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  • Our investigation is not the first to

    2018-10-22

    Our investigation is not the first to document integrin expression in the cornea. Others have shown differential expression of these adhesion molecules across the corneal epithelium. The reason for the altered expression pattern is not well understood but may be dependent on developmental and patho-physiological cues (Stepp et al., 1993; Stepp, 2006; Bystrom et al., 2009; Carter, 2009) and likely influenced by locally deposited ligands within the BM or stroma. Our results are congruent with previous studies that identified αvβ5 in the basal limbal epithelium with no αvβ3 in the corneal and limbal epithelium (Figs. 2G–I) (Lauweryns et al., 1993; Rayner et al., 1998). However, unlike the current study, those investigations were not supported by co-localization, functional, and molecular analyses. Interestingly, other integrins including α6, have been used to enrich for a minor population of small clonogenic cells that expressed LESC gpr55 agonist (Hayashi et al., 2008) and α9-integrin illuminated presumed LESC; although this marker was regarded less specific, due to its inability to distinguish between quiescent LESC and early transient amplifying cells (Pajoohesh-Ganji et al., 2004; Chen et al., 2004). If integrins such as αvβ5 are critical for supporting SC, then genetic ablation should have significant patho-physiological consequences. Indeed, keratinocytes from β5-integrin deficient mice have impaired adhesion and migration to VN, despite their normal development (Huang et al., 2000). In contrast, αv-depletion is embryonically lethal in 80% of mice, and live-born have major developmental and organ-dysfunction, dying soon after birth (Bader et al., 1998). To our knowledge the impact of deleting αv and/or β5 in the cornea has not been reported, although mice lacking αvβ5 develop blindness due to retinal dysfunction (Nandrot et al., 2004), and those that are devoid of epithelial-specific αv-integrin develop tumors of the conjunctiva and eyelid skin (McCarty et al., 2008). These findings suggest that integrin–ECM coupling is a mechanism that regulates differentiation, proliferation and transformation. A limitation that has hampered progress in the study of LESC is the lack of specific markers (Lavker and Sun, 2000). It is well documented that LESC are aligned along the basal limbal epithelium (Chen et al., 2004), and N-cadherin (Higa et al., 2012; Higa et al., 2009) and CK-15 (Yoshida et al., 2006; Higa et al., 2012; Davies et al., 2009; Figueira et al., 2007) are amongst the more reliable markers for these cells. Our report now identifies αvβ5 integrin as an additional LESC antigen. Although ours is the first comprehensive report on the selective expression of αvβ5 in limbal progenitor cells, in other organ systems, αv and αvβ5 were used to identify murine osteoclast precursors (Inoue et al., 1998) and contributed to endocrine progenitor cell migration in the developing pancreas (Cirulli et al., 2000). Furthermore, these integrins have been shown to partake in oligodendrocyte precursor cell migration, a process necessary for repairing the central nervous system during remyelination (Zhao et al., 2009); αvβ5 promotes muscle precursor cell adhesion (Sinanan et al., 2008) and is implicated in the development of endoderm from embryonic SC (Wong et al., 2010). In our investigation, human limbal epithelial cells were propagated from either explanted tissue or enzymatic-dissociation of corneal–limbal rims (Koizumi et al., 2002). These two methods have advantages and disadvantages. Explant-derived cells are more likely to retain a specific phenotype since the tissue location is selected. Moreover, these cells have a short-term supply of niche-specific signals during their expansion, but the time required to reach confluence in primary culture is longer and contamination from stromal fibrocytes can be an issue. In contrast, cells cultivated after dispase-dissociation expand more readily as the initial yield is higher, they also exhibit greater structural integrity and are thought to be superior morphologically to cells expanded from tissue segments (Koizumi et al., 2002); however our data would suggest otherwise (Figs. 3C and D). Alternative enzyme systems such as trypsin have been used to detach limbal epithelial cells from their native repository; however cells isolated by this method rapidly lose their SC phenotype (Vascotto and Griffith, 2006). When collagenase is used, limbal epithelial progenitors, as well as their closely associated mesenchymal niche cells are detached, and when co-cultured, LESC function and phenotype are effectively maintained (Chen et al., 2011), further evidence that stromal cells and niche factors are required for effective ex vivo expansion. The premise for using dispase in our study was to pan for a pure population of αvβ5+/− limbal epithelial cells free of contaminating corneal mesenchymal cells (Chen et al., 2011) as well as vascular endothelial cells (Caiado and Dias, 2012) and T-lymphocytes (Luzina et al., 2009) which are known to express this integrin. However, we acknowledge that dispase is not as effective at releasing all basal limbal epithelial progenitors as is collagenase (Chen et al., 2011), therefore we may not have collected the entire population of LESC using our method.