aluation of Human-Derived Feeder Layers for Ex Vivo aluation of Human-Derived Feeder Layers for Ex Vivo Cultivation of Corneal and Oral Epithelium for Cultivation of Corneal and Oral Epithelium for Ocular Surface Reconstruction Ocular Surface Reconstruction No financial conflict of interests Sandhya M. Sharma, Thomas Fuchsluger, Reza Dana, Ula Jurkunas Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston MA. [email protected]
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Evaluation of Human-Derived Feeder Layers for Ex Vivo Cultivation of Corneal and Oral Epithelium for Ocular Surface Reconstruction No financial conflict.
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Evaluation of Human-Derived Feeder Layers for Ex Vivo Evaluation of Human-Derived Feeder Layers for Ex Vivo Cultivation of Corneal and Oral Epithelium for Cultivation of Corneal and Oral Epithelium for
Ocular surface damage caused by infections, chemical burns, trauma, and various autoimmune diseases could lead to corneal limbal stem cell deficiency (LSCD).
Depletion of limbal stem cell causes corneal neovascularization, scarring, chronic inflammation, and loss of vision.
Current treatments for this condition involve transplanting cornea from cadaver donor or transplanting limbal stem cells from the contralateral eye. These transplants subject the patients to lifelong immunosuppression (if allogeneic) and could risk causing LSCD in the donor eye (if autologous).
In this study we investigated the use of ex vivo cultivated stem cells from autologous tissue such as the oral mucosa and the limbus as a potential source of epithelium for corneal transplantation. We evaluated the use of different types of feeder layers of human origin to support stem cell growth, which would eliminate the risks associated with using xenogeneic mouse 3T3 feeder cells that are currently used for propagation stem cells.
Oral and limbal epithelial cells were isolated by microdissection followed by digestion with dispase and trypsin/EDTA
The cells were seeded on amniotic membrane and co-cultured for three weeks with;
- human dermal fibroblast - human bone marrow fibroblast - mouse 3T3 fibroblast feeder layers, and - no feeder layer
The cultivated cells were examined by immunohistochemistry for differentiated (CK3) and putative stem cell markers (Iβ1)
These epithelial cells were then examined for their colony forming efficiency (C
FE)
Relative transcripts levels of putative stem cell markers; ABCG2 and p63 were accessed by quantitative RT-PCR.
All the results were compared to similar examination of cells co-cultured with mouse 3T3 fibroblasts.
METHODS
No feeder 3T3 Fibroblast Dermal Fibroblast Bone Marrow Fibroblast
IHC: PI / CK-3
Isotype control
200 µm 200 µm 200 µm 200 µm
200 µm
RESULTS
Cytokeratin-3 Expression in Corneal Limbal Cells
No Feeder
3T3
Dermal
Bone Marrow
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1
pixe
l
Quantification of pixels
No feeder 3T3 Fibroblast Dermal Fibroblast Bone Marrow Fibroblast
Isotype control
IHC: PI / Integrin-β1
200 µm
200 µm 200 µm 200 µm 200 µm
RESULTS
Integrin-β1 Expression in Corneal Limbal cells
No Feeder
3T3
Dermal
Bone Marrow
Quantification of pixels
00.20.40.60.8
11.21.41.61.8
2
1
pixe
l
No feeder 3T3 Fibroblast Dermal Fibroblast Bone Marrow Fibroblast
IHC: PI / CK-3
200 µm 200 µm 200 µm
Isotype control
200 µm
200 µm
RESULTS
Cytokeratin-3 Expression in Oral cells
00.5
11.5
22.5
33.5
44.5
5
1
No Feeder
3T3
Dermal
Bone Marrow
Quantification of pixels
pixe
l
No feeder 3T3 Fibroblast Dermal Fibroblast Bone Marrow Fibroblast
IHC: PI / Integrin-β1
200 µm 200 µm 200 µm 200 µm
Isotype control
200 µm
RESULTS
Integrin-β1 Expression in Oral cells
No Feeder
3T3
Dermal
Bone Marrow
Quantification of pixels
00.40.81.21.6
22.42.83.2
1
pixe
l
% C
olon
y Fo
rmin
g E
fficie
ncy
0
2
4
6
8
10
12
14
16*
*n.s.
0
2
4
6
8
10
12
14
16*
*n.s.n.s.
No Feeder
3T3
Dermal
Bone Marrow
No Feeder
3T33T3
Dermal
Bone Marrow
Dermal
Bone Marrow
Comparison of Colony Forming Efficiency in cultivated cells
RESULTS
% C
olon
y Fo
rmin
g E
fficie
ncy
0
2
4
6
8
10
12
14
16 **
n.s.n.s.
No Feeder
3T33T3
Dermal
Bone Marrow
Dermal
Bone Marrow
Limbal cells Oral cells
RESULTS
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Rela
tive
mRN
A Ex
pres
sion
*n.s
* *n.s
** n.s
** n.s* n.s*
No Feeder
3T3
Dermal
Bone Marrow
No Feeder
3T33T3
Dermal
Bone Marrow
Dermal
Bone Marrow
Comparison of mRNA level of p63 and ABCG2 in limbal cells
p63 ABCG2
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Rela
tive
mRN
A Ex
pres
sion
* n.s
n.s
* n.s
n.s
* n.s** n.s* n.s*
No Feeder
3T3
Dermal
Bone Marrow
No Feeder
3T33T3
Dermal
Bone Marrow
Dermal
Bone Marrow
Comparison of mRNA level of p63 and ABCG2 in oral cells
RESULTS
p63 ABCG2
Stratified epithelial sheets of oral and limbal epithelium expressing putative stem cell and corneal epithelial markers were successfully cultured on amniotic membrane with human-derived feeder layers.
Dermal fibroblast feeder seemed to preserve stem cell like phenotype of epithelial cells, which was comparable to cells grown on 3T3 feeder layer.
Human dermal fibroblast feeder layer may be used as a substitute for the cultivation of transplantable epithelial, as minimizing the use of animal-derived products may make these cell sheets suitable for human transplantation.
CONCLUSION
Massachusetts Lions Eye Research Fund (UJ) New England Cornea Transplantation Fund (UJ)Cornea Transplantation Research Fund (UJ and RD)