TISSUE-SPECIFIC STEM CELLS

Transcription Factor TCF4 Maintains the Properties of Human

Corneal Epithelial Stem Cells
RONG LU,a,b YANGLUOWA QU,b,c JIAN GE,a LILI ZHANG,b,d ZHITAO SU,b STEPHEN C. PFLUGFELDER,b DE-QUAN LIb
a
Zhongshan Ophthalmic Center, State Key Laboratory of Ophthalmology, Sun Yat-Sen University, Guangzhou,
People’s Republic of China; bOcular Surface Center, Department of Ophthalmology, Cullen Eye Institute, Baylor
College of Medicine, Houston, Texas, USA; cThe Eye Institute, Xiamen University, Xiamen, People’s Republic of
China; dDepartment of Ophthalmology, the Affiliated Hospital of Qingdao University Medical College, Qingdao,
People’s Republic of China

Key Words. Adult stem cell • Corneal epithelium • TCF4 • Survivin • Proliferation

ABSTRACT
TCF4, a key transcription factor of Wnt signaling system,
has been recently found to be essential for maintaining
stem cells. However, its signaling pathway is not well elu-
cidated. This study was to explore the functional roles and
signaling pathway of TCF4 in maintaining adult stem cell
properties using human corneal epithelial stem cells as a
model. With immunofluorescent staining and real-time
polymerase chain reaction, we observed that TCF4 was
exclusively expressed in the basal layer of human limbal
epithelium where corneal epithelial stem cells reside.
TCF4 was found to be well colocalized with ABCG2 and
p63, two recognized epithelial stem/progenitor cell
markers. Using in vitro culture models of primary human
corneal epithelial cells, we revealed that TCF4 mRNA and
protein were upregulated by cells in exponential growth
stage, and RNA interference by small interfering RNA-
TCF4 (10-50 nM) transfection blocked TCF4 signaling
Disclosure of potential conflicts of interest is found at the end of this article.
and suppressed cell proliferation as measured by WST-1
assay. TCF4 silence was found to be accompanied by
downregulated proliferation-associated factors p63 and
survivin, as well as upregulated cyclin-dependent kinase
inhibitor 1C (p57). By creating a wound healing model in
vitro, we identified upregulation and activation of b-cate-
nin/TCF4 with their protein translocation from cytoplasm
to nuclei, as evaluated by reverse transcription-quantita-
tive real-time polymerase chain reaction, immunostaining,
and Western blotting. Upregulated p63/survivin and down-
regulated p57 were further identified to be TCF4 down-
stream molecules that promote cell migration and
proliferation in wound healing process. These findings
demonstrate that transcription factor TCF4 plays an im-
portant role in determining or maintaining the phenotype
and functional properties of human corneal epithelial stem
cells. STEM CELLS 2012;30:753–761

placement in culture; ability for self-renewal and functional

INTRODUCTION
Adult stem cells are small subpopulations of slow-cycling un-
differentiated resident cells with high proliferative capacity
and self-renewal ability, as well as pluripotent potential.
These cells can be differentiated into functionally mature cells
to regenerate all the cell types of the tissue where they are
located [1]. Adult stem cells exhibit unique characteristics,
including slow-cycling or long cell-cycle time during homeo-
stasis in vivo; small size and poor differentiation with primi-
tive cytoplasm; high proliferative potential after wounding or
tissue regeneration [2–5]. However, the underlying mecha-
nism by which these properties of adult stem cells are main-
tained has not been well elucidated.
Wingless (Wnt) has been demonstrated as a potent morph-
ogen that promotes the expansion of stem cell population and
maintains their precursor properties throughout development
by activating signaling process controlling cell proliferation
and body patterning [6–8]. There are several branches of
Wnt-mediated signaling cascade in mammals, the most promi-
nent is the canonical Wnt signaling pathway, which has been
implicated in a diverse array of cellular functions, such as

Author contributions: R.L.: conception and design, provision of study material, collection and/or assembly of data, data analysis and
interpretation, and manuscript writing; Y.Q.: provision of study material, collection and/or assembly of data, data analysis and
interpretation, and manuscript writing; J.G.: conception and design, financial support, manuscript writing, and final approval of
manuscript; L.Z. and Z.S.: provision of study material or patients and collection and/or assembly of data; S.C.P.: conception and design,
financial support, and manuscript writing; and D.-Q.L.: conception and design, financial support, collection and/or assembly of data, data
analysis and interpretation, manuscript writing, and final approval of manuscript.
Correspondence: Jian Ge, M.D., Ph.D., Zhongshan Ophthalmic Center, State Key Laboratory of Ophthalmology, Sun Yat-Sen
University, Guangzhou, People’s Republic of China. Telephone: þ86 20 87333209; Fax: þ86 20 87333271; e-mail: gejian@mail.sysu.
edu.cn; or De-Quan Li, M. D., Ph.D., Ocular Surface Center, Department of Ophthalmology, Cullen Eye Institute, Baylor College of
Medicine, 6565 Fannin Street, NC-205, Houston, Texas 77030, USA. Telephone: 713-798-1123; Fax: 713-798-1457; e-mail: dequanl@
bcm.tmc.edu Received October 14, 2011; accepted for publication December 20, 2011; first published online in STEM CELLS EXPRESS
January 9, 2012. V
C AlphaMed Press 1066-5099/2012/$30.00/0 doi: 10.1002/stem.1032

