Clusters of Corneal Epithelial Cells Reside Ectopically in
Human Conjunctival Epithelium
Satoshi Kawasaki, Hidetoshi Tanioka, Kenta Yamasaki, Norihiko Yokoi, Aoi Komuro,
and Shigeru Kinoshita
PURPOSE. The ocular surface is covered by two biologically
distinct epithelia: corneal and conjunctival. The expression of
keratin12 (K12) is currently considered a hallmark of cornea-
type differentiation. In the current study, the biological fea-
tures of K12-positive cells in human bulbar conjunctival epi-
thelium were examined.
METHODS. Human conjunctival tissues were subjected to inves-
tigate the K12-positive cells in conjunctiva by immunostaining,
in situ hybridization, Western blot analysis, reverse transcrip-
tase–polymerase chain reaction (RT-PCR), and fluorescence-
activated cell sorting (FACS). Gene expression profiling of
these cells was performed with introduced amplified-fragment
length polymorphism (iAFLP). To determine the presence of
stem- or progenitor cells, immunostaining and colony-forming
assays were performed.
RESULTS. Western blot analysis, RT-PCR revealed that K12 was
expressed in conjunctival epithelium. Immunostaining analysis
showed that K12-positive cells reside mainly in clusters in
conjunctival epithelium. FACS analysis showed that 0.2% to
1.7% of conjunctival epithelial cells collected from the inferior
bulbar conjunctiva were K12 positive. iAFLP analysis revealed
that the gene expression patterns of these cells were highly
similar to that of corneal epithelial cells. p63 and ABCG2 were
expressed beneath the K12-positive cells. Some colony-form-
ing cells expressed K12.
CONCLUSIONS. The K12-positive cells appear to be ectopically
residing, self-maintaining corneal epithelial cells in the con-
junctival epithelium. (Invest Ophthalmol Vis Sci. 2006;47:
Corneal epithelial cells are continuously supplied from the
limbus where their stem cells reside.1–5Conjunctival epithelial
stem cells were found primarily at the conjunctival fornix by
colony-forming assay in humans5and by in vivo label-retaining
experiments in rabbits6and mice.7,8More recently, time-lapse
studies in green fluorescent protein (GFP) mice conjunctiva
disclosed the uniform distribution of stem cells in the bulbar
he ocular surface is covered by two different types of
epithelia: the conjunctival and the corneal epithelium.
conjunctiva.9In addition, the mucocutaneous junction con-
junctiva has been shown to contain stem cells that migrate
toward the fornix.10,11Thus, the site where conjunctival epi-
thelial stem cells reside remains controversial.
Besides their anatomic segregation, the two types of ocular
epithelium possess unique tissue- and cytological properties.
For example, conjunctival epithelium does, while corneal ep-
ithelium does not, contain mucin-secreting goblet cells.12Our
previous gene expression analysis13,14disclosed that many
genes are differentially expressed by these epithelia. Wei et
al.15reported that rabbit conjunctival and corneal epithelial
cells belong to two separate lineages. Based on these observa-
tions, corneal and conjunctival epithelial cells appear to be
The current dogma is that the expression of K3/12 is
thought to be a hallmark of epithelia with cornea-type differ-
entiation1,15–20and to be indispensable for corneal epithelial
homeostasis.21However, K3 is also expressed in other epithe-
lia, including snout,16gingiva, and tongue22and palpebral
conjunctiva.6Also, it has been reported that bovine bulbar
conjunctival epithelial cells expressed trace amounts of K3 and
were induced to express K3/12 by inoculation onto corneal
basement membrane.23Similar findings were made on cul-
tured rabbit conjunctival epithelial cells.6These reports
strongly suggest that the actual expression patterns of K3/12 in
ocular surface epithelium are not as clear cut as the current
We investigated expression of K12 in human conjunctival
epithelium. We found that K12-positive cells are present in this
tissue primarily as clusters and appear to possess cellular fea-
tures highly similar to corneal epithelial cells. Furthermore,
they seem to have their own stem- or progenitor cells. Based
on our observations, we postulated that the K12-positive cells
in conjunctival epithelium are ectopically residing corneal ep-
This study was approved by the Committee for Ethical Issues on
Human Research of Kyoto Prefectural University of Medicine and was
performed in accordance with the tenets of the Declaration of Hel-
Normal conjunctival tissues were obtained from otherwise healthy
eyes at cataract or conjunctivochalasis surgery. The resected normal
conjunctivae from the patients with cataract (n ? 10; three men and
seven women; mean age, 69.0 ? 13.1 years) were 3 ? 3 mm2in size
and located at inferior bulbar conjunctiva 5 mm distant from the
surgical limbus. The resected conjunctivae from the patients with
conjunctivochalasis (n ? 10; three men and seven women; mean age,
71.6 ? 8.7 years) were 3 to 6 mm (vertical) ? 15 mm (horizontal) and
located at the inferior bulbar conjunctiva, at least 2 mm distant from
the limbus. Prior informed consent was obtained from all subjects after
a detailed explanation of the procedures. Cadaveric corneas were
obtained from the Northwest Lions’ EyeBank (Seattle, WA). Permission
to use the donated corneas for research was obtained from all donor
From the Department of Ophthalmology, Kyoto Prefectural Uni-
versity of Medicine, Kyoto, Japan.
