ABSTRACT

The neuromuscular junction (NMJ) has been the model of choice to understand the principles of communication at chemical synapses. Following groundbreaking experiments carried out over 60 years ago, many studies have focused on the molecular mechanisms underlying the development and physiology of these synapses. This Review summarizes the progress made to date towards obtaining faithful models of NMJs in vitro. We provide a historical approach discussing initial experiments investigating NMJ development and function from Xenopus to mice, the creation of chimeric co-cultures, in vivo approaches and co-culture methods from ex vivo and in vitro derived cells, as well as the most recent developments to generate human NMJs. We discuss the benefits of these techniques and the challenges to be addressed in the future for promoting our understanding of development and human disease.

Introduction

At the beginning of the last century the ‘neuron doctrine’ proposed by Ramón y Cajal revolutionized the emerging field of neuroscience. The doctrine firmly established individual neurons as the basic anatomical and physiological unit of the nervous system, and prompted many questions regarding how neurons communicate with one another and how the space between them is crossed by the signals responsible for that communication. We now know that neurons make complex structures called synapses that allow chemical transmission of signals among cells. The neuromuscular junction (NMJ) is a tripartite chemical synapse of the peripheral nervous system. It allows the transmission of motor information that governs muscle contraction from the central nervous system to the muscle fibers. The NMJ comprises a motoneuron on the presynaptic side, a muscle cell on the post-synaptic side and a terminal Schwann cell (tSC) wrapping the NMJ structure. A fourth cell type, a kranocyte, which caps the NMJ and plays a role in synapse regeneration, has recently been identified in rodents (Court et al., 2008) (Fig. 1). Fatt and Katz showed that end-plate potentials (EPPs), which correspond to the chemically induced change in electric potential of the motor end plate, originate from the spontaneous or electrically evoked release of acetylcholine (ACh) from the nerves (Fatt and Katz, 1951). Furthermore, Katz and colleagues proposed the ‘quantal hypothesis’, stating that ACh is released from the nerve terminal in packets or ‘quanta’. The spontaneous release of one quantum results in a miniature EPP (mEPP) in the post-synaptic cell, whereas the synchronized release of multiple ACh quanta leads to EPPs. Synaptic vesicles accumulate at discrete zones of the nerve terminal called ‘active zones’ (Couteaux and Pecot-Dechavassine, 1970). A functional definition of active zones came a few years later, when it was shown that vesicles dock and fuse at the membrane, and their contents are exocytosed in response to nerve depolarization (Heuser et al., 1979). Taken together, these pioneering studies on NMJs provided the basis for our current knowledge of the intricate mechanisms underlying synaptic transmission.

Fig. 1.

The stages of NMJ formation in mice. (A) During development, prior to and independent of innervation, AChRs are clustered in the prospective region of muscle. Thus, the muscle cell plays an instructive role from a very early stage of development, in positioning and restricting synapses to a central band in the muscle, and building a post-synaptic density prior to axonal contact. (B) Axons do not contact muscle fibers at random locations, but rather in a narrow central region of the muscle fiber characterized by the presence of aneural ‘pre-patterned’ AChR clusters (Lin et al., 2001; Yang et al., 2001). Axons then become apposed to AChR clusters. (C) Additionally three or four nuclei accumulate under the post-synaptic membrane in the muscle cells (which are defined as multinucleated syncytia formed during development by fusion of mononucleated precursor cells) and become specialized for transcription of synaptic proteins. Concomitantly, folds form in the muscle membrane increasing the area of contact between the post-synaptic membrane and the extracellular matrix (Guarino et al., 2019). (D) Muscle fibers are poly-innervated until the end of the second week after birth in rodents. This is when the multiple innervations of individual post-synaptic densities are reduced to a single innervating motor axon through a process called synapse elimination (Sanes and Lichtman, 2001). AChR, acetylcholine receptor; adult AChR, α2βεδ; E, embryonic day; embryonic AChR, α2βγδ; MuSK, muscle-specific kinase; P, post-natal day; tSC, terminal Schwann cell.

Fig. 1.

The stages of NMJ formation in mice. (A) During development, prior to and independent of innervation, AChRs are clustered in the prospective region of muscle. Thus, the muscle cell plays an instructive role from a very early stage of development, in positioning and restricting synapses to a central band in the muscle, and building a post-synaptic density prior to axonal contact. (B) Axons do not contact muscle fibers at random locations, but rather in a narrow central region of the muscle fiber characterized by the presence of aneural ‘pre-patterned’ AChR clusters (Lin et al., 2001; Yang et al., 2001). Axons then become apposed to AChR clusters. (C) Additionally three or four nuclei accumulate under the post-synaptic membrane in the muscle cells (which are defined as multinucleated syncytia formed during development by fusion of mononucleated precursor cells) and become specialized for transcription of synaptic proteins. Concomitantly, folds form in the muscle membrane increasing the area of contact between the post-synaptic membrane and the extracellular matrix (Guarino et al., 2019). (D) Muscle fibers are poly-innervated until the end of the second week after birth in rodents. This is when the multiple innervations of individual post-synaptic densities are reduced to a single innervating motor axon through a process called synapse elimination (Sanes and Lichtman, 2001). AChR, acetylcholine receptor; adult AChR, α2βεδ; E, embryonic day; embryonic AChR, α2βγδ; MuSK, muscle-specific kinase; P, post-natal day; tSC, terminal Schwann cell.

Neuromuscular junctions have since been the most intensively studied synapses, because of their comparatively large size, relative simplicity and accessibility. Indeed, among specific features, neurotransmitter release at this synapse is mediated by multiple docking sites present in long active zones in frogs or several active zones in mammals. In contrast, synaptic transmission in the brain is associated with a single active zone per synapse (Biro et al., 2005; Silver et al., 2003). Another important distinction between NMJs and central synapses is that the post-synaptic element at an NMJ only receives cholinergic inputs, whereas post-synaptic neurons receive a large variety of inputs, which makes the analysis of the response to a single neurotransmitter more difficult to interpret. Therefore, the NMJ remains an excellent model for studying the mechanisms of neurotransmission, and is well suited to study synaptic formation, maintenance and function. Furthermore, the NMJ is at the core of a number of human pathologies that all have the perturbation of neurotransmission in common (Box 1).

Box 1. Neuromuscular junction disorders

Neuromuscular diseases (NMD) refer to a vast group of conditions that perturb or even progressively block the control of muscles. NMDs can originate from motoneuron defects, e.g. as in amyotrophic lateral sclerosis (ALS) and in spinal muscular atrophy (SMA), or from a defect of the neuromuscular junction itself, e.g. as in congenital myasthenic syndromes (CMS), and in autoimmune diseases such as myasthenia gravis (MG). NMJ diseases have a wide range of origins, including genetic mutations, antibodies or even toxins that interfere with the control of signaling between the presynaptic nerve ending and the post-synaptic muscle membrane, and/or the formation of the synapse.

Myasthenia gravis (MG) is one of the most common diseases of the NMJ. This autoimmune disorder of the neuromuscular transmission is increasingly considered as a syndrome more than as a single disease. At least three different antibodies to AChRs, MuSK or LRP4 have been identified as the cause of the typical weakness and fatigability associated with MG (Higuchi et al., 2011; Hoch et al., 2001; Lindstrom et al., 1976). In addition, Lambert-Eaton myasthenic syndrome (LEMS) is caused by auto-antibodies against voltage-gated calcium channels (Lennon et al., 1995).

As well as these autoimmune diseases, there is also a large family of genetic conditions that affect the functioning of the NMJ. Remarkable progress in molecular genetics during recent decades has allowed the identification of an increasing number of mutations in genes encoding proteins of the NMJ that drive pathogenesis of several inherited diseases. Mutations in key human genes, such as DOK7, RAPSN, MUSK, LAMB2, COLQ, AGRN and genes encoding AChRs, can cause different forms of CMS (Beeson et al., 2006; Engel et al., 2015; Hantai et al., 2013; Ohkawara et al., 2014; Verschuuren et al., 2016).

Here, we provide an update on the different in vitro models that have been investigated to increase our understanding of the normal and pathological development of NMJ. We present a focus on the recent findings on humanized in vitro models, which have been made possible using human pluripotent stem cells, and discuss the promises and challenges of these new models.

The neuromuscular junction: a model system for studying synaptic development and function

A great deal of information on how synapses form has been obtained through studying muscle cell lines (mostly the murine C2C12 cell line) that form spontaneous acetylcholine receptor (AChR) aggregates, and co-cultures of muscle cells and motoneurons (Sanes and Lichtman, 2001). Early studies were carried out in cultures of neurons and muscles from Xenopus laevis (Anderson et al., 1977) or from chick (Frank and Fischbach, 1979), laying the groundwork for understanding the mechanisms of in vitro NMJ formation and the study of NMJ disorders (discussed below). These datasets have been extended in vivo with the emergence of transgenic mice, along with the use of non-mammalian animal models (Box 2). Here, we briefly describe the steps in NMJ formation as well as the essential molecular and structural markers of NMJ formation and maturation (Figs 1 and 2). For more detailed discussion of the mechanisms of synapse formation, we refer the reader to recent reviews (Burden et al., 2018; Li et al., 2018).

Box 2. Contributions of non-mammalian model organisms for NMJ understanding

In parallel to the in vitro models for NMJ described in this Review, non-mammalian model organisms such as Drosophila and zebrafish have also been instrumental in identifying key components present at the pre- and post-synaptic levels, and understanding the mechanisms that underpin NMJ assembly. These organisms provide experimental advantages, including genetic and molecular traceability, amenability to functional studies, and increased experimental throughput compared with traditional mammalian models.

Although the NMJ of Drosophila has provided insights into the development, function and plasticity of synapses, the molecular composition and physiology of this NMJ is most similar to mammalian glutamatergic excitatory synapses and no orthologues of key molecules involved in the mammalian NMJ have been identified in Drosophila. In contrast, the use of zebrafish NMJ has continuously increased in popularity over recent decades, mainly because of the transparency of the embryonic and larval stages. This renders zebrafish particularly amenable to optical time-lapse studies in vivo. In combination with large-scale genetic screens, in the late 1990s Nüsslein-Vohlhard and colleagues identified genetic mutants based on morphological abnormalities and abnormal behaviors (Granato et al., 1996; Haffter et al., 1996). Subsequent identification of the genes shed light on the molecular mechanisms underpinning the motor system; many of the locomotion mutations mapped to genes coding for synaptic proteins in the NMJs that had also been identified in mammals, such as MuSK, Dok7, Lrp4, Rapsyn or specific subunits of AChR (Marangi et al., 2001; Ono et al., 2004). These examples illustrate how these approaches complement the advances obtained using human patients or mammalian animal models, although divergence in some pathways were observed.

Fig. 2.

Muscle, neuronal and synaptic hallmarks of the mature NMJ. The main markers that can be used to characterize the mature NMJ are shown. ACh, acetylcholine; AChE, acetylcholinesterase; AChR, acetylcholine receptor; AZ, active zone; BL, basal lamina; ColQ, collagen Q; Dok7, downstream of tyrosine kinase 7; LRP4, low-density lipoprotein receptor-related protein 4; MuSK, muscle-specific kinase; tSC, terminal Schwann cell; VDCC, voltage-dependent Ca2+ channel; VGSC, voltage-gated Na+ channel.

Fig. 2.

Muscle, neuronal and synaptic hallmarks of the mature NMJ. The main markers that can be used to characterize the mature NMJ are shown. ACh, acetylcholine; AChE, acetylcholinesterase; AChR, acetylcholine receptor; AZ, active zone; BL, basal lamina; ColQ, collagen Q; Dok7, downstream of tyrosine kinase 7; LRP4, low-density lipoprotein receptor-related protein 4; MuSK, muscle-specific kinase; tSC, terminal Schwann cell; VDCC, voltage-dependent Ca2+ channel; VGSC, voltage-gated Na+ channel.

Cellular components in synapse formation

NMJ development requires extensive communication among the three key components of the tripartite synapse: presynaptic motoneurons, post-synaptic muscle fibers and tSCs. NMJ formation is closely intertwined with myogenesis and innervation occurs between embryonic days (E) 12.5 and E14 in mice, immediately after the first wave of myotube formation. This timing varies slightly among specific muscles and their locations (Fig. 1).

