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1 From the Department of Ophthalmology, Bascom Palmer Eye Institute, and the 2 Department of Cell Biology and Anatomy, University of Miami School of Medicine, Miami, Florida.
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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METHODS. HLE cells were expanded on intact (i.e., remaining devitalized amniotic epithelium) or epithelially denuded AM (EDTA-treated). Cx43 expression and 24-hour 5-bromo-2'-deoxyuridine-5'monophosphate (BrdU) labeling index (percentage) were determined by double immunostaining. GJIC was investigated by a scrape-loading dye transfer assay. In a subset of cultures Cx43 and K3 keratin as well as BrdU-retaining nuclei were analyzed in the stratified epithelium obtained 5 days after subcutaneous transplantation in NIH bg-nu-xidBR mice of AM cultures continuously labeled with BrdU for 7 days.
RESULTS. The outgrowth rate, overall, was significantly higher on EDTA-treated AM than on intact AM (P < 0.05). Cx43 was expressed in 12.4% ± 14.5% (n = 5) on intact and 57.5% ± 18.2% (n = 5) on EDTA-treated AM (P < 0.05). The BrdU labeling index was 2.4% ± 0.9% (n = 5) for the intact AM group, which was significantly less than 22.5% ± 8.2% (n = 5) for EDTA-treated AM (P < 0.05). BrdU-labeled cells did not express Cx43. The dye transfer assay revealed reduced GJIC on both AM-cultured groups compared with the control culture on plastic (P < 0.002). GJIC on intact AM (17%) was reduced compared with that on EDTA-treated AM (27%; P = 0.42). After xenotransplantation, the basal layer of the stratified epithelium was Cx43 and K3 keratin negative and retained BrdU on intact AM, resembling characteristics of the limbal basal epithelium in vivo. In contrast, that of EDTA-treated AM was Cx43 and K3 keratin positive without BrdU retention, resembling characteristics of the corneal epithelium in vivo.
CONCLUSION. These data indicate that denudation of the devitalized amniotic epithelium to expose its basement membrane might be a microenvironmental cue to promote TAC differentiation. The model system described herein is ideal for future exploration of the exact mechanistic operation in the microenvironmental niche that maintains the "stemness" of limbal SCs as well as in the signal that promotes corneal TAC differentiation.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Clinically, limbal epithelial cells expanded in amniotic membrane (AM) cultures can restore the corneal surface with partial and total limbal SC deficiency.11 12 13 Experimentally, AM has been shown to be an ideal substrate that preferentially expands the outgrowth of limbal biopsy specimens.14 15 Such ex vivo expanded limbal epithelial cells are devoid of K3/K12 expression and remain slow cycling (Ref. 14 and manuscript submitted). Recently, we further noted that such ex vivo expanded limbal epithelial cells show no Cx43 expression and gap junction–mediated intercellular communication (GJIC; Ref. 16 and manuscript submitted). It should be noted that AM used in these studies was intact—that is, it retained a monolayer of devitalized amniotic epithelial cells. Koizumi et al.15 recently reported that rabbit corneal and limbal epithelial cells grow much faster when cultured on epithelially denuded AM compared with those on intact AM with a layer of devitalized amniotic epithelium. Their morphologic results led to the conclusion that denuded AM with the exposed basement membrane is more suitable to support corneal epithelial cell growth for further transplantation.
In this study we investigated the differences in proliferative activity, Cx43 expression and GJIC of human limbal epithelial (HLE) cells expanded as a monolayer on cryopreserved, intact and epithelially denuded AM. Our data suggest that intact AM (i.e., retaining devitalized AM epithelium) is a more favorable microenvironment for the expansion of SC-containing limbal epithelium than is EDTA-denuded AM (i.e., exposed AM basement membrane) with respect to the preservation of a Cx43-negative and GJIC-deficient phenotype. This notion is further supported by phenotypic characteristics of the expanded cell population after the induction of stratification through xenotransplantation into nude mice. The potential clinical significance of these two culture conditions for limbal epithelial cell expansion is further discussed.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human Tissue Preparation
Human tissue was handled according to the Declaration of
Helsinki. Corneoscleral tissues from human donor eyes were obtained
from the Florida Lions Eye Bank (Miami, FL) immediately after the
central corneal button had been used for corneal transplantation. The
tissue was rinsed three times with DMEM containing 50 µg/mL
gentamicin and 1.25 µg/mL amphotericin B. After careful removal of
excessive sclera, iris, and corneal endothelium, the remaining tissue
was placed in a culture dish and exposed to Dispase II (1.2 U/mL in
Mg2+- and Ca2+-free Hanks’
balanced salt solution) at 37°C under humidified 5%
CO2 for 5 to 10 minutes. After one rinse with
DMEM containing 10% FBS, the scleral rim was trimmed to obtain limbal
tissue cubes of approximately 1 x 1.5 x 2.5-mm size.
