REVIEW

Neuromuscular disease modeling on a chipJeffrey W. Santoso1 and Megan L. McCain1,2,*

ABSTRACTOrgans-on-chips are broadly defined as microfabricated surfaces ordevices designed to engineer cells into microscale tissues with native-like features and then extract physiologically relevant readouts at scale.Because they are generally compatible with patient-derived cells, thesetechnologies can address many of the human relevance limitations ofanimal models. As a result, organs-on-chips have emerged as apromising new paradigm for patient-specific diseasemodeling and drugdevelopment. Because neuromuscular diseases span a broad range ofrare conditions with diverse etiology and complex pathophysiology, theyhave been especially challenging to model in animals and thus are wellsuited for organ-on-chip approaches. In this Review, we first brieflysummarize the challenges in neuromuscular disease modeling withanimal models. Next, we describe a variety of existing organ-on-chipapproaches for neuromuscular tissues, including a survey of cellsources for both muscle and nerve, and two- and three-dimensionalneuromuscular tissue-engineering techniques. Although researchershavemade tremendous advances inmodeling neuromuscular diseaseson a chip, the remaining challenges in cell sourcing, cell maturity, tissueassembly and readout capabilities limit their integration into the drugdevelopment pipeline today. However, as the field advances, models ofhealthy and diseased neuromuscular tissues on a chip, coupled withanimal models, have vast potential as complementary tools formodeling multiple aspects of neuromuscular diseases and identifyingnew therapeutic strategies.

KEY WORDS: Skeletal muscle, Motor neurons, Amyotrophic lateralsclerosis, Induced pluripotent stem cells, Tissue engineering,Microfluidic devices

IntroductionNeuromuscular diseases collectively affect 160 per 100,000 peopleworldwide and are generally characterized by progressive motorimpairment and muscular atrophy (Deenen et al., 2015). Althoughthese conditions have diverse etiologies, they each affect one ormore components of the motor unit (see Box 1, Fig. 1). For decades,animal models, especially humanized mice (De Giorgio et al., 2019;Nair et al., 2019; Aartsma-Rus and van Putten, 2020), have been thegold standard for neuromuscular disease modeling. More recently,non-mammalian models, such as fruit flies (Lloyd and Taylor,2010), Caenorhabditis elegans (Sleigh and Sattelle, 2010) andzebrafish (Babin et al., 2014), have also been used for

neuromuscular disease modeling. Although these simpler modelsare limited by their lower conservation with human genetics,anatomy and physiology compared to mice, they are beneficialbecause of their lower cost, rapid growth rate, tractable anatomy andease of genetic manipulation. In general, animal models captureimportant hallmarks of their human disease counterparts and thusare invaluable for understanding disease progression on an organ-and organism-level scale. However, disease phenotypes in animalscan vary widely from humans in terms of progression, severity andother characteristics (De Giorgio et al., 2019; Aartsma-Rus and vanPutten, 2020; Babin et al., 2014).

Another limitation of animal models is that it is difficult, if notimpossible, to recapitulate the genotypic heterogeneity and allelicvariation observed in individuals with neuromuscular diseases withoutgenerating an unreasonable number of animal strains (Juneja et al.,2019; Morrice et al., 2018). Even monogenic neuromuscular diseases,such as spinal muscular atrophy (SMA), are difficult to model inanimals due to patient-specific genotypic features. SMA is anautosomal recessive disease caused by inactivating mutations in theSMN1 gene, which encodes the survival of motor neuron (SMN)protein (Li, 2017). SMN plays a role in protein homeostasis,cytoskeletal assembly, endocytosis, metabolism and many otherprocesses in motor neurons (Chaytow et al., 2018). SMN shortageor dysfunction causes deficits in axonogenesis, migration,electrophysiology and many other features, leading to neuromuscularjunction (NMJ) degeneration and motor neuron death (Laird et al.,2016; McGovern et al., 2015). A second gene, SMN2, also producesSMN, but at ∼20% of the levels transcribed from fully functionalSMN1 (Bowerman et al., 2017; Jedrzejowska et al., 2009). SMA hasbeenmodeled inmice (Hsieh-Li et al., 2000),Drosophila (Spring et al.,2019), zebrafish (McWhorter et al., 2003) andC. elegans (Briese et al.,2009) by deleting the endogenous Smn gene and overexpressing thehuman SMN2 gene. However, the severity and progression of SMAlargely depends on the number of SMN2 copies in a patient(Bowerman et al., 2017; Jedrzejowska et al., 2009), a patient-specificfeature of the disease that is nearly impossible to faithfully recapitulatein animals. The only treatment options for SMA are the gene therapydrugs Spinraza (Dangouloff and Servais, 2019) and Zolgensma(Zuroske, 2019), both of which are extremely expensive and thusimpractical for many individuals.

Compared to SMA, several neuromuscular diseases have a moreheterogeneous genetic etiology, which is even more challenging tomodel in animals. For example, Charcot-Marie-Tooth (CMT)diseases have been linked to 870 mutations in over 80 genes(McCorquodale et al., 2016), such as PMP22,MPZ, GJB1 orMFN2(Morena et al., 2019; Saporta et al., 2011). This genetic heterogeneitypartially explains the wide range of age of onset and diseasesymptoms, which usually involve involuntary contraction of limbsand loss of sensation due to axon demyelination. CMT has beenmodeled in zebrafish and other animal models by introducing amutation in a single gene known to cause a specific subtype of CMTdisease, such as mfn2 (Chapman et al., 2013) or prps1 (Pei et al.,2016). However, owing to the vast genetic heterogeneity of CMT

1Laboratory for Living Systems Engineering, Department of BiomedicalEngineering, USC Viterbi School of Engineering, University of Southern California,Los Angeles, CA 90089, USA. 2Department of Stem Cell Biology and RegenerativeMedicine, Keck School of Medicine of USC, University of Southern California, LosAngeles, CA 90033, USA.

*Author for correspondence (mlmccain@usc.edu)

M.L.M., 0000-0003-1908-6783

This is an Open Access article distributed under the terms of the Creative Commons AttributionLicense (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use,distribution and reproduction in any medium provided that the original work is properly attributed.

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diseases, it is infeasible to generate animal models that represent allmutations (Juneja et al., 2019). Largely due to a lack of modularmodel systems, CMT diseases still lack clinical data supporting anyeffective treatment beyond physical therapy and pain management(McCorquodale et al., 2016). Mouse models of CMT have alsodemonstrated that impaired development of the NMJ precedessynaptic deficits (Sleigh et al., 2013; Spaulding et al., 2016),suggesting that microscale models of the motor unit might be usefulfor elucidating the pathophysiology of this broad group of diseases.Amyotrophic lateral sclerosis (ALS) is another neuromuscular

disease that introduces unique challenges for modeling in animals

because it can be either inherited (10%) or sporadic (90%) (Boylan,2015). In ALS, over 50 genes either directly cause motor neurondeath or alter key functions, such as vesicle trafficking, axonalstructure and cytoskeletal stability (Boylan, 2015; Seminary et al.,2018; Shi et al., 2018). The most commonly affected genes areC9ORF72, SOD1, TARDBP and FUS, usually occurring in somekind of combination (Lattante et al., 2015; Nguyen et al., 2018).Animal models of ALS have been generated by expressing amutated version of one of these human genes in mice (Ripps et al.,1995; Zhang et al., 1997; