Reconstruction of damaged cornea by autologous transplantation of epidermal adult stem cells.

Abstract

It is crucial for the treatment of severe ocular surface diseases such as Stevens-Johnson syndrome (SJS) and ocular cicatricial pemphigoid (OCP) to find strategies that avoid the risks of allograft rejection and immunosuppression. Here, we report a new strategy for reconstructing the damaged corneal surface in a goat model of total limbal stem cell deficiency (LSCD) by autologous transplantation of epidermal adult stem cells (EpiASC).
EpiASC derived from adult goat ear skin by explant culture were purified by selecting single cell-derived clones. These EpiASC were cultivated on denuded human amniotic membrane (HAM) and transplanted onto goat eyes with total LSCD. The characteristics of both EpiASC and reconstructed corneal epithelium were identified by histology and immunohistochemistry. The clinical characteristic of reconstructed corneal surface was observed by digital camera.
Ten LSCD goats (10 eyes) were treated with EpiASC transplantation, leading to the restoration of corneal transparency and improvement of postoperative visual acuity to varying degrees in 80.00% (8/10) of the experimental eyes. The corneal epithelium of control groups either with HAM transplantation only or without any transplantation showed irregular surfaces, diffuse vascularization, and pannus on the entire cornea. The reconstructed corneal epithelium (RCE) expressed CK3, CK12, and PAX-6 and had the function of secreting glycocalyx-like material (AB-PAS positive). During the follow-up period, all corneal surfaces remained transparent and there were no serious complications. We also observed that the REC expressed CK1/10 weakly at six months after operation but not at 12 months after operation, suggesting that the REC was derived from grafted EpiASC.
Our results showed that EpiASC repaired the damaged cornea of goats with total LSCD and demonstrated that EpiASC can be induced to differentiate into corneal epithelial cell types in vivo, which at least in part correlated with down-regulation of CK1/10 and upregulation of PAX-6.

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Reconstruction of damaged cornea by autologous transplantation
of epidermal adult stem cells
Xueyi Yang,1,2 Nicanor I. Moldovan,3 Qingmei Zhao,2 Shengli Mi,2 Zhenhui Zhou,4 Dan Chen,5 Zhimin Gao,2
Dewen Tong,2 Zhongying Dou2
1Department of Life Sciences, Luoyang Normal University, Luoyang, China; 2Shaanxi Branch of National Stem Cell Engineering
Center, Northwest A & F University, Yangling, China; 3Davis Heart and Lung Research Institute, Ohio State University, Columbus,
OH,; 4Department of Animal Science and Veterinary, Beijing Agricultural Vocation College, Beijing, China; 5Electron Microscopic
Center, School of Basic Medicine, Fourth Military Medical University, Xi’an, China
Purpose: It is crucial for the treatment of severe ocular surface diseases such as Stevens-Johnson syndrome (SJS) and
ocular cicatricial pemphigoid (OCP) to find strategies that avoid the risks of allograft rejection and immunosuppression.
Here, we report a new strategy for reconstructing the damaged corneal surface in a goat model of total limbal stem cell
deficiency (LSCD) by autologous transplantation of epidermal adult stem cells (EpiASC).
Methods: EpiASC derived from adult goat ear skin by explant culture were purified by selecting single cell-derived
clones. These EpiASC were cultivated on denuded human amniotic membrane (HAM) and transplanted onto goat eyes
with total LSCD. The characteristics of both EpiASC and reconstructed corneal epithelium were identified by histology
and immunohistochemistry. The clinical characteristic of reconstructed corneal surface was observed by digital camera.
Results: Ten LSCD goats (10 eyes) were treated with EpiASC transplantation, leading to the restoration of corneal
transparency and improvement of postoperative visual acuity to varying degrees in 80.00% (8/10) of the experimental
eyes. The corneal epithelium of control groups either with HAM transplantation only or without any transplantation
showed irregular surfaces, diffuse vascularization, and pannus on the entire cornea. The reconstructed corneal epithelium
(RCE) expressed CK3, CK12, and PAX-6 and had the function of secreting glycocalyx-like material (AB-PAS positive).
