Cultured human corneal epithelial stem/progenitor cellsderived from the corneal limbus

Naoki Yamamoto & Koji Hirano & Hajime Kojima & Mariko Sumitomo &

Hiromi Yamashita & Masahiko Ayaki & Koki Taniguchi & Atsuhiro Tanikawa &

Masayuki Horiguchi

Received: 26 March 2010 /Accepted: 19 August 2010 /Published online: 16 September 2010 / Editor: J. Denry Sato# The Society for In Vitro Biology 2010

Abstract Stem/progenitor cells of the human cornealepithelium are present in the human corneal limbus, andseveral corneal epithelial stem/progenitor cell markers havebeen reported. Recently, the neurotrophin family receptorswere reported to be useful markers of corneal epithelial stem/progenitor cells. Therefore, we examined an enzymaticseparation method for obtaining corneal epithelial stem/progenitor cells and measuring the change in the expressionof low-affinity neurotrophin receptor p75 (p75NTR), areceptor belonging to the neurotrophin family. As a result,it was found that our separation method preserved cellviability. Furthermore, p75NTR was mainly observed in

epithelial basal cells as were the corneal epithelial stem/progenitor markers p63 and integrin β1. p75NTR was alsoobserved in the cultured cells, but its frequency decreasedwith passage. In conclusion, we propose that our culturemethod will enable the culture of corneal stem cells and thatit is a useful tool for elucidating the molecular basis of theniche that is necessary for the maintenance of epithelial stemcells in the corneal limbus. Furthermore, we conclude thatp75NTR is a useful cell marker for evaluating the character-istics of stem/progenitor cells in culture.

Keywords Human corneal epithelium . Corneal limbus .

Low-affinity neurotrophin receptor p75 (p75NTR) . p63 .

Integrin β1

Introduction

The cornea is comprised of five layers, the cornealepithelium, Bowman’s membrane, the corneal stroma,Descemet’s membrane, and the corneal endothelium.Several types of non-differentiated cells are present in thecorneal epithelium: stem cells with self-replication compe-tence; progenitor cells produced by the asymmetric divisionof stem cells; and transient amplifying (TA) cells, which areproduced by the differentiation of progenitor cells (Qi et al.2008). As the corneal epithelium is damaged by exposureto ultraviolet rays and oxygen (Higa et al. 2005), itundergoes constant turnover; i.e., TA cells from the basallayer of the corneal epithelium replace the corneal epithelialcells that detach from the surface of the corneal epitheliumat a high rate under steady-state conditions. Homeostasis ofthe corneal epithelium is governed by a small subpopula-tion of corneal epithelial stem/progenitor cells located in thebasal epithelium layer of the limbus. (Qi et al. 2007).

N. Yamamoto (*) :M. Sumitomo :H. Yamashita :K. TaniguchiLaboratory of Molecular Biology and Histochemistry,Fujita Health University Joint Research Laboratory,1-98 Dengakugakubo, Kutsukake-cho,Toyoake, Aichi 470-1192, Japane-mail: naokiy@fujita-hu.ac.jp

K. Hirano :A. Tanikawa :M. HoriguchiDepartment of Ophthalmology,Fujita Health University School of Medicine,1-98 Dengakugakubo, Kutsukake-cho,Toyoake, Aichi 470-1192, Japan

H. KojimaDivision of Pharmacology, Biological Safety Research Center,Japanese Center for the Validation of Alternative Methods(JaCVAM), National Institute of Health Sciences,1-18-1, Kamiyoga,Setagaya-ku, Tokyo 158-8501, Japan

M. AyakiDepartment of Ophthalmology,Showa University Fujigaoka Rehabilitation Hospital,2-1-1, Fujigaoka, Aoba-ku,Yokohama 227-8518, Kanagawa, Japan

In Vitro Cell.Dev.Biol.—Animal (2010) 46:774–780DOI 10.1007/s11626-010-9344-9

Corneal epithelial stem/progenitor cells, which are veryslow-cycling cells (Cotsarelis et al. 1989), move from thelimbus to the central region of the cornea, where TA cellsare found (Buck 1985).

The low-affinity neurotrophin receptor p75 (p75NTR) isone of various markers of self-renewing tissue stem cells(Kunimura et al. 1998; Hahn et al. 2005; Yamamoto et al.2007), and p75NTR-positive cells isolated from humanesophageal epithelial cells, such as slow-cycling cells andrelatively immature keratinocytes, showed several pheno-typic characteristics of tissue stem cells (Okumura et al.2003).

