Cytokeratin 15 Can Be Used to Identify the Limbal Phenotype in Normal and Diseased Ocular Surfaces

Satoru Yoshida; Shigeto Shimmura; Tetsuya Kawakita; Hideyuki Miyashita; Seika Den; Jun Shimazaki; Kazuo Tsubota

doi:10.1167/iovs.06-0574

Abstract

purpose. To elucidate the expression pattern of K15, K19, K14, and K12 in human and mouse ocular surface epithelium as putative markers of epithelial phenotype.

methods. Immunohistochemical staining with specific antibodies for K15, K19, K14, and K12 was performed in human donor cornea tissue and normal ICR mouse corneas, with emphasis on localization of immunopositive cells. Immunohistochemistry was performed in a limbus-deficient mouse model as well as in clinical samples of pannus surgically removed from a thermal burn and a patient with Saltzmann’s dystrophy. Staining patterns were classified as limited to the most basal layer (K^bas), basal and suprabasal layers (K^bas-sup), predominantly in suprabasal layers (K^sup) and negative staining (K⁻).

results. In human conjunctival epithelium, strong expression of K15 was observed in basal cells, whereas K19 was expressed in both basal and suprabasal layers (K15^bas/K19^bas-sup/K12⁻). Limbal epithelial cells were K15^bas-sup/K19^bas-sup/K12^sup, whereas epithelial cells in the central cornea were K15⁻/K19^bas-sup/K12^bas-sup. In contrast, the mouse ocular surface demonstrated a different expression pattern of K15 and K19 than did the human tissue in the conjunctiva (K15^bas-sup/K19^bas/K12⁻) and the limbus (K15^bas-sup/K19^bas/K12^sup). Neither K15 nor K19 was expressed in the central mouse cornea (K15⁻/K19⁻/K12^bas-sup). Similar cytokeratin expression was observed in conjunctivalized corneas in mice and in surgically removed pannus tissue.

conclusions. Although the expression of K15 and K19 differ in humans and mice, specific staining patterns can be used to characterize the epithelial phenotype in normal and diseased ocular surface.

Keratins (cytokeratins and hair keratins) are a family of cytoskeletal component proteins of epithelial cells. Cytokeratins are divided into two subfamilies: type I (acidic) and type II (basic to neutral). Usually, at least one member of the type I family and one member of the type II family are coordinately expressed in each epithelial cell, and together they form intermediate filaments responsible for the structural integrity of epithelial cells.¹ ²Cytokeratins also seem to play a critical role in tissue differentiation, and the different patterns of cytokeratin expression in epithelia is often used as markers of differentiation.³ ⁴ ⁵For example, it is widely known that differentiated human corneal epithelial cells express cytokeratin 3 (K3, type II) and K12 (type I).⁶ ⁷ ⁸ ⁹The cornea-specific expression of K12 has also been found in mice.¹⁰ ¹¹ ¹²In addition to the predominant expression of K3 and K12, other cytokeratins including K14 and K19 are expressed as minor components of the cytoskeleton in basal and/or suprabasal human corneal epithelial cells.² ⁶ ¹³ ¹⁴ ¹⁵

In the skin, K19 has been proposed as a marker for stem cells in the skin hair follicle and also for proliferative keratinocytes in the basal layer.¹⁶ ¹⁷In human ocular surface epithelia, K19 is a minor cytoskeletal component of the corneal epithelium, but it is one of the major components in the conjunctival epithelium where K19 expression is reported to be uniform.² ⁶ ¹³ ¹⁴ ¹⁵ ¹⁸ ¹⁹Several studies have reported that K19 expression is found in all layers of the limbal epithelium, which becomes patchy progressively toward the center of the cornea and finally disappears in the center.² ⁶ ¹⁵K14/K5 expression is believed to be a marker for mitotically active, proliferative basal cells of stratified epithelia.⁷Indeed, K14 expression in the basal layer of corneal epithelium has been reported in humans, mice, and rats.⁷ ¹¹ ¹⁴ ²⁰K15 is another type I cytokeratin expressed in stratified epithelia, with several histologic studies reporting the basal expression of K15 in the epidermis.²¹ ²² ²³Kasper et al.⁶detected K15 protein in corneal and conjunctival epithelium by two-dimensional gel electrophoresis⁶ ; however, the localization of K15 in ocular surface epithelia remains unknown.