The composition of AChRs changes with development as follows: nicotinic AChRs are pentamers composed of five subunits in a stoichiometry of two α-, one β-, one δ- and one γ-subunit in the embryo. These are replaced by a ε-subunit at the postnatal age (Changeux et al., 1992). Within 3 weeks of birth, AChR clusters adopt a complex, pretzel-like configuration in mice (Sanes and Lichtman, 2001). The maturation of the NMJ leads to an increase in number, stability, density and clustering of AChRs in the post-synaptic compartment, as well as the differentiation of the presynaptic membrane into active zones with proper synaptic proteins and associated synaptic vesicles (Burden et al., 2018). The overall process reflects strong reciprocal signaling between pre- and postsynaptic structures.

Another crucial component involved in the formation of the NMJ is the tSC. Although the very first steps in synapse formation do not require the presence of tSCs, these cells are necessary for maintenance and maturation of NMJs (Ko and Robitaille, 2015). Ablation of tSCs after NMJ maturation at P30 in mice, leads to NMJ fragmentation and neuromuscular transmission deficits (Barik et al., 2016).

Molecular components of NMJ development and function

Over the past decade, extensive progress has been made in understanding the molecular mechanisms involved in NMJ formation. Here, we summarize the current knowledge on some of the key molecules and signaling pathways that are indispensable for NMJ formation and maintenance (Figs 1 and 2).

MuSK-Lrp4-agrin signaling

MuSK (muscle-specific kinase), a transmembrane receptor tyrosine kinase and its co-receptor LRP4 (low-density lipoprotein receptor-related protein 4) are crucial for the aggregation of AChRs before and after innervation. Prior to innervation, the MuSK/LRP4 complex accumulates in the muscle central region where it induces AChR clustering. With innervation, LRP4 binds a neural isoform of agrin (AGRN), a heparan sulfate proteoglycan secreted by the axon terminal (Ferns et al., 1993; Kim et al., 2008; Ruegg and Bixby, 1998; Zhang et al., 2008). Agrin interaction with LRP4 results in the formation of tetrameric agrin-LRP4 complexes that promote activation and transphosphorylation of MuSK, leading to AChR clustering and NMJ formation (Zong et al., 2012). AChR clusters that are apposed to nerve terminals persist and grow, while the non-apposed ones disassemble (Wallace, 1989). Mutations in the human AGRN gene cause congenital myasthenic syndrome (CMS), with distal muscle weakness and atrophy (Nicole et al., 2014).

More recently, two studies have independently revealed that the muscle LRP4 also acts as a retrograde signal for presynaptic differentiation (Wu et al., 2012; Yumoto et al., 2012). MuSK activation requires Dok7 (docking protein 7; an adaptor protein expressed by the muscle), which is indispensable for MuSK activity and therefore AChR clustering. The binding of Dok7 to the cytoplasmic juxtamembrane region of MuSK strongly enhances the tyrosine kinase activity of MuSK, leading to its activation (Hamuro et al., 2008; Inoue et al., 2009). MuSK-deficient mice show neither muscle pre-patterning of AChRs nor NMJ formation, and die perinatally from respiratory failure (Okada et al., 2006). More recently, it has been shown that the postnatal knockdown of Dok7 gene in mice causes structural defects in NMJs and motor dysfunction, supporting a role for Dok7 in NMJ maintenance (Eguchi et al., 2016).

AChR-rapsyn

Beginning around the time of birth, fetal AChRs (α2βγδ) are gradually replaced by the adult subtype of AChRs (α2βεδ). Although the physiological significance of this developmental switch remains to be fully understood, different studies have suggested a role of the γ-subunit during the NMJ formation and more specifically in innervation patterning (Koenen et al., 2005; Takahashi et al., 2002). For example, deletion of the mouse AChR γ-subunit leads to perinatal lethality associated with an absence of pre-patterned AChR clusters during initial stages of neuromuscular synaptogenesis (Koenen et al., 2005; Takahashi et al., 2002). A key effector in AChR clustering is the protein rapsyn, a cytoplasmic scaffolding protein expressed in the muscle, which binds directly to AChR and anchors AChR in the post-synaptic membrane (LaRochelle et al., 1990; Sobel et al., 1978). Rapsyn-deficient mice fail to develop post-synaptic specializations (Gautam et al., 1995), indicating that rapsyn is essential for the tethering of AChRs to the post-synaptic apparatus. Although rapsyn assembles with AChRs in the secretory pathway (Marchand et al., 2002; Moransard et al., 2003), the exact mechanisms downstream of MuSK/Dok7 that stimulate rapsyn-induced AChR clustering remain to be determined.

Extracellular components

In addition to these core molecules that are essential for NMJ formation, a number of important regulators also participate in this process. Wnts are a group of glycoproteins secreted at the NMJ by the muscle fiber, motoneurons and tSCs. Several Wnt ligands bind to the cysteine-rich domain (CRD) in MuSK (Barik et al., 2016), and play a role in axon extension and branching, as well as in AChR prepatterning (Li et al., 2018). Both canonical and non-canonical planar cell polarity (PCP) Wnt pathways are affected in transgenic mice in which the CRD in MuSK is deleted (Messeant et al., 2017).

Dystroglycan (DG) has a dual function: it organizes extracellular matrix (ECM) scaffolds and stabilizes AChR clusters. DG is formed of two subunits that are generated by proteolytic cleavage of a single precursor protein. The extracellular subunit is necessary to organize a functional scaffold in the basal lamina including perlecan, acetylcholinesterase/ColQ and laminin (Jacobson et al., 2001; Peng et al., 1998). DG is also a receptor for agrin (Gee et al., 1994), although – unlike Lrp4 – it does not activate MuSK.

The synaptic basal lamina contains many components that have important regulatory roles in myogenesis and synaptogenesis. ECM molecules help guide the process of innervation by motor neurons and are crucial to formation of the post-synaptic density as well as organizing and maintaining functional apposition of the pre- and post-synaptic elements (Maselli et al., 2012; Rogers and Nishimune, 2017). Key junctional basal lamina components include collagens [such as collagen IV (α3)(α4)(α5) and (α5)2(α6) trimers, collagen XIII and ColQ), glycoproteins (such as laminin isoforms α4β2γ1, α5β2γ1 and α2β2γ1, and nidogen 2) and heparan sulfate proteoglycans (HSPGs), such as perlecan and agrin (Fox et al., 2007). The laminin β2 chain is considered as an active zone organizer and binds to the voltage-dependent calcium channels (VDCC) located in the active zones (Nishimune et al., 2004). The interaction between laminin β2 and VDCCs leads to VDCC clustering, which is the first step toward a mature active zone. In laminin β2 knockout mice, the number of active zones and synaptic vesicles are reduced along with a reduction in neurotransmitter release. Mice lacking ColQ, collagen XIII, collagen IV (α3)(α4)(α5), (α5)2(α6) trimers or collagen VI also show immature nerve terminals and/or NMJs (Cescon et al., 2018; Sigoillot et al., 2016; Zainul et al., 2018). Networks of laminins and collagens are connected to one another by nidogen and are anchored to the sarcolemma and intracellular cytoskeleton by binding to laminin receptors: integrins and, most importantly, DG (Fox et al., 1991; Jacobson et al., 2001).

Evolution of in vitro models of NMJs

For over one century, different models and approaches have been developed to decipher the mechanisms involved in the formation, maintenance and function of NMJs. Meanwhile, increased incidence and prevalence of inherited neuromuscular diseases require the development of pertinent pathological models for deciphering the pathophysiology underlying these diseases and developing effective treatments (Bhatt, 2016). Although animal models have been crucial for the NMJ field, they have certain limitations, such as ethical issues involving animal welfare, their capacity to fully reproduce some human pathological phenotypes and in their complexity, which hinders the identification of cell-specific mechanisms, including anterograde and retrograde signals. Researchers have tried to overcome these hurdles by developing in vitro models to recreate the NMJs. In the following section, we focus on the contribution and the evolution of these in vitro models of the NMJ (Fig. 3).

Fig. 3.

Different technical approaches to studying the NMJ in vitro. (A) Organ explant culture. (B) Dissociated explant culture. (C) Mixed cell and explant culture. (D) Dissociated cell culture. (C,D) Instead of explants or dissociated cells, immortalized cell lines or motoneurons (MNs)/muscle cells generated from induced pluripotent stem cells can be used. *Chimeric model possible (different species of origin for muscle cells and MNs).

Fig. 3.

Different technical approaches to studying the NMJ in vitro. (A) Organ explant culture. (B) Dissociated explant culture. (C) Mixed cell and explant culture. (D) Dissociated cell culture. (C,D) Instead of explants or dissociated cells, immortalized cell lines or motoneurons (MNs)/muscle cells generated from induced pluripotent stem cells can be used. *Chimeric model possible (different species of origin for muscle cells and MNs).

The first models of co-cultures: organ explant cultures

Historically, the first neuromuscular junction obtained in vitro was reported by R. G. Harrison in 1959 (Harrison, 1959). By cultivating medullary cord and axial mesoderm explants from clotted frog lymph between two glass slides, Harrison observed striated and contracting muscle fibers. As the frog muscle cells do not contract spontaneously, he concluded that the contractions were nerve induced. As the embryos are easy to manipulate, collect and dissect, amphibians have been extensively used as animal models for generating in vitro models of neuromuscular junctions (Anderson et al., 1977; Kidokoro et al., 1980; Peng and Nakajima, 1978; Swenarchuk et al., 1990). Among amphibians, two species stand out – Xenopus laevis and Ambystoma mexicanum – which belong to the anuran and the urodele orders, respectively. Xenopus laevis offers a number of advantages, most notably cells that are inexpensive to culture and mature rapidly (Peng et al., 1998; Swenarchuk et al., 1990). Within 3 days of co-culture, myotomal muscle cells are seen to be contracting due to innervation by neurons (Anderson et al., 1977). Cells from Ambystoma mexicanum, like Xenopus cells, can be cultivated at room temperature and in a basic cell culture medium complemented with 1% horse serum or fetal bovine serum (Anderson et al., 1977). Ambystoma cells differ from Xenopus cells in terms of the time of maturation. Ambystoma cells require a longer period to generate mature myofibers and establish functional synapses (Swenarchuk et al., 1990).

In parallel, a number of other species from chick to rodents have been used to build models for NMJs. These models were justified in part by the lack of tools to study cell biology in frog. Burgen and colleagues used rat phrenic nerve-diaphragm explants to investigate the action of the botulinum toxin (Burgen et al., 1949) (Fig. 3A). In these first studies, models were formed by removing a section of embryonic spinal cord with the phrenic nerve and muscle still attached. These explants were maintained as ‘organ cultures’.

Dissociated explants cultures and dissociated cells cultures

The next breakthrough was the development of physically separated explants at difference with the previous system (Fig. 3B). This strategy allowed the observation of newly formed NMJs (James and Tresman, 1968). In these dissociated explants cultures, most of the NMJ models aimed to visualize and understand the process of neuromuscular synapse formation and also to decipher the mode of action of pharmacological drugs (Evans, 1969). At the same time, the first serial electron microscope studies allowed detailed observations of the ultrastructure of mouse neuromuscular synapses in culture (Bornstein et al., 1968). Crain and colleagues showed that explant cultures of rat spinal cord and skeletal muscle tissues from rat and human formed functional NMJs (Crain et al., 1970).

The development of dissociated neuronal cell culture, where isolated neurons were grown on a single layer along with the availability of inverted microscopes, facilitated electrophysiological recordings in vitro (Scott et al., 1969). Using these tools, Fischbach and colleagues demonstrated that co-cultures of cells dissociated from chick skeletal muscle with those from chick spinal cord produced functional synaptic contacts (Fischbach, 1970) (Fig. 3C). These inter-species models, also called chimeric models, confirmed that NMJ formation and function is conserved among a variety of species.