STEM CELLS 2012;30:753–761 www.StemCells.com

754
stem cell proliferation and self-renewal; stem cell activation,
fate determination and differentiation; as well as aging and
senescence [9, 10].
The TCF/LEF family is a group of transcription factors that
bind to DNA through a high mobility group domain. They are
involved in the Wnt signaling pathway, where they recruit the
coactivator b-catenin to enhance elements of genes they target
[11–13]. Four TCF genes, TCF1, LEF1, TCF3 and TCF4, exist
in mammals. Indeed, TCF3 and TCF4 are mostly related to stem
cell property maintenance, while the TCF-1 locus acts as an in-
testinal tumor suppressor primarily due to the production of a
truncated dominant negative isoform of TCF-1, which antago-
nizes TCF-4 in stem cell renewal. Stabilized b-catenin acts as a
transcriptional cofactor for TCF3, TCF4, TCF1, and Lef1, as
well as other DNA-binding proteins. That the accumulated nu-
clear b-catenin binds TCF family members is a hallmark of ca-
nonical Wnt signaling pathway [14]. Many affected genes are
regulated by TCF activation, such as survivin, cyclin D1, and c-
Myc [15–17]. Nevertheless, little is known about the functional
role and underlying mechanisms by which TCF transcription fac-
tors maintain the adult stem cells properties.
The ocular surface is an ideal region to study adult stem cell
biology because of the unique spatial arrangement of stem cells
and transient amplifying cells. It has been known that corneal
epithelial stem cells are located in the basal layer of human cor-
neal limbus [18–20]. The compartmentalization of the corneal
epithelial stem cells within the limbus provides a valuable oppor-
tunity to study the behavior of adult stem cells [20, 21]. With
microarray analysis, we have observed that transcription factor
TCF4 was one of the most upregulated genes in rapidly adherent
progenitor cells isolated from limbal basal epithelium by colla-
gen IV adhesion technique, and b-catenin/TCF4 Wnt signaling is
an important pathway upregulated in the stem/progenitor cell
populations [22]. However, little is known yet on TCF4 molecu-
lar biology in corneal epithelial stem cells. The present study
was to explore the expression, localization, activation, and func-
tional role of TCF4 as well as its signaling molecules in main-
taining human corneal epithelial stem cells using multiple in
vitro models of primary human corneal epithelial cultures.
MATERIALS AND METHODS
Materials and Reagents
Cell culture dishes, plates, centrifuge tubes, and other plastic
ware were purchased from Becton, Dickinson and Company
(Franklin Lakes, NJ, http://www.bdbiosciences.com). Nunc
Lab-Tec II eight-chamber slides were from Nalge Nunc Inter-
national Corp (Naperville, IL, http://www.nalgenunc.com).
Dulbecco’s modified Eagle’s medium, Ham F-12, HEPES,
amphotericin B, gentamicin, and 0.25% trypsin/EDTA solution
were from Invitrogen-GIBCO BRL (Grand Island, NY, http://
www.invitrogen.com). Fetal bovine serum (FBS) was from
Hyclone (Logan, UT). Primary antibodies against TCF4 and
survivin were from Santa Cruz Biotechnology (Santa Cruz, CA,
http://www.scbt.com). Antibodies against b-catenin and b-actin
were from Cell Signaling Technology Inc (Beverly, MA, http://
www.cellsignal.com). ABCG2 mAb (clone BXP-21) was from
Millipore (Billerica, MA, http://www.millipore.com). Antibod-
ies against p57 and p63 (clone 4A4), and HRP-conjugated sec-
ondary antibodies (goat anti-mouse, goat anti-rabbit, and rabbit
anti-goat) were from Thermo Fisher Scientific (Fremont, CA,
http://www.thermofisher.com). Fluorescein Alexa-Fluor 488- or
594-conjugated secondary antibodies (donkey anti-goat IgG or
goat anti-mouse IgG) were from Molecular Probes (Eugene,
TCF4 Maintains Corneal Epithelial Stem Cells
OR, http://www.invitrogen.com). Nuclear Extract kit came
from Active Motif (Carlsbad, CA, http://www.activemotif.
com). Ready gels, Precision Plus Protein Unstained Standards
and Precision Protein Streptactin-AP Conjugate came from
Bio-Rad (Hercules, CA, http://www.bio-rad.com). The BCA
protein assay kit was from Pierce Chemical (Rockford, IL).
Ready-To-Go you-Prime First-Stand Beads were purchased
from GE Health Care (Piscataway, NJ, http://www3.gehealth
care.com). Cell Proliferation Reagent WST-1 was from Roche
Applied Scence (Mannheim, Germany, https://www.roche-
applied-science.com). Taqman Gene Expressin Assays and
Real-time PCR Master Mix kits were from Applied Biosystems
(Foster City, CA, http://www.appliedbiosystems.com).
RNeasy Mini Kit and Hiperfect transfection reagent from Qia-
gen (Valencia, CA, http://www.qiagen.com). Antibody to
c-tubulin, bovine insulin, human transferring, sodium selenite,
hydrocortision, human epidermal growth factor, cholera toxin
A subunit, propidium iodide (PI), and all other reagents came
from Sigma-Aldrich (St. Louis, http://www.sigmaaldrich.com).
Donor Corneal Tissues and Primary
Human Corneal Epithelial Cultures
Fresh human corneoscleral tissues (in 72 hours post-mortem),
which did not meet the criteria for clinical use, from donors
aged 23-64 years were obtained from the Lions Eye Bank of
Texas (Houston, TX). Human tissues were handled according
to the tenets of the Declaration of Helsinki. Donor corneoscleral
tissues were cut through the central cornea or peripheral limbus,
then frozen and sectioned using a previously described method
[23, 24]. To evaluate differential gene expressions, a 9-mm tre-
phine was used to separate central cornea from the limbal tis-
sue, and both corneal and limbal epithelia were directly
scrapped with a keratome and immediately lysed in Qiagen
RLT lysis buffer before RNA extraction.
Primary human corneal epithelial cells (HCECs) were
established from limbal explants using a previously described
method [25, 26]. In brief, each limbal rim was cut into 12
equal pieces (approximately 2
2 mm size each). Two
pieces with their epithelial side up were directly placed into a
well of six-well culture plates, or one piece into a well of 12-
well plates or eight-chamber slides and they were covered
with a drop of FBS overnight. The explants were then cul-
tured in a supplemented hormonal epidermal medium contain-
ing 5% FBS (SHEM) at 37 C under 5% CO2 and 95%
humidity. The medium was renewed every 2-3 days.
RNA Interference
To explore a functional role of TCF4 in proliferation, RNA inter-
ference was performed using small interfering RNA (siRNA)
according to a previous Fast-Forward Transfection method using
HiperFect transfection reagent [27]. In brief, primary HCECs (6

104 cells per cm2) in 6- or 12-well were transfected with
annealed double-stranded siRNA specific for TCF4 (siRNA-
TCF4) at different concentrations (10, 25, and 50 nM), and a
noncoding sequence siRNA-fluorescein (siRNA-F) was used as a
negative control (also served as a visible monitor for transfection
efficiency). The transfected cells were incubated for 48 hours for
RNA extraction and immunofluorescent staining. For WST-1
cell proliferation assay, the mixture of siRNA and Hiperfect rea-
gent was added in 96-well plate for 5-10 minutes incubation at
room temperature, and HCECs were then seeded at 6,000 per
well, which made total volume 100 ll per well, and incubated
for additional 48 hours before used for WST assay.
WST-1 Cell Proliferation Assay
WST-1 assay was performed according to the manufacturer’s
protocol as our previous report [28]. In brief, 10 ll of WST-1
Lu, Qu, Ge et al.
Figure 1. TCF4 localization in basal layer of human limbal epithelium. (A): Representative images showing TCF4 immunofluorescent staining
(green) with propidium iodide (red) counterstaining in human central and peripheral cornea and limbus. (B): Reverse transcription-quantitative
real-time polymerase chain reaction displayed TCF4 mRNA expression by corneal and limbal epithelia. Data shown as mean 6 standard devia-
tion, n ¼ 3; **, p < .01. (C): Representative images showing double immunofluorescent staining of TCF4 (green) with p63 or ABCG2 (red).
cell proliferation agent was added to each well containing
100 ll cell culture. The cells were then incubated for 2 hours
at 37 C in a 5% CO2 incubator. The plate was measured at a
wavelength of 450 nm with a reference wavelength 690 nm
in an Tecan Infinite M200 microplate reader (M€annedorf,
Switzerland, http://www.tecan.com).
In Vitro Wound Healing Model of HCECs
Primary HCECs were cultured in 6- or 12-well plates or 8-
chamber slides until 90% confluent. Wound incisions were
made by scraping cells in 2 mm wide area following the lines
drawn on the back of the plates or slides. The cultures were
carefully observed and photographed until the wounding area
was completely healed in 72 hours. Cultures at different time
periods of wound healing were used for RNA extraction, im-
munofluorescent staining, or Western blot analysis.
Immunofluorescent Staining and Laser Scanning
Confocal Microscopy
Immunofluorescent staining was performed as previously
described [23, 29]. Corneal frozen sections or cultured corneal
epithelial cells were fixed with cold methanol (for p63, b-cat-
enin, and TCF4) or freshly prepared 2% paraformaldehyde
(for ABCG2 staining) at 4 C for 10 minutes. Cultured cells
were permeabilized with 0.2% Triton X-100 in phosphate-buf-
fered saline (PBS) at room temperature for 10 minutes. After
blocking with 10% normal goat or 20% donkey serum in PBS
for 30 minutes, primary antibodies against TCF4 (1:100), b-
catenin (1:50), p63 (1:300), and ABCG2 (1:100) were applied
and incubated for 2 hours at room temperature. A secondary
antibody, Alexa Fluor 488-conjugated donkey anti-goat IgG
(1:300) or 594-conjugated goat anti-mouse IgG (1:300), was
then applied and incubated in a dark chamber for 1 hour and
followed by counterstaining with a DNA-binding dye PI
(2 lg/ml in PBS) for 5 minutes. After washing with PBS,
Antifade Gel/Mount and a coverslip were applied. Sections
were examined and photographed with the laser scanning con-
focal microscopy (LSM 510, Zeiss, Thornwood, NY, http://
www.zeiss.com).
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755
Total RNA Extraction, Reverse Transcription and
Relative Quantitative Real-Time PCR
Total RNA was isolated from tissue or cells using RNeasy Mini
kit (Qiagen) according to the manufacturer’s protocol, quantified
by NanoDrop ND-1000, and stored at 80 C. As previously
described [30, 31], the first strand cDNA was synthesized by
reverse transcription (RT) from 1 lg of total RNA using Ready-
To-Go You-Prime First-Strand Beads, and the real-time PCR was
performed in the Mx3005PTM system (Stratagene) with a 20 ll
reaction volume containing 5 ll of cDNA, 1 ll of TaqMan Gene
Expression Assay for b-catenin (Assay ID Hs99999168_ml),
TCF4 (Hs00162613_ml), survivin (Hs00153353_ml), p63
(Hs00186613_ml), p57 (Hs00175938_m1) or glyceraldehyde-
3-phosphate dehydrogenase (GAPDH, Hs99999905_ml), and 10
ll TaqMan Gene Expression Master Mix (Applied Biosystems,
http://www.appliedbiosystems.com). The thermocycler parameters
were 50 C for 2 minutes, 95 C for 10 minutes, followed by 40
cycles of 95 C for 15 seconds, and 60 C for 1 minute. A nontem-
plate control was included to evaluate DNA contamination. The
results were analyzed by the comparative threshold cycle (Ct)
method and normalized by a housekeeping gene GAPDH [31, 32].
Western Blotting Assay
Western blot analysis was performed using a previously
reported method [33]. Primary HCECs with wound model at
different time points were collected for extraction of cytoplas-
mic and nuclear proteins using nuclear extract kit from Active
Motif according to the manufacture’s protocol. Other cultures
were lysed with RIPA buffer. Protein concentration of these
extracts was measured by a Micro BCA protein assay kit.
Equal amount of protein (50 lg per lane) was mixed with 6

