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Human Limbal Progenitor Cells Expanded on Intact Amniotic Membrane Ex Vivo
Martin Grueterich, MD;
Scheffer C. G. Tseng, MD, PhD
Arch Ophthalmol. 2002;120:783-790.
ABSTRACT
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Background The transplantation of human limbal epithelium on amniotic membrane
as a substrate is a new treatment for limbal stem cell deficiency. Limbal
epithelial stem cells are characterized by a slow cell cycle and the lack
of K3 keratin and connexin 43 (Cx43), a gap junction protein. We investigated
Cx43 expression, gap junction intercellular communication (GJIC), and proliferative
activity of limbal epithelium expanded on amniotic membrane.
Methods Connexin 43 expression and bromodeoxyuridine (BrdU) incorporation were
determined by immunohistology. The GJIC was investigated by a scrape-loading
dye transfer assay. Expression of Cx43 and K3 keratin as well as BrdU-retaining
nuclei were also analyzed after xenotransplantation in nude mice.
Results Limbal epithelium showed mean ± SD 12.4% ± 14.5% positive
units of Cx43 expression and a low BrdU labeling index of 2.4% ± 0.9%
(n = 5), of which the latter was due to slow cycling, as proved by its increase
to 62.0% ± 9.5% after continuous BrdU labeling for 5 days. Most of
the expanded epithelium did not show GJIC (83%), significantly more than that
grown on plastic (6%; P<.002). Basal cells of
the stratified epithelium after xenotransplantation did not express Cx43 and
K3 keratin, but their nuclei retained BrdU.
Conclusion These results support the hypothesis that intact amniotic membrane preferentially
preserves and expands Cx43-negative, keratin K3–negative, and GJIC-deficient
limbal epithelium, a phenotype resembling that of stem cell–containing
limbal basal epithelial cells in vivo.
Clinical Relevance Intact amniotic membrane is a suitable substrate for bioengineering
limbal epithelia for ocular surface reconstruction.
INTRODUCTION
THE STEM CELLS (SCs) of the corneal epithelium are located exclusively
at the limbus, the anatomic junction between the cornea and the conjunctiva,1 and serve as the ultimate source for corneal epithelial
regeneration under normal and injured conditions.2-3
These cells were initially identified in the entire basal layer of the limbal
epithelium by the lack of cornea-specific K3 keratin expression.1
Subsequently, a fraction of these limbal-basal epithelial cells were found
to have a long cell cycle4 and high clonogenicity,5-6 ie, general characteristics of SCs.
When limbal SCs proliferate, they self-renew and/or give rise to transient
amplifying cells (TACs) located in the corneal basal epithelium. Unlike SCs,
TACs have a short cell cycle and a limited proliferative capacity (ie, a shorter
lifespan). The mechanism governing the balance between SC self-renewal and
SC differentiation into TACs remains unclear. One explanation of how SCs in
general maintain their "stemness" is the fact that they are located in a microenvironmental
"niche." Within such a niche, SCs lack gap junction intercellular communication
(GJIC), which enables SCs to be sequestered from more differentiated TACs.7-8
Gap junctions are specialized cell membrane structures forming intercellular
channels that are composed of a variety of transmembrane proteins (polypeptides)
called connexins (Cx).9-10 Gap
junctions play an important role in direct cell-cell communication, which
affects cell proliferation, differentiation, and apoptosis.9-13
Connexin 43 and Cx50 are the only 2 Cx found in the human ocular surface
epithelium so far.14-16
Under normal atraumatic conditions, expression of Cx43 is noted in the basal
cell layer of the human corneal but not limbal epithelium, suggesting that
the expression of Cx43 de notes the differentiation of SCs into corneal TACs.
Wolosin and coworkers16 proposed that the apparent
incongruity of Cx expression may endow limbal epithelial SCs with the property
of stemness in this microenvironmental niche so that they can be segregated
from further differentiated TACs.
When limbal epithelial SCs are partly or totally destroyed, the corneal
surface will invariably be covered by the migrating conjunctival epithelium,
a pathologic entity found in a number of ocular surface disorders.17 Clinical transplantation of limbal epithelial SCs
from an autologous or allogeneic source is necessary to restore vision and
a normal corneal surface.18-19
Transplantation of preserved intact amniotic membrane alone has recently been
shown to restore such damaged corneal surfaces in patients with partial limbal
stem cell deficiency, ie, the limbus has been partially destroyed.20-21 This result suggests that transplanted
amniotic membrane helps expand residual limbal epithelial SCs in vivo. Promising
results of transplanting limbal epithelial SCs expanded on amniotic membrane
in culture have recently been reported for treating partial or total limbal
SC deficiency in human patients.22-26
These findings prompt us to examine the hypothesis that amniotic membrane
may help maintain and expand limbal epithelial SCs ex vivo by serving as a
substrate mimicking their microenvironmental niche. Herein we provide experimental
evidence that limbal epithelial cells ex vivo expanded on intact amniotic
membrane are indeed largely devoid of Cx43 expression, lack GJIC, and are
slow cycling. On xenotransplantation into nude mice, these expanded cells
yield a stratified epithelium whose basal layer remains negative to Cx43 and
K3 keratin expression and retained bromodeoxyuridine (BrdU) labels, resembling
their in vivo counterpart.
MATERIALS AND METHODS
HUMAN TISSUE PREPARATION
Human tissue was handled according to the Declaration of Helsinki. Corneoscleral
tissue from human donor eyes was obtained from the Florida Lions Eye Bank,
Miami, directly after the central corneal button had been used for corneal
transplantation. The tissue was rinsed 3 times with Dulbecco modified Eagle
medium (GIBCO BRL, Grand Island, NY) containing 50-mg/mL gentamicin (GIBCO
BRL) and 1.25-mg/mL amphotericin B (GIBCO BRL). 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 magnesium- and calcium-free
Hank balanced saline solution [GIBCO BRL]) at 37°C under humidified 5%
carbon dioxide for 5 to 10 minutes. After 1 rinse with Dulbecco modified Eagle
medium containing 10% fetal bovine serum (GIBCO BRL), the scleral rim was
trimmed to obtain limbal tissue cubes of approximately 1 x 1.5 x
2.5 mm.
HUMAN LIMBUS CULTURES ON AMNIOTIC MEMBRANE
Amniotic membrane with the epithelial side facing up was fastened onto
a culture insert (Milipore Corp, Bedford, Mass) as previously reported.27 On the center of the amniotic membrane an explant
was placed and cultured in a medium made of an equal volume of HEPES-buffered
Dulbecco modified Eagle medium (GIBCO BRL) containing bicarbonate and Ham
F12 (GIBCO BRL). The medium was supplemented with 0.5% dimethylsulfoxide (Sigma-Aldrich
Corp, St Louis, Mo), 2-ng/mL mouse epidermal growth factor (Sigma-Aldrich
Corp), 5-µg/mL insulin, 5-µg/mL transferrin, 5-ng/mL selenium
(Sigma-Aldrich Corp), 0.5-µg/mL hydrocortisone (Sigma-Aldrich Corp),
30-ng/mL cholera toxin A subunit (Sigma-Aldrich Corp), 5% fetal bovine serum
(GIBCO BRL), 50-µg/mL gentamicin (GIBCO BRL), and 1.25-µg/mL amphotericin
B. Cultures were incubated at 37°C under 5% carbon dioxide and 95% air,
and the medium was changed every 2 to 3 days. When human limbal epithelium
cultures almost reached confluent growth, they were subjected to qualitative
dye transfer assay or incubated with 10µM BrdU (Boehringer-Mannheim
Corp, Indianapolis, Ind) for 24 hours and fixed in cold methanol for immunostaining.
XENOTRANSPLANTATION
All procedures were performed according to the Association for Research
in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic
and Vision Research. Details of this technique have been described previously.28 Briefly, nearly confluent human limbal epithelial
cultures were labeled with BrdU for 7 days and transplanted to the subcutaneous
plane of the abdomen of NIH-bg-nu-xidBR mice. After 5 days, mice were killed
and the tissue including implanted amniotic membrane was removed and embedded
in optimal cutting temperature compound for cryosectioning. A total of 6 cultures
was transplanted.
IMMUNOSTAINING
Immunostaining was performed as previously described.28
Briefly, frozen sections were fixed and preincubated with 5% bovine serum
albumin (Sigma-Aldrich Corp) to block nonspecific staining. Sections were
incubated with a mouse anti-Cx43 (1:200) (Chemicon International Inc, Temecula,
Calif), AE-5 (anti-K3) (1:100) (ICN Pharmaceuticals, Costa Mesa, Calif), or
anti-BrdU (1:1000) (Boehringer-Mannheim Corp) monoclonal antibody for 45 minutes
followed by a fluorescein isothiocyanate–conjugated secondary antibody
(goat anti–mouse IgG at 1:200) (Sigma-Aldrich Corp), mounted with an
antifade solution (Vectashield; Vector Laboratories, Burlingame, Calif), and
analyzed with a fluorescence microscope (Axiophot; Carl Zeiss Inc, Oberkochen,
Germany).
