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Yi-Jen Hsueh, Hung-Chi Chen, David Hui-Kang Ma; Epithelial progenitors are preserved in collagenase-digested oral mucosa. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5314. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Cultivated oral mucosal epithelial transplantation (COMET) has been demonstrated to be beneficial in wound healing for patients with severe ocular surface damages. However, animal source materials, such as fetal bovine serum (FBS) and mouse 3T3 feeder cell layers, are essential for fabrication of epithelial sheets, but with the risks of xenogeneic infection. In this study, we have developed a novel animal derived component free (ADCF) culture procedure (modified protocol).
Human oral mucosal tissue removed during oral surgeries were digested either by 1% collagenase A or 1.2 U dispase II/0.25% trypsin EDTA. The yielded cell pellets were seeded on denuded amniotic membrane (DAM) with or without co-culture with 3T3 cells. BrdU lebal retention, colony formation assay (CFA), immunostaining and Western blot for stem/progenitor cell markers (p63 and p75) and differentiation markers (keratins 3 and 8) were performed. Finally, involvement of Wnt and ILK signaling were studied.
Immunostaining patterns of differentiation markers (K3+ and K8-) in the oral mucosal epithelial cells (OMEC) cultivated by the modified protocol were similar to those by the original protocol. Colony formation efficiency and immunostaining results of p75NTR featured better progenitor cell characteristics in the OMEC cultivated by the modified protocol. Results of BrdU labeling showed higher proliferative status in the epithelial sheets from the modified protocol. ILK signal was also demonstrated to be more active in the epithelial sheets from the modified protocol, with β-catenin signal and its downstream proliferation modulating factors (cyclin and p27KIP1) altered significantly. Likewise, silence of ILK lead to inhibition of nuclear β-catenin, suppressed expression of p63, and reduced alteration of proliferation modulating factors, indicating that ILK/β-catenin pathway was involved.
The improved protocol of non-animal origins is supposed to replace the conventional method, with success application in ocular surface reconstruction, and further application in reconstruction of epidermal or epithelial defects elsewhere in the human body.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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