Human Limbus Cultures on Amniotic Membrane
Preserved human AM was kindly provided by Bio-Tissue (Miami,
FL). AM was preserved according to the method described by Lee and
Tseng.17
Briefly, AMs derived from cesarean section
placentas were rinsed in PBS containing 100 U/mL penicillin with 0.2
mg/mL streptomycin and stored in a solution of 50% DMEM and 50%
glycerol at -80°C for at least 3 months. With this preservation
method, both amniotic epithelium and stromal mesenchymal cells lose
their viability and proliferative capacity.18
After
thawing at room temperature, AM with the epithelial side facing up was
fastened onto a culture insert, as previously reported.19
Fifty percent of the membranes used for limbal cultures were treated
with 0.1% sterile EDTA solution for 30 minutes and then gently
scrubbed with an epithelial scrubber (Amoils Epithelial Scrubber;
Innova, Innovative Excimer Solutions, Inc., Toronto, Ontario, Canada),
to remove the amniotic epithelium without breaking the underlying
basement membrane. With this method, 90% to 100% of the epithelium
could be removed. On the center of the AM, an explant was placed and
cultured in a medium made of an equal volume of HEPES-buffered DMEM
containing bicarbonate and Ham’s F12. The medium was supplemented with
0.5% dimethyl sulfoxide, 2 ng/mL mouse epidermal growth factor (EGF),
5 µg/mL insulin, 5 µg/mL transferrin, 5 ng/mL selenium, 0.5 µg/mL
hydrocortisone, 30 ng/mL cholera toxin A subunit, 5% FBS, 50 µg/mL
gentamicin, and 1.25 µg/mL amphotericin B. Cultures were incubated at
37°C under 5% CO2 and 95% air, and the medium
was changed every 2 to 3 days. Each time the medium was changed, the
outgrowth area was measured. When HLE cultures had almost reached
confluent growth, defined as 270° of the circular outgrowth reaching
the plastic culture ring or no further growth noticed after 4 weeks of
culture, they were subjected to a semiquantitative dye transfer assay
or incubated with 10 µM BrdU for 24 hours followed by fixation in
cold methanol for immunostaining.
Xenotransplantation
All procedures were performed according to the ARVO Statement
for the use of Animals in Ophthalmic and Vision Research. NIH
bg-nu-xidBR mice, which have no thymus-derived T-cells, T-independent B
lymphocytes, and natural killer cells, aged 6 to 10 weeks were
purchased from Charles River Laboratories (Wilmington, MA). The animals
were housed under temperature-, humidity-, and light- (12-hour light
cycle; lights on at 7:00 AM) controlled conditions in filter-covered
cages in a laminar flow–equipped room and kept on standard chow and
water ad libitum. Before surgery, animals were anesthetized with
intramuscular injection of 0.1 mL ketamine (35 mg/kg) and xylazine (5
mg/kg). Nearly confluent HLE cultures on intact and EDTA-treated AM
were labeled with BrdU for 7 days to label rapid and slow-cycling cells
and transplanted to the subcutaneous plane of the abdomen of NIH
bg-nu-xidBR mice for a chase of 5 days, during which time only
slow-cycling cells will retain the label. The mice were killed by
craniocervical dislocation after intramuscular injection of 0.3 mL
ketamine (35 mg/kg) and xylazine (5 mg/kg). The tissue, including
implanted AM, was removed and embedded in optimal cutting temperature
(OCT) compound for cryosectioning. Twelve cultures (six per condition)
were transplanted.
Immunostaining
Frozen sections of 3 µm obtained from specimens after
xenotransplantation were fixed in cold methanol for 20 minutes at
-20°C followed by a 10-minute incubation in 0.1% Triton X-100 in
PBS. After three rinses with PBS for 7 minutes each and preincubation
with 5% BSA to block nonspecific staining, sections were incubated
with a rabbit polyclonal anti-Cx43 (1:200), AE-5 (mouse anti-K3;
1:100), or mouse anti-BrdU (1:1000) monoclonal antibody for 45 minutes.
