Ex Vivo Expansion of Oral Mucosal Epithelial Stem Cells on Freeze Dried Amniotic Membrane for...
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- Slide 1
- 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 The authors have no financial interest to
disclose Cairo University, Egypt
- Slide 2
- 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). Kobtan WCC VII
2015
- Slide 3
- 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) Kobtan
WCC VII 2015
- Slide 4
- Aim of the work The aim of the present study is to attempt to
expand OMECs from oral biopsies on freeze-dried amniotic membrane
(FD- AM). Kobtan WCC VII 2015
- Slide 5
- 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. Kobtan WCC VII 2015
- Slide 6
- 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 37C and were
maintained for 24 days. Outgrowth of ECs was assessed using light
microscopy and IHC. 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.
AB
- Slide 7
- After 24 days in submerged culture, the cells grew into a
healthy, confluent sheet (arrow head). Culture insert is indicated
by the arrow. A. Phase contrast microscopy Kobtan WCC VII 2015
Results
- Slide 8
- Light microscopic examination of TS in the oral epithelial
cultures on FD-dAM under submerged conditions on day 24 showed 25
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. B.
Haematoxylin and Eosin Kobtan WCC VII 2015
- Slide 9
- 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). C. Immunohistochemistry for K3 Kobtan WCC VII
2015
- Slide 10
- Positive control: normal limbal epithelium shows positive
immunostaining for K12 in all cell layers except the basal layer
(Magnification x400) D. Immunohistochemistry for K12 Cultured oral
epithelium with negative immuno-staining for K12 in all cell layers
(Magnification x400) Kobtan WCC VII 2015
- Slide 11
- 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 immuno- staining (Magnification x400). Kobtan WCC VII
2015
- Slide 12
- 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. Kobtan WCC VII 2015