Evaluation of Human-Derived Feeder Layers for Ex Vivo Cultivation of Corneal and Oral Epithelium for 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.

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.

 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 (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. METHODS

No feeder3T3 Fibroblast Dermal FibroblastBone Marrow Fibroblast IHC: PI / CK-3 Isotype control 200 µm RESULTS Cytokeratin-3 Expression in Corneal Limbal Cells No Feeder 3T3 Dermal Bone Marrow pixel Quantification of pixels

No feeder 3T3 FibroblastDermal Fibroblast Bone Marrow Fibroblast Isotype control IHC: PI / Integrin-β1 200 µm RESULTS Integrin-β1 Expression in Corneal Limbal cells No Feeder 3T3 Dermal Bone Marrow Quantification of pixels pixel

No feeder3T3 FibroblastDermal Fibroblast Bone Marrow Fibroblast IHC: PI / CK µm Isotype control 200 µm RESULTS Cytokeratin-3 Expression in Oral cells No Feeder 3T3 Dermal Bone Marrow Quantification of pixels pixel

No feeder3T3 FibroblastDermal Fibroblast Bone Marrow Fibroblast IHC: PI / Integrin-β1 200 µm Isotype control 200 µm RESULTS Integrin-β1 Expression in Oral cells No Feeder 3T3 Dermal Bone Marrow Quantification of pixels pixel

Comparison of Colony Forming Efficiency in cultivated cells RESULTS Limbal cells Oral cells

RESULTS Comparison of mRNA level of p63 and ABCG2 in limbal cells p63ABCG2

Comparison of mRNA level of p63 and ABCG2 in oral cells RESULTS p63ABCG2

 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) FUNDING