In the present study, K15 was expressed by limbal and conjunctival epithelia, but not by corneal epithelium, in both humans and mice. Furthermore, human limbal epithelium uniquely showed K15⁺ cells in the suprabasal layers, allowing the distinction of the limbus from conjunctiva. The limbal phenotype can further be characterized by multiple staining with K19 and K12. The pattern of K15 expression, together with other known markers such as ABCG2,²⁴ ²⁵ ²⁶Cx43,²⁶ ²⁷and vimentin,⁶ ¹⁴ ²⁸ ²⁹can be used to identify basal cells of the limbal area in normal and diseased tissue.

Material and Methods

Mouse Corneas

Specific pathogen-free adult ICR mice (n = 10) were purchased from CLEA Japan, Inc., Tokyo, Japan). All animals were handled in full accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and institutional guidelines. To produce total limbal deficiency, we denuded the corneal epithelium including the limbal area with an ophthalmic knife. Re-epithelialization of the scraped cornea was monitored by fluorescein staining. In another group of mice, the ocular surface was air dried for 15 minutes at room temperature under topical anesthesia. After 2 to 4 weeks, the mice were killed by cervical dislocation and the eyes were excised and embedded in 4% carboxy methyl cellulose (CMC; Finetec Co., Ltd., Tokyo, Japan) for immunohistochemical staining. Normal, untreated mice were used as the control.

Human Cornea Samples

Normal human corneas (n = 9) were obtained from Northwest Lions Eye Bank (Seattle, WA) and used as the normal control for immunohistochemistry. Clinical samples of pannus tissue were obtained during surgery from a thermal burn patient and a patient with Saltzmann’s nodular degeneration. Written informed consent was obtained from each patient before surgery. Excised tissue was immediately embedded in OCT compound (Tissue-Tek; Sakura Finetek, Co. Ltd., Tokyo, Japan) and prepared for immunohistochemistry. The study protocols involving patients and donor eyes were in compliance with the Declaration of Helsinki.

Immunohistochemistry

Immunocytochemistry was performed as described previously.³⁰In brief, whole mouse eye or segments of human sclerocorneal tissue were embedded in 4% CMC. Fresh frozen sections (5–10 μm thick) were air dried, fixed in 4% paraformaldehyde for 10 minutes, and then incubated in fixative (Morphosave; Ventana Medical Systems, Tucson, AZ) for 15 minutes. Blocking was performed with 10% donkey or goat serum in phosphate-buffered saline (PBS) for 30 minutes. Sections were then incubated with primary antibodies for 1 hour at room temperature. The primary antibodies used in this study are summarized in Table 1 . Immunoreactivity of primary antibodies was visualized with secondary antibodies conjugated with FITC, Cy3 (Jackson ImmunoResearch Laboratories, West Grove, PA) and Alexa 488 (Invitrogen Corp., Carlsbad, CA). After they were washed with PBS, the sections were mounted (Permafluor; Beckman Coulter Inc., Miami, FL). Images were observed by a microscope (Axioplan 2; Carl Zeiss Inc., Thornwood, NY) equipped with a digital camera (Axiocam; Carl Zeiss Inc.). PAS staining was performed according to standard procedures.

Staining patterns of keratin were classified as limited to the most basal layer (K^bas), basal and suprabasal layers (K^bas-sup), predominantly in suprabasal layers (K^sup) and negative staining (K⁻).

