Anti-inflammatory potential of human corneal stroma-derived stem cells determined by a novel in vitro corneal epithelial injury model

Figure 7 Effect of co-culture with corneal-stroma derived stem cells-amniotic membrane constructs on human corneal epithelial cells response after injury. Human corneal epithelial cells (hCEC) were treated with an injury model consisting of 30 s ethanol treatment followed by stimulation with 1 ng/mL IL-1β for 72 h. During IL-1β stimulation hCEC were co-cultured with either amniotic membrane (AM) only or a corneal-stroma derived stem cells-AM construct. A: PrestoBlue viability assay after 72 h. Data represented relative to reading for hCEC only, no injury control; B: Lactate dehydrogenase cytotoxicity assay performed on cell supernatants after 72 h treatment. Data displayed as percentage cytotoxicity and relative to cell viability; C: Concentration of IL-6 in the supernatant 72 h after injury. Data displayed relative to cell viability; D: Concentration of IL-8 in the supernatant 72 h after injury. Data displayed relative to cell viability; E-H: RT-qPCR performed to show mRNA expression of IL1B (E), TNF (F), IL6 (G), and CXCL8 (H). Expression of each target gene normalised to GAPDH and represented relative to hCEC only, no injury. Data for all graphs shown as mean ± SEM of three independent experiments with five replicates each. Statistical significance analysed by two-way ANOVA. Significance compared to non-injured, same group, represented by ^aP ≤ 0.05, ^bP ≤ 0.01, ^dP ≤ 0.0001. Significance compared to hCEC only, no injury represented by ^cP ≤ 0.05, ^fP ≤ 0.01, ^hP ≤ 0.001, ^jP ≤ 0.0001. Significance compared to hCEC, injury represented by ^eP ≤ 0.05, ^lP ≤ 0.01, ⁿP ≤ 0.001, ^vP ≤ 0.0001. AM: Amniotic membrane; CSSC: Corneal-stroma derived stem cells; hCEC: Human corneal epithelial cells; IL: Interleukin.