The purpose of this study is to assess the utility of a cellculture method involving enzyme processing in serum-freemedium for evaluating the expression of p75NTR in thecorneal limbal epithelium and subcultured cells as a markerof immature/mature human corneal epithelial cells.

Materials and Methods

Human corneal tissue. Human corneal preparations andprimary corneal epithelial cells were harvested from threedonor eyes independently (Asian; male; age range, 43–56 yr). The donor corneas were obtained from theNorthwest Lion Eye Bank (Seattle, WA) in accordancewith the provisions of the Declaration of Helsinki forresearch involving human tissue.

Immunohistochemistry of corneal tissue. A part of theanterior ocular segment including the corneal tissue wasfixed in SUPER FIX rapid fixative solution (KuraboIndustries, Osaka, Japan), which is used for morphologicalresearch in ophthalmology (for the lens, retina, and so on)(Yamamoto et al. 2008). The cells of the outer layer of thecorneal epithelium sometimes become detached whenformalin fixative solution is used, which is most commonlyused for tissue fixation, but in this study the structure of theeyeball including not only the corneal epithelium but alsothe corneal stroma, corneal endothelium, ciliary body, andiris was preserved using SUPER FIX rapid fixativesolution. Briefly, the corneal tissue was fixed for approx-imately 60 min, and 3-μm paraffin sections were preparedfrom the fixed corneal tissue in the usual manner andincubated with anti-human p63 antibody (ready to use,Nichirei corp., Tokyo, Japan), anti-human integrin β1antibody (1:100, Millipore Corp., Billerica, MA), andanti-human p75NTR antibody (1:100, Alomone Labs Ltd.,Israel) for 1 h at 37°C. The secondary antibodies andworking dilutions were as follows: Alexa Fluor® 488-labeled anti-mouse goat IgG antibody (for integrin β1 andp63, 1:1,000, Invitrogen Corp., Carlsbad, CA) and AlexaFluor® 594-labeled anti-rabbit goat IgG antibody (for

p75NTR, 1:1,000, Invitrogen), which were incubated withthe sections for 1 h at 37°C. DAPI (VECTASHIELD H-1200; Vector Laboratories, Burlingame, CA) was used fornuclear staining. A fluorescence microscope (Power BX-51, Olympus, Tokyo, Japan) was used for observation. Inaddition, the corneal tissue was observed in detail byhematoxylin and eosin (H. E.) staining.

Cell culture. Normal human corneal epithelial (HCE) cellswere separated from the corneal limbus of the peripheralcorneal region after Descemet’s membrane and the cornealendothelium had been removed. The corneal limbus wasthen cut into eight equal pieces and carefully separatedfrom the underlying stroma using 0.25% collagenase andAccumax (cell aggregate dissociation medium, InnovativeCell Technologies, Inc. San Diego, CA) at 37°C for 30 min(Grant et al. 2005), before being washed twice withphosphate-buffered saline solution (PBS; Sigma, St. Louis,MO) and diluted ten times in the cell-lotion solution (JUJIFIELD Inc., Tokyo, Japan) using a low-adhesion cellcentrifuge tube (Stemful, SUMITOMO BAKELITE Co.,Ltd., Tokyo, Japan). In addition, we compared our methodwith the standard method; i.e., using dispase at 37°C for60 min (Koizumi et al. 2002; Touhami et al. 2002;Nakamura et al. 2006). The HCE cells were cultured inserum-free PCT corneal epithelium medium (CnT-20medium, CELLnTEC Advanced Cell Systems, Bern,Switzerland) supplemented with 50 units/ml penicillin and50 μg/ml streptomycin (Sigma, St. Louis, MO) at 37°C in a5% CO2-humidified incubator (Chirila et al. 2008).

To compare the culture conditions, the HCE cells werecultured in a 35-mm culture dish (BD Biosciences, SanJose, CA) (1×104 cells/dish) coated with collagen(TOYOBO Co., Ltd., Osaka, Japan) or FNC CoatingMix® (FNC; cell attachment improvement media, AthenaEnvironmental Sciences, Inc., Baltimore MD) (Engler et al.2009) in CnT-20 medium, and the cells were thensubcultured with TrypLE™ Select (Cell dissociationreagents, Invitrogen) (Li et al. 2008) or trypsin (Kuraboindustries LTD).

