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Cell Biology International (2009) 33, 861–866 (Printed in Great Britain)
Corneal epithelial-like transdifferentiation of hair follicle stem cells is mediated by pax6 and β-catenin/Lef-1
Ke Yanga, Zilin Jiangb, Dong Wangc, Xiaohua Liana* and Tian Yanga*
aDepartment of Cell Biology, Third Military Medical University, Gaotanyan, Chongqing 400038, PR China
bBioengineering College of Chongqing University, Chongqing 400038, PR China
cDepartment of Ultrasound of XinQiao Hospital, Third Military Medical University, Chongqing 400038, PR China


Several types of adult stem cells are capable of transdifferentiaton into other types of tissues. The hair follicle bulge area is an abundant and easily accessible source of pluripotent adult stem cells. We demonstrate that the bulge KSCs have the potential for transdifferentiation into corneal epithelial-like cells. Bulge KSCs isolated by collagen type IV adhesiveness possessed the highest colony formation efficiency (CFE), and expressed specific markers (CD34 and α6-integrin). The isolated cells transdifferentiate into corneal epithelial-like cells in conditioned medium containing corneal limbus soluble factors, including their specific marker, keratin12. The transdifferentiation depends on upregulation of pax6 and downregulation of β-catenin and Lef-1. Furthermore, overexpression of pax6 in bulge KSCs induced their expression of k12. The expressions of β-catenin and Lef-1 were not suppressed in the pax6-transfected bulge KSCs, but which were downregulated pax6-transfected cells cultured in the conditioned medium. Bulge KSCs may have potential therapeutic application as cell source for the construction of bioengineered corneas.

Keywords: Hair follicle stem cells, Cornea, Pax6, β-Catenin, Lef-1, Transdifferentiation, k12.

*Corresponding authors. Tel.: +86 23 68752259; fax: +86 23 65463056.

1 Introduction

Adult stem cells have the unique capacity to self-renew and differentiate into specified cell lineages. A recent series of experiments suggest that several slow cycling adult stem cells reside in the bulge of hair follicle (Taylor et al., 2000). Moreover, bulge stem cells are multipotent, and can contribute to the formation of different types of cells in hair follicle, including those of outer root sheath, inner root sheath, hair shaft, sebaceous gland and epidermis. Other studies have demonstrated that nestin-positive cells located in the bulge region can give rise to neural cells, smooth muscle cells, melanocytes, and endothelial cells (Amoh et al., 2004, 2005). The bulge of adult mouse whisker hair follicles contains neural crest multipotent stem cells (Sieber-Blum et al., 2004). These findings suggest that the hair follicle bulge is a unique area serving as a local reservoir for pluripotent adult stem cells. Furthermore, most of hair follicle bulge cells are keratinocyte stem cells (KSCs) for hair follicle self-renewing and cycle (Ohyama et al., 2006).

It is now well established that the niche plays an important role in the proliferation and differentiation of stem cells in several tissues. Soluble factors are among the most important components in niche. Recent studies show that the soluble factors from neural microenvironment induce neural-like changes in mesenchymal stem cells (Rivera et al., 2006). The cornea is composed of three layers: the outer epithelium, the stroma, and the endothelium. The corneal epithelium is maintained by stem cells located at the basal layer of the corneal epithelium in a region known as the limbus (Cotsarelis et al., 1989). Inducing differentiation of human embryonic stem cells into corneal epithelial-like cells can be achieved by replicating factors located in the limbal stem cell niche in a vitro system (Ahmad et al., 2007). We have investigated the potential of the bulge KSCs to transdifferentiate into corneal epithelial-like cells by treatment with limbus soluble factors.

Many attempts have been made to reconstruct the corneal epithelium reconstruction using cells from other tissues, such as oral mucosal epithelial progenitor cells (Hayashida et al., 2005). However, the molecular mechanisms of the corneal epithelial-like differentiation remain unclear yet. The transcription factor, pax6, seen as the master regulator of eye development, plays a critical role in determining the early stage of cell differentiation in eye development, and involving in the corneal epithelium lineage-specific differentiation (Ramaesh et al., 2005). In addition, Wnt signaling is involved in the final decision and differentiation of stem cells, including hair follicle stem cells. The Wnt-activated transcription factor complex, β-catenin/Lef-1, has an important role in hair follicle formation (Huelsken et al., 2001). We have characterized the transdifferentiation of bulge KSCs into corneal epithelial-like cells in vitro at the cellular and molecular level. Importantly, both pax6 and β-catenin/Lef-1 signal processing contributed to the transdifferentiation.