SDS reducing sample buffer and boiled for 5 minutes before
loading. The proteins were separated on an SDS polyacryl-
amide gel and transferred electronically to PVDF membranes.
The membranes were then blocked with 5% nonfat milk in tris-
buffered saline (50 mM Tris [pH 7.5], 0.9% NaCl, and 0.1%
Tween 20) for 1 hour at room temperature and incubated with
primary antibodies to TCF4 (1:100), b-cantenin (1:1,000), p57
(1:500), p63 (1:1,000), or b-actin (1:2,000) overnight at 4 C.
The membranes were incubated with horseradish peroxidase-
756 TCF4 Maintains Corneal Epithelial Stem Cells

conjugated goat anti-rabbit or anti-mouse IgG (1:2,000) for 1

hour at room temperature. The signal bands were detected with
enhanced chemiluminescence reagent, and the images were
acquired by Kodak image station 2000R (Eastman Kodak, New
Haven, CT).

Statistical Analysis
The Student’s t test or analysis of variance with Tukey’s post
hoc testing was used for statistical comparisons. p  .05 was
considered statistically significant. All of these tests were per-
formed using the GraphPad Prism 5.0 software (Graph-Pad
Prism, Inc., San Diego, CA, http://www.graphpad.com).

RESULTS
TCF4 is Exclusively Expressed by Limbal Basal
Cells Where Stem Cells Reside
The immunofluorescent staining on corneal limbal tissue fro-
zen sections revealed that TCF4 protein was exclusively
immunolocalized at basal cells of limbal and peripheral cor-
neal epithelia, where corneal epithelial stem cells reside. As
shown in Figure 1A, TCF4 immunoreactivity was primarily
located in cytoplasm and nuclei of basal cells at limbal epi-
thelium, and the numerous TCF4-positive cells were inter-
spersed with patches of TCF4 negative cells. TCF4-positive
cells decreased and cluster-like dispersed in the basal layer of
peripheral corneal epithelium. There was no TCF4 immunore-
activity detected in the suprabasal and superficial layers of
limbal epithelium, nor in all layers of corneal epithelium. To
verify this unique pattern of TCF4 expression in cornea and
limbus, the levels of TCF4 mRNA of central corneal or
limbal epithelia were evaluated by RT-quantitative real-time
PCR (qPCR) with GAPDH as an internal control. The results
confirmed that levels of TCF4 mRNA expression by limbal
epithelium were significantly higher (4.957 6 0.52-fold, p <
.05, n ¼ 3) than that in corneal epithelium (Fig. 1B).
TCF4 Protein was Colocalized with Corneal
Epithelial Progenitor Markers, ABCG2 and
p63 in the Basal Layer of Limbal Epithelium
ABCG2 and p63 have been accepted as stem cell-associated
markers or progenitor cell markers of keratinocytes including
corneal epithelial cells [34, 35]. As shown in Figure 1C, the dou-
ble immunostaining of TCF4 and p63 indicated that both pro-
teins were coimmunolocalized in limbal basal epithelium, and
the most TCF4-positive cells also expressed p63. The pattern of
coimmunolocalization of TCF4 and ABCG2 was different from
TCF4 and p63. Although both TCF4 and ABCG2 were immuno-
located within limbal basal layer, some cells expressing strong
ABCG2 were weakly positive to TCF4, and other cells express-
ing weak ABCG2 were strongly positive to TCF4. This colocali-
zation pattern of TCF4 with p63 and ABCG2 indicates that
TCF4 may play an important role in maintaining stemness prop-
erties of corneal epithelial stem cells.
TCF4 Signaling was Related to the Growth Stage
and Proliferation Capacity of HCECs
To evaluate the role of TCF4 in cell proliferation and differen-
tiation, the primary HCECs were collected in different growing
stages with 70% or 100% confluence for immunofluorescent
staining and RT-qPCR. As shown in Figure 2A, TCF4 antibody
was found to stain cell cytoplasm, and strong immunoreactivity
was preferentially expressed by small size cells in primary
HCECs. The 70% confluent culture where cells are in exponen-
Figure 2. TCF4 expression in human corneal epithelial cells (HCECs)
at different growth stages. (A): Representative images showing TCF4 im-
munofluorescent staining (green) with propidium iodide (red) counterstain-
ing in HCECs at different growth stages (70% and 100% confluence). (B):
Percentages of TCF4-positive (TCF4þ) cells in 70% and 100% confluent
cultures. (C): Reverse transcription-quantitative real-time polymerase
chain reaction displayed the expression levels (relative fold of mRNA) of
b-catenin, TCF4, p63, survivin, and p57 by 70% confluent HCECs com-
pared with 100% confluent cells. Data shown as mean 6 standard devia-
tion, n ¼ 3; *, p < .05; **, p < .01; ***, p < .001.
tial growth contained significantly higher percentage of TCF4-
positive cells (38.47 6 2.51%, p < .05, n ¼ 3) than 100%
confluent culture (7.65 6 1.96%, n ¼ 3) (Fig. 2B). RT-qPCR
results shown in Figure 2C confirmed the finding by TCF4 im-
munofluorescent staining. TCF4 mRNA expressed significantly
