The failure of the neuromuscular junction (NMJ) is a key component of degenerative neuromuscular disease, yet how NMJs degenerate in disease is unclear. Human induced pluripotent stem cells (hiPSCs) offer the ability to model disease via differentiation toward affected cell types, however, the re-creation of an in vitro neuromuscular system has proven challenging. Here we present a scalable, all-hiPSC-derived co-culture system composed of independently derived spinal motor neurons (MNs) and skeletal myotubes (sKM). In a model of C9orf72 -associated disease, co-cultures form functional NMJs that can be manipulated through optical stimulation, eliciting muscle contraction and measurable calcium flux in innervated sKM. Furthermore, co-cultures grown on multi-electrode arrays (MEAs) permit the pharmacological interrogation of neuromuscular physiology. Utilization of this co-culture model as a tunable, patient-derived system may offer significant insights into NMJ formation, maturation, repair, or pathogenic mechanisms that underlie NMJ dysfunction in disease.
Results
Creation of stable optogenetic motor neuronshiPSC lines were reprogrammed from fibroblasts collected from a healthy control (Line C), a C9orf72 G 4 C 2 expansion carrier diagnosed with behavioral variant FTD (bvFTD, Line A), and genetically corrected isogenic clones (isoA80, isoA51). All hiPSC lines were previously characterized Pribadi et al., 2016). To generate MNs, we followed a previously published protocol (Du et al., 2015) to produce populations of Hb9 + MNs via an adherent-or suspension-based culture protocol within 25 days, and abundant ChAT expression by day 30. ( Figure 1A-E ). We additionally generated isogenic, optogenetic reporter lines via co-transduction with two lentiviruses to enable optogenetic control of Hb9 + MNs ( Figure S1A ). iPSC clones were screened (see Methods, Figure S1B-E ), resulting in selection of a single pair of stable isogenic clones (Opto-A1, Opto-isoA80-8). Suspension-based methodology yielded highly pure, Hb9 + MN spheres ( Figure 1F-G ), and was adopted in subsequent experiments.
Spontaneous myotube contractions in 2D culture are mediated via gap junctionsWe generated hiPSC-sKM following our previously published protocol . Differentiated sKM showed mature sarcomeric organization by Titin staining and electron microscopy ( Figure 1H-I ), and spontaneous contractions in dense cultures. Prior to establishing co-cultures, we posited that sKM would need to be dissociated in order to distinguish NMJ-mediated contractions from spontaneous contractions. Upon dissociation and re-plating of contractile sKM, we observed a cessation in contractile activity of re-plated myotubes, although some sKM still displayed spontaneous calcium oscillations ( Figure S2A-B ). We reasoned this cessation may be due to the loss of gap junction connections, whose proteins are expressed in sKM during development (Merrifield and Laird, 2016), and were putatively observed via electron microscopy ( Figure S2C-D ).Evidence for gap junctions in sKM wa...