Chimeric cell cultures

Chicks, frogs and rodents are the most common species used as sources of tissues for studying NMJ formation. Chicks offer easy access and manipulation of the embryo (Frank and Fischbach, 1979; Johnson et al., 1981), and have been used in chimeric models with rat and mouse cells. However, as mammals, the rodents Mus musculus (mouse) and Rattus norvegicus (rat) provide greater insight into human development or pathology than amphibians or birds (Ionescu et al., 2016). Mouse myoblasts can differentiate into fused and striated myofibers 2-4 days after plating (Ionescu et al., 2016). Functional synapses form 4 days later when they are cultured with murine neuronal explants (i.e. 2 days for contact between axons and myotubes followed by 2 days for synapse maturation) (Vilmont et al., 2016) (Fig. 3D). However, mouse co-cultures are rather inefficient systems, with few synapses forming, in contrast to frog co-cultures (Sanes and Lichtman, 1999).

Chimeric cell cultures models have provided answers to the origin – muscle or nerve – of a number of molecules expressed at the NMJ. For example, species-specific antibodies, one targeting specifically the HSPG from anuran species and the second targeting the HSPG from urodela species, have identified that HSPGs originate from the muscle (Swenarchuk et al., 1990). In the same vein, the development of transgenic animals brought new tools in the field of in vitro NMJ modeling. For example, Hb9::GFP transgenic mice express GFP in differentiated motoneurons (Wichterle et al., 2002). The use of Hb9::GFP mouse cells allow for easy visualization and identification of motoneurons in culture (Ionescu et al., 2016; Umbach et al., 2012).

Limitations and next challenges for dissociated cell models

Embryonic spinal cord explants provide motoneurons in situ, within their 3D multicellular environment (Ionescu et al., 2016). Alternatively, dissociated cells from embryonic spinal cord or myotome allow one to enrich specifically for motoneurons or muscle cells (Fischbach, 1972; Nakajima et al., 1980). Clonal and immortalized cell lines are often used to provide a large, convenient and homogenous population of cells ready to culture. However, these cells may harbor abnormal phenotypes that are not completely representative of healthy cells in vivo, either because of the immortalization process they have been through or the fact that they are cancer cells. For example, G-8 clonal myogenic cells have a modal chromosome number of 75 instead of 40 (Christian et al., 1977). Nevertheless, muscle cell lines are used largely to study the mechanism of post-synaptic differentiation and more specifically, the mechanisms of AChRs aggregation. For example, C2C12 cells are a subclone originally isolated by Blau (Blau et al., 1983) from a murine cell line generated in 1977 by Yaffe and Saxel (Yaffe and Saxel, 1977). C2C12 cells are useful to study early muscle differentiation, including myoblast fusion as well as AChR aggregation. However, C2C12 cells do not fully differentiate and do not form aggregates of acetylcholinesterase (AChE). A more recently established murine muscle cell line called MLCL forms AChE clusters in addition to the AChR clusters (Cartaud et al., 2004). Finally, collaborations between biologists and physicists lead to the emergence of new culture devices such as microfluidic devices with the objectives of better modeling NMJ pathologies, as well as providing a more suitable system for some mechanistic studies (Machado et al., 2019; Uzel et al., 2016; Zahavi et al., 2015). These developments are discussed below.

Criteria for characterizing in vitro NMJs

The establishment of reliable protocols for co-cultures led many groups to consider developing humanized in vitro models of NMJs to study human neuromuscular diseases. Whichever in vitro model is used, a number of criteria have to be met to ascertain that an NMJ model is representative of those in vivo in terms of structure and function, and therefore a valid model. The criteria list that has been used is described in the next few paragraphs and is summarized in Tables 1 and 2.

Table 1.

Non-human in vitro models of NMJ – physiological criteria

Non-human in vitro models of NMJ – physiological criteria
Non-human in vitro models of NMJ – physiological criteria
Table 2.

Non-human in vitro models of NMJ – molecular criteria and summary of the studies

Non-human in vitro models of NMJ – molecular criteria and summary of the studies
Non-human in vitro models of NMJ – molecular criteria and summary of the studies

NMJ morphology

In the first instance, different presynaptic, synaptic and post-synaptic hallmarks can be used as physiological and morphological clues indicative of normal NMJs. Some of them can be detected by immunolabeling of key synaptic proteins (Das et al., 2007; Dorchies et al., 2001; Umbach et al., 2012; Vilmont et al., 2016) (Fig. 2). For skeletal muscle, the first indication of complete differentiation is the morphology of the myofibers. NMJ morphology is dependent on the species as well as the stage of NMJ differentiation and the innervated muscle. Gillingwater and colleagues developed a standardized ‘NMJ morph’ tool to study NMJ morphology from tissue samples, highlighting these specific parameters in function of the species (Jones et al., 2017; Jones et al., 2016). The best approach to ascertain the presence of a synapse is through electron microscopy (EM). EM allows the visualization of a contact between axon terminals and muscle cells, the presence of synaptic vesicles in the presynaptic element and post-synaptic densities apposed to presynaptic elements (reviewed by Slater, 2017). However, it remains to be shown that this synapse is functional; i.e. muscle contractions induced by nerve activity and the presence of spontaneous/evoked transmission.

Muscle contractions

Striations and spontaneous contractions are also characteristics acquired during differentiation of myoblasts into myotubes in mammals. Mouse, rat and human muscle cells can contract in aneural mono-culture, unlike amphibians (Guo et al., 2014). These contractions are helpful in assessing the differentiation of the cells. Generally, two types of muscle cell contractions can be observed in co-culture models: small twitches independent of innervation (which are short in length and time) and contractions induced by innervation, which are longer and stronger (Ionescu et al., 2016). Innervation-dependent contractions are also characterized by their synchrony from the innervation of several myotubes by axonal branches of the same motoneuron (Vilmont et al., 2016). These two types of twitching movements can easily be distinguished by adding the drugs tetrodotoxin (which inhibits voltage-gated sodium channels) or d-tubocurarine (an antagonist of AChR that prevents ACh binding to its receptor) to the culture medium (Vilmont et al., 2016) (Table S1). The innervation-independent twitches are blocked only by tetrodotoxin, whereas the contractions induced by innervation are abolished by both drugs.

Functional analysis

Electrophysiological demonstration of spontaneous and evoked neurotransmission is the most detailed way to characterize the functionality of a NMJ model. mEPPS can be recorded by a microelectrode in the motoneuron and another in the myotube close to the point of contact. This criterion alone is an unequivocal evidence of a synapse, although it may not be fully mature. A functional neuron can be identified upon electrical stimulation of the motoneuron and evocation of EPPs (Nelson et al., 1993). Alternatively, EPPs can be evoked and synapses revealed by applying glutamate from a microelectrode directly onto glutamatergic motoneurons (Puro et al., 1977).

To assess whether the EPPs or contractions are dependent on neurotransmission and AChRs, different toxins or drugs can be used. As mentioned previously, d-tubocurarine is a toxin blocking the AChR functionally, and thus blocking twitching, EPPs and mEPPs mediated by nicotinic AChR (Chipman et al., 2014; Kidokoro et al., 1980). A common test is to record muscle cell twitching, spontaneous EPPs or evoked EPPs, and then add d-tubocurarine in the medium. This addition of d-tubocurarine eliminates twitching and EPPs in the muscle cells, but not the motorneuron action potential (Umbach et al., 2012). This inhibition is reversible and when the d-tubocurarine is removed from the medium, the muscle fibers should re-start twitching or expressing mEPPs and EPPs almost immediately (Anderson et al., 1977; Das et al., 2007; Dorchies et al., 2001; Puro et al., 1977; Umbach et al., 2012; Vilmont et al., 2016).

The first humanized in vitro models of the NMJ

As the prevalence of neurodegenerative and muscular disorders is rising, and available treatments for these diseases are scarce, it is a matter of urgency to investigate new treatment strategies. Understanding the pathophysiology underlying diseases, such as amyotrophy lateral sclerosis (ALS) or congenital myasthenic syndrome (CMS), is a crucial step for early diagnosis and effective treatment. Therefore, the remaining challenge was to generate a human in vitro NMJ adapted to mimic in vivo biology with the aim to decipher pathological mechanisms and perform functional analysis.

In 1970, Peterson and Crain demonstrated that neuromuscular connections could be formed between explants of embryonic spinal cord from rodents and skeletal muscle tissue from adult humans (Crain et al., 1970). Formed with human myofibers, these cultures were maintained up to 7 weeks and electrophysiological analysis indicated that synaptic connections between rodent neurons and human muscle were functional and could be blocked with d-tubocurarine. They also demonstrated an accumulation of acetylcholinesterase (AChE) at sites of synaptic contact (Crain et al., 1970). Extending this first demonstration, Peterson and Crain observed pathological features in long-term co-cultures with human dystrophic muscle. In co-cultures maintained for more than 4 months, unusual focal myofibrillar lesions in a substantial number of innervated mature Duchenne muscular dystrophy (DMD) muscle fibers were described (Peterson et al., 1986). However, the complexity of preparing these co-cultures from human muscle biopsies has restricted their use.

An important breakthrough in the field of NMJ in vitro models came with the development of co-cultures between dissociated human primary skeletal muscle cells, derived from satellite cells, with rodent embryonic spinal cords (Table 3). Kobayashi and Askanas were the first to demonstrate that monolayer cultures of adult human muscle cells can be innervated by embryonic rat spinal cord neurons and that the differentiation of human skeletal muscle cells, as well as their electrophysiological properties, are greatly enhanced by innervation (Askanas et al., 1987; Kobayashi et al., 1987). Importantly, dorsal-root ganglia were essential for achieving functional innervation, as demonstrated by the presence of regular contractions and well-organized acetylcholinesterase-positive contacts (Askanas et al., 1987; Kobayashi et al., 1987). Further electrophysiological studies also demonstrated mEPPS and EPPs in these co-cultures were abolished by application of AChR antagonists, such as d-tubocurarine or α-bungarotoxin (α-BTX), which also blocks the contractions of human myotubes in these chimeric co-cultures. These studies also highlight that innervation can occur without the original basal lamina (Kobayashi and Askanas, 1985). Finally, the development of these chimeric models of NMJ provided an important basis for pathological studies described for myotonic dystrophy (Kobayashi et al., 1990), DMD (Park-Matsumoto et al., 1991), McArdle's myopathy (Martinuzzi et al., 1993), spinal muscular atrophy (SMA), ALS and facioscapulohumeral muscular dystrophy (FSHD) (Braun et al., 1995). Several of these studies revealed some pathological phenotypes. Thus, human skeletal muscle from children affected by a severe form of SMA began to display degeneration only when co-cultured with embryonic rat spinal cords (Braun et al., 1995). Despite these important advances, the only human tissue that could be used in these co-cultures was skeletal muscle cells. Recent advances in stem cell biology have fueled the prospect of generating the main components of the NMJ, offering new perspectives for pathobiological studies and therapeutic development for neuromuscular diseases. Thus, the availability of human pluripotent stem cells (hPSCs), either of embryonic stem cell (ESC) or induced pluripotent stem cell (iPSC) origin, has made the generation of human muscle and nerve cells possible. The first protocols developed from hPSCs concerned the specification of spinal motoneurons. Based on developmental insights generated from animal studies and those gained on mouse ESCs (Wichterle et al., 2002), much progress has been made on the capacity to convert hPSCs into spinal motoneurons. Li and colleagues were among the first to attempt co-cultures between hPSC-derived spinal motoneurons and murine skeletal muscle cells C2C12 (Li et al., 2005) (Table 4). After 3 weeks of culture, contractions were observed only in the presence of hESC-derived motoneurons and immunostaining analysis revealed the presence of clusters of AChR juxtaposed to neurite contacts (Li et al., 2005). Since then, this approach has been extended to disease-specific hiPSC-derived motoneurons such as SMA (Yoshida et al., 2015). In this context, the use of SMA-hiPSC derived motoneurons led to an impaired AChR clustering on C2C12 that precedes motoneuron loss (Yoshida et al., 2015). However, it is important to emphasize the relatively low rate of differentiation of hPSCs into hPSC-derived motoneurons observed in these studies, with only 25-30% of motoneurons generated on average.

Table 3.

In vitro models of NMJ – human source of skeletal muscle cells

In vitro models of NMJ – human source of skeletal muscle cells
In vitro models of NMJ – human source of skeletal muscle cells
Table 4.