higher levels (2.51 6 0.32-fold, p < .01, n ¼ 3) by younger
cells in 70% confluence than that in 100% confluent culture,
which contained more quiescent and differentiated cells. Inter-
estingly, TCF4 signaling-associated molecules were also found
to be regulated in the different growth stages. b-Catenin,
known as an activator of TCF4, was expressed at higher
mRNA level by 70% confluent HCECs than 100% confluent
cells. The mRNA expression of p63 and survivin, two prolifer-
ation-associated factors were also upregulated while the mRNA
of cyclin-dependent kinase inhibitor 1C (p57), a proliferation
inhibitor, was downregulated in 70% confluent HCECs, com-
pared with 100% confluent cells (Fig. 2C).
Cell Proliferative Capacity is Impaired in TCF4
Silenced HCECs by siRNA Interference
To explore a functional role of TCF4 in cell proliferation,
RNA interference was performed to silence TCF4 gene by
Lu, Qu, Ge et al.
siRNA-TCF4 in HCECs. Immunofluorescent staining showed
a successful knockdown of TCF4 gene by siRNA-TCF4 in 48
hours, when TCF4 protein production was almost completely
suppressed in comparison of cells transfected with siRNA-F,
a noncoding negative control siRNA (Fig. 3A). TCF4 mRNA
expression was largely blocked by siRNA-TCF4 at all three
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757
different concentrations (10, 25, and 50 nM), when compared
with untreated control or siRNA-F transfected cells (Fig. 3C).
WST-1 cell proliferation assay was performed using HCECs
in 96-well plates with or without transfection of siRNA-TCF4
or siRNA-F. We observed that WST proliferation index was
dose-dependently (10-50 nM) suppressed by TCF4-siRNA in
48-hour growth period, when compared with untreated or
siRNA-F control groups (p < .05, n ¼ 5) (Fig. 3B).
To identify the molecular signaling through which TCF4
controls proliferative capacity, several potential TCF4 related
factors were evaluated at mRNA levels by RT-qPCR after
TCF4 knockdown (Fig. 3C). As anticipated, b-catenin, an
upstream activate factor of TCF4, did not show any change at
mRNA levels after TCF4 gene silenced. Interestingly, p63
and survivin were found to be downregulated significantly at
mRNA levels after TCF4 knockdown (p < .05) (Fig. 3C).
The extent of this decrease was dependent to the doses of
siRNA-TCF4 (10-50 nM). By contrast, p57 mRNA was sig-
nificantly upregulated following TCF4 silence. These findings
suggest that p63 and survivin, two proliferation-associated
genes, and a proliferation inhibitor p57 are potential down-
stream target molecules of TCF4. The downregulated p63/sur-
vivin and upregulated p57 may be the signaling pathway
through which cell growth was suppressed by TCF4 gene
knockdown.
TCF4 and its Upstream b-Catenin were Activated
with Nucleus Translocation in Wound Healing
Model of HCECs
To further confirm that TCF4 is an important transcription
factor determining and promoting cell proliferative capacity,
we created an in vitro wound healing model in HCECs. Strik-
ingly, we observed marked translocalization of TCF4 protein
from cytoplasm to nuclei when cells migrated and proliferated
during wound healing process. As shown in Figure 4A, immu-
nofluorescent staining displayed TCF4-nuclear translocation at
the wound edge as early as 4 hours after wounding, and the
TCF4 nuclear-positive cells increased significantly during
wounding process until wound area was completely healed in
48 hours when the TCF4 nuclear-positive cells were largely
decreased. Interestingly, the similar pattern of nuclear translo-
cation of b-catenin was observed in wounding area, especially
at the wound edge during wound healing process (Fig. 4A).
These results provided the evidence that TCF4 and its
upstream b-catenin were activated with nucleus translocation
during wound healing process in HCECs.
These findings were further substantiated by Western blot
analysis, which showed quantified changes of cytoplamic and
nuclear protein levels of TCF4 and b-catenin. Both TCF4 and
b-catenin cytoplasmic protein decreased from 4 hours and
remained the lower level until 48 hours after wound (Fig.
4B). Conversely, the nuclear protein levels of TCF4 and b-
Figure 3. Effects of TCF4 silence by siRNA interference on prolif-
eration of human corneal epithelial cells (HCECs). (A): Representa-
tive images showing TCF4 immunofluorescent staining (green) with
propidium iodide (red) counterstaining in HCECs transfected with
siRNA-TCF4 and siRNA-F. (B): WST-1 assay showing the cell pro-
liferation index in 48 hours after siRNA-TCF4 transfection. Data
shown as the mean and standard deviation of results; ns, no signifi-
cance; *, p < .05; **, p < .01; ***, p < .001; n ¼ 3, compared with
siRNA-F transfection. (C): Reverse transcription-quantitative real-