2 Materials and methods

2.1 Enrichment of putative bulge KSCs

Experimental procedures and protocols were approved by the Animal Care and Use Committee of The Third Military Medical University. FAD medium containing low-glucose DMEM (Gibco) and Ham's F12(3:1) (Gibco), 10% FCS (Hyclone), 1% penicillin–streptomycin (Gibco), hydrocortisone (Sigma–Aldrich), insulin (Sigma–Aldrich), adenine (Sigma–Aldrich), cholera toxin (Sigma–Aldrich) and epidermal growth factor (Sigma–Aldrich) was made. The rat vibrissa follicles in anagen phase were dissected under a stereomicroscope. The dermal sheath was separated and treated with dispase for 1h at 37°C. The bulge regions of the hair follicles were carefully excised and incubated with a mixture of trypsin (Gibco) and EDTA (Gibco) for 15min. The separated cells were collected and seeded in culture flasks, which had been previously smeared with collagen IV. The cells adhered to collagen type IV for 5min, and non-adhering cells were seeded in another flask. α6-integrin expression of bulge KSCs adhering to type IV collagen was detected by flow cytometry.

The CFE (colony-forming efficiency) was used to assess the proliferative potential of the isolated cells (Jones and Watt, 1993). In brief, the equivalent numbers of adhering cells and non-adhering cells (500 cells/plate) were cultured in FAD culture medium for 2 weeks. The colonies were fixed with 4% formaldehyde and stained with 1% rhodamine B (Sigma) and >32 cells were scored under a microscope.

2.2 Bulge KSCs' cultures in conditioned medium

Bulge KSCs were cultured with limbus tissue conditioned medium in vitro. The limbus tissue of New Zealand rabbit was obtained from eyes removed immediately after death and washed in phosphate buffer solution (PBS). After careful removal of corneal endothelium under a surgical microscope, the limbal rings were placed on ice and their wet weight was rapidly measured. The tissue pieces were homogenized by adding L-DMEM and incubated on ice for 10min. The homogenate was centrifuged for 10min at 10,000g at 4°C, and the supernatant was collected, filtered with a filter of 0.22μm and stored at −80°C. Bulge KSCs of passage 3 were seeded in 6-wells' culture dishes, and the medium substituted with a 1:1 mixture of L-DMEM containing tissue extracts and FAD medium for conditioned induced group, and a 1:1 mixture of L-DMEM and FAD medium for control group.

2.3 Overexpression of pax6 in bulge KSCs

The plasmid expressing rat Pax6 (pAct/Pax6) was generously gifted by Dr. Masaharu Sakai (Hokkaido University Graduate School of Medicine, Sapporo, Japan). The full-length pax6-cDNA was successfully cloned into expression plasmid pcDNA3.1. For transient transfection in bulge KSCs, pcDNA3.1/Pax6 vectors were prepared in serum-free medium containing Tfx™ reagent (Promega). Cells were added to the mixture and incubated for 1h. Overexpression of pax6 was confirmed by RT-PCR. For further experiments, the cells overexpressing pax6 were cultured in limbal tissue conditioned medium, according to aforementioned method.

2.4 Reverse transcription-PCR analysis

Total RNA was isolated by TRIzol kit (Invitrogen) according to the manufacturer's instructions. Approximately 1μg of total RNA was reverse transcribed to cDNA using RT Kit (promega). The PCR was carried out using standard procedures. Primers:

β-actin: forward (5′-CGTAAAGACCTCTATGCCAACA-3′),


pax6: forward: (5′-CCTCCTTTACATCGGGTTCCAT-3′),


k12: forward: (5′-GAAACCGAGGGTGGATACTGC-3′),


β-catenin: forward: (5′-TCCGCATGGAGGAGATGATTG-3′),


Lef-1: forward: (5′-TCTCCTTTAGCGTACACTCG-3′),


2.5 Immunofluorescence

Cells were seeded on poly-d-lysine coated coverslips and fixed with acetone for 10min at room temperature. Non-specific reactions were blocked with goat serum and bovine serum albumin for 30min. Cells were incubated with primary antibodies, and stained with FITC-conjugated and TRITC-conjugated secondary antibodies. Images were taken on a fluorescence microscope (Leica). The following primary antibodies were used: anti-pax6 (R&D, 1:500), anti-keratin12 (k12, Santa Cruz, 1:1000), anti-CD34 (Santa Cruz, 1:500), anti-α6-integrin (Santa Cruz, 1:400), anti-β-catenin (Santa Cruz, 1:500), anti-lymphoid enhancer binding factor 1 (Lef-1, Chemicon, 1:500).

2.6 Statistical analysis

Results were analyzed using the unpaired Student's t-test. A P value <0.05 was considered statistically significant. Results are provided as mean±SEM from at least 3 independent experiments.