In vitro models of NMJ – human source of motoneurons

In vitro models of NMJ – human source of motoneurons
In vitro models of NMJ – human source of motoneurons

To circumvent this limitation, motoneurons enriched by FACS or directly induced from fibroblasts (hi-motoneurons), without passing through an induced pluripotent stem cell stage, have been used (Du et al., 2015; Liu et al., 2016; Singh Roy et al., 2005). When co-cultured with primary rodent skeletal muscle cells, both sources of motoneurons led to the colocalization of synaptic markers such as synapsin with AChR clusters. Electrophysiological analyses also revealed spontaneous end-plate currents detected in murine myotubes when co-cultured with hi-motoneurons generated from human fibroblasts by overexpression of four transcription factors (SOX11, NGN2, ISL1 and LHX3). This electrophysiological activity can be blocked by using either d-tubocurarine, tetrodotoxin or a specific inhibitor of voltage-dependent sodium channels in muscle: ω-connotoxin. When applied to a pathological condition such as ALS, profound dysfunction in the ability of patient-specific hi-motoneurons to control functional muscle maturation is observed (Liu et al., 2016). Although promising, these first descriptions were all based on chimeric models and to reach the ‘holy grail’ of a ‘real’ human NMJ in vitro model, both co-culture components should be of human origin.

Towards entirely humanized NMJ in vitro models

Over the past decade, the literature has documented fully humanized in vitro models of NMJs based on motoneurons mainly derived from hPSCs cultured with human primary skeletal muscle cells (Afshar Bakooshli et al., 2019; Marteyn et al., 2011; Santhanam et al., 2018; Shimojo et al., 2015; Steinbeck et al., 2016; Vila et al., 2019). Although all these studies diverged in their protocols, they all described the presence of AChR clusters in close proximity to neuritic processes, as well as muscle twitching after 2 or 3 weeks of co-culture. This indicates to some extent the functionality of human primary skeletal muscle cells when co-cultured with human motoneurons (Table 5). To go further ahead in the functional characterization of this system, Steinbeck and colleagues were the first to combine hPSC-based in vitro models of NMJs with optogenetics (Steinbeck et al., 2016). The authors used purified hESC-derived motoneurons stably expressing channelrhodopsin 2 (ChR2) coupled to the GFP under the human synapsin promoter. After 6-8 weeks of co-culture with human primary skeletal muscle, muscle twitches were observed and quantified after light stimulation. The addition of vecuronium, an antagonist of the nicotinic AChR, completely blocked the light-induced muscle twitches. Calcium imaging and electrophysiological analyses also confirmed these results. As purification of hPSC-derived motoneurons could lead to the selection of physiologically irrelevant motoneurons, these datasets were reproduced using motoneurons generated with a recent protocol that allows the efficient conversion of hPSC into a homogenous population of spinal motoneurons without using a purification step (Maury et al., 2015). Interestingly, the authors also demonstrated that their ‘all human’ NMJ cultures could be used in the pathological context of myasthenia gravis (Box 1) (Steinbeck et al., 2016).

Table 5.

In vitro models of NMJ – human source of skeletal muscle cells and motoneurons

In vitro models of NMJ – human source of skeletal muscle cells and motoneurons
In vitro models of NMJ – human source of skeletal muscle cells and motoneurons

Despite these developments, several challenges remain. The first limitation is the source of the human skeletal muscle cells. All the models described are based on the use of human primary skeletal muscle cells, which exhibit substantial donor variation in terms of differentiation (Nikolic et al., 2017). The development of an ‘all human’ NMJ model in which both neuron and muscle components are derived from the same individual is of great interest for modeling disease of genetic etiology. With the recent development of protocols allowing the conversion of hPSCs into skeletal muscle cells (reviewed by Pourquie et al., 2018), it is possible to produce co-cultures between motoneurons and skeletal muscle cells derived from hiPSCs from the same donor (Demestre et al., 2015; Maffioletti et al., 2018; Mazaleyrat et al., 2020; Osaki et al., 2018; Picchiarelli et al., 2019; Puttonen et al., 2015). Although the maturation level of these co-cultures generated from ‘embryonic-like’ pluripotent stem cells is still questionable, the detection of AChR clustering, contractions and calcium imaging suggest an acceptable level of functionality of these systems.

Another limitation stems from the fact that most of these studies have used a 2D cell culture system that does not mimic the native tissue structure. Consequently, different attempts have been made recently to evaluate the possibility of developing an ‘all human’ NMJ in vitro model by taking advantage of the advent of 3D cultures and microfluidic lab-on-a-chip technologies (Afshar Bakooshli et al., 2019; Maffioletti et al., 2018; Osaki et al., 2018; Santhanam et al., 2018; Vila et al., 2019). Microfluidics allows precise control over the cell microenvironment, providing an interesting platform for studying the NMJ. Motoneurons and muscle can be in two different chambers connected by axons, which enables spatiotemporal control over the cells. Analysis of the localized actions at the soma, along the axon and at the NMJ can be performed. Santhanam and colleagues were among the first to apply microfluidics to human motoneurons and muscle cells. Two weeks of co-culture were sufficient to observe muscle contractions after electrical stimulation of the motoneurons that can be blocked by treatment with d-tubocurarine (Table S1). However, the percentage of contracting myotubes is lower than the number of NMJs determined immunocytochemically, suggesting that some of the latter may not be mature enough to be functional (Santhanam et al., 2018).

Another approach relies on the use of 3D co-cultures with muscle cells in hydrogels, such as the commercially available basement membrane extract Matrigel followed by differentiating the motoneurons on the surface. 3D cultures tend to be functionally more relevant with respect to contractile muscle. Collagen I is a major component of the native ECM and harbors adequate properties needed for contraction and force generation. However, Collagen I has less stability, degrades faster and appears to be a less effective solution for long-term cultures (Leikina et al., 2002). In contrast to Collagen I, Matrigel seems to be better adapted for muscle differentiation (Grefte et al., 2012). Other alternatives to collagen are fibrin, fibrinogen or thrombin (Wu et al., 2005). Such approaches have recently been applied to the development of fully humanized NMJ in vitro models in which muscle cell laden hydrogels were formed in specific molds with pillar structures at each extremity sufficient. These then created a continuous uniaxial tension and directed orientation of cells along the axis of tension (Afshar Bakooshli et al., 2019; Maffioletti et al., 2018). A comparative analysis of 2D versus 3D co-cultures reveals a faster and more efficient functional innervation in 3D versus 2D co-cultures when reviewed after 2 weeks in culture. The determination was performed using calcium imaging, optogenetics and electrophysiological recordings (Afshar Bakooshli et al., 2019). A more mature expression profile of AChR isoforms was observed in 3D co-cultures with an increased expression of the adult AChR ε isoform in comparison with 2D cultures. Nonetheless, the small size of muscle fibers, as well as the properties of action potentials recorded in these co-cultures when compared with in vivo mammalian models, suggests that the 3D human NMJs co-cultures are not fully mature and that additional cues might be necessary to ensure further maturation of tissues (Afshar Bakooshli et al., 2019). Increasing culture time, adding trophic factors or providing other cell types have been suggested. Recently, complex isogenic multilineage 3D co-cultures have been developed containing skeletal muscle cells, vascular endothelial cells, pericytes and motoneurons, all derived from hiPSCs (Maffioletti et al., 2018). Whereas these complex co-cultures were mainly characterized at the morphological level, these results open new perspectives for the development of humanized organoid-like platforms for studying NMJ development and disease modeling.

Two studies have also described the combination of 3D system and microfluidics (Osaki et al., 2018; Vila et al., 2019). These approaches have the advantage that motoneurons and muscle cells can be compartmentalized while providing spatial guidance cues and signal gradients using 3D hydrogels for better integration. The study uses engineered functional contractile hiPSC-derived skeletal muscle cells co-cultured with hiPSC-derived motoneurons. Interestingly, such approaches are compatible for studying and validating the therapeutic potential of compounds for neuromuscular disorders such as ALS (Osaki et al., 2018).

Finally, 3D bioprinting is an emerging field that could be relevant for the development of in vitro NMJ systems. A recent study described the feasibility of using bio-printed 3D hydrogel skeletons to support culture, differentiation and alignment of human primary skeletal muscle cells compatible with the functional integration of immortalized human neural progenitor cells (Kim et al., 2020). Interestingly, these bio-printed constructs with neural cells facilitate rapid innervation and mature into organized muscle tissue, restoring normal muscle function in a rodent model of muscle defect injury (Kim et al., 2020). This development can be pursued in the future for improving the maturity of NMJ systems and highlights an area of considerable interest in the tissue-engineering field.

Future perspectives

For over one century, the field of research around NMJs has grown constantly. Recent advances in stem cell biology have stimulated the development of innovative models of human NMJs. Despite initial successes in developing human motoneuron-muscle cells co-cultures, stem-cell derived NMJs remain immature in comparison with the human and chimeric NMJ models. One challenge is the limited survival of these co-cultures. The first chimeric mouse-human co-cultures could survive for more than 6 months (Peterson et al., 1986), whereas human stem-cell based co-cultures often do not survive beyond 4 weeks. However, a recent protocol of human stem cell co-differentiation enables the co-culture to survive up to 7 months (Mazaleyrat et al., 2020). Another challenge for future studies is to find a different method for generating spinal motoneurons. Among the motoneuron lineages in vivo, seven different lower motoneuron subtypes exist, each of them innervating a different muscle group. Most of the NMJ models from iPSCs in the currently published studies used iPS cell-derived general motoneurons. Efforts are under way to characterize the subtypes obtained and to improve the motoneuron differentiation protocols in order to obtain specific motoneuron subtypes (Hester et al., 2011; Lukovic et al., 2017). Similarly, iPS cell-derived muscle fibers are so immature that they cannot be classified as muscle fiber type I or II. Future work to obtain well-characterized motoneurons and muscle fibers subtypes will be particularly important when it comes to modeling neuromuscular pathologies, as all NMJs are not affected in the same way (Nicole et al., 2014). Additionally, tSCs are still a missing partner in current in vitro models. Protocols enabling the differentiation of iPS cells into myelinated Schwann cells are available (Liu et al., 2012; Ziegler et al., 2011). To date, there are no protocols for generating tSCs. These cells, together with complex and specialized basal laminas, will likely be required to reach a fully mature in vitro human NMJ model. Future research on 3D in vitro human NMJ models will be important in this effort. Recently, Faustino Martins and colleagues developed an elegant strategy to derive neuromuscular organoids from hiPSCs. Over a period of 50 to 100 days, these organoids reached a size of 5-6 mm in diameter and displayed an elongated morphology with neural tissue on one side and muscle cells on the other. In this system, electron microscopy confirmed the presence of presynaptic nerve terminals containing synaptic vesicles and active zones: tSCs and post-synaptic muscle fibers with organized sarcomeres and junctional folds. Importantly, these NMJs were functional, as demonstrated by the detection of muscle contraction after 50 days of culture. The functionality could be inhibited with d-tubocurarine, as well as spontaneous calcium oscillations and electrical activities (Faustino Martins et al., 2020). When employed with patient-specific stem cells together with CRISPR/Cas9 genome editing to restore or create mutations identified in patients, these new models will provide more reliable models of human NMJs in vitro and of neuromuscular diseases. Finally, to address the needs of new therapeutic approaches for neuromuscular diseases, standardized and miniaturized human in vitro NMJ models are a long-awaited drug-screening tool.

Acknowledgements

I-Stem is part of the Biotherapies Institute for Rare Diseases (BIRD) supported by the Association Française contre les Myopathies-Téléthon. The authors thank Dr Salvatore Carbonetto and Dr Amit Sandra for constructive comments and proofreading of the manuscript.

Footnotes

Funding

S.B.’s PhD thesis was supported by an Agence Nationale de la Recherche grant (HUMANISM) and by Association Française contre les Myopathies-Téléthon. J.T.’s PhD thesis was supported by the Region Ile-de-France.