Results

Cytokeratin Expression on the Human Ocular Surface

We first examined the expression pattern of K15, K19, and K12 on the human ocular surface. As shown in Figures 1B and 2A , strong K15 expression was observed in the basal layer of the conjunctiva, while K19 was expressed in both the basal and suprabasal layers (Fig 2A) . Because K12 was negative (Fig 1B) , the conjunctival epithelial phenotype was K15^bas/K19^bas-sup/K12⁻. Further into the limbus, K12 expression appeared mainly in the suprabasal layers, although weak staining was observed in the basal cells as well (Figs. 1D 1E) . K19 expression was similar to that in the conjunctiva; however, K15 staining was distinct from the conjunctiva, with positive cells found in the suprabasal layers as well (Figs. 2A 2C) . The number of K15⁺ layers varied in different sections, even in samples from the same donor (Figs. 1D 2C 2F) . The limbal epithelial phenotype can thus be represented as K15^bas-sup/K19^bas-sup/K12^sup. The central corneal epithelium was uniformly K12⁺ and K15⁻ (Fig. 1C) . In addition, as previously reported by Chen et al.,³¹we found various levels of K19⁺ cells in the central corneal epithelium, although the expression level was lower than in the conjunctiva (Fig. 2B) . The phenotype of the central cornea was therefore K15⁻/K19^bas-sup/K12^bas-sup.

We further compared the expression of K15 with that of K14, another basal cell marker. Although K14 expression in the corneal epithelium is considered to be restricted to the basal layer,¹¹ ¹⁴ ²⁰ ²⁸ ³²we also found K14 staining in the suprabasal layers of corneal and conjunctival epithelium (Figs. 2D 2E 2F) . Nevertheless, K14 expression was strongest in the K15⁺ basal cells of the limbal and conjunctival epithelium, as well as in K15⁻ corneal basal cells.

Cytokeratin Expression in the Mouse Ocular Surface

We further examined the expression pattern of cytokeratins in mice, and found a contrasting staining pattern in the conjunctiva compared with human tissue. K15 was expressed in all layers of the conjunctival epithelium (Figs. 3A 3C 3D) , which was similar to the expression of K19 in human conjunctiva. In contrast, K19 was observed only in the most basal layer of the conjunctival epithelium, mirroring that of K15 expression in humans (Figs. 3B 3C) . In other words, K19 and K15 showed a reciprocal pattern in humans and mice (K15^bas-sup/K19^bas/K12⁻). As expected, the limbal epithelium was positive for K12 in the suprabasal layer (K15^bas-sup/K19^bas/K12^sup). Cells from the central to midperipheral cornea were predominantly K15 negative (Figs. 3E 3F 3G) . K19 staining was also negative in the central mouse cornea (Fig. 3F) , whereas K12 was positive in the basal and suprabasal layers (K15⁻/K19⁻/K12^bas-sup).

The expression pattern of K14 in mouse cornea and conjunctiva was similar to human tissue. Strong expression of K14 was observed in the basal and suprabasal cells from the conjunctiva to the limbus (Figs. 3D 3G) . Suprabasal cells in the central area also expressed K14, but the expression level was weak compared with that in the conjunctiva (Fig. 3G) .

Cytokeratin Pattern in Identifying Epithelial Phenotype in Pathologic Tissue

Furthermore, we examine the expression pattern of these cytokeratins in conjunctivalized mouse corneas. Debrided corneal epithelium, as well as corneas subjected to severe drying demonstrated the conjunctival phenotype K19^bas/K15^bas-sup/K12⁻) in the central cornea (Figs. 4A 4B 4D) . PAS-positive goblet cells were also observed, confirming the conjunctival phenotype (Fig. 4E) .

We finally examined the expression pattern of K15, K19, and K12 in two clinical pannus specimens. In the thermal burn patient, excised tissue clearly showed the conjunctival phenotype K15^bas/K19^bas-sup/K12⁻ (Fig. 5) . In contrast, pannus tissue from the patient with Saltzmann’s nodular degeneration showed patchy staining of all 3 cytokeratins (Fig. 6B) . High magnification revealed both the conjunctival phenotype K15^bas/K19^bas-sup/K12⁻ and the limbal phenotype K15^bas-sup/K19^bas-sup/K12^sup in the same field of view (Figs. 6D 6E 6F) , suggesting that the epithelium extending into the clear cornea includes basal limbal epithelial cells.

Discussion

Several disorders in humans and mice are caused by deficiencies in cytokeratin genes, suggesting that cytoskeletal proteins have important functions in maintaining cellular integrity.³³In addition, cytokeratins are often used in the characterization of epithelial phenotype and differentiation and in the diagnosis of carcinomas. K15 is a type I cytokeratin reported in basal keratinocytes of the epidermis²¹and has also been proposed as a marker of stem cells in the hair follicle bulge.³⁴ ³⁵However, reports on the expression of K15 in the ocular surface are scarce,⁶ ²¹and the expression pattern remains unclear. In this study, we demonstrated the unique expression pattern of K15 in the basal human and mouse corneal and conjunctival epithelium. Different epithelial phenotypes were shown to express unique patterns of K15, K19, and K12 expression (summarized in Table 2 ).