Frozen cultured human corneal epithelial cells (HCEC-2cells), at passage 2, were purchased from Kurabo industriesLTD (Osaka, Japan). The cells were then thawed andcultured in a 35 mm culture dish coated with FNC inEpiLife™ KG2 medium (Kurabo); M-stars C medium,which includes fetal bovine serum (ArBlast Co., Ltd.,Hyogo, Japan); or CnT-20 medium, supplemented withantibiotics at 37°C in a 5% CO2-humidified incubator. Eachmedium was changed every 2 d, and the cells weresubcultured with TrypLE™ Select.

Immunofluorescence staining and flow cytometric analy-sis. The cells separated from the corneal tissue, the fixed

EPITHELIUM CULTURE FROM THE CORNEAL LIMBUS 775

HCE cells cultured at passage 2 or 4, and the HCEC-2 cellscultured at passage 1 were incubated with anti-human p63antibody, anti-human integrin β1 antibody, anti-humanp75NTR antibody, anti-human TrkA antibody (1:100, SantaCruz Biotechnology, Inc., Santa Cruz, CA), and anti-humankeratin 3 (cytokeratin 3) antibody (1:100, ENZO LifeSciences International, Inc., Farmingdale, NY) at 4°C for30 min. The cells were then washed with PBS and incubatedwith the Alexa Fluor® 488-labeled secondary antibody at 4°Cfor 30 min. Next, the cells were washed with PBS and thenobserved under a fluorescence microscope or subjected toflow cytometry (fluorescence-activated cell sorting (FACS),FACSCan, BDBiosciences). All experiments were performedthree times.

Results

Marker expression in the corneal limbal region. Pigmentwas observed in the corneal limbal region, which containscorneal epithelial stem/progenitor cells (Fig. 1A). Thecorneal limbal preparation demonstrated melanocytes con-taining melanin near to the basal region of the cornealepithelium during H. E. staining (Fig. 1B). These cells wereslightly smaller and demonstrated a higher cell density thantheir neighboring cells (Fig. 1C). The p63 and integrin β1-positive cells were mainly found in the corneal epithelial

basal cell layer, and these cells were also positive forp75NTR. However, the cells on the surface of the cornealepithelium in the corneal limbus were negative for thesemarkers (Fig. 1D–I).

Comparison of culture conditions. After 7 d culture, mostof the non-coated culture dishes HCE cells were notattached (Fig. 2A). However, in the dishes coated withcollagen, the HCE cells multiplied to 2.1±0.2×105 from1×104 cells/dish (Fig. 2B). Furthermore, in the dishescoated with FNC, the HCE cells multiplied to 3.0±0.2×105

from 1×104 cells/dish (Fig. 2C). During the cell passage,most of the HCE cells were not attached when trypsin wasused (Fig. 2D), but the HCE cells multiplied to 3.0±0.2×105 from 1×104 cells/dish when TrypLE™ Select was used(Fig. 2E). When corneal epithelial cells were separatedfrom the corneal limbus using the standard method,keratocytes were often mixed in with them (Fig. 2F).

Morphology of HCE cells. The commercial HCEC-2 cellswere distorted at passage 2 regardless of whether they werecultured in EpiLife™ KG2 serum-free medium or M-StarsC. Cell growth arrested at passage 3, and the HCEC-2 cellscould not be subcultured after passage 3. In CnT-20medium, the HCEC-2 cells maintained their form untilpassage 3, but they could not be subcultured after passage4. On the other hand, the HCE cells reproduced aggres-sively at passage 3, and their polygonal form was

A C

D E F

G H I

BFigure 1 The structure shown(palisades of Vogt) is present inthe corneal limbus between thecornea and conjunctiva (A).Sections of the human anteriorocular segment were stainedwith H. E. (B, the bar indicates1 mm), and the area in the blacksquare has been highly magni-fied in (C). The corneal limbusdemonstrated melanocytes(arrowheads) containing mela-nin near the basal region (greendotted line) of the cornealepithelium. Staining of thecorneal limbus with threeantibodies to stem/progenitormarkers; p63 (D), p75NTR (E,H), integrin β1 (G), mergedimage of p63 and p75NTR (F),and merged image of integrinβ1 and p75NTR (I). The surfaceof the corneal epithelium ishighlighted by a white dottedline. The bars indicate 50 μm.

