Fig. 2

Development and morphological characterization of myoneurovascular tri-culture. A Schematic demonstrating our cell culture approach. Myoneurovascular triculture was generated by differentiating human iPSC-derived myocytes for 7 days in differentiation media to allow formation of myotubes before adding human iPSC-derived motor neurons to the culture. The combined neuromuscular culture was maintained for a further 7 days in coculture media before adding HUVEC endothelial cells. Finally, the three cell types were cultured for another 7 days in triculture media. B Human iPSC-derived myocytes (MYO), motor neurons (MN), and primary endothelial cells (EC) were cultured in various combinations under optimized conditions for 21 DIV and phenotypically characterized using cell-specific markers. Myocytes were stained for F-actin using phalloidin, motor neurons were stained for expression of hypo-phosphorylated neurofilament H using SMI-35 antibody, and CD31 was used as marker for endothelial cells. Neurons and endothelial cells did not exhibit any significant morphological change when grown individually (MYO + MN/MYO + EC) or together (MYO + MN + EC) on a bed of pre-differentiated myocytes. However, the iPSC myocytes demonstrated a more elongated and aligned architecture when cultured in the presence of motor neurons (MYO + MN and MYO + MN + EC), as compared to when grown only in the presence of ECs (MYO + EC) or as a monoculture (MYO). Scale bar — 250 µm. C Morphological parameters of myocytes were monitored to investigate the neurovascular implications on myocyte development. Cellular fusion leading to polynucleated myofibers was analyzed by measuring myocyte fusion index. Motor neurons significantly increased MFI (MYO + MN) over myocyte monocultures as compared to endothelial cell-myocyte coculture group (MYO + EC). Of note, addition of endothelial cells on neuromuscular coculture group (MYO + MN + EC) further enhanced myocyte fusion, but this effect was not significant in comparison to fusion index observed in neuromuscular cocultures (MYO + MN). Further, simultaneous coculture of motor neurons and endothelial cells contributed to elongation of myofibers but had no effect on their thickness. n = 3/group, p < 0.05 for significance