time polymerase chain reaction showing the effects of siRNA-TCF4
(10-50 nM) transfection on mRNA expression of b-catenin, TCF4,
p63, survivin, and p57 with untreated and transfected cells with non-
coding sequence siRNA-F as control, n ¼ 5. Abbreviations: siRNA,
small interfering RNA; siRNA-F, small interfering RNA-fluorescein.
758
Figure 4. TCF4 and b-catenin activation with nucleus translocation during wound healing process of human corneal epithelial cells in vitro.
(A): Representative images of double immunofluorescent staining of TCF4 (green) and b-catenin (red) showing their nucleus translocation pro-
cess at different time points after wound. (B): Western blot analysis showing changed levels of TCF4 and b-catenin in isolated cytoplasmic and
nuclear proteins, indicating their nucleus translocation during wound healing process.
catenin increased from 4 hours and remained elevated up to
48 hours. All these results revealed that TCF4 was activated
and transported into the nucleus where it bound b-catenin to
activate target genes and thus promote cell migration and pro-
liferation to repair wound in HCECs.
TCF4 Promotes Wound Healing in HCECs
Through Upregulation of p63/Survivin and
Downregulation of p57
To explore the signaling pathways of TCF4 that promotes
wound healing, we evaluated its potential downstream mole-
cules regulated during wound healing process. Under our
wounding condition, normal HCECs were observed to be ca-
pable of repairing a 2-mm wide wounding area within 48
hours (Fig. 5A). RT-qPCR results showed that TCF4 expres-
sion was significantly upregulated during wound healing pro-
cess in HCECs (Fig. 5B). Compared with untreated control of
HCECs, the mRNA levels of TCF4 was significantly
increased at 8 hours, reached peak levels by fourfold high at
16 hours after wounding, and the upregulated mRNA levels
lasted more than 24 hours before decreased to above-normal
levels in 48 hours when the wound has been healed. The
expression of b-catenin, a TCF4 activator in Wnt signaling
pathway, was found to respond quickly at its mRNA levels
that increased to peak at 4 hours and decreased to normal in
16 hours after wound. The upregulation of b-catenin mRNA
was earlier but lasted shorter than TCF4 upregulation. The
upregulation pattern of b-catenin and TCF4 indicated that
Wnt signaling pathway was activated in wound healing pro-
cess. Moreover, we observed the relevant regulation of prolif-
eration-associated genes, p63, survivin, and p57. After wound,
p63 mRNA increased significantly in 8 hours, reached peak
by threefold at 16 hours, and then gradually reduced back to
normal level within 48 hours, a pattern similar to TCF4. Sur-
vivin mRNA was increased slowly at 16 hours after wound,
but strongly stimulated to peak levels by ninefold at 24 hours,
and remained higher expression more than 48 hours after the
TCF4 Maintains Corneal Epithelial Stem Cells
wound has been healed. Interestingly, the expression of p57, a
proliferation inhibitor, decreased significantly during 8-16
hours after wound. The time period of p57 mRNA downregu-
lation was associated with upregulation of p63 and survivin
genes.
This regulated pattern of b-catenin/TCF4 and prolifera-
tion-associated factors at transcriptional expression was fur-
ther confirmed at protein level by Western blot analysis,
which was performed using cell lysates from cultures with
wound healing model up to 72 hours after wound. As shown
in Figure 5C, we observed the markedly increased protein
production of b-catenin, TCF4, p63, and survivin but
decreased p57 protein levels during wound healing process,
especially from 16 hours to 24 or 48 hours, respectively. By
contrast, a house keeping protein b-actin protein was not sig-
nificantly changed during these time points.
The regulation pattern of these proliferation-associated
factors following TCF4 upregulation in wound healing pro-
cess supported aforementioned RNA interference results that
TCF4 gene knockdown by siRNA-TCF4 suppressed cell pro-
liferation with downregulated p63/survivin, and upregulated
p57. These findings suggest that TCF4 controls corneal epi-
thelial cell proliferation through its downstream signaling
molecules p63, survivin, and p57, the balance between upreg-
ulated p63/survivin and downregulated p57.
DISCUSSION
TCF4 is a basic helix-turn-helix transcription factor. Mamma-
lian TCF4 is highly expressed in the midbrain, intestine, and
mammary epithelium [36–39]. In late embryonic and early
neonatal gut, TCF4 was found to be present in the prolifera-
tive intervillus pockets and essential for maintenance of the
progenitor compartment of gut epithelium [40, 41]. TCF4 was
also found to be essential for maintaining stemness in skin
epithelial stem cells [11]. However, it is still not clear through
Lu, Qu, Ge et al. 759