For BrdU and Cx43 double labeling, confluent cultures were incubated
with 10µM BrdU in the same culture medium for 24 hours. These cultures
on amniotic membrane were prepared as flat-mount samples. After samples were
treated with 2N hydrochloric acid at 37°C for 45 minutes and neutralized
in boric acid (pH 8.5), 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 a diaminobenzidine-peroxidase
staining protocol (Vectastain Elite Kit; Vector Laboratories). 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 (within the major outgrowth area) were counted per specimen.
The labeling index for BrdU was expressed as the number of positive-labeled
nuclei divided by the number of all nuclei multiplied by 100%. We defined
1 U of Cx43 expression as all cells in one x400 field expressing Cx43.
That is, 0.5 U was defined as 50% of cells expressing Cx43. We counted 100
fields per sample for a total of 5 samples and reported their mean and SD.
SEMIQUANTITATIVE DYE TRANSFER ASSAY
We used the scrape-loading dye transfer assay originally described by
El-Fouly et al29-30 and discussed
further by Trosko et al.30 For positive control
we cultured human limbal epithelium from an explant on plastic dishes for
14 days. Human limbal epithelium on plastic or amniotic membrane was rinsed
with sterile phosphate-buffered saline. One milliliter of lucifer yellow plus
rhodamine-dextran (0.5 mg/mL) in phosphate-buffered saline 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 phosphate-buffered saline to prevent high background fluorescence. Cultures
were fixed in 4% formalin and epifluorescence was examined with a microscope
(Axiophot; Carl Zeiss Inc) equipped with a UV light source. A rhodamine filter
set was used to identify red fluorescence 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 18 scrape lines (6 scrape lines per culture for 3 separate
cultures). The percentage of the entire length of all 6 scrape lines per culture
was measured where we observed dye transfer in more than 4 cell rows away
from the initial loaded cells.
RESULTS
Cx43 EXPRESSION OF LIMBAL AND CORNEAL EPITHELIA IN VIVO
The limbus, ie, the transitional zone between human conjunctiva and
cornea, had a multilayered epithelium with the basal layer arranged in a palisade
pattern (Figure 1A). The limbal
epithelium was situated on top of a loose and vascular connective tissue (Figure 1A, arrowheads), while the corneal
epithelium lay on top of the dense Bowman layer (ie, a thick basement membrane; Figure 1A, black arrows) with a subjacent
dense avascular stroma. Immunostaining showed that the expression of Cx43
was absent in the basal layer, but positive in the suprabasal layers, of the
limbal epithelium (Figure 1B and
C). In central cornea sections, Cx43 was predominantly expressed in the basal
layer and with a less intensity in more superficial layers (Figure 1D). This finding confirmed that reported by Matic et al14 and Wolosin and coworkers.16
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Figure 1. Connexin 43 (Cx43) expression
in corneolimbal zone in vivo. A, Typical stratified epithelium, with a compact
basal cell layer at the limbus arranged in a palisade pattern (arrowheads).
Corneal basal cells lay on top of the dense Bowman layer (arrows) (hematoxylin-eosin,
bar indicates 100 µm). B, Connexin 43 fluorescence was absent in the
basal layer (arrowheads) but detected in suprabasal cell layers of the limbus.
Asterisks indicate the basement membrane (immunostaining, bar indicates 100
µm). C, Distinct punctate fluorescence (arrows) was noted in the basal
cells of the peripheral cornea, but not in the limbus (area between arrowheads
and the basement membrane [asterisks]) (immunostaining, bar indicates 50 µm).
D, Central cornea shows strong Cx43 staining predominantly in the basal layer
and with less intensity in the superficial layers (bar indicates 100 µm;
inset, original magnification x1000).
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GENERAL MORPHOLOGIC FEATURES
Human limbal epithelium outgrowth was detected after 1 week from the
border of a limbal explant (Figure 2A)
and reached confluence (ie, 22 mm in diameter) after an average duration of
3 to 4 weeks (Figure 2B, nearly
reaching the insert edge). The outgrowth consisted of a sheet of small, compact,
and uniform cells with an approximate 1:1 nucleus-cytoplasm ratio in the majority
of cells (Figure 2C); the leading
edge of the outgrowth built a bulge consisting of both limbal epithelial cells
and amniotic epithelial cells (Figure 2B,
arrows). On cross section, the majority of the outgrowth consisted of a monolayer
of cuboidal cells (Figure 1 2D,
black arrows) growing on top of amniotic epithelial cell debris (Figure 2D, black arrowheads).
