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.

Figure 1

Expression of stem cell and differentiation markers in murine hair follicle (HF) and cornea. (A, B): Expression of K15, a putative epithelial stem and progenitor cell marker, in the HF bulge and basal cells. (C): Selective expression of K15 in the basal cells of limbal epithelium. (D, E): Expression of α6 integrin, a putative stem and progenitor cell marker, in HF bulge and basal cells. (F): Expression of α6 integrin in the basal cells of the corneal epithelium. (G): Lack of expression of K12 and Pax6 in the murine vibrissae HF. (H): Expression of K12 (green fluorescence) in murine corneal epithelium. (I): Expression of Pax6 (red fluorescence) in the cell nuclei of corneal epithelium throughout all the epithelial cell layers. Nuclear staining was performed with propidium iodide (red) or 4′,6-diamidino-2-phenylindole (blue). Magnification: 200× (E, I, H), 100× (B, C, F, G), 40× (A, D). Abbreviations: bg, bulge; dpa, dermal papilla; ep, epithelium; hs, hair shaft; irs, inner root sheath; ma, matrix; ors, outer root sheath; sg, sebaceous gland; st, stroma.

Figure 2

Isolation of hair follicle (HF) bulge cells. (A): Isolated HF after enzymatic digestion of the collagen capsule. (B): Dissection of the isolated HF into three fragments: S1, sebaceous gland-containing section; S2, bulge-containing section; S3, dermal papilla and matrix-containing section. (C): Colony-forming assay performed with cells derived from S1, S2, and S3 on a 3T3 feeder cell layer; rhodamin B staining after 2 weeks of culture. (D): Fluorescence-activated cell sorting (FACS) of isolated HF cells using an antibody to α6 integrin; the α6 integrin-positive cell population accounts for about 5% of the total cells. (E, F): Clonogenic capacity of α6 integrin-positive (E) and α6 integrin-negative (F) cell populations obtained by FACS. (G): Calculation of the colony-forming efficiency (CFE) of an α6 integrin-positive cell population after cell sorting in comparison with the CFE of the bulge cells after mechanical dissection. The bar chart demonstrates the mean number of colonies ± standard deviation from five experiments. Magnification: 100× (E, F), 40× (A, B). Abbreviations: bg, bulge; dpa, dermal papilla; hs, hair shaft; irs, inner root sheath; ors, outer root sheath; sg, sebaceous gland.

Figure 3

Clonal enrichment of hair follicle (HF) bulge cells. (A): Cellular outgrowth of HF bulge explants after 7-10 days of culture on a 3T3 feeder layer. (B): Expression of K15, an epithelial stem cell marker, in the outgrowing cells (arrow) of a bulge tissue explant. (C): Light microscopy of a holoclone obtained by either mechanical dissection or fluorescence-activated cell sorting on a 3T3 feeder layer. (D): K15-positive basal cells (arrow) are present in the culture of a holoclone. (E): K10-positive cells (arrow) are present in the culture of a holoclone, confined to a suprabasal layer of the central focus of stratification. (F): Lack of K12 expression within a holoclone. Nuclear staining was performed with propidium iodide (red). Magnification: 100× (D–F), 40× (A–C).

Figure 4

Effect of various culture conditions (matrices and conditioned media [CM]) on growth of clonally enriched, subcultivated hair follicle stem cells. (A–D): Light microscopic appearance of clonal cells subcultivated on type IV collagen (A), laminin-1 (B), laminin-5 (C), and fibronectin (D).Cultivation on type IV collagen and laminin-5 resulted in formation of regular epitheloid cell layers (A, C), whereas laminin-1 and fibronectin adversely affected cell adhesion and growth (B, D). (E–H): Light microscopic appearance of clonal cells subcultivated on laminin-5 in various CM from central corneal fibroblasts (CCF-CM) (E), peripheral corneal fibroblasts (PCF-CM) (F), limbal fibroblasts (LF-CM) (G), and 3T3 fibroblasts (3T3F-CM) (H). LF-CM and 3T3F-CM induced formation of regularly arranged, epitheloid cell sheets (G, H), whereas CCF-CM and PCF-CM resulted in rather irregular growth patterns of cells (E, F). Magnification: 100× (A–D), 200× (E–H).

Figure 5

Effect of various conditioned media (CM) on differentiation of clonally enriched hair follicle stem cells subcultivated on laminin-5. (A–D): Expression of K10, an epidermal differentiation marker, in clonal cells exposed to CM from central corneal fibroblasts (CCF-CM) (A), peripheral corneal fibroblasts (PCF-CM) (B), limbal fibroblasts (LF-CM) (C), and 3T3 fibroblasts (3T3F-CM) (D). (E–H): Expression of K12, a corneal differentiation marker, in clonal cells exposed to CCF-CM (E), PCF-CM (F), LF-CM (G), and 3T3F-CM (H). The lowest expression of K10 (C) and the highest expression of K12 (G) was observed in cells cultured in LF-CM. Nuclear staining was performed with propidium iodide (red) or 4′,6-diamidino-2-phenylindole (blue). Magnification: 100×. (I–K): Quantitative determination of K10, K12, and Pax6 mRNA expression levels of clonal cells cultured in various CM using real-time reverse transcription-polymerase chain reaction technology. The expression level was normalized against β-actin expression. The lowest expression of K10 (I) and the highest expression of K12 (J) and Pax6 (K) were observed in cells cultured in LF-CM. Statistical significance was assessed using the Mann-Whitney test for nonparametric analysis (*p < .05, **p < .005). (L, M): Determination of K12, K10, and Pax6 protein levels (arrows) in cells cultured in different CM by Western blot analysis based on a β-actin loading control. (L): Representative Western blot of cells cultured in CCF-CM, PCF-CM, LF-CM, and 3T3F-CM; MW, molecular weight marker. (M): Densitometric analysis of specific immunoreactive bands. Data were normalized to β-actin and represent the mean of two independent experiments.

Figure 6

Phenotypic appearance of cell sheets subcultivated in limbal fibroblast conditioned medium on fibrin gels coated with laminin-5. (A, C, E): Light and electron microscopic appearance of a two-layered epithelial cell sheet after 1 week of culture. (B, D, F): Light and electron microscopic appearance of a multilayered cell sheet after 2 weeks of culture. The cells show ultrastructural signs of epithelial differentiation such as apical microvilli, keratin filaments, desmosomes, and hemidesmosome. (G): Expression of α6 integrin (green fluorescence), a putative stem and progenitor cell marker, in the basal cell layer of the epithelial sheet. (H): Expression of K15 (green fluorescence), a putative stem cell marker, in a few single basal cells of the epithelial sheet (arrows). (I): Expression of K10 (green fluorescence), a marker of epidermal differentiation, in the superficial cell layer. (J): Expression of K12 (green fluorescence), a marker of corneal differentiation, throughout all epithelial cell layers. (K): Expression of Pax6 (red fluorescence), a transcription factor for K12, in nuclei of epithelial cells. (L): Colocalization of K12 (green) and Pax6 (red) expression in epithelial cells. Magnification: 40× (A, B), 100× (G, I, J, K), 200× (H, L). Scale bars, 5 μm (C, D) and 1 μm (E, F). Nuclear staining was performed with propidium iodide (red) or 4′,6-diamidino-2-phenylindole (blue). Abbreviations: amv, apical microvilli; ds, desmosomes; hds, hemidesmosomes; kf, keratin filaments; no, nucleolus; nu, nucleus.