After three washes with PBS for 15 minutes, the sections were incubated
with an FITC-conjugated secondary antibody (goat anti-rabbit or
anti-mouse IgG at 1:200) for 45 minutes. After three additional PBS
washes (15 minutes each), they were mounted with an anti-fade solution
(Vectashield, Vector Laboratories, Burlingame, CA) and analyzed with a
fluorescence microscope (Axiophot; Carl Zeiss, Oberkochen, Germany).
For BrdU and Cx43 double-labeling, confluent cultures were incubated with 10 µM BrdU in the same culture medium for 24 hours. These cultures on AM were prepared as flatmount samples. After samples were air dried, rehydrated in PBS for 5 minutes, treated with 2 N HCl at 37°C for 45 minutes to denature DNA and neutralized in boric acid (pH 8.5) for 20 minutes, incorporated BrdU and Cx43 expression were detected by immunostaining with a mouse anti-BrdU antibody (1:1000) and a mouse anti-Cx43 antibody (1:200) followed by an ABC kit (Vectastain Elite) protocol (DAB-peroxidase staining). Samples were counterstained with hematoxylin. Under magnification of x400, positive nuclei were counted among the total nuclei within the entire field, and a total of 16 fields were counted per specimen. The labeling index for BrdU was expressed as the number of positively labeled nuclei/the number of all nuclei x 100%. If all cells in one x400 field expressed Cx43, we defined it as 1 unit of Cx43 expression. That is, if 50% of cells expressed Cx43, 0.5 unit was defined. We counted 100 fields per sample for a total of 10 samples (n = 5 per condition) and reported their mean ± SD. Counterstaining on nude mice specimens to analyze BrdU retaining cells was performed with propidium iodide.
Semiquantitative Dye Transfer Assay
We used the scrape-loading dye transfer assay originally
described by El-Fouly et al.20
and Trosko et
al.21
For a positive control, we cultured HLE cells from
an explant on plastic dishes for 14 days. HLE cells on plastic or AM
were rinsed with sterile PBS. One milliliter lucifer yellow plus
rhodamine-dextran (0.5 mg/mL) in PBS was added to the culture dish. A
sterile scalpel blade was applied with gentle pressure to cut the
cells. Six scrape lines were placed in different areas per culture.
Dishes were left in a dark room for 3 minutes. Cells were rinsed
extensively with PBS to prevent high background fluorescence. Cultures
were fixed in 4% formalin and epifluorescence was examined using a
fluorescence microscope (Axiophot; Zeiss) equipped with a UV light
source. A rhodamine filter set was used to identify the red color of
the primary loaded cells along the scrape line (absorbency 555 nm,
emission 580 nm). Fluorescence filter sets were used to detect green
fluorescence of lucifer yellow, which was transferred through gap
junctions (absorbency 428 nm, emission 536 nm). We analyzed a total
number of 54 scrape lines (6 scrape lines per culture for three
separate cultures per condition; intact AM, EDTA-AM, control). The
percentages of the entire length of all six scrape lines per culture
were measured when we observed dye transfer more than four cell rows
away from the initially loaded cells.
Statistical Analysis
A Greenhouse-Geisser corrected test of interaction between
groups and the linear time component was used to evaluate the
statistical significance between intact and EDTA-treated AM groups. The
Mann-Whitney test was applied to compare the means of the cell density
in both conditions. Data from the proliferation as well as from the
Cx43 expression assay was analyzed by an unpooled variance approximate
t-test. An ANOVA was applied to analyze the dye transfer
assay. The Fisher exact test was used to compare samples with and
without label-retaining basal cells after xenotransplantation.
P < 0.05 was considered statistically significant.