Figure 5. TCF4 in wound healing model of human corneal epithelial cells (HCECs) in vitro. (A): Representative phase images showing a 2-
mm wide wound area was healed within 48 hours. (B): Reverse transcription-quantitative real-time polymerase chain reaction data showing the
expression levels (relative fold of mRNA) of b-catenin, TCF4, p63, survivin, and p57 by HCECs at different time points after wound. Data
shown as the mean and standard deviation of results, *, p < .05; **, p < .01; ***, p < .001; n ¼ 3. (C): Western blot results showing the pro-
tein levels of b-catenin, TCF4, p63, survivin, and p57 by HCECs at different time points after wound.

what signaling pathway TCF4 maintains these adult epithelial
stem cells. Corneal epithelial stem cells have been identified
to reside at the corneal limbus for more than 2 decades [18–
20], but it is little known how TCF4 plays an important role
in maintaining the properties of corneal epithelial stem cells.
The present study revealed that TCF4 was exclusively local-
ized in the basal layer of human limbal epithelium and well
colocalized with ABCG2 and p63, two recognized epithelial
stem cell/progenitor markers [34, 35]. Using different in vitro
culture models of primary HCECs, we evaluated the differen-
tial expression of TCF4 by cells in exponential growth and
quiescent confluent stages, investigated the TCF4 signaling
and proliferative capacity in TCF4-silenced cultures, and
explored TCF4 activation and downstream signaling mole-
cules in wound healing process. All these findings identified
the novel role and potential signaling pathways of TCF4 in
maintaining corneal epithelial stem cells.
TCF4 Plays an Important Role in Maintaining the
Phenotype of Corneal Epithelial Stem Cells
There is no report on TCF4 expression in ocular surface until
our recent findings through human genome microarray analy-
sis for human corneal epithelial progenitor populations iso-
lated from donor limbal tissues [22], as well as through the
cultured corneal epithelial progenitors in a low calcium se-
rum-free medium CnT-20 that promotes ex vivo expansion of
corneal epithelial progenitor cells via b-catenin/Tcf4/survivin
signaling, a novel intrinsic pathway [42]. The present study
revealed that TCF4 was uniquely expressed by the limbal ba-
sal cells and may determine the phenotype of corneal epithe-
lial stem cells (Fig. 1). The immunofluorescent staining dem-
onstrated that TCF4 was exclusively localized in a
www.StemCells.com
subpopulation of basal layers of limbal epithelium where cor-
neal epithelial stem cells reside, but absent in the suprabasal
limbal and full central corneal epithelia. TCF4 was found to
be colocalized with major epithelial progenitor markers p63
and ABCG2 in limbal basal cells. Interestingly, the most
TCF4 positive basal cells consistently expressed p63, while
some basal cells expressing strong TCF4 were weakly posi-
tive to ABCG2, or vice versa. This differential pattern of
TCF4 colocalization with p63 and ABCG2 may suggest the
different functions of these proteins. TCF4 may have similar
function to p63 that promotes epithelial cell proliferation [43]
but may be less common to ABCG2 that protects stem cells
from toxic damage as a multidrug resistant gene [35, 44].
This observation was further substantiated by RT-qPCR that
message RNA expression of TCF4 was statistically higher in
limbal than corneal epithelia isolated from donor tissues.
These findings revealed that TCF4 expression may represent a
characteristic of corneal epithelial stem cells.
Our previous studies [45, 46] have shown that the corneal
epithelial cells in confluent cultures are more differentiated
than younger growing cells in less confluent condition. When
comparing the cells in different growth stages, the number of
TCF4 immunoreactivity-positive cells was statistically greater
in exponentially grown cells with 70% confluence than that in
quiescent differentiated cells with 100% confluence (Fig. 2).
This finding was confirmed by RT-qPCR at transcriptional
levels that showed higher TCF4 expression accompanied by
upregulated mRNA levels of several proliferation factors in
cells with exponential growth. b-Catenin that binds and acti-
