Fig. 1
Immunophenotyping of f-LSCs. a The immunological profile of fibroblast-like limbal stem cells (f-LSCs) was assessed by means of flow cytometry before (left panel) and after (right panel) 48 h of cytokine-treatment (IL-1β, IL-6, and IFNγ, 500 U/ml) for the human leukocyte antigens (HLA-DR), the immune T-activator molecule programmed death-1 (PD-1, CD279) and its ligands (programmed death-ligand 1/2 (PDL-1/2)), the negative regulator of T cell response B7-H4 and the costimulatory molecules CD80 (B7-1) and CD86 (B70/B7-2). All histogram plots include the percentage of expression of each protein as a representative value of five independent experiments. b qRT-PCR shows the expression of several immunosuppressive and tolerogenic markers (HLA-G, transforming growth factor beta (TGF-β), indoleamine-pyrrole 2,3-dioxygenase (IDO), cyclooxygenase-2 (COX-2), hepatocyte growth factor (HGF), PDL-1/2, Fas, Fas-L, monocyte chemotactic protein 1 (MCP-1), interleukin-6 (IL-6), and autoimmune regulator (AIRE)) compared to the bone marrow-derived mesenchymal stem cell (BM-MSC) positive control (upper panel). The same markers were tested in f-LSCs after cytokine stimulation. The fold change for each gene was calculated with the Delta Delta Ct method using the Rotor Gene Q software and the expression was normalized for the housekeeping gene β-actin. c f-LSCs were cultured with the abovementioned Th1 cytokines for 48 h in T-25 flasks. After acutase-mediated dissociation, 106 cells were used for AO/EB staining. Pictures show the viability of untreated and cytokine treated cells using a fluorescein filter in a 40× objective. d Proliferation assay (MTT) at 48 h for untreated and cytokine-treated f-LSCs. All data/pictures are representative of at least five independent experiments. Values on the bars are shown as mean ± SE; *p < 0.05, **p < 0.02, ***p < 0.01