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Results |
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Semiquantitative Dye Transfer Assay
To evaluate whether immunohistochemically detected Cx43 was indeed
assembled into functioning gap junction channels, we performed a
semiquantitative dye transfer assay using the previously described
scrape-loading technique.20
HLE cells expanded on intact
AM did not show any dye transfer from the scraped area to the adjacent
cells in most of the scrape lines performed (Figs. 5A
5B)
. When semiquantitated, HLE cells cultured on intact AM showed dye
transfer to adjacent cells in 17% of the total length of 18 scrape
lines (three cultures and six scrapes per sample). When the same sample
was subjected to subsequent immunostaining, we did not detect any Cx43
expression in these areas (Fig. 5C)
. However, in those areas that
revealed focal GJIC (Figs. 5D
5E)
, we noted positive expression of
Cx43 (Fig. 5F , punctate staining). HLE cells expanded on EDTA-treated
AM showed a similar result, except that we noted a slightly increase of
communicating areas, amounting to 27% of the total length of 18 scrape
lines (P = 0.42). As a positive control, we also scrape
loaded the outgrowth of HLE grown on plastic and found 94% of the
total length of 18 scrape lines showing dye transfer to adjacent cells
(Figs. 5G
5H)
. The positive GJIC correlated with positive expression
of Cx43 of the same area (Fig. 5I)
. This amount of GJIC was
significantly higher than amounts in both AM groups (P = 0.002).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our data show that HLE expanded on intact AM had a significantly slower
outgrowth rate than those expanded on EDTA-denuded AM (Fig. 2)
. This
difference was due to a delayed onset of growth of cultures on intact
AM and an increase of the growth rate on EDTA-treated AM in the
late-culture phase. This finding was consistent with the data published
by Koizumi et al.,15
using rabbit limbal and corneal
explants. Because 3T3 fibroblast feeder layers were used in their
coculture system15
but not in ours, we thus speculate that
the fibroblast feeder layer does not play a major role in yielding such
a difference in proliferative activity. One may speculate that such a
different growth rate is caused by a mechanical resistance of a firmly
adhered amniotic epithelium against expanding HLE that had to grow over
them (Figs. 1E
1F)
. However, another alternative explanation may be
that HLE’s proliferative activity was lower on intact AM, as shown by
a significantly lower BrdU-labeling index on intact AM than on
EDTA-denuded AM (Fig. 4)
. Koizumi et al.15
as well as
Meller et al.,14
from our laboratory, demonstrated
desmosomal structures between HLE and the underlying amniotic
epithelium, using electron microscopy. We have recently conducted
further analyses and noted that both integrins 
6 and 3 were not
expressed, whereas integrins ß4 and ß1 were expressed by HLE in
contact with devitalized amniotic epithelial cells. This was in great
contrast with the positive expression of integrins 6ß4 and
3ß1 by HLE when growing on denuded amniotic basement membrane
(Grueterich et al., manuscript in preparation). This information
strongly suggests that HLE uses different adhesion complexes when
growing on intact versus EDTA-treated AM.
We further confirmed that the lower labeling index of HLE on intact AM was a result of a slow cell cycle and not of postmitotic differentiation, in that continuous BrdU labeling for 6 days caused a 30-fold increase in the labeling index (Fig. 4D) . It should be noted that the BrdU labeling index was calculated at different time points, because both culture conditions grew at different growth rates. We naturally had the concern that, in cultured cells, proliferative activity slows when the cells approach confluence or plateau growth. However, we believe this concern would not have affected the interpretation of our data because the same growth condition was used for both groups during the labeling experiment. Immunofluorescent visualization of incorporated BrdU of HLE nuclei confirmed that the number of BrdU-positive nuclei was indeed higher in areas where amniotic epithelial cells were absent than in those where they were present (Fig. 4E 4F) . We thus conclude that HLE expanded on intact AM maintains a slower cell cycle—one feature of epithelial SCs in vivo. This finding was also consistent with previous findings by Meller et al. (Ref. 14 and manuscript submitted).
The next natural question is whether the rapid cell cycle and the higher outgrowth rate of HLE on EDTA-denuded AM represent a rapid self-renewal of limbal progenitor cells or their actual differentiation into TAC. Matic et al.8 first proposed the theory that the absence of Cx43-containing gap junctions and GJIC is a mechanism by which SCs maintain their "stemness" in their specialized microenvironment, and expression of Cx43 activates GJIC that is needed for corneal TAC synchrony. We thus examined the expression of Cx43 and GJIC and noted that HLE cells expanded on intact AM were largely devoid of Cx43, a phenotype resembling that of the SC-containing basal limbal epithelium in vivo. Removal of the amniotic epithelium by EDTA mechanical debridement exposed the amniotic basement membrane and promoted the HLE to adopt a phenotype that showed significantly more Cx43 expression (Fig. 3) . In both groups of AM, the patchy, positive GJIC was consistent with subsequent immunolocalization of Cx43 on the same samples (Fig. 5) .