During the follow-up period, all corneal surfaces remained transparent and there were no serious complications. We also
observed that the REC expressed CK1/10 weakly at six months after operation but not at 12 months after operation,
suggesting that the REC was derived from grafted EpiASC.
Conclusions: Our results showed that EpiASC repaired the damaged cornea of goats with total LSCD and demonstrated
that EpiASC can be induced to differentiate into corneal epithelial cell types in vivo, which at least in part correlated with
down-regulation of CK1/10 and upregulation of PAX-6.
The integrity of the normal ocular surface depends on its
epithelial stem cells located at the limbus, termed limbal stem
cells (LSCs). LSCs serve as the ultimate source for
maintenance and regeneration of the corneal epithelium
[1-3]. Damage to the limbus can result in limbal stem cell
(LSC) deficiency, characterized by conjunctivalization,
vascularization, and chronic inflammation (reviewed in
Espana et al.) [4]. Transplantation of cultivated LSCs for
reconstructing the ocular surface of total LSC deficiency is
one recently developed treatment [5-8]. Damaged corneas
have been successfully reconstructed by transplanting
autologous limbal epithelial cells [7,8] but not in severe ocular
surface diseases such as Stevens-Johnson syndrome (SJS) and
ocular cicatricial pemphigoid (OCP). In the case of severe
ocular diseases, the bilateral damage to LSCs and the immune
response to heterologous corneal epithelial transplantation are
the most challenging clinical entities. Therefore, our goal was
Correspondence to: Professor Zhongying Dou, Northwest A & F
University, Shaanxi Branch of National Stem Cell Engineering
Center, Yangling, 712100, China; Phone: 86-29-87080068; FAX:
86-29-87080068; email: douzhongying@china.com
to examine whether epidermal stem cells possess the capacity
to activate corneal genetic programs in response to corneal
stromal stimuli.
It is widely accepted that epidermis and corneal
epithelium are derived from ectoderm during embryogenesis.
A comparison of the corneal epithelium and follicular
epidermis illustrates that keratinocyte stem cells of these two
systems share several important attributes (reviewed in Taylor
et al.) [9]. These two cell types express the same markers such
as protein 63 (P63), β1-integrin, and cytokeratin 19 (CK19)
[10-12]. Ferraris et al. and Pearton et al. [13,14] have shown
that the transient amplifying (TA) cells of the central corneal
epithelium can be converted into the epidermis and its
appendages. This reprogramming occurs by means of a multi-
step process that involves the de-differentiation of TA cells
into primitive stem cells followed by re-differentiation into
hair follicles and associated multipotent stem cells [14]. Liang
and Bickenbach [15,16] have demonstrated that somatic
epidermal adult stem cells (EpiASC) have the ability to
produce cells of different lineages during development and
suggested that EpiASC may have pluripotentiality similar to
Molecular Vision 2008; 14:1064-1074 <http://www.molvis.org/molvis/v14/a127>
Received 28 June 2007 | Accepted 30 May 2008 | Published 5 June 2008
© 2008 Molecular Vision
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embryonic stem cells (ESC). These authors co-cultured
EpiASC with embryonic stem cells or transfected
keratinocytes with Octamer-4 (Oct4) and found that EpiASC
could alter their cell lineage protein expression to that of a
more primitive cell type [17,18]. Thus, the microenvironment
of the organ’s structure induces cells of various origins and
degrees of stemness to undergo a 'functional adaptation' to fit
into the overall role of the host organ [19]. This implies that
EpiASC might convert into corneal cell types when
recombined by the corneal stroma in vivo and also suggests
that transplantation of EpiASC may be a good way of solving
the clinical problems encountered in severe ocular diseases.
