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S. Kolli, S. Ahmad, M. Lako, F. C. Figueiredo; Ex vivo Expansion of Oral Mucosa Epithelial Stem Cells for the Treatment of Total Bilateral Limbal Stem Cell Deficiency. Invest. Ophthalmol. Vis. Sci. 2008;49(13):6058. doi: https://doi.org/.
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The treatment of total limbal stem cell deficiency (LSCD) has been revolutionised using limbal tissue grafts generated by the ex vivo expansion of small autologous limbal biopsies. This approach prevents the problems associated with whole tissue grafts including potential iatrogenic donor LSCD (if autologous) and the need for potent immunosuppression (if allogeneic). However, in a proportion of patients, total bilateral LSCD exists where there are no remaining LSCs to expand. Recently, the possibility of using ex vivo expanded autologous oral mucosal epithelium to treat LSCD has been suggested as a viable alternative in these patients.
Oral mucosa biopsies were obtained from healthy volunteers (n=5). The epithelium was separated from the stroma and then treated with trypsin to generate a suspension of cells or used whole as an explant to try and generate epithelial colonies and sheets. The cultures were carried out on either 3T3 fibroblast feeder layers or human amniotic membrane (HAM). The oral epithelial colonies were then examined for colony forming efficiency (CFE) and the expression of LSC associated markers including ΔNp63, ABCG2 and cytokeratin 3 by real time reverse transcription PCR and compared with the results from limbal epithelial colonies cultured under identical conditions. The ex vivo expanded oral epithelial sheets were then examined histologically, with electron microscopy and immunohistochemistry for a variety of epithelial cytokeratins and the results compared with similar examinations of normal oral mucosal epithelium and corneal epithelium.
Epithelial colonies can be generated from oral mucosa using either explant or suspension culture techniques on either 3T3 fibroblasts or HAM. The CFE and the expression of putative SC markers indicated the presence of significant epithelial SCs in oral mucosa. Generated oral epithelial sheets on HAM look very different from native oral mucosa but show remarkable phenotypic similarity to corneal epithelium both in terms of morphology and cytokeratin expression.
Ex vivo expansion of oral mucosal epithelium produces epithelial sheets which are rich in SC properties and appear phenotypically "cornea-like" in many respects. These laboratory finding provide support for the recent use of oral mucosa derived epithelial sheets in the clinical management of severe bilateral LSCD.
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