Supported by Grants 15791001 and 16390502 from the Japanese
Ministry of Education, Science, Culture and Sports; a grant from the
Japanese Ministry of Health, Labor and Welfare; and research funds
from the Kyoto Foundation for the Promotion of Medical Science.
Submitted for publication August 17, 2005; revised November 26,
2005; accepted February 15, 2006.
Disclosure: S. Kawasaki, None; H. Tanioka, None; K. Ya-
masaki, None; N. Yokoi, None; A. Komuro, None; S. Kinoshita,
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked “advertise-
ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Corresponding author: Satoshi Kawasaki, Department of Ophthal-
mology, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Hi-
rokoji-agaru, Kawaramachi-dori, Kamigyo-ku, Kyoto, 602-0841, Japan;
Investigative Ophthalmology & Visual Science, April 2006, Vol. 47, No. 4
Copyright © Association for Research in Vision and Ophthalmology
Corneal and conjunctival tissues were embedded in OCT compound
(Tissue-Tek; Sakura Finetechnical Co. Ltd., Tokyo, Japan) and snap
frozen with liquid nitrogen. Sections were placed on glass slides for
immunostaining and in situ hybridization or on slides (Penfoil; Leica
Microsystems, Co., Ltd., Wetzlar, Germany) for laser microdissection.
Sections or cells were dried and fixed at 4°C with Zamboni’s fixative.
Then they were incubated in blocking solution (1% BSA in 0.01 M PBS),
incubated in the primary antibodies (Table 1), washed with 0.01 M
PBS, incubated again with the corresponding fluorescence-labeled sec-
ondary antibody, immersed in mounting medium, and covered with
Western Blot Analysis
Conjunctival tissue was stretched on a filter paper epithelial side up.
With a spatula, the epithelium was mechanically scraped from the
tissue with special care taken to avoid breaking the underlying stroma.
The collected epithelium was lysed in a buffer containing 25 mM
Tris-HCl (pH 7.4), 0.6 M KCl, 1% Triton X-100, and a protease inhibitor
cocktail (Complete Mini; Roche Diagnostics, Penzberg, Germany). Af-
ter centrifugation, pellets were solubilized in sample buffer containing
25 mM Tris-HCl (pH 6.8), 2% SDS, 5% ?-mercaptoethanol, 10% glycerol,
and 0.005% bromphenol blue (BPB). The samples were electrophoresed,
transferred to a polyvinylidene difluoride (PVDF) membrane (HybondP;
GE Healthcare, Piscataway, NJ) and immunostained with anti-K12 anti-
body (1:1000, sc-17098; Santa Cruz Biotechnology, Santa Cruz, CA).
Fluorescence-Activated Cell Sorting
The conjunctival epithelium was separated from underlying stroma
with 1.2 U/mL dispase24and further disintegrated with 0.05% trypsin/
EDTA. After fixation with 4% paraformaldehyde, the cells were incu-
bated in a blocking buffer containing 0.1 M PBS and 0.5% BSA. After
incubation in a permeabilization buffer (0.5% saponin in the blocking
buffer), the cells were immunostained with anti-K12 antibody (1;100,
sc-17098) or normal goat IgG. After washing in 0.1 M PBS, the cells
were stained with Alexa488 anti-goat IgG (Invitrogen, Carlsbad, CA)
and analyzed by fluorescence-activated cell sorting (FACS; FACSCali-
ber, BD Biosciences, San Jose, CA).