3 Results

3.1 Enrichment and characteristics of bulge KSCs

Bulge KSCs can be directly enriched from the bulge cells of rat vibrissa follicle rapidly adhering to collagen type IV, owing to the characteristics of keratinocyte stem cells (Kim et al., 2004; Zhou et al., 2004). The cells adhering to collagen type IV grew clonogenic (Fig. 1A and B). To examine the enriched cell populations, flow cytometric analysis was done after treatment with the anti-α6-integrin antibody. Adherent cells expressed high levels of α6-integrin, the marker of epidermal stem cells (Fig. 1F). Cells expressing high levels of α6-integrin (Fig. 1C) also expressed high levels of CD34 (Fig. 1D). CFE was used to measure the proliferative ability of these populations (Fig. 1E); that of adherent cells was 21.5±0.8%, which is significantly higher than that of non-adhering cells (6.6±0.5%, P<0.01), indicating high purity.

Fig. 1

Culture and characterization of bulge KSCs. (A) Cultured bulge KSCs for 2 days adhered to collagen IV. (B) Cultured bulge KSCs confluenced on day 10. (C and D) Double immunocytochemistry showed that cells co-expressed specific markers, α6-integrin and CD34. (E) CFE of adherent and non-adherent cells. CFE values are the mean±SEM. *P<0.01. (F) FACS analysis showed that 95.1% of cells were α6-integrin-positive. Scale bars: 50μm. Data are representative of results in 3 independent experiments.

3.2 Corneal epithelial-like transdifferentiation of bulge KSCs

The specified tissue soluble factors can control the fate specification of adult stem/progenitor cells (Rivera et al., 2006). We assessed if soluble factors derived from corneal limbus can induce a corneal epithelial-like phenotype in bulge KSCs. After conditioned culture for 7 days, the bulge KSCs began to become flattened. The intercellular space of induced cells became narrower compared to the control (Fig. 2A and B). Bulge KSCs were positively stained with keratin 12 (k12), which is a specific keratin found only in corneal epithelium (Fig. 2E and F). In addition, pax6 was expressed in both undifferentiated and differentiated cells (Fig. 2C and D).

Fig. 2

Corneal epithelial-like transdifferentiation of bulge KSCs in conditioned medium. (A) Phase-contrast images of cells in control group. (B) Phase-contrast images of cells in conditioned induced group. The cells became much flatter. Immunostaining of cells in control group (C, E, G, I) and conditioned culture group on day 7 (D, F, H, J). Pax6 expression both in control group (C) and in conditioned culture group (D); k12 expression lacking in control group (E) but was expressed in the conditioned culture group (F); nuclear and cytoplasm expression of β-catenin in the control group (G), but cytoplasm and cell membrane expression in conditioned culture group (H); Lef-1 expression in control group (I), which was markedly decreased in the conditioned culture group (J). (K) mRNA transcript analysis of the conditioned culture cells from day 0 to 7. RNA was extracted at indicated times and analyzed for the expression of pax6, k12, β-catenin and Lef-1. Data are representative of results in 3 independent experiments.

3.3 Corneal epithelial-like transdifferentiation of bulge KSCs and pax6 and Wnt signaling

mRNA expression of the specific marker, k12, was detected in corneal epithelial-like transdifferentiation. Cells began to express k12 on day 3, increasing until day 7 (Fig. 2K). Although pax6 was expressed in undifferentiated and differentiation cells, upregulation of endogenous pax6 mRNA level was seen on day 3 (Fig. 2K), suggesting that it became activated during the process. To investigate Wnt signaling during corneal epithelial-like transdifferentiation of bulge KSCs, expression levels of β-catenin and Lef-1 were followed. Expression of β-catenin mRNA transiently decreased on day 3 of transdifferentiation, increasing from day 5 to day 7 (Fig. 2K). However, expression level of Lef-1, which interacts with β-catenin in canonical Wnt pathway, gradually decreased from day 0 to day 7 (Fig. 2K). These results suggest that the Wnt signaling pathway was inhibited during transdifferentiation. β-catenin protein accumulated in the nucleus and cytoplasm in undifferentiated cells, while it was located in the cell membrane and cytoplasm in differentiated cells (Fig. 2G and H). Lef-1 protein expression was downregulated in differentiated cells, coincident with an altered tendency in mRNA expression (Fig. 2I and J). The results suggest that the transdifferentiation of bulge KSCs into corneal epithelial-like cells depends on the upregulation of pax6 and downregulation of β-catenin/Lef-1.

3.4 Effect of pax6 in corneal epithelial-like transdifferentiation of bulge KSCs

Regarding the effect of pax6 in the transdifferentiation, the expression of k12 after transient transfection of pax6 in bulge KSCs at 48h was confirmed by RT-PCR (Fig. 3A). Although corneal epithelial-like transdifferentiation was detected, the expressions of β-catenin and Lef-1 were not suppressed (Fig. 3B and C). Expression of β-catenin increased, but Lef-1 did not change (Fig. 3B and C). However, the expressions of β-catenin and Lef-1 were downregulated in the pax6-transfected bulge KSCs cultured in conditioned medium within 3 days. The results strongly suggest that pax6 mediated activation of cornea-specific k12 does not contribute to Wnt signaling.

