title: 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 alpha6 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, alpha6 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.
authors.affiliation: Department of Ophthalmology, University of Erlangen-Nuremberg, Germany. ewa.blazejewska@uk-erlangen.de
authors.forename: Ewa Anna
authors.initials: EA
authors.lastname: Blazejewska
authors.forename: Ursula
authors.initials: U
authors.lastname: Schlötzer-Schrehardt
authors.forename: Matthias
authors.initials: M
authors.lastname: Zenkel
authors.forename: Björn
authors.initials: B
authors.lastname: Bachmann
authors.forename: Erik
authors.initials: E
authors.lastname: Chankiewitz
authors.forename: Christina
authors.initials: C
authors.lastname: Jacobi
authors.forename: Friedrich E
authors.initials: FE
authors.lastname: Kruse
mesh_terms: D000818:Animals; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects*; D054338:Cell Transdifferentiation; D002478:Cells, Cultured; D024141:Collagen Type IV / Q000378:metabolism; D003315:Cornea / Q000166:cytology*; D017077:Culture Media, Conditioned / Q000494:pharmacology*; D019573:Epithelium, Corneal / Q000166:cytology*; D005136:Eye Proteins / Q000235:genetics; D005347:Fibroblasts / Q000378:metabolism* / Q000648:ultrastructure; D005353:Fibronectins / Q000378:metabolism; D005434:Flow Cytometry; D018859:Hair Follicle / Q000166:cytology*; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D039503:Integrin alpha6 / Q000378:metabolism; D007797:Laminin / Q000378:metabolism; D016850:Limbus Corneae / Q000166:cytology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008853:Microscopy; D046529:Microscopy, Electron, Transmission; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D000097794:Kalinin
chemical_list: D015815:Cell Adhesion Molecules; D024141:Collagen Type IV; D017077:Culture Media, Conditioned; D005136:Eye Proteins; D005353:Fibronectins; D018398:Homeodomain Proteins; D039503:Integrin alpha6; D007797:Laminin; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D012097:Repressor Proteins; D000097794:Kalinin; C000605730:PAX6 protein, human; C000605732:Pax6 protein, mouse; C121346:laminin 1