Download Ex Vivo Expansion of Oral Mucosal Epithelial Stem Cells on Freeze

Ex Vivo Expansion of Oral Mucosal Epithelial Stem
Cells on Freeze Dried Amniotic Membrane for
Potential Use in Ocular Surface Reconstruction
Dina Kobtan MD , Hatem Kobtan MD FRCS
Mervat Elansary MD, Nancy Elguindy MD
Cairo University, Egypt
The authors have no financial interest to disclose
Introduction
 Stem cells (SCs) for the corneal epithelium reside in the
basal layer of limbus (Nishida, 20003).
 Limbal SCs (LSCs) are supported by a unique stromal
microenvironment called the stem cell niche. Destructive
loss of LSCs and/or dysfunction of their stromal
environment render many corneas with a clinical entity
called limbal stem cell deficiency (LSCD) (Grueterich et al,
2003).
 Ex vivo cultivated corneal epithelial transplantation (CCET)
has gained general acceptance as an effective treatment
modality for LSCD (Koizumi et al, 2001; Shimazaki et al,
2002) .
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 The ideal therapy for unilateral LSCD is the use of autologous
CCET (using a minimal biopsy obtained from the contralateral
healthy eye) (Schwab et al, 2000; Tsai et al, 2000).
 In bilateral LSCD, allogenic limbus from living related or
cadaveric donors needs to be transplanted (directly or after ex
vivo expansion) onto the affected eye (Frucht-Pery et al,
1998).
This
necessitates
long-term
systemic
immunosuppression to prevent graft rejection (Cauchi et al,
2008).
 Cultured oral mucosal epithelial cells (OMECs) have been
successfully utilized in the treatment of LSCD in a technique
called cultivated oral mucosal epithelial transplantation
(COMET) (Chu, 2000; Nakamura et al, 2004)
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Aim of the work
The aim of the present study is to attempt to expand OMECs
from oral biopsies on freeze-dried amniotic membrane (FDAM).
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Materials and Methods
 In the current study, oral biopsies were obtained from 3
potential COMET candidates after obtaining an informed
consent. The oral biopsies were cultured as explants on FD
denuded AM (dAM) without using feeder cells or airlifting for
24 days.
 Epithelial outgrowth was assessed using phase contrast
microscopy, haematoxylin and eosin (HE) staining, and
immunohistochemistry (IHC) for keratin 3 (K3), keratin 12 (K 12)
and p63.
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A
B
Oral mucosal biopsy harvest. local
anesthetic is injected into the donor site,
an incision at the apex of the outline is
made with a steel blade, a forceps is used
to handle the tissue on the submucosa
side of the graft. A: The resultant elliptical
shaped raw area, B: Harvested oral
mucosal tissue.
Explant culture. After careful dissection of
each oral biopsy under a microscope, an
explant (arrow head) was placed directly
on the center of each AM fastened onto
the culture insert. Cultures were
submerged in culture media, incubated at
37°C and were maintained for 24 days.
Outgrowth of ECs was assessed using light
microscopy and IHC.
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Results
A. Phase contrast microscopy
After 24 days in submerged culture,
the cells grew into a healthy, confluent
sheet (arrow head). Culture insert is
indicated by the arrow.
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B. Haematoxylin and Eosin
Light microscopic examination of TS in the oral epithelial cultures on FD-dAM under
submerged conditions on day 24 showed 2–5 layers of stratified epithelium (arrow
head). The oral explant remained attached to the AM throughout the culture duration.
The insert shows the oral explant after 24 days in culture.
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C. Immunohistochemistry for K3
Positive control: Normal limbal
epithelium
shows
positive
immunostaining for K3 in all cell
layers except the basal layer
(Magnification x400) .
Cultured oral epithelium with
positive
cytoplasmic
K3
immunostaining mainly in the
superficial cell layer (Magnification x400) .
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D. Immunohistochemistry for K12
Positive
control:
normal
limbal
epithelium
shows
positive
immunostaining for K12 in all cell layers
except the basal layer (Magnification x400)
Cultured oral epithelium with negative
immuno-staining for K12 in all cell layers
(Magnification x400)
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D. Immunohistochemistry for p63
Normal limbal epithelium shows
positive immunostaining for p63 in
the basal and suprabasal cell layers
(Magnification x400).
Cultured oral epithelium with
positive p63 nuclear immunostaining (Magnification x400) .
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Conclusion
 We have cultured oral mucosal epithelial cells (ECs) from biopsy-derived oral
mucosal tissues on sterilized FD-AM. The use of the explant culture
technique circumvented the dependence on feeder cells, which eliminates
the risk of xenogeneic contamination.
 We were able to observe nuclear expression of p63 (used here to identify
the presence of ECs which had not terminally differentiated) within the cells
of the stratified layers. K3, the marker of corneal and oral ECs was found to
be expressed by cultured cells. This suggests that cell sheets derived from
OMECs showed similar characteristics to normal corneal epithelium. Our
cultured cells were negative for the corneal epithelium-specific K12 and
hence do not acquire the corneal phenotype after culture.
 We should investigate how long these cultivated oral ECs can maintain the
ocular surface.
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