Background

Limbal epithelial stem/progenitor cells (LEPC), responsible for the continuous renewal of the corneal epithelium, reside in a highly specialized and complex environment. It comprises specific extracellular matrix components and supporting limbal niche cells at the corneo-scleral limbus (Schermer et al., 1986; Cotsarelis et al., 1989; Ordonez et al., 2012; Mei et al., 2012). Damage or injury to this stem/progenitor cell reservoir can lead to corneal neovascularization, chronic inflammation, and stromal scarring associated with corneal opacity and loss of vision (Kenyon and Tseng, 1989; Sangwan and Tseng, 2001; Dua et al., 2010). Cultured limbal epithelial transplantation (CLET) has been applied as a therapeutic approach in various clinical centers and the overall success rates of autologous CLET for unilateral cases with a follow-up period of at least 24 months were reported to amount to 72-76% (Rama et al., 2010; Tsai et al., 2010; Utheim et al., 2013; Holland et al., 2015). In the case of bilateral, 34 months of follow-up showed a 70% success rate after allogeneic central penetrating limbo-keratoplasty in conjunction with conjunctivoplasty, mitomycin C, and amniotic membrane transplantation (Eberwein et al., 2012). However, long-term corneal regeneration often proved less satisfactory. This might be caused by the low quality of the graft or inadequate properties of transplanted progenitor cells (Daya et al., 2005; Rama et al., 2010; Pellegrini et al., 2014; Nakamura et al., 2016). These limitations underscore the need for developing novel standardized LEPC culture techniques that ensure the preservation of the stem/progenitor cell phenotype and function during cultivation and after transplantation.

Laminins (LNs) are the best-characterized extracellular matrix constituents present in basement membranes (BM) of adult stem cell niches, including the limbal stem cell niche, where they influence cell behaviors, such as cell adhesion, differentiation, and phenotype stability. Various reports have shown heterogeneity of LN isoform expression in BMs of ocular surface epithelia. Especially LN chains α5, β1, β2, and γ1 are preferentially localized to the BM of the limbal epithelium compared to that of the corneal epithelium (Ljubimov et al., 1995; Schlötzer-Schrehardt et al., 2007; Polisetti et al., 2017) (Figure 1). We have reported earlier that LN alpha 5-containing isoforms as well as the bioactive fragment of human LN-511 (LN-511 E8) support efficient ex vivo expansion of LEPC (Polisetti et al., 2017).


Figure 1. Expression of laminin chains in the limbal stem cell niche in situ. A. Quantitative real-time polymerase chain reaction (qRT-PCR) primer assays showing higher expression levels of laminin α2 (LAMA2), α4 (LAMA4), α5 (LAMA5), β2 (LAMB2), β3 (LAMB3), and γ2 (LAMC2) in microdissected limbal epithelial stem/progenitor cell (LEPC) clusters compared with basal corneal epithelial cell (BCEC) populations; laminin α1 (LAMA1), α3 (LAMA3), β1 (LAMB1), β4 (LAMB4), γ1 (LAMC1) showed no differential expression patterns. B. Immunofluorescence analyses of corneoscleral tissue sections showing differential staining patterns of laminin α2, α5, β2, β3, γ2, and γ3, but similar staining patterns of laminin α1, α3, β1, and γ1 in the basement membranes of corneal and limbal epithelia; laminin α4 was largely negative in epithelial basement membranes. C. Immunofluorescence double labeling of laminin (LN) α5 (green) and cytokeratin (CK)15, N-Cadherin, p63α, integrin α6, integrin α3, and integrin β1 (red); nuclear counterstaining with DAPI (blue); scale bar  =  20 µm. Reprinted from Polisetti et al., 2017, licensed under a CC BY 4.0.

Here, we present an optimized protocol for isolation and expansion of LEPC. The isolation protocol is similar to the protocol provided by Chen et al. (2011) with few modifications, but we expanded LEPC on LN-511E8-coated plates to improve proliferation and preservation of a stem/progenitor cell phenotype (Polisetti et al., 2017).