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Comparative Analysis of Human-Derived Feeder Layers with 3T3 Fibroblasts for the Ex Vivo Expansion of Human Limbal and Oral Epithelium

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Abstract

Corneal transplantation with cultivated limbal or oral epithelium is a feasible treatment option for limbal stem cell deficiency (LSCD). Currently utilized co-culture of stem cells with murine 3T3 feeder layer renders the epithelial constructs as xenografts. To overcome the potential risks involved with xenotransplantation, we investigated the use of human-derived feeder layers for the ex vivo expansion of epithelial (stem) cells. Human limbal and oral epithelium was co-cultured with mouse 3T3 fibroblasts, human dermal fibroblasts (DF), human mesenchymal stem cells (MSC), and with no feeder cells (NF). Cell morphology was monitored with phase-contrast microscopy, and stem cell characteristics were assessed by immunohistochemistry, real-time PCR for p63 and ABCG2, (stem cell markers), and by colony-forming efficiency (CFE) assay. Immunohistochemical analysis detected positive staining for CK3 (cornea specific marker) and Iβ1 and p63 (putative stem cell markers) in all culture conditions. The level of Iβ1 and p63 was significantly higher in both limbal and oral cells cultured on the 3T3 feeder, as compared to the MSC or NF group (p < 0.01). This level was comparable to the cells cultured on DF. Expression of p63 and ABCG2 in limbal and oral epithelial cells in the 3T3 and DF groups was significantly higher than that in the MSC or NF group (p < 0.01). No statistical difference was detected between 3T3 and DF groups. The CFE of both limbal and oral cells co-cultured on 3T3 fibroblasts was comparable to cells grown on DF, and was significantly higher than that of cells co-cultured with MSC or NF (p < 0.01). Epithelial cells grown on a DF feeder layer maintained a stem cell-like phenotype, comparable to cells grown on a 3T3 feeder layer. In conclusion, DF provides a promising substitute for 3T3 feeder cells during cultivation of xenobiotic-free corneal equivalents.

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Acknowledgements

We thank Dr. Shigeru Kinoshita, Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan, for his mentorship in this project and allowing us to adopt his cell cultivation protocols. This work was supported by Center of Excellence in Women’s Health Award, Harvard Medical School (UVJ), New England Corneal Transplant Research Award (UVJ), Massachusetts Lions Eye Research Fund (UVJ), Cornea Donor Research Fund (UVJ and RD).

Disclosures

The authors have no affiliations with any organization or entity having a declared financial or personal interest in the subject matter or materials discussed.

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Authors and Affiliations

  1. Schepens Eye Research Institute, 20 Staniford Street, Boston, MA, 02114, USA

    Sandhya M. Sharma, Thomas Fuchsluger, Kishore R. Katikireddy, Reza Dana & Ula V. Jurkunas

  2. Massachusetts Eye and Ear Infirmary, 243 Charles Street, Boston, USA

    Reza Dana & Ula V. Jurkunas

  3. Department of Ophthalmology, Harvard Medical School, Boston, MA, 02114, USA

    Sandhya M. Sharma, Thomas Fuchsluger, Kishore R. Katikireddy, Reza Dana & Ula V. Jurkunas

  4. Institute of Human Genetics, Newcastle University, Central Parkway, Newcastle upon Tyne, NE1 3BZ, UK

    Sajjad Ahmad

  5. Center for Human Cell Therapy, Immune Disease Institute, 3 Blackfan Circle, Boston, MA, 02115, USA

    Myriam Armant

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  1. Sandhya M. Sharma
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  2. Thomas Fuchsluger
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Correspondence to Ula V. Jurkunas.

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Sharma, S.M., Fuchsluger, T., Ahmad, S. et al. Comparative Analysis of Human-Derived Feeder Layers with 3T3 Fibroblasts for the Ex Vivo Expansion of Human Limbal and Oral Epithelium. Stem Cell Rev and Rep 8, 696–705 (2012). https://doi.org/10.1007/s12015-011-9319-6

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