References

Afshar Bakooshli
,
M.
,
Lippmann
,
E. S.
,
Mulcahy
,
B.
,
Iyer
,
N.
,
Nguyen
,
C. T.
,
Tung
,
K.
,
Stewart
,
B. A.
,
van den Dorpel
,
H.
,
Fuehrmann
,
T.
,
Shoichet
,
M.
, et al. 
(
2019
).
A 3D culture model of innervated human skeletal muscle enables studies of the adult neuromuscular junction
.
eLife
8
,
e44530
.
Anderson
,
M. J.
,
Cohen
,
M. W.
and
Zorychta
,
E.
(
1977
).
Effects of innervation on the distribution of acetylcholine receptors on cultured muscle cells
.
J. Physiol.
268
,
731
-
756
.
Askanas
,
V.
,
Kwan
,
H.
,
Alvarez
,
R. B.
,
Engel
,
W. K.
,
Kobayashi
,
T.
,
Martinuzzi
,
A.
and
Hawkins
,
E. F.
(
1987
).
De novo neuromuscular junction formation on human muscle fibres cultured in monolayer and innervated by foetal rat spinal cord: ultrastructural and ultrastructural--cytochemical studies
.
J. Neurocytol.
16
,
523
-
537
.
Barik
,
A.
,
Li
,
L.
,
Sathyamurthy
,
A.
,
Xiong
,
W. C.
and
Mei
,
L.
(
2016
).
Schwann Cells in Neuromuscular Junction Formation and Maintenance
.
J. Neurosci.
36
,
9770
-
9781
.
Bellmann
,
J.
,
Goswami
,
R. Y.
,
Girardo
,
S.
,
Rein
,
N.
,
Hosseinzadeh
,
Z.
,
Hicks
,
M. R.
,
Busskamp
,
V.
,
Pyle
,
A. D.
,
Werner
,
C.
and
Sterneckert
,
J.
(
2019
).
A customizable microfluidic platform for medium-throughput modeling of neuromuscular circuits. Biomaterials 225, 119537.
Beeson
,
D.
,
Higuchi
,
O.
,
Palace
,
J.
,
Cossins
,
J.
,
Spearman
,
H.
,
Maxwell
,
S.
,
Newsom-Davis
,
J.
,
Burke
,
G.
,
Fawcett
,
P.
,
Motomura
,
M.
, et al. 
(
2006
).
Dok-7 mutations underlie a neuromuscular junction synaptopathy
.
Science
313
,
1975
-
1978
.
Bhatt
,
J. M.
(
2016
).
The Epidemiology of Neuromuscular Diseases
.
Neurol. Clin.
34
,
999
-
1021
.
Biro
,
A. A.
,
Holderith
,
N. B.
and
Nusser
,
Z.
(
2005
).
Quantal size is independent of the release probability at hippocampal excitatory synapses
.
J. Neurosci.
25
,
223
-
232
.
Blau
,
H. M.
,
Chiu
,
C. P.
and
Webster
,
C.
(
1983
).
Cytoplasmic activation of human nuclear genes in stable heterocaryons
.
Cell
32
,
1171
-
1180
.
Bornstein
,
M. B.
,
Iwanami
,
H.
,
Lehrer
,
G. M.
and
Breitbart
,
L.
(
1968
).
Observations on the appearance of neuromuscular relationships in cultured mouse tissues
.
Z Zellforsch Mikrosk Anat
92
,
197
-
206
.
Braun
,
S.
,
Croizat
,
B.
,
Lagrange
,
M. C.
,
Warter
,
J. M.
and
Poindron
,
P.
(
1995
).
Constitutive muscular abnormalities in culture in spinal muscular atrophy
.
Lancet
345
,
694
-
695
.
Burden
,
S. J.
,
Huijbers
,
M. G.
and
Remedio
,
L.
(
2018
).
Fundamental Molecules and Mechanisms for Forming and Maintaining Neuromuscular Synapses
.
Int. J. Mol. Sci.
19
,
490
-
509
.
Burgen
,
A. S.
,
Dickens
,
F.
and
Zatman
,
L. J.
(
1949
).
The action of botulinum toxin on the neuro-muscular junction
.
J. Physiol.
109
,
10
-
24
.
Cartaud
,
A.
,
Strochlic
,
L.
,
Guerra
,
M.
,
Blanchard
,
B.
,
Lambergeon
,
M.
,
Krejci
,
E.
,
Cartaud
,
J.
and
Legay
,
C.
(
2004
).
MuSK is required for anchoring acetylcholinesterase at the neuromuscular junction
.
J. Cell Biol.
165
,
505
-
515
.
Cescon
,
M.
,
Gregorio
,
I.
,
Eiber
,
N.
,
Borgia
,
D.
,
Fusto
,
A.
,
Sabatelli
,
P.
,
Scorzeto
,
M.
,
Megighian
,
A.
,
Pegoraro
,
E.
,
Hashemolhosseini
,
S.
, et al. 
(
2018
).
Collagen VI is required for the structural and functional integrity of the neuromuscular junction
.
Acta Neuropathol.
136
,
483
-
499
.
Changeux
,
J. P.
,
Galzi
,
J. L.
,
Devillers-Thiery
,
A.
and
Bertrand
,
D.
(
1992
).
The functional architecture of the acetylcholine nicotinic receptor explored by affinity labelling and site-directed mutagenesis
.
Q. Rev. Biophys.
25
,
395
-
432
.
Chipman
,
P. H.
,
Zhang
,
Y.
and
Rafuse
,
V. F.
(
2014
).
A stem-cell based bioassay to critically assess the pathology of dysfunctional neuromuscular junctions
.
PLoS ONE
9
,
e91643
.
Christian
,
C. N.
,
Nelson
,
P. G.
,
Peacock
,
J.
and
Nirenberg
,
M.
(
1977
).
Synapse formation between two clonal cell lines
.
Science
196
,
995
-
998
.
Court
,
F. A.
,
Gillingwater
,
T. H.
,
Melrose
,
S.
,
Sherman
,
D. L.
,
Greenshields
,
K. N.
,
Morton
,
A. J.
,
Harris
,
J. B.
,
Willison
,
H. J.
and
Ribchester
,
R. R.
(
2008
).
Identity, developmental restriction and reactivity of extralaminar cells capping mammalian neuromuscular junctions
.
J. Cell Sci.
121
,
3901
-
3911
.
Couteaux
,
R.
and
Pecot-Dechavassine
,
M.
(
1970
).
[Synaptic vesicles and pouches at the level of “active zones” of the neuromuscular junction]
.
C R Acad. Hebd Seances Acad. Sci. D
271
,
2346
-
2349
.
Crain
,
S. M.
,
Alfei
,
L.
and
Peterson
,
E. R.
(
1970
).
Neuromuscular transmission in cultures of adult human and rodent skeletal muscle after innervation in vitro by fetal rodent spinal cord
.
J. Neurobiol.
1
,
471
-
489
.
Das
,
M.
,
Rumsey
,
J. W.
,
Gregory
,
C. A.
,
Bhargava
,
N.
,
Kang
,
J. F.
,
Molnar
,
P.
,
Riedel
,
L.
,
Guo
,
X.
and
Hickman
,
J. J.
(
2007
).
Embryonic motoneuron-skeletal muscle co-culture in a defined system
.
Neuroscience
146
,
481
-
488
.
Demestre
,
M.
,
Orth
,
M.
,
Fohr
,
K. J.
,
Achberger
,
K.
,
Ludolph
,
A. C.
,
Liebau
,
S.
and
Boeckers
,
T. M.
(
2015
).
Formation and characterisation of neuromuscular junctions between hiPSC derived motoneurons and myotubes
.
Stem Cell Res.
15
,
328
-
336
.
Dorchies
,
O. M.
,
Laporte
,
J.
,
Wagner
,
S.
,
Hindelang
,
C.
,
Warter
,
J. M.
,
Mandel
,
J. L.
and
Poindron
,
P.
(
2001
).
Normal innervation and differentiation of X-linked myotubular myopathy muscle cells in a nerve-muscle coculture system
.
Neuromuscul. Disord.
11
,
736
-
746
.
Du
,
Z. W.
,
Chen
,
H.
,
Liu
,
H.
,
Lu
,
J.
,
Qian
,
K.
,
Huang
,
C. L.
,
Zhong
,
X.
,
Fan
,
F.
and
Zhang
,
S. C.
(
2015
).
Generation and expansion of highly pure motor neuron progenitors from human pluripotent stem cells
.
Nat. Commun.
6
,
6626
.
Eguchi
,
T.
,
Tezuka
,
T.
,
Miyoshi
,
S.
and
Yamanashi
,
Y.
(
2016
).
Postnatal knockdown of dok-7 gene expression in mice causes structural defects in neuromuscular synapses and myasthenic pathology
.
Genes Cells
21
,
670
-
676
.
Engel
,
A. G.
,
Shen
,
X. M.
,
Selcen
,
D.
and
Sine
,
S. M.
(
2015
).
Congenital myasthenic syndromes: pathogenesis, diagnosis, and treatment
.
Lancet Neurol.
14
,
420
-
434
.
Evans
,
M. H.
(
1969
).
Differences between the effects of saxitoxin (paralytic shellfish poison) and tetrodotoxin on the frog neuromuscular junction
.
Br. J. Pharmacol.
36
,
426
-
436
.
Fatt
,
P.
and
Katz
,
B.
(
1951
).
An analysis of the end-plate potential recorded with an intracellular electrode
.
J. Physiol.
115
,
320
-
370
.
Faustino-Martins
,
J. M.
,
Fischer
,
C.
,
Urzi
,
A.
,
Vidal
,
R.
,
Kunz
,
S.
,
Ruffault
,
P. L.
,
Kabuss
,
L.
,
Hube
,
I.
,
Gazzerro
,
E.
,
Birchmeier
,
C.
, et al. 
(
2020
).
Self-Organizing 3D Human Trunk Neuromuscular Organoids
.
Cell Stem Cell
26
,
172
-
186.e176
.
Ferns
,
M. J.
,
Campanelli
,
J. T.
,
Hoch
,
W.
,
Scheller
,
R. H.
and
Hall
,
Z.
(
1993
).
The ability of agrin to cluster AChRs depends on alternative splicing and on cell surface proteoglycans
.
Neuron
11
,
491
-
502
.
Fischbach
,
G. D.
(
1970
).
Synaptic potentials recorded in cell cultures of nerve and muscle
.
Science
169
,
1331
-
1333
.
Fischbach
,
G. D.
(
1972
).
Synapse formation between dissociated nerve and muscle cells in low density cell cultures
.
Dev. Biol.
28
,
407
-
429
.
Fox
,
J. W.
,
Mayer
,
U.
,
Nischt
,
R.
,
Aumailley
,
M.
,
Reinhardt
,
D.
,
Wiedemann
,
H.
,
Mann
,
K.
,
Timpl
,
R.
,
Krieg
,
T.
,
Engel
,
J.
, et al. 
(
1991
).
Recombinant nidogen consists of three globular domains and mediates binding of laminin to collagen type IV
.
EMBO J.
10
,
3137
-
3146
.
Fox
,
M. A.
,
Sanes
,
J. R.
,
Borza
,
D. B.
,
Eswarakumar
,
V. P.
,
Fassler
,
R.
,
Hudson
,
B. G.
,
John
,
S. W.
,
Ninomiya
,
Y.
,
Pedchenko
,
V.
,
Pfaff
,
S. L.
, et al. 
(
2007
).
Distinct target-derived signals organize formation, maturation, and maintenance of motor nerve terminals
.
Cell
129
,
179
-
193
.
Frank
,
E.
and
Fischbach
,
G. D.
(
1979
).
Early events in neuromuscular junction formation in vitro: induction of acetylcholine receptor clusters in the postsynaptic membrane and morphology of newly formed synapses
.
J. Cell Biol.
83
,
143
-
158
.
Gautam
,
M.
,
Noakes
,
P. G.
,
Mudd
,
J.
,
Nichol
,
M.
,
Chu
,
G. C.
,
Sanes
,
J. R.
and
Merlie
,
J. P.
(
1995
).
Failure of postsynaptic specialization to develop at neuromuscular junctions of rapsyn-deficient mice
.
Nature
377
,
232
-
236
.
Gee
,
S. H.
,
Montanaro
,
F.
,
Lindenbaum
,
M. H.
and
Carbonetto
,
S.
(
1994
).
Dystroglycan-alpha, a dystrophin-associated glycoprotein, is a functional agrin receptor
.
Cell
77
,
675
-
686
.
Granato
,
M.
,
van Eeden
,
F. J.
,
Schach
,
U.
,
Trowe
,
T.
,
Brand
,
M.
,
Furutani-Seiki
,
M.
,
Haffter
,
P.
,
Hammerschmidt
,
M.
,
Heisenberg
,
C. P.
,
Jiang
,
Y. J.
, et al. 
(
1996
).
Genes controlling and mediating locomotion behavior of the zebrafish embryo and larva
.
Development
123
,
399
-
413
.
Grefte
,
S.
,
Vullinghs
,
S.
,
Kuijpers-Jagtman
,
A. M.
,
Torensma
,
R.
and
Von den Hoff
,
J. W.
(
2012
).
Matrigel, but not collagen I, maintains the differentiation capacity of muscle derived cells in vitro
.
Biomed. Mater.
7
,
055004
.
Guarino
,
S. R.
,
Canciani
,
A.
and
Forneris
,
F.
(
2019
).
Dissecting the extracellular complexity of neuromuscular junction organizers
.
Front. Mol. Biosci.
6
,
156
.
Guo
,
X.
,
Das
,
M.
,
Rumsey
,
J.
,
Gonzalez
,
M.
,
Stancescu
,
M.
and
Hickman
,
J.
(
2010
).