776 YAMAMOTO ET AL.

maintained until passage 4 in CnT-20 medium. However, atpassage 6, they became hypertrophic or extended (Fig. 3).

The change in marker expression with passage. The cellsseparated from the corneal limbus were stained with eachantibody, and were counted the number of positive cellsunder a fluorescence microscope. We found that p63-positive cells accounted for 40.4±2.1% (Fig. 4A), integrinβ1-positive cells for 34.4±2.0% (Fig. 4C), p75NTR-positive

cells for 28.4±3.1% (Fig. 4E), TrkA-positive cells for 24.8±3.2% (Fig. 4G), and keratin-positive cells for 70.3±2.7%(Fig. 4I) of all cells. Of the HCEC-2 cells, we found thatp63-positive cells accounted for 0.7±0.5%, integrin β1-positive cells for 97.1±2.0%, p75NTR-positive cells for 3.3±1.2%, TrkA-positive cells for 8.3±1.5%, and keratin-positivecells for 98.3±2.7% (data not shown).

The proportions of cultured HCE cells expressing thesemarkers at passages 2 and 4 were analyzed using FACS.

Figure 3 Morphology ofHCEC-2 cells and HCE cells.The HCEC-2 cells becamedistorted cells from passage 2onwards when cultured inEpiLife™ KG2 medium orM-Stars C medium, but theymaintained their form untilpassage 3 when cultured inCnT-20 medium. On the otherhand, the HCE cells reproducedaggressively at passage 3 andmaintained their polygonal cellform until passage 4, but theirform changed at passage 6. Thebars indicate 100 μm.

A B C

D E F

Figure 2 Comparison ofculture condition. Most of theHCE cells in the non-coatedculture dishes were not attached(A). The HCE cells grew morerapidly in the dishes coated withFNC (C) than in those coatedwith collagen (B). During thecell passage, most of the HCEcells did not attach when trypsinwas used (D), but they didattach and grow whenTrypLE™ Select was used (E).When the standard method wasused, keratocytes (arrowheads)were often mixed in with theHCE cells (F). The bars indicate100 μm.

EPITHELIUM CULTURE FROM THE CORNEAL LIMBUS 777

p63-positive cells accounted for 78.6±2.4% of the cells atpassage 2 and 49.6±4.7% of those at passage 4 (Fig. 4B),and the integrin β1-positive cells accounted for 98.7±0.4%and 97.0±1.1% (Fig. 4D), the p75NTR-positive cellsaccounted for 70.2±5.0% and 7.8±0.8% (Fig. 4F), theTrkA-positive cells accounted for 85.5±5.3% and 23.8±2.0% (Fig. 4H), and the keratin 3-positive cells accounted for98.6±0.5% and 98.2±0.5% of the cells at passages 2 and 4,respectively (Fig. 4J). The numbers of integrin β1 andkeratin 3-positive cells were not affected by passage number.

Discussion

Characteristics of the corneal limbus region. Severalstudies have reported that corneal epithelial stem/progenitorcells are present in the corneal limbus; where limbalpapillary structures (palisades of Vogt) are located(Schermer et al. 1986; Cotsarelis et al. 1989; Higa et al.2005; Li et al. 2007). It is considered that the stem cellniche model applies to the corneal limbus because themelanocytes in the corneal limbus are smaller than thenearby non-melanin cells (Li et al. 2007), adhere to eachother, and are attached to corneal epithelial stem/progenitorcells through N-cadherin (Hayashi et al. 2007). Corneallimbus cells were also found to be positive for p63, aknown corneal epithelial stem/progenitor cell marker;

integrin β1 (CD29) (Nakamura et al. 2006; Liu et al.2007; Utheim et al. 2009), which is an epithelial stem/progenitor cell marker; and p75NTR (CD271), which isexpressed by basal cells (TA cells) but not by cell that havedifferentiated from TA cells in the center of cornea (Qi et al.2007; Yamamoto et al. 2007; Qi et al. 2008). Theexpression of these cell markers was localized to the basalregion of the corneal epithelium (no such expression wasfound for the cells on the surface of the corneal limbus), anextremely unique region in which stem/progenitor cells,TA cells, and differentiated corneal epithelial cells arefound.