Fluorescence In Situ Hybridization
A specific region for K12 mRNA (nucleotide positions 1337-1792 in
NM000223) was amplified and cloned into a T-overhang vector
(pGEM-T Easy Vector; Promega, Madison, WI). After confirmation by
sequencing, the plasmid was digested with restriction enzyme and
used to prepare a sense or antisense digoxigenin (DIG)-labeled RNA
Frozen conjunctival sections (10 ?m) were fixed with 4% parafor-
maldehyde and incubated in a 0.2-?g/mL proteinase-K solution. Then,
they were acetylated with 0.25% acetic anhydride in 0.1 M triethanol-
amine (TEA; pH 8.0), washed with PBS, and dehydrated with a graded
series of ethanol. After 30-minute air drying, they were incubated in
hybridization buffer containing 50% formamide, 0.3 M NaCl, 20 mM
Tris-HCl (pH 7.5), 5 mM EDTA, 10% dextran sulfate, 1? Denhardt
solution (Wako Pure Chemical Industries, Ltd., Osaka, Japan), 500
ng/mL salmon sperm DNA (Invitrogen), 0.5 mg/mL yeast tRNA (Roche
Diagnostics), and 10 mM dithiothreitol [DTT] plus 10 ng/mL of the
sense or antisense probe. After an 18-hour incubation at 60°C, the
sections were washed twice at 52°C for 30 minutes in 0.5? SSC and
50% formamide and then washed twice for 15 minutes at room tem-
perature in 0.2? SSC. After a 30-minute incubation in a blocking
buffer, the sections were incubated in horseradish peroxidase (HRP)-
labeled anti-DIG antibody (1:100; Roche Diagnostics) solution. After
signal intensification by tyramide signal amplification (Biotin-TSA kit;
Perkin-Elmer Life Sciences Inc., Boston, MA), the sections were incu-
bated with Alexa488-labeled streptavidin (Invitrogen).
Reverse Transcriptase–Polymerase Chain
Reaction and Real-Time PCR
RNAs were extracted from corneal or conjunctival epithelium, reverse-
transcribed, amplified with primer pairs against the genes (Table 2),
and electrophoresed in 2% agarose gels. Southern blot analysis was
performed to validate the results.
Real-time PCR was performed to quantitate the relative gene ex-
pression of K12 and ribosomal RNA (for normalization) using a se-
quence-detection system (Prism 7000 Sequence Detection System;
Applied Biosystems, Ltd. [ABI], Tokyo, Japan). (Sequences for these
primers and internal probes were not disclosed.)
For introduced amplified fragment length polymorphism (iAFLP) anal-
ysis, K12-positive and K12-negative cells were individually harvested
from three individual conjunctivas (Fig. 1) by using a laser-microdis-
section device (AS LMD; Leica Microsystems).
Gene Expression Analysis by iAFLP
Corneal or conjunctival epithelial cells were mechanically peeled from
five corneas or five conjunctivas. RNAs were extracted from these cells
or from six microdissected samples (TRIzol reagent; Invitrogen).
Comprehensive gene expression profiles were examined with the
iAFLP method of Kawamoto et al.25slightly modified. Briefly, double-
stranded cDNA was synthesized with a pUC119-based vector primer,
as described previously,26and digested with MboI for subsequent
adaptor ligation. Small aliquots (approximately one-sixth) of all di-
gested cDNAs were pooled to obtain a reference sample to connect the
data among the different sample sets. Each of the cDNA samples,
including the reference sample, was ligated with an individual length
polymorphic adaptor (TTnew33-TTnew45 adaptors for individual sam-
ples and TTnew48 adaptor for the reference sample). Five different
cDNA samples and the reference sample were pooled to make four
sample sets in total. After PCR amplification with AntVpPst and
T7revBam primers, the four sample sets were digested with BamHI,
ligated with the T7-3000 adaptor, and amplified by PCR with a fluo-
rescent-labeled MA20 primer and a gene-specific primer. Gene-specific
primers (288 genes) were designed to analyze genes that were domi-
nantly and/or specifically expressed in corneal epithelial cells (for gene
selection, we referred to the Bodymap database; http://bodymap.
ims.u-tokyo.ac.jp/). Each amplified product was electrophoresed on a
fluorescence autosequencer (ABI3100 DNA analyzer; ABI) and the
results were analyzed on computer (Genescan and Genotyper soft-
ware; ABI). The resultant gene expression data were further analyzed
with Cluster and Treeview software.27All oligomers and adaptors
except for gene-specific primers used in iAFLP analysis are listed in
TABLE 1. List of Antibodies
Animal Source Dilution
Mono: monoclonal antibody, Poly: polyclonal antibody; PROGEN,
PROGEN Biotechnik GmbH, Heidelberg, Germany; Novocastra, Novo-
castra Laboratories Ltd, Newcastle, UK; Santa Cruz, Santa Cruz Biotech-
nology Inc., Santa Cruz, CA; NC, not commercially available; KAMIYA,
Kamiya Biomedical Company, Seattle, WA.
1360Kawasaki et al.
IOVS, April 2006, Vol. 47, No. 4
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