Background

Homeostasis of the corneal epithelium is regulated by limbal epithelial stem/progenitor cells (LEPC) located at a specific anatomic location referred to as the limbal stem cell niche (Gonzalez et al., 2018). It is characterized by limbal vasculature, a specific extracellular matrix composition (ECM), and surrounding non-epithelial limbal niche cells (LNCs) (Shortt et al., 2007; Ordonez et al., 2012; Polisetti et al., 2016) (Figure 1). Limbal mesenchymal stromal cells (LMSC), a cellular component of the limbal stem cell niche, have been shown to support corneal epithelial regeneration during wound healing, and the maintenance of the LEPC phenotype, both in vitro and in vivo (Dziasko et al., 2014; Nakatsu et al., 2014; Li et al., 2018; Polisetti et al., 2016). In addition, LMSC were shown to have potent immunomodulatory, anti-inflammatory, and anti-angiogenic properties, making them potentially attractive tools for clinical use (Funderburgh et al., 2016; Veréb et al., 2016; Al-Jaibaji et al., 2019; Polisetti et al., 2021). Thus, the co-cultivation of LEPC with LMSC might represent an improved strategy to generate cell transplants for patients suffering from limbal stem cell deficiency (Rama et al., 2017; Ghareeb et al., 2020). Previously, LMSC have been isolated either by enzymatic digestion of limbal tissue (dispase, collagenase, or in combination) or by explant culture of limbal tissue followed by enrichment using cell type-specific media (Chen et al., 2011; Xie et al., 2012; Li et al.,2012; Li et al., 2014; Chen et al., 2015; González et al., 2013; Polisetty et al., 2008; Xiao et al., 2020). The main disadvantage of these methods is the contamination by other cell types (Polisetty et al., 2008; Li et al., 2012). Collagenase digestion of limbal tissue results in cell clusters consisting of 80% epithelial cells and 20% LMSC, whereas a combination of dispase and collagenase yields clusters composed of approximately 95% LMSC (Li et al., 2012). Thus, current protocols for LMSC purification require culturing of at least one passage to eliminate contaminating cells. Thus, research on freshly isolated LMSC has been hampered by the lack of a good protocol for isolating this cell type.

Here, we present an optimized protocol for the isolation of LMSC from organ-cultured corneal samples by means of fluorescence-activated cell sorting (FACS), using CD90 as a selective marker (Polisetti et al., 2022).

Figure 1. Localization of limbal niche cells in situ. A. Triple immunostaining analysis of limbal tissue sections showing melanocytes [melan-A⁺ (red) vimentin⁺ (cyan) cells, arrow heads] in close contact with clusters of cytokeratin (CK)15⁺, CK14⁺, CK19⁺ (green) limbal epithelial progenitor cells (LEPC), whereas sub-epithelial stromal cells [vimentin⁺ cells (cyan), arrows] were in close association with basal limbal epithelial cells, and not with more superficial CK3⁺ cells (green). Dashed line represents the basement membrane (BM), and nuclear counterstaining was done with 4′,6-diamidino-2-phenylindole (DAPI, blue). B. Double immunostaining of limbal sections showing the co-localization of CD90 (green) and vimentin (cyan) in the sub-epithelial stromal cells (white arrows), which were in close association with basal layers of limbal epithelium (dotted line represents the BM), as well as blood vessels of the limbal stroma (yellow arrows). The limbal sections also show the co-localization of CD117 (green) and melan A (red) in the melanocytes (arrow heads) at the basal layer of the limbal epithelium. Nuclear counterstaining with DAPI (blue). C. Immunofluorescence analysis of cultured limbal clusters showing the expression of keratins (PCK, green) and vimentin (cyan) in epithelial cells, melan-A (red) and vimentin (cyan) expression in melanocytes (arrow heads), and only vimentin expression in stromal cells (arrows). Double immunostaining of cultured limbal clusters showing the CD90⁺ stromal cells (green, arrow) at the edge of clusters and also in between E-cadherin⁺ epithelial cells (red, dashed line represents the edge of the cluster), whereas melan-A⁺ melanocytes were located between the cells (red, arrow heads). Nuclear counterstaining with DAPI (blue). Reprinted from Polisetti et al. (2022), licensed under a CC BY 4.0.