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. Author manuscript; available in PMC: 2018 May 1.
Published in final edited form as: Gut. 2017 Feb 21;67(5):827–836. doi: 10.1136/gutjnl-2016-312940

Figure 4. Effect of aging and pro-inflammatory cytokines on enteric neural stem cells.

Figure 4

Using an ex vivo organotypic culturing system to measure proliferation, a nearly 3 fold reduction of EdU+ cells (green) were found per ganglia area in old compared to young mice (A). Using CD49b magnetic beads, ENSCs were isolated from a Wnt1-cre;tdTomato mouse (fluorescently labelled neural-crest cells) and cultured with conditioned media prepared with LMMP from young (Young CM) or old (Old CM) mice. Old CM was also cultured with either IL-6 neutralizing antibody or an IgG control. TUNEL assay revealed over 2-fold increase in numbers of TUNEL+ cells (green) when ENSCs (red) were cultured with Old CM compared to Young CM indicating increased apoptosis due to factors in the aged ENS microenvironment. This increase in TUNEL-positivity was reversed with the addition of IL-6 neutralizing antibody but not IgG control (B and C). When cultured with recombinant IL-6 at increasing concentrations, ENSCs demonstrated a statistically significant increase in TUNEL+ cells (D) at the highest concentration (100 ng/ml). The addition of soluble IL-6 Rα (200 ng/ml) augmented the number of TUNEL+ cells when added to IL-6 at a lower concentration (50 ng/ml). *p<.05 by t-test for (A); *p<.05, **p<.005 by one-way ANOVA with Bonferroni’s multiple comparisons test for (C) and (D). Scale bars, 50 μm.