It is commonly accepted that in humans, K14 is expressed in the basal layer of the corneal and conjunctival epithelium, and K19 is expressed in basal limbal cells and all layers of the conjunctival epithelium. However, we found K14 in all layers of each stratified epithelia, although the expression level in the suprabasal layers of the corneal epithelium was lower than that in the basal layer. Because the expression of K14 spans the entire length of the ocular surface, it is not useful as a marker to distinguish limbal cells from conjunctival and corneal basal cells. The expression pattern of K19 is also controversial,¹⁹ ³¹and we found that it is expressed through all layers of the human conjunctival and corneal epithelium, including the limbal area. However, the expression of K19 in the central corneal epithelium showed variation among individuals. Another report by Kasper et al.⁶also showed K19 expressed in all layers of the limbal epithelium.

The difference in the immunohistochemical staining pattern is probably due to variations in technical procedure, which includes the method of tissue processing, the sensitivity of antibodies used, and conditions for visualization. For example, overfixation often leads to loss or reduction of antigen reactivity. Indeed, staining patterns of p63, another gene often used to stain the limbal epithelium, varies greatly in tissue-processing methods.²⁰We have confirmed the expression pattern of the cytokeratins demonstrated in this study by using several antibodies for K15, K19, or K14 (Table 1 , Supplementary Fig. S1). In addition to K14, we found that K5, a type II partner of K14, is expressed in all layers of the epithelia (Supplementary Fig. S1), suggesting that the variation in staining mainly depends on the difference in tissue processing rather than the type of antibody used. The condition of the tissue used for immunocytochemistry may also affect keratin expression, because Di Iorio et al.³⁶reported that the expression pattern of other stem cell markers depended on the condition of the donor corneas used. However, the elapsed time between death and use of the corneas used in our study was standardized at approximately 5 days (range, 3.5–8.5), and samples were fixed immediately after use for surgery. The variation of cytokeratin expression did not seem to be associated with the variation in elapsed time from death, but rather on the location within a specific sample.

Recently, Kawasaki et al.³⁷reported that the K12-positive cells appear to be ectopically residing, self-maintaining corneal epithelial cells in the conjunctival epithelium. We were unable to find such cells in our samples, probably because we did not specifically look for such cell clusters. However, it would be interesting to re-examine such K12-positive clusters in the conjunctiva for K15 expression in the basal and suprabasal layers.

In mice, although expression of K12 and K14 was similar to that in human tissue, strong expression of K15 and K19 was found respectively in the suprabasal and basal layers of the limbal and conjunctival epithelium. The expression pattern of K15 and K19 in mice was exactly the opposite of what was found in human tissue. The data suggest that the functions of these cytokeratins are switched between both species. Both K15 and K19 are type I acidic cytokeratins with undefined type II partners. Because K15 and K19 are not expressed in the central cornea (K15⁻/K19⁻/K12^bas-sup), both can be used to demonstrate conjunctivalization in mice. The possibility that antibodies for K15 and K19 cross-react respectively with K19 and K15 in mice cannot be completely ruled out. However, this is highly unlikely when the amino acid sequences of the cytokeratins are compared in both species.

Although cytokeratin expression alone is not sufficient to identify stem cells or transient amplifying (TA) cells, the expression profile of several key cytokeratins can be used to characterize epithelial phenotype in normal and diseased tissue. We have also identified K15 as a key cytokeratin that, unlike K14, is not expressed in differentiated corneal epithelium, and can also be used to differentiate limbal phenotype from the conjunctiva. Indeed, the human limbal phenotype K15^bas-sup/K19^bas-sup/K12^sup was found in surgically removed tissue that was clinically diagnosed as invading conjunctival epithelium. Therefore, this staining combination may be used to identify residual limbal structures in limbal deficient eyes.

