Fig. 7

Growth factor expression pattern in myoneurovascular triculture. A Cell culture supernatant from the various groups was taken after 21 DIV and monitored for the secretion of growth factors like amphiregulin, insulin growth factor-binding proteins (IGFBP) 3, 4, and 6 subtypes. Motor neuron-myocyte coculture significantly increased production of amphiregulin, which is a potent mitogen of neural stem cells and has been shown to promote neurite outgrowth. IGFBPs regulate the activity of insulin-like growth factors (IGFs), wherein IGBP-4 is specifically inhibitory to IGF-1 and IGF-2. We observed high levels of extracellular IGFBP-4 when endothelial cells were cocultured with myocytes (MYO + EC) compared to other groups indicating that such a system can result in inhibiting IGF-1 and IGF-2-mediated effects on cells. Although not significant, the presence of motor neurons was found to increase levels of IGFBP-6, which is a regulator of IGF-2 signaling pathway. IGFBP-6 is significantly upregulated in spinal motor neurons post injury and has been found to be neuroprotective. We did not observe any significant difference in IGFBP-3 expression among the different experimental groups. Kruskal–Wallis test followed by Dunn's multiple comparison test was performed, n = 3/group was analyzed, and p < 0.05 was considered for significance. B Agrin expression by different cell combinations after 21 DIV was quantified from cell culture supernatants using an Agrin-specific ELISA assay. Endothelial cells alone (MYO + EC) as well as in combination with motor neurons (MYO + MN + EC) lead to elevated levels of Agrin expression as compared to myocyte monocultures. Ordinary one-way ANOVA with Tukey’s multiple comparison test was performed. n = 6/group was analyzed, and p < 0.05 was considered for significance