No financial conflict of interests

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Evaluation of Human-Derived Feeder Layers for Ex
Vivo Cultivation of Corneal and Oral Epithelium
for Ocular Surface Reconstruction
Sandhya M. Sharma, Thomas Fuchsluger, Reza Dana,
Ula Jurkunas Schepens Eye Research Institute,
Department of Ophthalmology, Harvard Medical
School, Boston MA. sandhya.sharma_at_schepens.harvar
d.edu

• No financial conflict of interests

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INTRODUCTION

• 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.

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METHODS

• 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 (Iß1)
• These epithelial cells were then examined for
  their colony forming efficiency (CFE)
• 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.

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RESULTS
Cytokeratin-3 Expression in Corneal Limbal Cells
IHC PI / CK-3
No feeder
3T3 Fibroblast
Dermal Fibroblast
Bone Marrow Fibroblast
Quantification of pixels
pixel
Isotype control
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RESULTS
Integrin-ß1 Expression in Corneal Limbal cells
IHC PI / Integrin-ß1
Quantification of pixels
Isotype control
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RESULTS
Cytokeratin-3 Expression in Oral cells
IHC PI / CK-3
No feeder
3T3 Fibroblast
Dermal Fibroblast
Bone Marrow Fibroblast
Quantification of pixels
Isotype control
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RESULTS
Integrin-ß1 Expression in Oral cells
IHC PI / Integrin-ß1
No feeder
3T3 Fibroblast
Dermal Fibroblast
Bone Marrow Fibroblast
Quantification of pixels
pixel
Isotype control
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RESULTS
Comparison of Colony Forming Efficiency in
cultivated cells
Oral cells
Limbal cells
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RESULTS
Comparison of mRNA level of p63 and ABCG2 in
limbal cells
p63
ABCG2
10
RESULTS
Comparison of mRNA level of p63 and ABCG2 in oral
cells
p63
ABCG2
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CONCLUSION

• 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.

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FUNDING
Massachusetts Lions Eye Research Fund (UJ)  
  New England Cornea Transplantation Fund
(UJ)Cornea Transplantation Research Fund (UJ and
RD)