vates TCF4 [12], p63 that associates with cell proliferation
[47], and survivin that is a potential target gene of b-catenin/
TCF4 [48] were all found to be upregulated in 70% confluent
younger cells. Interestingly, cycline-dependent kinase inhibitor
760
p57 was conversely decreased in these younger cells, consist-
ent with a previous report that p57 was downregulated by
Wnt signaling activation [36]. Ultimately, the increased p63
and survivin with decreased p57 may contribute to the prolif-
erative capacity of these younger cells that contained more
progenitors with exponential growth.
TCF4 Activation Promotes Cell Proliferation Via
Nuclear Translocation
TCF4 protein, as the pivotal transcription factor for the ca-
nonical Wnt pathway, is responsible for transactivation of cell
proliferation and survival genes [49, 50]. To investigate the
underlying mechanism of TCF4 signaling pathway, we cre-
ated wound healing model of HCECs in vitro (Fig. 4). Strik-
ingly, we observed increasing translocalization of TCF4 from
cytoplasm into nucleus activated by wound condition. The im-
munofluorescent staining clearly showed the dynamic process
of TCF4 nucleus translocation in cells at the area of wound-
ing edges. The key for TCF4 activation is known to be bound
by its activator b-catenin in the nucleus. With double immu-
nofluorescent staining, we observed a similar pattern of b-cat-
enin protein translocation from cytoplasm to nucleus in cells
at wounding edges. Western blot analysis using isolated cyto-
plasmic and nuclear proteins gave a quantitative evidence for
the nuclear translocation of both b-catenin and TCF4. These
results suggest that TCF4 is mainly quiescent and localizes in
cytoplasm under normal circumstance, but TCF4 would be
activated through translocation into nucleus and then trigger
target genes that promote cell migration and proliferation dur-
ing wound healing process. Our findings are consistent with a
previous report that TCF4 activation was induced by injury
[51].
TCF4 Controls Cell Proliferation Through Balanced
Signaling Pathways with p63, Survivin, and p57
To explore signaling pathways of TCF4 in regulating cell pro-
liferation, RNA interference and subsequent WST prolifera-
tion assay were performed in HCEC cultures (Fig. 3). We
observed that TCF4 gene knockdown sharply inhibited cell
proliferation, suggesting an important role of TCF4 in main-
taining proliferative capacity of human corneal epithelial stem
cells. Interestingly, TCF4-silenced HCECs expressed lower
p63 and survivin but higher p57, indicating that TCF4, p63,
survivin, and p57 were involved in maintaining proliferative
capacity of human corneal stem cells. Nuclear factor p63 has
been accepted as a proliferation-related factor and a stem
cell-associated or progenitor marker [34, 43, 47, 52, 53]. Sur-
vivin is a bifunctional member of the inhibitor of apoptosis
gene family that counteracts cell death and controls mitotic
progression [54]. It has been documented to be regulated by
TCF/b-catenin in cancer cells [48, 55, 56] and stem cells [57,
58]. Survivin is also found to be a potential target gene of the
b-catenin/TCF signaling axis, coupling increased cell prolifer-
ation to enhance cell survival in intestinal crypt cells [56]. As
a member of Cip/Kip family, p57 specifically inactivates G1
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CONCLUSION
We have explored a unique pattern of TCF4 expression that
is exclusively localized in the basal layer of human limbal ep-
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markers ABCG2 and p63. TCF4 was found to be expressed
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ACKNOWLEDGMENTS
We thank the Lions Eye Bank of Texas for their great support in
providing human corneoscleral tissues. This study was supported
by DOD CDMRP FY06 PR064719 (DQL), NIH Grant EY11915
(SCP), National Natural Science Foundation of China
(30901634), Fight for Sight (YQ), Research to Prevent Blind-
ness, Oshman Foundation, William Stamps Farish Fund.
DISCLOSURE OF POTENTIAL
CONFLICTS OF INTEREST
The authors indicate no potential conflicts of interest.
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