Formation and characterisation of neuromuscular junctions between hiPSC derived motoneurons and myotubes

Abstract

Striated skeletal muscle cells from humans represent a valuable source for in vitro studies of the motoric system as well as for pathophysiological investigations in the clinical settings. Myoblasts can readily be grown from human muscle tissue. However, if muscle tissue is unavailable, myogenic cells can be generated from human induced pluripotent stem cells (hiPSCs) preferably without genetic engineering. Our study aimed to optimize the generation of hiPSCs derived myogenic cells by employing selection of CD34 positive cells and followed by distinct, stepwise culture conditions. Following the expansion of CD34 positive single cells under myogenic cell culture conditions, serum deprived myoblast-like cells finally fused and formed multinucleated striated myotubes that expressed a set of key markers for muscle differentiation. In addition, these myotubes contracted upon electrical stimulation, responded to acetylcholine (Ach) and were able to generate action potentials. Finally, we co-cultured motoneurons and myotubes generated from identical hiPSCs cell lines. We could observe the early aggregation of acetylcholine receptors in muscle cells of immature co-cultures. At later stages, we identified and characterised mature neuromuscular junctions (NMJs). In summary, we describe here the successful generation of an iPS cell derived functional cellular system consisting of two distinct communicating cells types. This in vitro co-culture system could therefore contribute to research on diseases in which the motoneurons and the NMJ are predominantly affected, such as in amyotrophic lateral sclerosis or spinal muscular atrophy.

Making muscle: skeletal myogenesisin vivoandin vitro

“…For this reason, there have been several efforts to establish a co-culture system of PSC-derived myogenic cells with rat embryonic spine or with PSCderived motoneurons. This system improved the maturation of PSC-derived myofibers, as evidenced by AchR clustering and electrophysiological recordings (Demestre et al, 2015;Puttonen et al, 2015;Rohwedel et al, 1998b); however, electrophysiological analysis suggested immaturity of neuromuscular junction activity (Puttonen et al, 2015;Vyskočil and Vrbová, 1993).…”

“…Notably, PDGFRα + /VEGFR2 − cells isolated from differentiating PSC cultures have been proposed to correspond to putative paraxial mesoderm progenitors, including premyogenic cells (Darabi et al, 2012(Darabi et al, , 2008Sakurai et al, 2009Sakurai et al, , 2012. Myogenic progenitors have also been isolated from mouse or human PSC cultures using CD106 (VCAM1, SM/C2.6) (Chang et al, 2009;Mizuno et al, 2010), CD34 (Demestre et al, 2015), CD56 (NCAM1) (Young et al, 2016), CD56 and CD73 (Barberi et al, 2007), CD82 (Uezumi et al, 2016), CXCR4 (CD184) and c-Met (Borchin et al, 2013). However, the usefulness of these markers is limited by their overall poor lineage specificity.…”

“…Mature striated myotubes have been generated in vitro from mESCs spontaneously differentiated from EBs (Hirsch et al, 1998;Rohwedel et al, 1998b), as well as from Pax7-reprogrammed hPSCs (Skoglund et al, 2014). Demestre et al (2015) compared these two methods in their ability to produce human actinin + myotubes and reported that myotubes generated by Pax7-mediated reprogramming were slower to mature. Millimeter-long myofibers exhibiting MyHC + and titin + striations and containing large numbers of nuclei have also been generated from mouse and human PSCs in adherent, serum-free conditions (Chal et al, 2015) (Fig.…”

Advances and Current Challenges Associated with the Use of Human Induced Pluripotent Stem Cells in Modeling Neurodegenerative Disease

“…Human iPSC-derived motor neurons have been cocultured with skeletal muscle from various human and nonhuman sources as a means to study neuromuscular junction formation and function [Demestre et al, 2015;Puttonen et al, 2015;Santhanam et al, 2018]. Such models can be used to test the efficacy of pharmacological agents as well as to model synaptic breakdown in cases of neuromuscular disorders, such as spinal muscular atrophy [Yoshida et al, 2015].…”

Myogenic progenitor specification from pluripotent stem cells

“…Interestingly, these authors were able to detect expression of the secondary myogenesis marker Nfix [69], transition from the embryonic slow myosin to the perinatal isoform, and an overall high myofibril organization. Multiple groups have interpreted the incomplete electrophysiological maturation as a consequence of the lack of innervation and establishment of co-culture protocols could represent an alternative for overcoming this issue [155]. Interestingly, lack of maturation seems to be a less relevant issue for cell therapy applications.…”