Neuromuscular junction formation between human stem-cell-derived motoneurons and rat skeletal muscle in a defined system. Tissue Eng, Part C Methods 16, 1347-1355.
Guo
,
X.
,
Gonzalez
,
M.
,
Stancescu
,
M.
,
Vandenburgh
,
H. H.
and
Hickman
,
J. J.
(
2011
).
Neuromuscular junction formation between human stem cell-derived motoneurons and human skeletal muscle in a defined system
.
Biomaterials
32
,
9602
-
9611
.
Guo
,
X.
,
Greene
,
K.
,
Akanda
,
N.
,
Smith
,
A.
,
Stancescu
,
M.
,
Lambert
,
S.
,
Vandenburgh
,
H.
and
Hickman
,
J.
(
2014
).
In vitro Differentiation of Functional Human Skeletal Myotubes in a Defined System
.
Biomater Sci.
2
,
131
-
138
.
Haffter
,
P.
,
Granato
,
M.
,
Brand
,
M.
,
Mullins
,
M. C.
,
Hammerschmidt
,
M.
,
Kane
,
D. A.
,
Odenthal
,
J.
,
van Eeden
,
F. J.
,
Jiang
,
Y. J.
,
Heisenberg
,
C. P.
, et al. 
(
1996
).
The identification of genes with unique and essential functions in the development of the zebrafish, Danio rerio
.
Development
123
,
1
-
36
.
Hamuro
,
J.
,
Higuchi
,
O.
,
Okada
,
K.
,
Ueno
,
M.
,
Iemura
,
S.
,
Natsume
,
T.
,
Spearman
,
H.
,
Beeson
,
D.
and
Yamanashi
,
Y.
(
2008
).
Mutations causing DOK7 congenital myasthenia ablate functional motifs in Dok-7
.
J. Biol. Chem.
283
,
5518
-
5524
.
Hantai
,
D.
,
Nicole
,
S.
and
Eymard
,
B.
(
2013
).
Congenital myasthenic syndromes: an update
.
Curr. Opin. Neurol.
26
,
561
-
568
.
Harrison
,
R. G.
(
1959
).
The outgrowth of the nerve fiber as a mode of protoplasmic movement
.
J. Exp. Zool.
142
,
5
-
73
.
Hester
,
M. E.
,
Murtha
,
M. J.
,
Song
,
S.
,
Rao
,
M.
,
Miranda
,
C. J.
,
Meyer
,
K.
,
Tian
,
J.
,
Boulting
,
G.
,
Schaffer
,
D. V.
,
Zhu
,
M. X.
, et al. 
(
2011
).
Rapid and efficient generation of functional motor neurons from human pluripotent stem cells using gene delivered transcription factor codes
.
Mol. Ther.
19
,
1905
-
1912
.
Heuser
,
J. E.
,
Reese
,
T. S.
,
Dennis
,
M. J.
,
Jan
,
Y.
,
Jan
,
L.
and
Evans
,
L.
(
1979
).
Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release
.
J. Cell Biol.
81
,
275
-
300
.
Higuchi
,
O.
,
Hamuro
,
J.
,
Motomura
,
M.
and
Yamanashi
,
Y.
(
2011
).
Autoantibodies to low-density lipoprotein receptor-related protein 4 in myasthenia gravis
.
Ann. Neurol.
69
,
418
-
422
.
Hoch
,
W.
,
McConville
,
J.
,
Helms
,
S.
,
Newsom-Davis
,
J.
,
Melms
,
A.
and
Vincent
,
A.
(
2001
).
Auto-antibodies to the receptor tyrosine kinase MuSK in patients with myasthenia gravis without acetylcholine receptor antibodies
.
Nat. Med.
7
,
365
-
368
.
Inoue
,
A.
,
Setoguchi
,
K.
,
Matsubara
,
Y.
,
Okada
,
K.
,
Sato
,
N.
,
Iwakura
,
Y.
,
Higuchi
,
O.
and
Yamanashi
,
Y.
(
2009
).
Dok-7 activates the muscle receptor kinase MuSK and shapes synapse formation
.
Sci. Signal.
2
,
ra7
.
Ionescu
,
A.
,
Zahavi
,
E. E.
,
Gradus
,
T.
,
Ben-Yaakov
,
K.
and
Perlson
,
E.
(
2016
).
Compartmental microfluidic system for studying muscle-neuron communication and neuromuscular junction maintenance
.
Eur. J. Cell Biol.
95
,
69
-
88
.
Jacobson
,
C.
,
Cote
,
P. D.
,
Rossi
,
S. G.
,
Rotundo
,
R. L.
and
Carbonetto
,
S.
(
2001
).
The dystroglycan complex is necessary for stabilization of acetylcholine receptor clusters at neuromuscular junctions and formation of the synaptic basement membrane
.
J. Cell Biol.
152
,
435
-
450
.
James
,
D. W.
and
Tresman
,
R. L.
(
1968
).
De novo formation of neuro-muscular junctions in tissue culture
.
Nature
220
,
384
-
385
.
Johnson
,
D. D.
,
Bailey
,
S.
and
Wenger
,
B. S.
(
1981
).
Trophic activity of sheep sciatic nerve extracts in skeletal muscle cultures from normal and dystrophic chick embryos: failure of dystrophic muscle to respond
.
Exp. Neurol.
73
,
421
-
429
.
Jones
,
R. A.
,
Reich
,
C. D.
,
Dissanayake
,
K. N.
,
Kristmundsdottir
,
F.
,
Findlater
,
G. S.
,
Ribchester
,
R. R.
,
Simmen
,
M. W.
and
Gillingwater
,
T. H.
(
2016
).
NMJ-morph reveals principal components of synaptic morphology influencing structure-function relationships at the neuromuscular junction
.
Open Biol.
6
.
Jones
,
R. A.
,
Harrison
,
C.
,
Eaton
,
S. L.
,
Llavero Hurtado
,
M.
,
Graham
,
L. C.
,
Alkhammash
,
L.
,
Oladiran
,
O. A.
,
Gale
,
A.
,
Lamont
,
D. J.
,
Simpson
,
H.
, et al. 
(
2017
).
Cellular and Molecular Anatomy of the Human Neuromuscular Junction
.
Cell Rep.
21
,
2348
-
2356
.
Kidokoro
,
Y.
,
Anderson
,
M. J.
and
Gruener
,
R.
(
1980
).
Changes in synaptic potential properties during acetylcholine receptor accumulation and neurospecific interactions in Xenopus nerve-muscle cell culture
.
Dev. Biol.
78
,
464
-
483
.
Kim
,
N.
,
Stiegler
,
A. L.
,
Cameron
,
T. O.
,
Hallock
,
P. T.
,
Gomez
,
A. M.
,
Huang
,
J. H.
,
Hubbard
,
S. R.
,
Dustin
,
M. L.
and
Burden
,
S. J.
(
2008
).
Lrp4 is a receptor for Agrin and forms a complex with MuSK
.
Cell
135
,
334
-
342
.
Kim
,
J. H.
,
Kim
,
I.
,
Seol
,
Y. J.
,
Ko
,
I. K.
,
Yoo
,
J. J.
,
Atala
,
A.
and
Lee
,
S. J.
(
2020
).
Neural cell integration into 3D bioprinted skeletal muscle constructs accelerates restoration of muscle function
.
Nat. Commun.
11
,
1025
.
Ko
,
C. P.
and
Robitaille
,
R.
(
2015
).
Perisynaptic Schwann Cells at the Neuromuscular Synapse: Adaptable, Multitasking Glial Cells
.
Cold Spring Harb. Perspect Biol.
7
,
a020503
.
Kobayashi
,
T.
and
Askanas
,
V.
(
1985
).
Acetylcholine receptors and acetylcholinesterase accumulate at the nerve-muscle contacts of de novo grown human monolayer muscle cocultured with fetal rat spinal cord
.
Exp. Neurol.
88
,
327
-
335
.
Kobayashi
,
T.
,
Askanas
,
V.
and
Engel
,
W. K.
(
1987
).
Human muscle cultured in monolayer and cocultured with fetal rat spinal cord: importance of dorsal root ganglia for achieving successful functional innervation
.
J. Neurosci.
7
,
3131
-
3141
.
Kobayashi
,
T.
,
Askanas
,
V.
,
Saito
,
K.
,
Engel
,
W. K.
and
Ishikawa
,
K.
(
1990
).
Abnormalities of aneural and innervated cultured muscle fibers from patients with myotonic atrophy (dystrophy)
.
Arch. Neurol.
47
,
893
-
896
.
Koenen
,
M.
,
Peter
,
C.
,
Villarroel
,
A.
,
Witzemann
,
V.
and
Sakmann
,
B.
(
2005
).
Acetylcholine receptor channel subtype directs the innervation pattern of skeletal muscle
.
EMBO Rep.
6
,
570
-
576
.
Larkin
,
L. M.
,
Van der Meulen
,
J. H.
,
Dennis
,
R. G.
and
Kennedy
,
J. B.
(
2006
).
Functional evaluation of nerve-skeletal muscle constructs engineered in vitro. In Vitro Cell. Dev. Biol. Anim. 42, 75-82.
LaRochelle
,
W. J.
,
Witzemann
,
V.
,
Fiedler
,
W.
and
Froehner
,
S. C.
(
1990
).
Developmental expression of the 43K and 58K postsynaptic membrane proteins and nicotinic acetylcholine receptors in Torpedo electrocytes
.
J. Neurosci.
10
,
3460
-
3467
.
Leikina
,
E.
,
Mertts
,
M. V.
,
Kuznetsova
,
N.
and
Leikin
,
S.
(
2002
).
Type I collagen is thermally unstable at body temperature
.
Proc. Natl. Acad. Sci. USA
99
,
1314
-
1318
.
Lennon
,
V. A.
,
Kryzer
,
T. J.
,
Griesmann
,
G. E.
,
O'Suilleabhain
,
P. E.
,
Windebank
,
A. J.
,
Woppmann
,
A.
,
Miljanich
,
G. P.
and
Lambert
,
E. H.
(
1995
).
Calcium-channel antibodies in the Lambert-Eaton syndrome and other paraneoplastic syndromes
.
N. Engl. J. Med.
332
,
1467
-
1474
.
Li
,
X. J.
,
Du
,
Z. W.
,
Zarnowska
,
E. D.
,
Pankratz
,
M.
,
Hansen
,
L. O.
,
Pearce
,
R. A.
and
Zhang
,
S. C.
(
2005
).
Specification of motoneurons from human embryonic stem cells
.
Nat. Biotechnol.
23
,
215
-
221
.
Li
,
L.
,
Xiong
,
W. C.
and
Mei
,
L.
(
2018
).
Neuromuscular Junction Formation, Aging, and Disorders
.
Annu. Rev. Physiol.
80
,
159
-
188
.
Lin
,
W.
,
Burgess
,
R. W.
,
Dominguez
,
B.
,
Pfaff
,
S. L.
,
Sanes
,
J. R.
and
Lee
,
K. F.
(
2001
).
Distinct roles of nerve and muscle in postsynaptic differentiation of the neuromuscular synapse
.
Nature
410
,
1057
-
1064
.
Lin
,
C. Y.
,
Yoshida
,
M.
,
Li
,
L. T.
,
Ikenaka
,
A.
,
Oshima
,
S.
,
Nakagawa
,
K.
,
Sakurai
,
H.
,
Matsui
,
E.
,
Nakahata
,
T.
and
Saito
,
M. K.
(
2019
).
iPSC-derived functional human neuromuscular junctions model the pathophysiology of neuromuscular diseases. JCI Insight 4, e124299.
Lindstrom
,
J. M.
,
Seybold
,
M. E.
,
Lennon
,
V. A.
,
Whittingham
,
S.
and
Duane
,
D. D.
(
1976
).
Antibody to acetylcholine receptor in myasthenia gravis. Prevalence, clinical correlates, and diagnostic value
.
Neurology
26
,
1054
-
1059
.
Liu
,
Q.
,
Spusta
,
S. C.
,
Mi
,
R.
,
Lassiter
,
R. N.
,
Stark
,
M. R.
,
Hoke
,
A.
,
Rao
,
M. S.
and
Zeng
,
X.
(
2012
).
Human neural crest stem cells derived from human ESCs and induced pluripotent stem cells: induction, maintenance, and differentiation into functional schwann cells
.
Stem Cells Transl. Med.
1
,
266
-
278
.
Liu
,
M. L.
,
Zang
,
T.
and
Zhang
,
C. L.
(
2016
).
Direct Lineage Reprogramming Reveals Disease-Specific Phenotypes of Motor Neurons from Human ALS Patients
.
Cell Rep
14
,
115
-
128
.
Lukovic
,
D.
,
Diez Lloret
,
A.
,
Stojkovic
,
P.
,
Rodriguez-Martinez
,
D.
,
Perez Arago
,
M. A.
,
Rodriguez-Jimenez
,
F. J.
,
Gonzalez-Rodriguez
,
P.
,
Lopez-Barneo
,
J.
,
Sykova
,
E.
,
Jendelova
,
P.
, et al. 
(
2017
).
Highly efficient neural conversion of human pluripotent stem cells in adherent and animal-free conditions
.
Stem. Cells Transl. Med.
6
,
1217
-
1226
.
Machado
,
C. B.
,
Pluchon
,
P.
,
Harley
,
P.
,
Rigby
,
M.
,
Gonzalez Sabater
,
V.
,
Stevenson
,
D. C.
,
Hynes
,
S.
,
Lowe
,
A.
,
Burrone
,
J.
,
Viasnoff
,
V.
, et al. 
(
2019
).
In vitro modelling of nerve-muscle connectivity in a compartmentalised tissue culture device
.
Adv. Biosys.
3
.
Maffioletti
,
S. M.
,
Sarcar
,
S.
,
Henderson
,