It is well known that EpiASC in vivo are harbored in
niches, which keep them in a state of quiescence and stemness
[20-22]. EpiASC are surrounded by a group of TA cells, which
become active and proliferate in culture [23,24]. TA cells may
play an important role in keeping EpiASC quiescent and in
protecting them from damage [9,25,26]. However,
purification of EpiASC is still problematic because of the
absence of a reliable, specific marker. Many researchers have
reported the isolation of EpiASC from the skin by digesting
the explants with enzymes such as trypsin and dispase
(reviewed in Kaur et al.) [27]. To address this purification
problem, we used a recently developed strategy to isolate and
purify EpiASC [28]. To identify whether EpiASC can convert
into corneal cell types when recombined by the corneal stroma
in vivo, we developed a method of culturing EpiASC on
denuded human amniotic membrane (HAM) and then
transplanted the resulting autologous EpiASC-HAM sheet
(EHS) onto the ocular surface of a goat model of limbal stem
cell deficiency (LSCD). The clinical outcome was assessed
after 30 months of follow up [29].
METHODS
Isolation and identification of epidermal stem cells from the
goat ear skin: The isolation and identification of epidermal
stem cells were performed as previously described [28].
Briefly, pieces of skin approximately 0.5 cm × 0.6 cm were
cut from the ear of Guanzhong dairy goats (three to four years
old). The tissues were rinsed three times with Medium 199
(Invitrogen-Gibco BRL, Grand Island, NY), which contained
50 μg/ml gentamicin and 1.25 μg/ml amphotericin B. After
the careful removal of cartilage and conjunctive tissue, the
remaining tissue was moved to a culture dish and cut into
pieces approximately 1 mm × 2 mm × 3 mm using a scalpel.
We then placed three to four pieces of the explant into each
well of a six-well TC-Plate (Hyclone, Logan, UT) and added
Medium 199 (Invitrogen-Gibco BRL, Grand Island, NY)
supplemented with 15% neonatal bovine serum (Baian
Biotechnology Corporation, Zhengzhou City, China), 5 μg/ml
insulin (Sigma-Aldrich Corporation, St. Louis, MO), 0.5 μg/
ml hydrocortisone, 40 μg/ml gentamicin, and 0.25 μg/ml
amphotericin B. The explants were cultured at 37 °C in a 5%
CO2 /95% air incubator. The medium was changed every two
to three days while the extent of each outgrowth was
monitored with a phase contrast microscope. We selected the
holoclones under dissecting microscope and treated them with
0.2% trypsin and 0.02% EDTA for approximately 10 min to
disperse each clone to single cells. The trypsin was then
inactivated by adding a medium containing 20% serum, and
cells were gently centrifuged and resuspended in a serum free
medium. The selected cells were seeded into culture dishes
coated with gelatin type B (Sigma) in a medium containing
80% Ham F12 (Invitrogen-Gibco BRL, Grand Island, NY),
20% GSFCM (goat skin fibroblast-conditioned medium), 1 -
2% BSA (Sigma-Aldrich, St. Louis, MO), 20 ng/ml EGF
(Sigma), 20 ng/ml IGF-1 (Sigma), 5 μg/ml insulin, 0.5 μg/ml
hydrocortisone, and 50 μg/ml penicillin and streptomycin.
Primary putative epidermal stem cells were left to grow
until they reached about 70% confluence in the culture plate.
Adherent cells were then harvested with 0.2% trypsin and
0.02% EDTA to dislodge the cells followed by rapid trypsin
neutralization to prevent cell damage. Isolated single cell
suspensions were subcultured on culture dishes coated with
gelatin at final plating density of 0.5–2 × 105 viable cells per
well. At each passage, cells from one well out of a set of
triplicate cultures were detached, pooled, centrifuged,
resuspended in fresh growth medium, seeded into three new
wells, and expanded until they reached the same level of
confluence. The growth medium was changed completely
every other day. The cultures were monitored under a Leica
Microsystems Wetzier microscope, LEICA MPS 60 (Leitz,
Wetzlar, Germany).
