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  • Introduction The stem cell niche is a

    2018-10-29

    Introduction The stem cell niche is a unique microenvironment that maintains long-term repopulation of a specific tissue by undergoing self-renewal of stem ha tag peptide and by producing progenies of increasingly differentiated cells. A large amount of knowledge concerning the regulation of stem cell self-renewal and differentiation by niche cells is described in a subpopulation of adult osteogenic and adult hematopoietic stem cells (HSCs) (Bianco, 2011; Calvi et al., 2003; Zhang et al., 2003). In particular, recent studies showed that specialized spindle-shaped N-cadherin (N-cad) expressing osteoblasts (SNO) are a key component of the bone marrow stem cell niche, where HSCs directly interact with SNO via N-cad interaction (Nilsson et al., 2001; Whetton and Graham, 1999; Zhang et al., 2003; Zhu and Emerson, 2004). The corneal epithelial stem cells are located in the cornea limbus, a ring of tissue surrounding the peripheral clear cornea (Cotsarelis et al., 1989; Schermer et al., 1986), and continue to supply corneal epithelial cells to the central cornea. Hayashi, et al. recently reported that limbal basal epithelial cells in the peripheral cornea express N-cad as a possible marker of putative epithelial stem cells, and that melanocytes may be associated with these cells through homotypic adhesion by N-cad in the human limbal epithelial stem cell niche (Hayashi et al., 2007). We recently found that 3T3 feeder cells lacking N-cad expression significantly lost the ability to support clonal growth and maintain basal Keratin 15 positive (K15+) limbal phenotype in epithelial sheets using colony forming assays and the duplex feeder system, an in vitro model of the limbal epithelium (Higa et al., 2009; Miyashita et al., 2008). Recently, Chen et al. demonstrated that isolation of human limbal progenitor cells using collagenase digestion dramatically maintained close association with their niche cells compared to dispase digestion (S.Y. Chen et al., 2011). These findings strongly suggest that a unique niche cell exists in the corneal limbus that regulates limbal epithelial progenitors/stem cells. Although there are several reports concerning the corneal limbal progenitor/stem cells, studies describing the limbal niche are few. In addition, whether corneal epithelial stem cells directly interact with subepithelial niche cells via N-cad interaction remains to be shown. In this paper, by using collagenase digestion we demonstrate that aquaporin1 (AQP1) positive niche-like cells exist immediately beneath the limbal epithelial basement membrane, and directly interact with N-cad positive limbal basal epithelial cells in a calcium-dependent manner.
    Material and methods
    Results
    Discussion In this study, we showed that large dendritic AQP1+ stromal cells exist beneath N-cad+ limbal basal progenitor/stem clusters by using collagenase digestion. Chen et al. recently reported that collagenase enables the isolation of the corneal limbal progenitors/stem cell microenvironment, including the basement membrane, compared with dispase isolation (S.Y. Chen et al., 2011). In addition, they also isolated cytokeratin and p63 negative, vimentin positive 5–10μm small diameter cells by collagenase isolation (S.Y. Chen et al., 2011). However, the vimentin+, AQP1+ dendritic cells that we isolated were approximately 30–50μm in diameter, which is several fold larger than the cells that they have reported (Fig. 4C, E). They also reported that collagenase isolation maintained basement membrane components such as laminin 5 (S.Y. Chen et al., 2011). Preservation of basement membrane components during isolation is important for rapid re-synthesis and deposition of it components in association with limbal epithelial progenitor cells and corneal endothelial cells (S.Y. Chen et al., 2011; Li et al., 2007). Although we also isolated limbal epithelial cells with stromal cells using different collagenase conditions, basement membrane components were still maintained after both collagenase and accutase digestions (Figs. 5 and 7). We used accutase for adhesion assays since it is milder than trypsin for preserving cadherin, and because we failed to isolate the large AQP1+ cells using trypsin (data not shown). Due to the slight difference in isolation protocol, there is the possibility that the cells we isolated were a completely different cell type that support limbal basal progenitor cells.