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Figure 2. Morphologic features of human
limbal epithelium expanded on intact amniotic membrane. A, Human limbal epithelium
started to grow from the border of the limbal explant after 1 week (arrows).
B, The outgrowth expanded to an area nearly confluent after 3 weeks (arrows).
C, The expanded cells appeared as a monolayer of small, uniform cells with
a nucleus-cytoplasm ratio of approximately 1:1 (phase contrast, bar indicates
100 µm). D, Cross sections showed a cell layer of expanded human limbal
epithelium (arrows) on top of amniotic epithelial cell debris (arrowheads)
(hematoxylin-eosin, bar indicates 50 µm).
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Cx43 EXPRESSION AND CELL CYCLE ANALYSIS OF EX VIVO EXPANDED HUMAN LIMBAL
EPITHELIUM ON INTACT AMNIOTIC MEMBRANE
After 3 weeks of culturing on amniotic membrane, the majority of expanded
human limbal epithelium did not express Cx43 regardless of whether the final
outgrowth area was 70%, 90%, or confluent (Figure 3A). Cells expressing Cx43 were calculated as 12.4% ±
14.5% positive units and were found in focal areas predominantly adjacent
to the explant and randomly scattered among the outgrowth area (Figure 3B). Positive Cx43 staining appeared in a punctate pattern
confined to the cell membrane of adjacent cells, compatible with the formation
of gap-junction channels (Figure 3B,
inset). To correlate Cx43 expression with the proliferative activity at the
same time, we labeled the S-phase of the cell cycle with BrdU, a thymidine
analogue, for 24 hours in nearly confluent cultures. The labeling index was
low, in the range of 2.4% ± 0.9% (n = 5) (Figure 3A and C, arrows). Areas with high BrdU uptake were found
predominantly near the explant or at the leading edge of the outgrowth and
were devoid of Cx43 expression (Figure 3C,
inset). To confirm that the nonlabeled cells are indeed slow cycling and not
postmitotic differentiated cells, we continuously incubated a set of 6 cultures
with BrdU for 5 days. As shown in Figure 3D, the BrdU labeling index increased to 62.0% ± 9.5% (n =
6). This result indicated that the majority of the expanded human limbal epithelium
on amniotic membrane was indeed slow cycling.
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Figure 3. Double labeling of connexin 43
(Cx43) expression and bromodeoxyuridine (BrdU) incorporation. A, Only a few
cells of the entire outgrowth incorporated BrdU (brown staining of the nucleus,
arrow). No cCx43 expression was noted at the cell membranes (bar indicates
50 µm). B, Focal area of Cx43 expression as shown by a punctate staining
pattern at the cell membranes (white arrows). Inset, High-power magnification
of a cell expressing Cx43 at the cell membrane (original magnification x1000).
C, After 24-hour labeling, the BrdU labeling index was low overall, except
near the explant (inset [same magnification as part C]), where higher BrdU
uptake was noted (arrows indicate areas of BrdU uptake; bar indicates 100
µm). D, A marked increase in BrdU uptake was noted after 5 days of continuous
BrdU labeling (bar indicates 100 µm).
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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 qualitative dye transfer
assay using a scrape-loading technique previously described.29
Human limbal epithelium expanded on intact amniotic membrane did not show
any dye transfer from the scraped area to the adjacent cells in most (83%)
of the scrape lines performed (n = 6 per sample) (Figure 4A and B). These areas did not express Cx43 after counterimmunostaining
(Figure 4C). In a total of 18 scrape
lines (3 cultures and 6 scrapes per sample) we found 3 patches, representing
17% of the entire length of all scrape lines, of dye transfer to neighboring
cells (Figure 4D and E). These areas
were also found to express Cx43 when subsequently counterimmunostained (Figure 4F, arrows). As a positive control,
we scrape-loaded the outgrowth of human limbal epithelium grown on plastic
and found marked dye transfer to adjacent cells in 94% of the entire length
of all 18 scrape lines (Figure 4G
and H). This difference was statistically significant (P<.002, analysis of variance).
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Figure 4. Gap junction intercellular communication
(GJIC) studies of amniotic membrane cultures and plastic cultures. A, Human
limbal epithelium on amniotic membrane showed that the primary loaded cells
incorporated rhodamine-dextran (red fluorescence). B, No transfer of lucifer
yellow could be detected from the initially loaded cells (arrows) to their
adjacent neighboring cells. C, Immunohistochemical detection of connexin 43
(Cx43) expression in the same area illustrated in A and B showed no Cx43 expression.