To confirm the EpiASC derived from interfollicular
epidermis, we dissected epidermis from dermis as previously
described [30] with the following modifications. Skin samples
were treated with 0.5% dispase II (Boehringer, Ingelheim,
Germany) for 14–16 h at 4 °C to separate the interfollicular
epidermis as a sheet from the underlying dermal tissue,
leaving the hair follicular from the infundibulum to the dermal
papillae within the dermis. The dermal surface was brushed
gently with curved forceps to release any loosely attached
basal keratinocytes, and then both interfollicular epidermis
and dermis were cultured in the same medium. The cultures
were monitored under a Leica Microsystems Wetzier
microscope LEICA MPS 60 (Leitz Wetzlar).
Preparation of human amniotic membrane and cultivation of
autologous epidermal stem cells on human amniotic
membrane: Human amniotic membranes (HAMs) were
obtained at the time of Cesarean section with proper informed
consent in accordance with the tenets of the Declaration of
Helsinki for research involving human subjects and upon
approval by the Institutional Review Board of the Northwest
A&F University (Shaanxi, Yangling, China). Under sterile
conditions, the membranes were washed with sterile
phosphate-buffered saline (PBS) containing antibiotics
(200 IU/ml penicillin and 200 IU/ml streptomycin) then were
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incubated with 2.5% dispase at 4 °C for 12–20 h to loosen
cellular adhesion followed by gentle scraping with a cell
scraper and washing three times with PBS. The denuded HAM
was frozen and stored at −80 °C in pure glycerol. Immediately
before use, the HAM was thawed, washed three times with
PBS containing antibiotics, cut into approximately 4 cm x 4
cm pieces, and flattened with the stromal side on the surface
of a nitrocellulose paper (NCP) framework.
We used passage three to four putative EpiASC as seed
cells. The EpiASC (1 × 105 cells/ml) were seeded on the
denuded HAM spread upon the NCP frame in a culture dish.
The culture was submerged in a serum-free medium as
described above for one week and then exposed to air by
lowering the medium level over a two week period. The
medium contained 80% F12, 20% GSFCM, 1%–2% BSA, 20
ng/ml EGF, 20 ng/ml IGF-1, 5 μg/ml insulin, 0.5 μg/ml
hydrocortisone, 50 μg/ml Vitamin C (Sigma), and penicillin
and streptomycin (50 μg/ml each). Cultures were incubated at
37 °C in a 5% CO2/95% air incubator for up to 21 days, and
the medium was changed every day.
Creation of total limbal stem cell deficiency model: The model
of total LSCD was created in the left eye of 30 goats using a
method previously described [28] with slight modifications.
Briefly, after anesthesia, an ocular surface injury was created
in one eye of each of the 26 healthy Guanzhong dairy goats
by excising all the conjunctival tissue with 5–7 mm of the
limbus and performing a superficial keratectomy of the central
corneal surface including limbal epithelial cells, and then the
limbus was burned by mechanical erasure with 1 N NaOH.
The goats received chloramphenicol and dexamethasone eye
drops twice a day during the first week and the necessary care
for 28 days after surgery. LSCD was verified using clinical
signs such as corneal haze, vascularization, epithelial
irregularity, and the presence of conjunctival goblet cells on
the corneal surface.
Transplantation of autologous cultivated epidermal stem cells
on human amniotic membrane sheets: Four weeks after the
ocular surface injury, the conjunctivalized ocular surfaces of
10 goats were surgically reconstructed by receiving
transplants of EpiASC-HAM sheets (EHS), eight animals
received HAM only transplantation (first control group), and
the remaining eight underwent removal of the fibrovascular
pannus without any transplantation (second control group)
[28]. After surgery, the eye was covered with sterile gauze.