The separation and culture method. In previous reports,two types of separation method have generally been used toobtain cells from the corneal limbus, an explant method inwhich the cells were cultured from a small sample ofcorneal limbus tissue (Kim et al. 2004; Liu et al. 2007; Qiet al. 2008) and an enzymatic method that involved usingdispase for 60 min (Koizumi et al. 2002; Touhami et al.2002; Nakamura et al. 2006). Using our method, cornealepithelial stem/progenitor cells were effectively separatedfrom the corneal limbus by 30-min enzyme processingwithout keratocyte contamination. Furthermore, in thesubculture stage, the adhesive properties of the cells andtheir viability were improved by using TrypLE™ Selectrather than trypsin. In addition, the polygonal form of thecells was maintained until passage 4.

A C E

B D F

I

J

H

G

Figure 4. The changes in marker expression with passage. Thecorneal epithelial stem/progenitor cell markers p63 (A), integrin β1(C), p75NTR (E), and TrkA (G), and the differentiated cornealepithelium cell marker keratin 3 (I) were observed in the corneallimbus separated cells. Therefore, the cultured HCE cells were

analyzed for p63 (B), integrin β1 (D), p75NTR (F), TrkA (H), andkeratin 3 (J) by FACS at passages 2 and 4. The numbers of p63,p75NTR, and TrkA-positive cells were decreased at passage 4. Thebars indicate 100 μm.

778 YAMAMOTO ET AL.

The expression of several markers. In the cells separatedfrom the corneal limbus, p75NTR was expressed inapproximately similar proportions to p63, integrin β1, andTrkA (Di Girolamo et al. 2008; Utheim et al. 2009).Furthermore, the corneal epithelial stem/progenitor cellsalso expressed p63 and ABCG2 (data not shown), butthey did not express keratin 3 (Schermer et al. 1986;Cotsarelis et al. 1989; Chen et al. 2004; Hayashi et al.2007). On the other hand, keratin 3-positive cells wereobserved at an approximately similar ratio to the stem/progenitor marker negative cells (Qi et al. 2008). As thenumber of cells expressing p63 and p75NTR decreasedwith each passage, we concluded that the cultured cellswere gradually differentiating into mature corneal epithe-lial cells. Therefore, because the number of cells express-ing integrin β1 on the basal membrane side did notchange, we concluded that the adhesion culture methodrather than the three-dimensional culture method was themost appropriate for these cells.

The expression of neurotrophin factor receptor. In thecultured cells, we found that the nerve growth factor(NGF) receptor was expressed together with the NGFhigh-affinity receptor TrkA and the NGF low-affinityreceptor p75NTR. It is believed that co-expression of thep75NTR and Trk receptors leads to NT signaling throughTrk receptors and the promotion of cell survival (Carter andLewin 1997; Dechant and Barde 1997). The neurotrophinfamily consists of four members: NGF, brain-derivedneurotrophic factor, neurotrophin-3 (NT-3), andneurotrophin-4/5 (NT-4/5), and NT-3 and NT-4/5 have asequence homology of at least 50%. A neurotrophin is akind of cytokine that is involved in maintaining theviability of nerves (Johansson et al. 1997), the promotionof axis regeneration (Hollowell et al. 1990), and the growthand development of the regeneration-promoting nervoussystem (Levi-Montalcini and Booker 1960). The receptorsfor this neurotrophin family are molecules of the Trk familyof tyrosine kinase receptors: NGF has a high affinity forTrk A, BNDF and NT-4/5 have high affinities for Trk B,and NT-3 has a high affinity for Trk C. p75NTR can bind toall four of these neurotrophins (Bothwell 1995; Bibel et al.1999; Dechant 2001) and has recently been renamedCD271.

Finally, a report about the signals relevant to theembryonic stem cell niche was recently published (Bendallet al. 2007). We expect that our findings will enable theelucidation of the molecular basis of the niche that isnecessary for the maintenance of epithelial stem cells forresearching the corneal limbus. Therefore, we conclude thatp75NTR is a cell marker that can be used to evaluate thecharacteristics of stem/progenitor cells produced using theadhesion culture method.

Acknowledgments This study received public welfare labor scienceresearch expenses subsidies (H19-medicine public-003: from 2007 to2009, and H22-medicine public-002: from 2010 to 2012) andassistance from the Ministry of Education, Culture, Sports, Science,and Technology in the form of Grants in Aid for Scientific ResearchJAPAN (19700324 and 22591970).

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