D, Primary loaded cells. E, Some focal areas within the outgrowth showed cell-cell
communication in a grapelike formation (white arrows, green fluorescence).
F, Immunochemical staining of the same area showed Cx43-expressing cells (brown
punctate staining) (black arrows). Dotted line indicates the scrape lines.
G, A positive control culture of human limbal epithelium grown on plastic
showed primary loaded cells at the scrape line. H, Pronounced GJIC to adjacent
cells (green fluoresence) was noted (A-H, bar indicates 100 µm).
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Cx43, K3 KERATIN EXPRESSION, AND BrdU LABELING AFTER XENOTRANSPLANTATION
We transplanted amniotic membrane with expanded human limbal epithelium
as a composite graft (n = 6) into the subcutaneous plane of NIH-bg-nu-xidBR
mice after 7 days of continuous BrdU labeling. Five days later, the resultant
epithelium was stratified to an average of 5 cell layers. Basal cells were
small and compact, whereas superficial cells appear more flat and squamous
(Figure 5A). Expression of Cx43
was absent throughout the entire epithelium (Figure 5B). Within the same section, a positive control could be
found in the mouse epidermis, which expressed Cx43 in large amounts (Figure 5B, inset). As basal cells did not
express Cx43, this phenotype resembled a limbal basal epithelial cell phenotype
in vivo; thus, we examined the expression of K3 keratin, which has been reported
to be absent also in the limbal basal epithelium.1
Our result showed that K3 keratin was indeed absent in the basal layer but
markedly expressed in the suprabasal and superficial layers of the resultant
stratified epithelium (Figure 5C).
Label-retaining (BrdU-positive) cells were exclusively identified in the basal
layer in direct contact with the underlying amniotic membrane (Figure 5D).
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Figure 5. Phenotypic studies of human limbal
epithelium on intact amniotic membrane after xenotransplantation. A, Stratified
epithelium with compact and relatively small basal cells. More superficial
cells were larger and squamous (hematoxylin-eosin). B, Immunostaining did
not show any connexin 43 (Cx43) expression throughout the entire epithelium.
Nuclei were stained with propidium iodide. A positive control was found in
the mouse epidermis in the same sample, which expressed a large amount of
Cx43 (inset [original magnification x200]). C, Keratin K3 was expressed
by suprabasal and superficial cells but not by the basal epithelial layer.
Nuclei were stained with propidium iodide. D, Incorporated bromodeoxyuridine
could be identified exclusively in the basal layer in direct contact with
the amniotic membrane (indicated by asterisks in all parts) (A-D, bar indicates
100 µm).
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COMMENT
In this study, we provide experimental evidence to support the hypothesis
that intact amniotic membrane preferentially preserves and expands human limbal
epithelial progenitor cells. After stratification in nude mice, the basal
layer of the resultant epithelium was devoid of Cx43 and K3 keratin expression
and retained a fraction of slow-cycling cells, resembling the phenotype of
the SC-containing human limbal basal epithelium in vivo. In the aggregate,
these data support the notion that amniotic membrane mimics the in vivo stromal
niche to maintain SC characteristics. Collectively, these data also explain
why amniotic membrane and ex vivo expanded limbal epithelium have been successful
as a new surgical strategy to reconstruct the corneal surface in patients
with limbal SC deficiency.22, 24-26,31
Matic et al14 and Wolosin and coworkers16 proposed the theory that noncommunication of limbal
basal epithelial cells in vivo is one feature of the microenvironmental niche
in which limbal SCs lie. We observed only 12.4% ± 14.5% of Cx43-positive
units in our expanded cell population after culturing for an average of 3
to 4 weeks on intact amniotic membrane (Figure
3A and B). We further proved that the lack of Cx43 expression indeed
reflected the lack of gap junction formation and GJIC by means of the well-established
scrape-loading dye transfer technique (Figure
4).29-30 Except for
3 localized areas composing up to 17% of the total length of all 18 scrape
lines, the majority of human limbal epithelium (83%) expanded on intact amniotic
membrane showed no GJIC. This value was significantly less than that observed
for human limbal epithelium on plastic (17 [94%] of 18 scrape lines) (Figure 4).