From the first postoperative day, the goats were treated with
topical preservative-free 1% methyl prednisolone acetate and
0.3% ofloxacin four times a day and with intramuscular
penicillin sulfate and streptomycin sulfate every day for a
week.
Histology and immunohistochemistry: To identify whether
epidermal stem cells expressed stem cell markers and to
compare the protein expression profile of normal goat cornea
with damaged and reconstructed corneal epithelium, the
resultant cells and corneas were collected, fixed in 4%
paraformaldehyde, and embedded in tissue Tek OCT
compound (Electron Microscopy Sciences, Washington, PA)
for sectioning. For hematoxylin and eosin (H&E), PAS, and
AB-PAS staining, slides were re-fixed, washed, and stained
by their respective methods. For immunofluorescent staining,
slides were re-fixed and rinsed three times (15 min each time)
in PBS that contained 0.5% Triton-X 100 and then incubated
with 2% BSA in PBS for 60 min at 37 °C. Primary antibodies
(for sources and working dilutions, see Table 1) were applied
to tissues for 2 h at 37 °C. After washing, fluorescein-
conjugated secondary antibodies were applied for 30 min at
37 °C. Immunofluorescence microscopy was performed with
a Nikon microscope (Nikon, Tokyo, Japan).
Immunocytochemistry staining was done by the PV-6002
Histostain™–Plus method, using kits from Beijing
Zhongshan Biological and Technological Co. (Beijing,
China). Sections were blocked with normal goat serum for 15
min at room temperature and incubated with primary
antibodies (Table 1) overnight at 4 °C. Cells were rinsed with
0.1 M PBS without calcium and magesium and incubated with
the secondary goat anti-mouse IgG for 15 min at 37 °C. The
samples were washed and then incubated with the
streptavidin/peroxidase work medium (S-A/HRP) for 15 min
at 37 °C. Cells were rinsed in 0.1 M PBS without calcium and
magesium, and the samples were finally incubated with the 3,
3′-diaminobenzidine (DAB) peroxidase substrate to give a
brown stain and counterstained with hematoxylin or with
chromogen solution, BCIP/NBT, to give a blue stain. After
washing with PBS, the mounted sections were examined and
TABLE 1. PRIMARY ANTIBODIES AND SOURCES
Antibodies
CK1/10
PAX-6
CK3
CK12
P63
Category
Mouse monoclonal
Mouse monoclonal
Mouse monoclonal
Mouse monoclonal
Mouse monoclonal
Dilution
50X
1000X
50X
100X
200X
Source
Abcam®.
Chemicon®
USBiological
Santa Cruz Biotechnology, Inc.
Santa Cruz Biotechnology, Inc.
The primary antibodies used in this study and their sources.
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photographed with an epifluorescent microscope, (Nikon,
Tokyo, Japan).
RESULTS
The characteristics and origin of epidermal adult stem cells:
We first cultured skin explants in a serum-based medium.
After five to six days in culture, we found epithelial-like cells
growing in a mosaic pattern surrounding the explant. Next,
small, blast-like cells with a high nuclear/cytoplasmic ratio
appeared on the surface of the epithelial-like cells. After 12–
15 days, these cells formed clones with diameters of
approximately 50–100
Immunohistochemistry showed that these primary EpiASCs
either in clusters or single were Oct4 positive (Figure 1B,C).
These clones were picked under the dissecting microscope,
trypsinized into single-cell suspensions, and cultured in a
serum-free medium on gelatin-coated dishes. Subsequently,
the adherent cells formed holoclones (Figure 1D). These
clones were expanded in vitro. Immunohistochemistry
showed that the holoclone cells strongly expressed CK19,
μm (Figure 1A).
Figure 1. The characteristics of epidermal adult stem cells. A: Primary EpiASC clones derived from goat ear skin formed cell clusters, 100X.
