Cell Adhesion Molecules and Stem Cell-Niche-Interactions in the Limbal Stem Cell Niche

Abstract

Interactions between stem cells and their microenvironment are critical for regulation and maintenance of stem cell function. To elucidate the molecular interactions within the human limbal epithelial stem/progenitor cell (LEPC) niche, which is essential for maintaining corneal transparency and vision, we performed a comprehensive expression analysis of cell adhesion molecules (CAMs) using custom-made quantitative real-time polymerase chain reaction (qRT-PCR) arrays and laser capture-microdissected LEPC clusters, comprising LEPCs, melanocytes, mesenchymal cells, and transmigrating immune cells. We show that LEPCs are anchored to their supporting basement membrane by the laminin receptors a3b1 and a6b4 integrin and the dystroglycan complex, while intercellular contacts between LEPCs and melanocytes are mediated by N-, P-, and E-cadherin together with L1-CAM, a member of the immunoglobulin superfamily (Ig)CAMs. In addition to the LEPC-associated heparan sulfate proteoglycans syndecan-2, glypican-3, and glypican-4, the IgCAM members ICAM-1 and VCAM-1 were found to be variably expressed on LEPCs and associated niche cells and to be dynamically regulated in response to chemokines such as interferon-c to enhance interactions with immune cells. Moreover, junctional adhesion molecule JAM-C accumulating in the subepithelial limbal matrix, appeared to be involved in recruitment of immune cells, while mesenchymal stromal cells appeared to use the nephronectin receptor integrin a8 for approaching the limbal basement membrane. In summary, we identified a novel combination of cell surface receptors that may regulate both stable and dynamic cell-matrix and cell-cell interactions within the limbal niche. The findings provide a solid foundation for further functional studies and for advancement of our current therapeutic strategies for ocular surface reconstruction. STEM CELLS 2016;34:203-219 SIGNIFICANCE STATEMENTThe study provides novel data on the molecular composition of the human limbal stem cell niche by identification of a combination of cell surface receptors that regulate both stable and dynamic cell-matrix and cell-cell interactions within the limbal niche. Most of the molecules identified, i.e. dystroglycan-1, integrin a8, nephronectin, syndecan-2, glypicans-3 and -4, L1-CAM, and JAM-C, have not been described before in the context of the limbal niche. In addition, the ICAM-1/ VCAM-1-mediated interaction of limbal epithelial progenitor cells with immune cells, which could be regulated by chemokines such as interferon-c, provides a novel view of the dynamic nature of the limbal niche. Moreover, we demonstrated the usability of surface molecules such as L1-CAM for isolation and enrichment of limbal progenitor and niche cells.Although being still on a rather descriptive level, the findings provide insights into molecular mechanisms of progenitor cell-niche anchorage mediated by integrins, cadherins, and dystrophin-associated proteins, and into dynamic physical interactions with immune cells mediated by immunoglob...

Laminin-511 and -521-based matrices for efficient ex vivo-expansion of human limbal epithelial progenitor cells

“…We have previously shown that LN chains α1, α2, α5, β1, β2, γ1 and γ3 are preferentially localized to the BM of the limbal epithelium compared to that of the corneal epithelium, where LN chains α3, β3 and γ2 predominate 31 . Our work has further suggested that LEPC are anchored to LN-α2 and -α5 in their niche by expression of LN receptors α3β1 and α6β4 integrins 32 . The heterogeneity of LN isoform expression in BMs of ocular surface epithelia, which has been also reported by other groups 33–35 , suggests a functional role for LN-α2 and -α5 containing isoforms in the limbal stem cell niche.…”

“…2A) 32 . To verify the purity of cell populations, we analyzed the expression profiles of established corneal epithelial (progenitor) markers, such as cytokeratin 3 (KRT3) and 15 (KRT15) and carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), as well as mesenchymal (stem cell) markers, such as intercellular cell adhesion molecule 1 (ICAM1), sex determining region Y –box 2 (SOX2), and stem cell factor receptor (KIT) by qPCR primer assays (n = 5).…”

“…Expression patterns of different LN chains were analyzed in LCM-dissected LEPC clusters, containing both epithelial stem/progenitor cells and associated niche cells 32 , and basal corneal epithelial cell (BCEC) populations by qPCR (n = 5). Quality control of amplified RNA and purity of dissected cell populations were as described previously 32 .…”

“…Quality control of amplified RNA and purity of dissected cell populations were as described previously 32 . All LN chains were expressed in both LEPC and BCEC populations except LN-γ3 ( LAMC3 ).…”

“…Because integrins α3β1and α6β4 have been previously reported to mediate anchorage of LEPC to their niche 32 , we analyzed the expression of these integrin subunits on the cell surface of cultured LEPC (n = 3) and evaluated the effect of integrin blocking on LEPC adhesion to LN-521, -511, -511-E8 and -332 (n = 4). As demonstrated by flow cytometry, almost 100% of LEPC expressed α3, α6, β1 and β4 integrins on their surface (Fig.…”