A. B. H.
,
Mannhardt
,
I.
,
Pinton
,
L.
,
Moyle
,
L. A.
,
Steele-Stallard
,
H.
,
Cappellari
,
O.
,
Wells
,
K. E.
,
Ferrari
,
G.
, et al. 
(
2018
).
Three-dimensional human iPSC-derived artificial skeletal muscles model muscular dystrophies and enable multilineage tissue engineering
.
Cell Rep
23
,
899
-
908
.
Marangi
,
P. A.
,
Forsayeth
,
J. R.
,
Mittaud
,
P.
,
Erb-Vogtli
,
S.
,
Blake
,
D. J.
,
Moransard
,
M.
,
Sander
,
A.
and
Fuhrer
,
C.
(
2001
).
Acetylcholine receptors are required for agrin-induced clustering of postsynaptic proteins
.
EMBO J.
20
,
7060
-
7073
.
Marchand
,
S.
,
Devillers-Thiery
,
A.
,
Pons
,
S.
,
Changeux
,
J. P.
and
Cartaud
,
J.
(
2002
).
Rapsyn escorts the nicotinic acetylcholine receptor along the exocytic pathway via association with lipid rafts
.
J. Neurosci.
22
,
8891
-
8901
.
Marteyn
,
A.
,
Maury
,
Y.
,
Gauthier
,
M. M.
,
Lecuyer
,
C.
,
Vernet
,
R.
,
Denis
,
J. A.
,
Pietu
,
G.
,
Peschanski
,
M.
and
Martinat
,
C.
(
2011
).
Mutant human embryonic stem cells reveal neurite and synapse formation defects in type 1 myotonic dystrophy
.
Cell Stem Cell
8
,
434
-
444
.
Martinuzzi
,
A.
,
Vergani
,
L.
,
Carrozzo
,
R.
,
Fanin
,
M.
,
Bartoloni
,
L.
,
Angelini
,
C.
,
Askanas
,
V.
and
Engel
,
W. K.
(
1993
).
Expression of muscle-type phosphorylase in innervated and aneural cultured muscle of patients with myophosphorylase deficiency
.
J. Clin. Invest.
92
,
1774
-
1780
.
Maselli
,
R. A.
,
Arredondo
,
J.
,
Ferns
,
M. J.
and
Wollmann
,
R. L.
(
2012
).
Synaptic basal lamina-associated congenital myasthenic syndromes
.
Ann. N. Y. Acad. Sci.
1275
,
36
-
48
.
Maury
,
Y.
,
Come
,
J.
,
Piskorowski
,
R. A.
,
Salah-Mohellibi
,
N.
,
Chevaleyre
,
V.
,
Peschanski
,
M.
,
Martinat
,
C.
and
Nedelec
,
S.
(
2015
).
Combinatorial analysis of developmental cues efficiently converts human pluripotent stem cells into multiple neuronal subtypes
.
Nat. Biotechnol.
33
,
89
-
96
.
Mazaleyrat
,
K.
,
Badja
,
C.
,
Broucqsault
,
N.
,
Chevalier
,
R.
,
Laberthonniere
,
C.
,
Dion
,
C.
,
Baldasseroni
,
L.
,
El-Yazidi
,
C.
,
Thomas
,
M.
,
Bachelier
,
R.
, et al. 
(
2020
).
Multilineage differentiation for formation of innervated skeletal muscle fibers from healthy and diseased human pluripotent stem cells
.
Cells
9
.
Messeant
,
J.
,
Ezan
,
J.
,
Delers
,
P.
,
Glebov
,
K.
,
Marchiol
,
C.
,
Lager
,
F.
,
Renault
,
G.
,
Tissir
,
F.
,
Montcouquiol
,
M.
,
Sans
,
N.
, et al. 
(
2017
).
Wnt proteins contribute to neuromuscular junction formation through distinct signaling pathways
.
Development
144
,
1712
-
1724
.
Michikawa
,
M.
,
Kobayashi
,
T.
and
Tsukagoshi
,
H.
(
1991
).
Early events of chemical transmission of newly formed neuromuscular junctions in monolayers of human muscle cells co-cultured with fetal rat spinal cord explants. Brain Res. 538, 79-85.
Moransard
,
M.
,
Borges
,
L. S.
,
Willmann
,
R.
,
Marangi
,
P. A.
,
Brenner
,
H. R.
,
Ferns
,
M. J.
and
Fuhrer
,
C.
(
2003
).
Agrin regulates rapsyn interaction with surface acetylcholine receptors, and this underlies cytoskeletal anchoring and clustering
.
J. Biol. Chem.
278
,
7350
-
7359
.
Nakajima
,
Y.
,
Kidokoro
,
Y.
and
Klier
,
F. G.
(
1980
).
The development of functional neuromuscular junctions in vitro: an ultrastructural and physiological study
.
Dev. Biol.
77
,
52
-
72
.
Nelson
,
P. G.
,
Fields
,
R. D.
,
Yu
,
C.
and
Liu
,
Y.
(
1993
).
Synapse elimination from the mouse neuromuscular junction in vitro: a non-Hebbian activity-dependent process
.
J. Neurobiol.
24
,
1517
-
1530
.
Nicole
,
S.
,
Chaouch
,
A.
,
Torbergsen
,
T.
,
Bauche
,
S.
,
de Bruyckere
,
E.
,
Fontenille
,
M. J.
,
Horn
,
M. A.
,
van Ghelue
,
M.
,
Loseth
,
S.
,
Issop
,
Y.
, et al. 
(
2014
).
Agrin mutations lead to a congenital myasthenic syndrome with distal muscle weakness and atrophy
.
Brain
137
,
2429
-
2443
.
Nikolic
,
N.
,
Gorgens
,
S. W.
,
Thoresen
,
G. H.
,
Aas
,
V.
,
Eckel
,
J.
and
Eckardt
,
K.
(
2017
).
Electrical pulse stimulation of cultured skeletal muscle cells as a model for in vitro exercise - possibilities and limitations
.
Acta Physiol. (Oxf)
220
,
310
-
331
.
Nishimune
,
H.
,
Sanes
,
J. R.
and
Carlson
,
S. S.
(
2004
).
A synaptic laminin-calcium channel interaction organizes active zones in motor nerve terminals
.
Nature
432
,
580
-
587
.
Ohkawara
,
B.
,
Cabrera-Serrano
,
M.
,
Nakata
,
T.
,
Milone
,
M.
,
Asai
,
N.
,
Ito
,
K.
,
Ito
,
M.
,
Masuda
,
A.
,
Ito
,
Y.
,
Engel
,
A. G.
, et al. 
(
2014
).
LRP4 third beta-propeller domain mutations cause novel congenital myasthenia by compromising agrin-mediated MuSK signaling in a position-specific manner
.
Hum. Mol. Genet.
23
,
1856
-
1868
.
Okada
,
K.
,
Inoue
,
A.
,
Okada
,
M.
,
Murata
,
Y.
,
Kakuta
,
S.
,
Jigami
,
T.
,
Kubo
,
S.
,
Shiraishi
,
H.
,
Eguchi
,
K.
,
Motomura
,
M.
, et al. 
(
2006
).
The muscle protein Dok-7 is essential for neuromuscular synaptogenesis
.
Science
312
,
1802
-
1805
.
Ono
,
F.
,
Mandel
,
G.
and
Brehm
,
P.
(
2004
).
Acetylcholine receptors direct rapsyn clusters to the neuromuscular synapse in zebrafish
.
J. Neurosci.
24
,
5475
-
5481
.
Osaki
,
T.
,
Uzel
,
S. G. M.
and
Kamm
,
R. D.
(
2018
).
Microphysiological 3D model of amyotrophic lateral sclerosis (ALS) from human iPS-derived muscle cells and optogenetic motor neurons
.
Sci. Adv.
4
,
eaat5847
.
Park-Matsumoto
,
Y. C.
,
Kameda
,
N.
,
Kobayashi
,
T.
and
Tsukagoshi
,
H.
(
1991
).
Developmental study of the expression of dystrophin in cultured human muscle aneurally and innervated with fetal rat spinal cord
.
Brain Res.
565
,
280
-
289
.
Peng
,
H. B.
and
Nakajima
,
Y.
(
1978
).
Membrane particle aggregates in innervated and noninnervated cultures of Xenopus embryonic muscle cells
.
Proc. Natl. Acad. Sci. USA
75
,
500
-
504
.
Peng
,
H. B.
,
Ali
,
A. A.
,
Daggett
,
D. F.
,
Rauvala
,
H.
,
Hassell
,
J. R.
and
Smalheiser
,
N. R.
(
1998
).
The relationship between perlecan and dystroglycan and its implication in the formation of the neuromuscular junction
.
Cell Adhes Commun
5
,
475
-
489
.
Peterson
,
E. R.
,
Masurovsky
,
E. B.
,
Spiro
,
A. J.
and
Crain
,
S. M.
(
1986
).
Duchenne dystrophic muscle develops lesions in long-term coculture with mouse spinal cord
.
Muscle Nerve
9
,
787
-
808
.
Picchiarelli
,
G.
,
Demestre
,
M.
,
Zuko
,
A.
,
Been
,
M.
,
Higelin
,
J.
,
Dieterle
,
S.
,
Goy
,
M. A.
,
Mallik
,
M.
,
Sellier
,
C.
,
Scekic-Zahirovic
,
J.
, et al. 
(
2019
).
FUS-mediated regulation of acetylcholine receptor transcription at neuromuscular junctions is compromised in amyotrophic lateral sclerosis
.
Nat. Neurosci.
22
,
1793
-
1805
.
Pourquie
,
O.
,
Al Tanoury
,
Z.
and
Chal
,
J.
(
2018
).
The Long Road to Making Muscle In Vitro
.
Curr. Top. Dev. Biol.
129
,
123
-
142
.
Puro
,
D. G.
,
De Mello
,
F. G.
and
Nirenberg
,
M.
(
1977
).
Synapse turnover: the formation and termination of transient synapses
.
Proc. Natl. Acad. Sci. USA
74
,
4977
-
4981
.
Puttonen
,
K. A.
,
Ruponen
,
M.
,
Naumenko
,
N.
,
Hovatta
,
O. H.
,
Tavi
,
P.
and
Koistinaho
,
J.
(
2015
).
Generation of functional neuromuscular junctions from human pluripotent stem cell lines
.
Front. Cell Neurosci.
9
,
473
.
Rogers
,
R. S.
and
Nishimune
,
H.
(
2017
).
The role of laminins in the organization and function of neuromuscular junctions
.
Matrix Biol.
57-58
,
86
-
105
.
Ruegg
,
M. A.
and
Bixby
,
J. L.
(
1998
).
Agrin orchestrates synaptic differentiation at the vertebrate neuromuscular junction
.
Trends Neurosci.
21
,
22
-
27
.
Sanes
,
J. R.
and
Lichtman
,
J. W.
(
1999
).
Development of the vertebrate neuromuscular junction
.
Annu. Rev. Neurosci.
22
,
389
-
442
.
Sanes
,
J. R.
and
Lichtman
,
J. W.
(
2001
).
Induction, assembly, maturation and maintenance of a postsynaptic apparatus
.
Nat. Rev. Neurosci.
2
,
791
-
805
.
Santhanam
,
N.
,
Kumanchik
,
L.
,
Guo
,
X.
,
Sommerhage
,
F.
,
Cai
,
Y.
,
Jackson
,
M.
,
Martin
,
C.
,
Saad
,
G.
,
McAleer
,
C. W.
,
Wang
,
Y.
, et al. 
(
2018
).
Stem cell derived phenotypic human neuromuscular junction model for dose response evaluation of therapeutics
.
Biomaterials
166
,
64
-
78
.
Scott
,
B. S.
,
Engelbert
,
V. E.
and
Fisher
,
K. C.
(
1969
).
Morphological and electrophysiological characteristics of dissociated chick embryonic spinal ganglion cells in culture
.
Exp. Neurol.
23
,
230
-
248
.
Shimojo
,
D.
,
Onodera
,
K.
,
Doi-Torii
,
Y.
,
Ishihara
,
Y.
,
Hattori
,
C.
,
Miwa
,
Y.
,
Tanaka
,
S.
,
Okada
,
R.
,
Ohyama
,
M.
,
Shoji
,
M.
, et al. 
(
2015
).
Rapid, efficient, and simple motor neuron differentiation from human pluripotent stem cells
.
Mol Brain
8
,
79
.
Sigoillot
,
S. M.
,
Bourgeois
,
F.
,
Karmouch
,
J.
,
Molgo
,
J.
,
Dobbertin
,
A.
,
Chevalier
,
C.
,
Houlgatte
,
R.
,
Leger
,
J.
and
Legay
,
C.
(
2016
).
Neuromuscular junction immaturity and muscle atrophy are hallmarks of the ColQ-deficient mouse, a model of congenital myasthenic syndrome with acetylcholinesterase deficiency
.
FASEB J.
30
,
2382
-
2399
.
Silver
,
R. A.
,
Lubke
,
J.
,
Sakmann
,
B.
and
Feldmeyer
,
D.
(
2003
).
High-probability uniquantal transmission at excitatory synapses in barrel cortex
.
Science
302
,
1981
-
1984
.
Roy N.
,
S.
,
Nakano
,
T.
,
Xuing
,
L.
,
Kang
,
J.
,
Nedergaard
,
M.
and
Goldman
,
S. A.
(
2005
).
Enhancer-specified GFP-based FACS purification of human spinal motor neurons from embryonic stem cells
.
Exp. Neurol.
196
,
224
-
234
.
Slater
,
C. R.
(
2017
).
The structure of human neuromuscular junctions: some unanswered molecular questions
.
Int. J. Mol. Sci.
 