Supported in part by a grant from the Advanced and Innovational Research Program in Life Sciences from the Ministry of Education, Culture, Sports, Science and Technology (TK), and a Grant-in-Aid for Scientific Research (SS).

Submitted for publication May 27, 2006; revised July 3, 2006; accepted August 30, 2006.

Disclosure: S. Yoshida, None; S. Shimmura, None; T. Kawakita, None; H. Miyashita, None; S. Den, None; J. Shimazaki, None; K. Tsubot a, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Shigeto Shimmura, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan; shige@sc.itc.keio.ac.jp.

Table 1.

View Table

Antibodies Used in the Study

Table 1.

Antibodies Used in the Study

Antigen	Clone Name/Code	Type	Host	Immunogen	Manufacturer
K12	sc-17101 (L-15)	Polyclonal	Goat	Mouse K12	Santa Cruz Biotechnology, Santa Cruz, CA
K15	LHK15	Monoclonal	Mouse	Human K15	Lab Vision, Fremont, CA
K15	PCK-153P	Polyclonal	Chicken	Human K15	CRP, Denver, CO
K19	RCK108	Monoclonal	Mouse	Human K19	Lab Vision Corp.
K19	RB-9021	Polyclonal	Rabbit	Human K19	Lab Vision Corp.
K19	A53-B/A2.26	Monoclonal	Mouse	Human K19	Chemicon International, Inc., Temecula, CA
K14	PRB-155P	Polyclonal	Rabbit	Mouse K14	CRP
K14	LL001^*	Monoclonal	Mouse	Human K14	Abcam Inc., Cambridge MA
K14	SPK14.2^*	Monoclonal	Mouse	Human K14	Abcam Inc.
K5	XM26^*	Monoclonal	Mouse	Human K5	Abcam Inc.
K5	PRB-160P^*	Polyclonal	Rabbit	Mouse K5	CRP

* Used in Supplementary Figure S1.

Figure 1.

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Expression of K15 and K12 in human corneal and conjunctival epithelium. (A) An overview of a human ocular surface immunostained with anti-K12 (green, FITC) and anti-K15 (red, Cy3). Boxes: magnified regions shown in conjunctiva (B), central cornea (C), and limbal area (D, D′, E). (D, D′) Images across the limbus; white arrow: same cell in each image. In the conjunctiva, K15 is expressed only in basal cells (B, K15^bas/K12⁻). In contrast, K15⁺ cells were found in the suprabasal layers of the limbus (A, D, D′, K15^bas-sup/K12^sup). Isolated K15⁺ cells were observed in the central area that were also K12⁺ (C). The expression of K15 was highest in basal cells of the limbal area (D′, arrowheads). These cells also expressed low levels of K12 (E, arrowheads). Scale bar: (A–D′) 200 μm; (E) 50 μm.

Figure 2.

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Expression pattern of K15, K19, and K14 in human corneal and conjunctival epithelium. Images of serial sections stained with anti-K19 (green, FITC) and anti-K15 (red, Cy3) antibodies (A–C) and with anti-K14 (green) and anti-K15 (red) antibodies (D–F). Conjunctiva (A, D) and central (B, E), or limbal area (C, F) of the cornea. Together with the expression of K15, a strong expression of K19 was observed in the basal and suprabasal layers of the conjunctival (K15^bas/K19^bas-sup) and limbal (K15^bas-sup/K19^bas-sup) epithelia (A, C, D, F). Cells weakly positive for K19 were also found in the central area (B, K15⁻/K19^bas-sup). K14 was observed in the conjunctiva and in the basal and suprabasal layers of the limbus and central corneal epithelium (D, E, F). Scale bar, 50 μm.

Figure 3.

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Expression of K12, K14, K19, and K15 in mouse corneal and conjunctival epithelium. Images of serial sections stained with anti-K12 (green, Alexa-488) and anti-K15 (red, Cy3) antibodies (A, E), anti-K12 (green) and anti-K19 (red) antibodies (B), anti-K19 (green) and anti-K15 (red) antibodies (C, F), and anti-K14 (green) and anti-K15 (red) antibodies (D, G). (A–D) Limbal area of mouse cornea and conjunctiva. (E–G) Central area of the cornea. Characteristic keratin expression patterns were observed in the central cornea (K15⁻/K19⁻/K12^bas-sup), the limbal area (K15^bas-sup/K19^bas/K12^sup), and the conjunctiva (K15^bas-sup /K19^bas/K12⁻). As in human tissue, strong expression of K14 was observed in the basal layer of the cornea and suprabasal cells of the conjunctiva (D, G). Moderate expression of K14 was observed in suprabasal cells in corneal epithelium (G). Scale bar, 50 μm.

