These findings suggest that attachment of LMs to basement membrane LNs is mediated by integrins a3?1 and a6?1. Effect of laminin isoforms on melanocyte adhesion, migration, and proliferation in vitro To analyze the effect of LN isoforms on LM function in vitro, we used recombinant human LNs containing 1 (LN-111), 2 (LN-211), 3 (LN-332), and 5 (LN-521, LN-511-E8) chains, since cell binding activities are largely determined by chains. promising experimental model for investigating the functional functions of melanocytes in the limbal stem cell niche and their suitability for developing advanced epithelial grafts for ocular surface surface reconstruction. test. Immunofluorescence double labeling of Melan-A (green) with c-Kit, nestin, Sox-10, MITF, TRP1, and HMB-45 (red); nuclear counterstaining with DAPI (blue). (CK15, cytokeratin 15; ICAM-1, intercellular cell adhesion molecule 1; LEPC, limbal epithelial progenitor cells; LMSC, limbal mesenchymal stromal cells; LM, limbal melanocytes; KRT, keratin; NT5E, 5-ecto nucleotidase; Sox10, sex related HMG box 10; TYRP1/TRP1, tyrosinase related protein 1; HMB-45, human melanoma black-45; MITF, micropthalmia associated transcription factor). A low concentration of trypsin (0.05%) was used to enzymatically separate epithelial cells from fibroblast-like and melanocyte-like cells. The remaining cell cultures still contained a large proportion of Edaravone (MCI-186) contaminating fibroblasts, which were vimentin+/Melan-A? by immunocytochemistry and ICAM-1+/Melan-A?/CD117? by flow cytometry (Fig.?1C, left column). After 3 cycles of treatment with geneticin, an inhibitor of protein synthesis, relatively real cultures of Melan-A+/vimentin+ melanocytes were obtained (Fig.?1C, right column). Flow cytometry showed that the small fraction of Melan-A+/ICAM-1+ cells increased from 3.8 to 78.3%, indicating that melanocytes partially express ICAM-119, and that Melan-A+/CD117+ cells increased from 1.4 to 99.2%, indicating an almost 100% pure melanocyte populace after geneticin treatment (Fig.?1C, right column)20. To verify the purity of LM cultures, expression profiles of known positive and negative melanocyte markers were analyzed around the Edaravone (MCI-186) mRNA and protein level in comparison with cultivated LEPCs and LMSCs. qPCR showed high expression levels of common melanocyte markers, including CD117/c-Kit (KIT), Melan-A (MLANA), and tyrosine-related protein (TYRP1)20,21, whereas corneal epithelial markers, such as cytokeratin 3 (KRT3) and cytokeratin 15 (KRT15), and mesenchymal stem cell markers, such as CD73 (NT5E), were not expressed in the enriched LM populations (Fig.?1D). Doubling labeling immunocytochemistry showed colocalization of Melan-A with c-Kit, nestin, SRY-box transcription factor 10 (Sox10), microphthalmia-associated transcription factor (MITF), TRP1, and HMB-45 (Fig.?1D). Extracellular environment of limbal melanocytes in situ Immunohistochemistry analyses Edaravone (MCI-186) of corneoscleral tissue sections showed that LMs were localized within the basal limbal epithelium in close association with LEPC clusters (Fig.?2A). LMs rested on a basement membrane which contained the LN chains 1, 2, 3, 5, 1, 2, 3, 1, 2 and, focally, 3 (Fig.?2B). They appeared to be anchored to the basement membrane by integrins 3, -6, and -1 expressed along their basal cell surface, whereas integrin-?4 appeared to be not expressed by LMs (Fig.?2C). Open in Rabbit Polyclonal to Tubulin beta a separate window Physique 2 Localization of melanocytes in the limbal niche in situ. (A) Immunofluorescence triple staining of corneoscleral tissue sections showing a cell cluster in the basal limbal epithelium made up of cytokeratin 15 (CK15)+ epithelial stem/progenitor cells (green), Melan-A+ melanocytes (red), and vimentin+ mesenchymal stromal cells (turquoise); nuclear counterstaining with 4,6\diamidino\2\phenylindole (DAPI, blue); scale bar?=?10?m; dotted line indicates basement membrane. (B) Immunofluorescence double labeling of corneoscleral tissue sections showing staining patterns of laminin (LN)-1, 2, 3, 5, 1, 2, 3, 1, 2 and 3 in the limbal basement membrane (green) in association with Melan-A+ melanocytes (red); nuclei are counterstained with DAPI (blue); scale bar?=?20?m. (C) Immunofluorescence double labeling showing staining patterns of integrin 3, 6, 1, and 4 (green) in the basal epithelial cell membranes in association with Melan-A+ melanocytes (red); nuclear counterstaining with DAPI (blue); scale bar?=?20?m. (D) Quantitative real-time polymerase chain reaction (qRT-PCR) primer assays showing relative expression levels of laminin chains in cultured limbal melanocytes (LM), limbal epithelial progenitor cells (LEPC) and limbal mesenchymal stromal cells (LMSC). Data are normalized to GAPDH and expressed as means (2?CT??1,000)??SEM (n?=?5). *test. (E) Flow cytometry analyses of cultured LMs showing expression of integrin 3 (ITGA3), integrin 6 (ITGA6), integrin 1 (ITGB1), and integrin 4 (ITGB4) or isotype control antibodies. Data (% of positive cells) are expressed as means??SEM (n?=?3). Differential gene expression analyses of cultivated LMs in comparison with cultivated LEPCs and LMSCs, derived from the same limbal clusters, showed that LMs predominantly expressed LN-1 (LAMA1), LN-?1 (LAMB1), LN-?2 (LAMB2), and LN-1 (LAMC1) (Fig.?2D), suggesting deposition of LN-111 in the limbal basement membrane. By contrast, LEPCs expressed Edaravone (MCI-186) mainly LN-3, 5, ?1, ?3, ?4, 1 and 2, suggesting secretion of LN-332 and LN-511, but potentially also of the rarer isoforms laminin 312 and laminin 512. LMSCs expressed LN-2, 4, ?1, ?2, 1 and 3, indicating contribution of LN-211/221 and LN-411/421 to the basement membrane (Fig.?2D). This differential expression.