18
.
Sobel
,
A.
,
Heidmann
,
T.
,
Hofler
,
J.
and
Changeux
,
J. P.
(
1978
).
Distinct protein components from Torpedo marmorata membranes carry the acetylcholine receptor site and the binding site for local anesthetics and histrionicotoxin
.
Proc. Natl. Acad. Sci. USA
75
,
510
-
514
.
Steinbeck
,
J. A.
,
Jaiswal
,
M. K.
,
Calder
,
E. L.
,
Kishinevsky
,
S.
,
Weishaupt
,
A.
,
Toyka
,
K. V.
,
Goldstein
,
P. A.
and
Studer
,
L.
(
2016
).
Functional connectivity under optogenetic control allows modeling of human neuromuscular disease
.
Cell Stem Cell
18
,
134
-
143
.
Swenarchuk
,
L. E.
,
Champaneria
,
S.
and
Anderson
,
M. J.
(
1990
).
Induction of a specialized muscle basal lamina at chimaeric synapses in culture
.
Development
110
,
51
-
61
.
Takahashi
,
M.
,
Kubo
,
T.
,
Mizoguchi
,
A.
,
Carlson
,
C. G.
,
Endo
,
K.
and
Ohnishi
,
K.
(
2002
).
Spontaneous muscle action potentials fail to develop without fetal-type acetylcholine receptors
.
EMBO Rep.
3
,
674
-
681
.
Umbach
,
J. A.
,
Adams
,
K. L.
,
Gundersen
,
C. B.
and
Novitch
,
B. G.
(
2012
).
Functional neuromuscular junctions formed by embryonic stem cell-derived motor neurons
.
PLoS ONE
7
,
e36049
.
Uzel
,
S. G.
,
Platt
,
R. J.
,
Subramanian
,
V.
,
Pearl
,
T. M.
,
Rowlands
,
C. J.
,
Chan
,
V.
,
Boyer
,
L. A.
,
So
,
P. T.
and
Kamm
,
R. D.
(
2016
).
Microfluidic device for the formation of optically excitable, three-dimensional, compartmentalized motor units
.
Sci. Adv.
2
,
e1501429
.
Verschuuren
,
J.
,
Strijbos
,
E.
and
Vincent
,
A.
(
2016
).
Neuromuscular junction disorders
.
Handb. Clin. Neurol.
133
,
447
-
466
.
Vila
,
O. F.
,
Uzel
,
S. G. M.
,
Ma
,
S. P.
,
Williams
,
D.
,
Pak
,
J.
,
Kamm
,
R. D.
and
Vunjak-Novakovic
,
G.
(
2019
).
Quantification of human neuromuscular function through optogenetics
.
Theranostics
9
,
1232
-
1246
.
Vilmont
,
V.
,
Cadot
,
B.
,
Ouanounou
,
G.
and
Gomes
,
E. R.
(
2016
).
A system for studying mechanisms of neuromuscular junction development and maintenance
.
Development
143
,
2464
-
2477
.
Wallace
,
B. G.
(
1989
).
Agrin-induced specializations contain cytoplasmic, membrane, and extracellular matrix-associated components of the postsynaptic apparatus
.
J. Neurosci.
9
,
1294
-
1302
.
Wichterle
,
H.
,
Lieberam
,
I.
,
Porter
,
J. A.
and
Jessell
,
T. M.
(
2002
).
Directed differentiation of embryonic stem cells into motor neurons
.
Cell
110
,
385
-
397
.
Witkowski
,
J. A.
and
Dubowitz
,
V.
(
1975
).
Growth of diseased human muscle in combined cultures with normal mouse embryonic spinal cord. J. Neurol. Sci. 26, 203-220.
Wu
,
X.
,
Chen
,
X.
and
Huang
,
B.
(
2005
).
[Relationship between fibrinogen content and the protracted inflammation of secretory otitis media]
.
Lin Chuang Er Bi Yan Hou Ke Za Zhi
19
,
449
-
450
.
Wu
,
H.
,
Lu
,
Y.
,
Shen
,
C.
,
Patel
,
N.
,
Gan
,
L.
,
Xiong
,
W. C.
and
Mei
,
L.
(
2012
).
Distinct roles of muscle and motoneuron LRP4 in neuromuscular junction formation
.
Neuron
75
,
94
-
107
.
Yaffe
,
D.
and
Saxel
,
O.
(
1977
).
Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle
.
Nature
270
,
725
-
727
.
Yang
,
X.
,
Arber
,
S.
,
William
,
C.
,
Li
,
L.
,
Tanabe
,
Y.
,
Jessell
,
T. M.
,
Birchmeier
,
C.
and
Burden
,
S. J.
(
2001
).
Patterning of muscle acetylcholine receptor gene expression in the absence of motor innervation
.
Neuron
30
,
399
-
410
.
Yoshida
,
M.
,
Kitaoka
,
S.
,
Egawa
,
N.
,
Yamane
,
M.
,
Ikeda
,
R.
,
Tsukita
,
K.
,
Amano
,
N.
,
Watanabe
,
A.
,
Morimoto
,
M.
,
Takahashi
,
J.
, et al. 
(
2015
).
Modeling the early phenotype at the neuromuscular junction of spinal muscular atrophy using patient-derived iPSCs
.
Stem Cell Reports
4
,
561
-
568
.
Yumoto
,
N.
,
Kim
,
N.
and
Burden
,
S. J.
(
2012
).
Lrp4 is a retrograde signal for presynaptic differentiation at neuromuscular synapses
.
Nature
489
,
438
-
442
.
Zahavi
,
E. E.
,
Ionescu
,
A.
,
Gluska
,
S.
,
Gradus
,
T.
,
Ben-Yaakov
,
K.
and
Perlson
,
E.
(
2015
).
A compartmentalized microfluidic neuromuscular co-culture system reveals spatial aspects of GDNF functions
.
J. Cell Sci.
128
,
1241
-
1252
.
Zainul
,
Z.
,
Heikkinen
,
A.
,
Koivisto
,
H.
,
Rautalahti
,
I.
,
Kallio
,
M.
,
Lin
,
S.
,
Haronen
,
H.
,
Norman
,
O.
,
Ruegg
,
M. A.
,
Tanila
,
H.
 et al.  (
2018
).
Collagen XIII is required for neuromuscular synapse regeneration and functional recovery after peripheral nerve injury
.
J. Neurosci.
38
,
4243
-
4258
.
Zhang
,
B.
,
Luo
,
S.
,
Wang
,
Q.
,
Suzuki
,
T.
,
Xiong
,
W. C.
and
Mei
,
L.
(
2008
).
LRP4 serves as a coreceptor of agrin
.
Neuron
60
,
285
-
297
.
Ziegler
,
L.
,
Grigoryan
,
S.
,
Yang
,
I. H.
,
Thakor
,
N. V.
and
Goldstein
,
R. S.
(
2011
).
Efficient generation of schwann cells from human embryonic stem cell-derived neurospheres
.
Stem Cell Rev. Rep.
7
,
394
-
403
.
Zong
,
Y.
,
Zhang
,
B.
,
Gu
,
S.
,
Lee
,
K.
,
Zhou
,
J.
,
Yao
,
G.
,
Figueiredo
,
D.
,
Perry
,
K.
,
Mei
,
L.
and
Jin
,
R.
(
2012
).
Structural basis of agrin-LRP4-MuSK signaling
.
Genes Dev.
26
,
247
-
258
.

Competing interests

The authors declare no competing or financial interests.