Figure 4.

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Expression of K12, K19, and K15 in conjunctivalized mouse cornea. Sections were stained with anti-K12 (green, Alexa-488) and anti-K15 (red, Cy3) antibodies (A–C), anti-K19 (green) and anti-K15 (red) antibodies (D), and PAS (E, F). (A, B, D, E) Central mouse cornea with conjunctivalized epithelium. (A) Low-magnification image; box: magnified region shown in (B). (C, F) Conjunctival positive control. Conjunctivalization of the cornea is verified by the conjunctival phenotype pattern (K15^bas-sup /K19^bas/K12⁻). PAS-positive goblet cells (black arrowhead) were also observed (E). Background anti-K12 staining was found in goblet cells (white arrowhead) in the conjunctiva (C) and conjunctivalized cornea epithelium (B). Scale bars: (A) 100 μm; (F) 50 μm.

Figure 5.

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Expression of K12, K19, and K15 in a thermal burn patient. (A) Slit micrograph of a vascularized pannus with central corneal phenotype. Hematoxylin-eosin–stained (C) and immunostained (D–F) areas corresponding to the boxed region in (B). Serial sections were double stained for K12 (green, negative) and K15 (red) (D), K12 (green, negative) and K19 (red) (E), or K19 (green) and K15 (green) (F). The tissue shows the conjunctiva phenotype K15^bas/K19^bas-sup/K12⁻. Scale bars: (B) 200 μm; (F) 50 μm.

Figure 6.

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Expression of K12, K19, and K15 in a Saltzmann’s nodular degeneration patient. (A) Presurgical slit micrograph of the pannus extending from the inferior nasal quadrant of the right eye. Hematoxylin-eosin–stained (C) and immunostained (D–F) images of the area corresponding to the boxed region in (B). Serial sections were double stained for K12 (green, negative) and K15 (red) (D), K12 (green, negative) and K19 (red) (E), or K19 (green) and K15 (green) (F). Both the conjunctival phenotype K15^bas/K19^bas-sup/K12⁻ and limbal phenotype K15^bas-sup/K19^bas-sup/K12^sup (D–F, boxes) were observed in this tissue. Scale bar, 50 μm.

Table 2.

View Table

Summary of K12, K14, K15, and K19 Expression in Human and Mouse Ocular Surface Epithelium

Table 2.

Summary of K12, K14, K15, and K19 Expression in Human and Mouse Ocular Surface Epithelium

	Conjunctiva			Limbal Area			Central Area
		Basal	Suprabasal	Basal	Suprabasal	Basal	Suprabasal
Human	K15^bas/K19^bas-sup/K12⁻			K15^bas-sup/K19^bas-sup/K12			K15⁻/K19^bas-sup/K12^bas-sup
K12	−	−	+/−	+/−	++	++
K15	+++	−	+++^*	++/+/−^*	−^{, †}	−^{, †}
K19	+++	++	+++	++	+	+
K14	+++	++	+++	++	+++
Mouse	K15^bas-sup/K19^bas/K12⁻			K15^bas-sup/K19^bas/K12^sup			K15⁻/K19⁻/K12^bas-sup
K12	−	−	+/−	+/−	++	++
K15	+	++	+	++	−	−
K19	++	−^{, ‡}	++	−^{, ‡}	−	−
K14	+++	++	+++	++	+++	+

* The number of K15-positive cells vary in different sections.

† Occasional positive cell.

‡ Background staining.

Supplementary Materials

Supplementary Figure S1 - PDF version - 76.4 KB

Supplementary Figure S1 - TIF version - 18.8 MB

K14 and K5 are expressed in all layers of the limbal epithelium.

The authors thank Kimie Kato for technical assistance.

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Figure 1.

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