B: Clusters of primary EpiASC positively express Oct4 (9E3), 200X. C: Single primary EpiASC express Oct4 (9E3), 200X. D: Subcultured
EpiASC formed holoclones in vitro, 100X. E: Subcultured EpiASC express CK19 (A53-B/A2), 200X. F: Subcultured EpiASC express P63
(4A4), 200X. G: Subcultured EpiASC express β1-integrin (W1B10), 200X. H: Subcultured EpiASC express CD34, 100X. I: Part of the
subcultured EpiASC expresses Oct4, 200X. The scale bar represents 100 μm.
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P63, β1-integrin, and CD34, a group of previously reported
EpiASC markers (Figure 1E-H). Surprisingly, we found
~50%of subcultured EpiASC still strongly expressing Oct4
(Figure 1). To confirm the origin of isolated EpiASC, we
separated the epidermis from the dermis by dispase II
treatment and cultured both of them in the same medium. After
three to four days, we found that cells that were derived from
the epidermis expand in a mosaic pattern (Figure 2A), which
is different from the fibroblast cells derived from dermis
(Figure 2B). After 8–10 days, cell clones were formed in the
surface of epithelial cells, which exhibited the same properties
as stem-like cell clones from total skin explant culture (Figure
2C). In contrast, the fibroblast cells became confluent, and no
stem-like cell clones were observed (Figure 2D). These results
showed that EpiASC are derived from the epidermis of the
skin.
Human amniotic membrane serves as a niche for epidermal
adult stem cells: Preserved HAM transplantation alone was
previously shown to be sufficient to reconstruct the corneal
surface in cases of partial LSCD [31,32], suggesting that
HAM may help expand the residual limbal epithelial stem
cells. Clinical and experimental studies have also shown that
ex vivo-expanded limbal epithelial cells on HAM are capable
of restoring the corneal surface in LSCD [32]. Meller et al.
[33] provided evidence that HAM cultures preferentially
preserve and expand limbal epithelial stem cells that maintain
their in vivo properties of slow cycling, label retention, and
undifferentiation. We proposed that HAM might be an ideal
matrix for ex vivo preservation and expansion of EpiASC. To
investigate this, we seeded EpiASC that were isolated from a
single cell (Figure 3A) on denuded HAM (Figure 3B). The
culture was submerged in a serum-free medium for one week
and then exposed to air by lowering the medium level over
two weeks. We found that EpiASC adhered to HAM within
20–30 min, forming EHS. EpiASC remained quiescent during
the first two days and then proliferated quickly during a further
three days culture. We also observed that EpiASC formed
clones in a similar way when co-cultured with 3T3 cells [data
not shown]. To promote EpiASC proliferation, we exposed
the surface of EHS to the air a week after culture and added
50 μg/ml Vitamin C to the medium. We found that EpiASC
proliferation was arrested and differentiation had begun. The
surface of EHS was covered with flattened cells, which
expressed CK1/10 (Figure 3C). When the EHS was digested
with 0.25% trypsin for 20–25 min, we found that a single cell
layer was firmly attached to the HAM. Staining with anti-P63
antibody revealed that P63-positive cells were surrounded by
groups of P63-negative cells (Figure 3D). This suggested that
in the serum-free culture conditions, EpiASC reconstructed
their niche on the surface of HAM. Immunostaining also
showed that the P63 expression pattern of the EHS (Figure
3E) was similar to the pattern observed within the basal
membrane layer of corneal epithelium (Figure 3F), further
evidence that HAM can serve as a niche for EpiASC and can
support EpiASC proliferation in vitro.
Figure 2. Morphological properties of
cells derived from the epidermis are
distinct from cells derived from the
dermis. A: After about 3 to 4 days in
culture, cells derived from the epidermis
expand in a mosaic way, self renewing
cells appeared on the surface of the cell
layer (red arrow). B: Shows fibroblast
cells derived from the dermis. C: After
about 8 to 10 days, stem-like cell clones
formed on the surface of the epithelial
cell layer (red arrow). D: After 8~10
days fibroblasts derived from the dermis
became confluent and stem-like cell
clones were not observed.
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