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Sanja Bojic, Dean Hallam, Pervinder Sagoo, Nuno Alcada, Harley Buck, Gustavo Figueiredo, Aya Amitai Lange, Biljana Ljujic, Ruby Shalom-Feuerstein, Alex J Shortt, Francisco Figueiredo, Majlinda Lako; Towards identification of novel limbal stem cell surface markers. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3862.
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© ARVO (1962-2015); The Authors (2016-present)
One of the main challenges in limbal stem cell (LSC) biology and transplantation is the lack of definitive cell surface markers which can be used to enrich LSCs and study them at the single cell level. This study was designed to identify and validate novel specific LSC markers.
Primary human limbal epithelial cells were generated from limbal suspension cultures. LSCs were clonally enriched on 3T3 feeder cells. Limbal epithelial cells at primary passage were stained with 242 PE labelled antibodies using the BD Lyoplate Human Cell Surface Marker Screening Panel. The expression of selected markers was monitored through subsequent passages and also during calcium induced differentiation and detected by flow cytometry. Colony forming efficiency (CFE) assay, BrdU labelling, Ki67 and pyronine Y staining were used to compare clonogenic and proliferative potential of sorted positive and negative populations in addition to qPCR and RNA-seq analysis. Freshly isolated human and mouse corneas were cryosectioned for the evaluation of potential marker expression ex vivo. The function of CD200 in vivo is currently being investigated in a mouse model of a total limbal stem cell deficiency.
One marker with high expression in LSC cultures (CD109: 36.03±4.92%, n=4) and one with low expression (CD200: 4.13±1.1%, n=10) were selected for further analysis. CD200 expression rapidly decreased through passages and calcium induced differentiation while CD109 decreased slowly as confirmed by flow cytometry. Both markers were exclusively located at the limbus and co-localised with ΔNp63 in both human and mouse cornea; CD200 localised to a small population of cells at the bottom of the limbal epithelial crypts, while CD109 was more abundantly distributed. Although there were no significant differences in CFE between positive and negative group for both markers, the CD200 positive population was exclusively able to form holoclones. Moreover, CD200 positive cells were slow cycling and contained all side population cells. Subpopulation of CD200 positive cells was proven to be in G0 phase of the cell cycle which corresponds to potential existence of quiescent stem cells.
Our data suggest that CD109 is a marker of transient amplifying cells while CD200 represent a putative LSC marker that will enable isolation of viable stem cells from limbal epithelial cell cultures.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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