We noted that the lack of Cx43 expression was neither dependent on the
stage of confluence nor restricted to the edge of the outgrowth. Wolosin and
coworkers16 found Cx43 expression and GJIC
in 9-day-old rabbit limbal epithelial cells cultured on a 3T3 fibroblast feeder
layer, and a shift from Cx43 to Cx50 expression after raising the culture
to the air-liquid interface to promote stratification. The fact that most
of our culture was actually devoid of Cx43 expression even after 3 weeks of
culturing underscores the striking difference between the amniotic membrane
culture and the 3T3 fibroblast feeder layer system.
It has been reported that gap junctions and Cx expression are dramatically
decreased in late G1 and S phases and reappear throughout the rest
of the cell cycle in regenerating hepatocyte cultures, and it has been suggested
that the down-regulation of GJIC might be an effective way to allow cell division
without interfering with the homeostatic balance within the nonproliferative
cell population.32-33 By the use
of BrdU labeling to identify rapid-cycling cells, we noted that Cx43 was not
expressed by both BrdU-labeled and nonlabeled cells (Figure 3A). Therefore, we ruled out that the lack of Cx43 expression
was a result of rapid proliferation. We actually noted that the labeling index
was overall low, in the range of 2.4% ± 0.9% after 24 hours of BrdU
labeling (Figure 3C). To further
prove that such a low labeling index was not caused by terminal differentiation,
we continuously labeled these cells for 6 days and found an approximately
30-fold increase of the labeling index, ie, to an average of 62% ±
9.5% (Figure 3D). Collectively,
these data confirmed our assertion that the amniotic membrane culture system
predominantly maintains and expands slow-cycling human limbal epithelium.
The data discussed so far were obtained from cultured human limbal epithelium
monolayers. To further investigate the phenotype of expanded human limbal
epithelium, we performed xenotransplantation of nearly confluent human limbal
epithelium on intact amniotic membrane to promote epithelial stratification.
This time we labeled human limbal epithelium on amniotic membrane continuously
for 7 days before xenotransplantation to identify slow-cycling, label-retaining
cells after a chasing period of 5 days. The resultant stratified epithelium
was devoid of Cx43 expression throughout all layers (Figure 5B), was devoid of keratin K3 expression in the basal layer
(Figure 5C), and retained BrdU labels
in the basal layer (Figure 5D).
All of these characteristics are found in the SC-containing basal limbal epithelium
in vivo.
In the present study we used intact amniotic membrane and found out
that most expanded human limbal epithelium was growing on top of devitalized
amniotic epithelial cells without direct contact with the underlying basement
membrane (Figure 2). This finding
was consistent with what has been recently reported by Koizumi et al34 with the use of rabbit limbal epithelial cells. Our
further study indicated that the separation of expanded human limbal epithelial
cells by amniotic epithelial cells was important to maintain such a phenotype
without Cx43 expression and GJIC, because denudation of amniotic epithelium
to expose amniotic basement membrane will promote a corneal epithelial phenotype.28 Future studies are also needed to elucidate the mechanism
by which the cell contact with the basement membrane may affect the cell cycle
and the expression of differentiation markers, imitating the differentiation
of limbal SCs to corneal TACs, of which the latter lie on a thick corneal
basement membrane. Furthermore, investigation into culturing conditions that
may optimize limbal epithelial SC expansion on amniotic membrane should be
fruitful for devising a clinical protocol of this new surgical procedure for
treating limbal stem cell deficiency.
AUTHOR INFORMATION
Submitted for publication July 17, 2001; final revision received February
3, 2002; accepted February 28, 2002.
This study was supported in part by Public Health Service Research Grant
EY 06819 from the Department of Health and Human Services, National Eye Institute,
National Institutes of Health, Bethesda, Md (Dr Tseng); in part by an unrestricted
grant from Research to Prevent Blindness Inc, New York, NY; and in part by
research fellowship grant GR 1814/1-1 from the Deutsche Forschungsgemeinschaft,
Bonn, Germany (Dr Grueterich).
Preserved human amniotic membrane was kindly provided by Bio-Tissue
(Miami, Fla).
Corresponding author: Scheffer C. G. Tseng, MD, PhD, Ocular Surface
Center and Ocular Surface Research & Education Foundation, 8780 SW 92nd
St, Miami, FL 33176 (e-mail: stseng{at}ocularsurface.com).
From the Department of Ophthalmology, Bascom Palmer Eye Institute,
University of Miami School of Medicine (Dr Grueterich), and Ocular Surface
Center and Ocular Surface Research & Education Foundation (Dr Tseng),
Miami, Fla.
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