Fig. 3

RT-PCR analysis of the pax6-transfected cells. RNA was extracted at 48 and 72h post-transfection. mRNA expression of k12 was detected at 48h (A). mRNA expression of β-catenin and Lef-1 was analyzed for pax6-transfected (B) and pax6-transfected with conditioned culture (C), respectively. β-catenin and Lef-1 were inhibited in pax6-transfected cells with conditioned culture (*P<0.05 vs 0h control), but not in pax6-transfected cells. Data are representative of results in 3 independent experiments.

4 Discussion

The discovery of stem cells in hair follicle bulge region has encouraged researches in regenerative medicine. Melanocyte stem cells, nestin-positive stem cell populations, have been identified in hair follicle bulge (Amoh et al., 2005). Our initial goal was to isolate the KSCs-enriched cell population from the bulge region of rat whisker follicle. The putative bulge KSCs were positive for both α6-integrin and CD34 markers (Trempus et al., 2003), and showed increasing in CFE compared with non-adherent cells.

Human embryonic stem cells cultured in conditioned medium from corneal limbal fibroblasts could differentiate into corneal epithelial-like cells which is evidence of a limbal mesenchymal (niche) role in corneal epithelial differentiation (Ahmad et al., 2007). Soluble factors derived from corneal limbus induced of k12 expression in bulge KSCs. K12 is generally believed to be the specific marker for mature corneal epithelial cells. We suggest that bulge KSCs differentiated into corneal epithelial-like cells. Some signaling molecules derived from the limbus played an important role in mediating corneal epithelial-like trandifferentiation. Therefore, bulge KSCs can be reprogrammed to differentiate into corneal epithelium in corneal epithelial stem cells niche.

Pax6 is deeply involved in controlling eye development, including the development of the cornea (Collinson et al., 2003; Ramaesh et al., 2004). The expression of pax6 was u-regulated by day 3, which is consistent with recent findings that transdifferentiation of corneal epithelium into epidermis is involved in downregulation of pax6 expression in basal corneal epithelium (Pearton et al., 2005). In corneal development, pax6 acts as the upstream regulator of k12 gene expression (Liu et al., 1999). We have shown that k12 expression is accompanied by upregulation of pax6 expression during transdifferentiation. Overexpression of pax6 in bulge cells switched on k12 expression. A similar result is that pax6-transfected embryonic stem cells expressed k12 and differentiated into corneal epithelial-like cells (Ueno et al., 2007), suggesting that pax6 is necessary for k12 promoter n.

Wnt signaling pathway is essential not only for embryogenesis, but also for stem cell differentiation in a range of epithelia. β-catenin, combined with Lef-1, is necessary for determining the differentiated of bulge KSCs into hair follicular lineage (Huelsken et al., 2001). However, normal development of corneal epithelium requires suppression of Wnt signaling. If Wnt signaling was activated in cornea development, the corneal epithelium would transform into stratified epidermis and hair follicle tissues. In corneal development, Wnt activity is suppressed in the limbal stroma region (Mukhopadhyay et al., 2006). We found that soluble factors derived from limbus induced bulge KSCs differentiation into corneal epithelial-like cells, and inhibited expressions of β-catenin and Lef-1. Upregulation of β-catenin expression on day 5 may attribute to the dual role of β-catenin in cell adhesion and signaling (Gottardi and Gumbiner, 2004). In corneal epithelium, β-catenin is one of the important proteins for cell–cell adherence junctions. β-catenin is mainly located in the nucleus before induced transdifferentiation, while it is in the cytoplasm and cell membrane after transdifferentiation, indicating that β-catenin-mediated transactivation is inhibited during transdifferentiation. From the analysis of pax6-transfected bulge stem cells, soluble factors derived from limbus, but not from pax6, are considered responsible for the suppression of β-catenin and Lef-1.

In conclusion, the process the transdifferentiation of bulge KSCs into corneal epithelial-like cells in limbus microenvironment depends on the upregulation of pax6 and the downregulation of β-catenin/Lef-1. Moreover, pax6 is essential for corneal epithelial transdifferentiation. During transdifferentiation, pax6 switches on the expression of k12, but does not contribute to the downregulation of β-catenin and Lef-1. Suppression of β-catenin/Lef-1 can be attributed to limbus soluble factors. These results provide a first step in the use of bulge KSCs to reconstruct damaged corneal epithelia.


We thank Dr. Masaharu Sakai, Hokkaido University Graduate School of Medicine, Japan for providing the plasmid pAct/Pax6. This work was supported by Chongqing Science and Technology Commission (No. CSTC 2004AC5074).


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Received 14 February 2008/1 December 2008; accepted 14 April 2009


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
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