Positive GJIC areas of the EDTA-denuded AM group were larger than those of the intact AM group (27% vs 17%), but this difference was not statistically significant and was not consistent with the aforementioned 10-fold difference in Cx43 expression. There are several possible reasons for this discrepancy. First, Cx43 detected by immunostaining had not yet assembled into connexon associations between neighboring cells, which are essential for gap junction conductance. Second, it is not known whether gap junctions in corneal and limbal epithelium also form heterotypic junctions (i.e., a gap junction channel composed of different Cx subtypes). If this were the case, other Cxs might respond differently to the two culture conditions described herein, which may explain why we found no difference in GJIC, even though Cx43 expression was 10 times higher in cultures on EDTA-treated AM.
To further explore the cell population derived with our culture system, we transplanted expanded HLE on AM as a composite subcutaneous graft in immunocompromised mice to promote stratification and differentiation. In addition to the nude gene, which results in an absence of thymus-derived T-cells, these mice have two other mutations important in regulating the function of the immune system. They are X-linked immune defect (xid), which affects the maturation of T-independent B-lymphocytes, and beige (bg), in which the homozygote is devoid of natural killer cells that are cytotoxic to tumor cells in vitro. In both conditions a nicely stratified epithelium was found with a relatively small and compact basal cell layer. The presence of the devitalized amniotic epithelium could not be discerned underneath the stratified HLE any longer, suggesting their partial or complete disintegration, as proposed by others.18 Our results showed that the basal layer of the resultant stratified epithelium on intact AM did not express Cx43, in contrast to HLE on EDTA-treated AM (Figs. 6C 6D) . In addition, we found that keratin K3 was absent in the basal layer on intact AM, whereas the basal layer in the EDTA-treated group expressed keratin K3 (Figs. 6E 6F) . Moreover, BrdU label-retaining nuclei were found in the basal layer of the epithelium on intact AM, but not at all in EDTA-treated AM cultures (Figs. 6G 6H) . That the amniotic epithelium and fibroblasts are devitalized and do not have any proliferative activity after the present method of preservation18 shows that the basal BrdU-labeled cells are derived from HLE. Collectively, these data indicate that a rapid cell cycle and positive expression of Cx43 and keratin K3 were promoted when HLE grew directly on the amniotic basement membrane, strongly supporting the notion that this culture condition promotes TAC differentiation. This interpretation, however, cannot be extrapolated to the method used by Koizumi et al.15 22 in which 3T3 fibroblasts feeder layers are routinely included. Nevertheless, in another report,13 they mentioned that corneal differentiation is promoted, but failed to provide evidence that limbal epithelial progenitor cells are actually preserved. Future studies are needed to show that denudation of the amniotic epithelium to expose the amniotic basement membrane is an important microenvironmental "cue" in promoting TAC differentiation. This hypothesis can be tested by transplanting ex vivo expanded HLE in a rabbit model of limbal SC deficiency that we have recently reported.23 If this were the case, we may understand why migration of the offspring of limbal SCs to the corneal basement membrane signifies the beginning of TAC differentiation. Future studies should also be extended to investigating which amniotic basement membrane component(s)—laminin-1, laminin-5, collagen VII, and fibronectin24 —is responsible for upregulation of Cx43 expression and thus, TAC differentiation. In this regard, previous studies have shown that rat hepatocyte cultures25 26 and human epidermal keratinocyte cultures27 show an increase in gap junction synthesis and GJIC when exposed to certain extracellular matrix components (e.g., glycosaminoglycans, proteoglycans, and laminin-5).
Similarly, it seams equally important to delineate the role of devitalized amniotic epithelial cells in endowing HLE with a status of slow cycling and poor differentiation—that is, features resembling limbal epithelial SCs in vivo. Whether this role is a simple masking of the amniotic basement membrane or a release of cytokines from the devitalized amniotic epithelium, comparable to a feeder layer system, should be further investigated. Collectively, we believe the model system described herein is ideal for future exploration of the exact mechanistic operation in the microenvironmental niche that maintains "stemness" in limbal SCs, and in the cue that promotes corneal TAC differentiation. This new knowledge will help us understand the pathogenesis of limbal SC deficiency that develops in various ocular surface diseases and how transplantation of limbal epithelial SCs can be better refined in the future.
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Footnotes |
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Submitted for publication June 19, 2001; revised September 4, 2001; accepted September 14, 2001.
Commercial relationships policy: N.
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: Scheffer C. G. Tseng, Bascom Palmer Eye Institute, William L. McKnight Vision Research Center, 1638 NW 10th Avenue, Miami, FL 33136; stseng{at}bpei.med.miami.edu.
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3ß1 with laminin 5 promotes gap junctional communication J Cell Biol 143,1735-1747This article has been cited by other articles:
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