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Title: Characterization of limbal explant sites: Optimization of stem cell outgrowth in in vitro culture
Authors: Pattama Ekpo
Naharuthai Inthasin
Sutthicha Matamnan
Patimaporn Wongprompitak
Methichit Wattanapanitch
Chawikan Boonwong
Chareenun Chirapapaisan
Panotsom Ngowyutagon
Mongkol Uiprasertkul
Pinnita Prabhasawat
Wiwit Tantibhedhyangkul
Faculty of Medicine, Siriraj Hospital, Mahidol University
Keywords: Agricultural and Biological Sciences;Biochemistry, Genetics and Molecular Biology;Multidisciplinary
Issue Date: 1-May-2020
Citation: PLoS ONE. Vol.15, No.5 (2020)
Abstract: © 2020 Ekpo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Simple limbal epithelial transplantation (SLET) and cultivated limbal epithelial transplantation (CLET) are proven techniques for treating limbal stem cell deficiency (LSCD). However, the precise regions that are most suitable for preparing explants for transplantation have not been identified conclusively. Accordingly, this in vitro study aimed at determining ideal sites to be selected for tissue harvest for limbal stem cell culture and transplantation. We evaluated cell outgrowth potential and the expression of stem cell markers in cultures from 48 limbal explants from five cadaveric donors. The limbal explants were generated from the three specific sites: Lcor (located innermost and adjacent to the cornea), Lm (middle limbus), and Lconj (located outermost adjacent to the conjunctiva). We found that explants from the Lconj and Lm sites exhibited higher growth potential than those from the Lcor site. Transcript encoding the stem cell marker and p63 isoform, ΔNp63, was detected in cells from Lm and Lconj explants; expression levels were slightly, though significantly (p-value < 0.05), higher in Lm than in Lconj, although expression of ΔNp63α protein was similar in cells from all explants. Differential expression of ATP-Binding Cassette Subfamily G Member 2 (ABCG2) did not reach statistical significance. Immunohistochemistry by indirect immunofluorescence analysis of limbus tissue revealed that the basal layer in explant tissue from Lconj and Lm contained markedly more stem cells than found in Lcor explant tissue; these findings correlate with a higher capacity for growth. Collectively, our findings suggest that explants from the Lconj and Lm sites should be selected for limbal cell expansion for both CLET and SLET procedures. These new insights may guide surgeons toward specific limbal sites that are most suitable for stem cell culture and transplantation and may ultimately improve treatment outcomes in the patients with LSCD.
ISSN: 19326203
Appears in Collections:Scopus 2020

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