CK12 was not expressed in any of the oral epithelial cultures in this study, and PAX6 and MUC16 were not significantly different for HOME co-cultured with limbal or epithelial fibroblasts our data suggests that more than just fibroblasts are required to direct HOME towards a more corneal-like phenotype.An advantage of feeder layer pre-expansion of epithelial cells over explant techniques for cell therapy, is the provision of sufficient cells for quality control testing e.g.…”

“…Other studies have shown that human embryonic stem cells and murine hair follicle stem cells can be directed towards a more corneal like lineage using a combination of extracellular matrix and limbal fibroblast conditioned media [27,28]. In these studies CK12 [27,28] and Pax6 [28] were upregulated.…”

Bone marrow mesenchymal stem cells differentiate into urothelial cells and the implications for reconstructing urinary bladder mucosa

“…When cultured under specific microenvironmental factors, stem cells have the potential to transdifferentiate into corresponding mature cells (Blazejewska et al 2009;Altschuler et al 2008). This approach has also been applied to BMSCs (Coleman et al 2007).…”

Concise Review: Limbal Epithelial Stem Cell Therapy: Controversies and Challenges

“…Conditions mimicking the in vivo LESC niche have been used to induce a more corneal phenotype in hair follicle stem cells, embryonic stem cells, and marrow mesenchymal stem cells. Limbal/corneal fibroblasts and extracellular matrix found in the limbus have been used to do this [45][46][47][48]. In vivo, limbal fibroblasts are closely associated with LESCs and appear to be an important component of the limbal stem cell niche [51].…”

“…In vivo, limbal fibroblasts are closely associated with LESCs and appear to be an important component of the limbal stem cell niche [51]. Blazejewska et al (2009) found that pax 6 (involved in eye development) and CK12, which are both expressed in the cornea, were upregulated when mouse hair follicle stem cells were cultured on laminin V with conditioned medium from limbal fibroblasts. Mesenchymal stem cells were found to express CK12 when cocultured with corneal stromal cells in a transwell system [48].…”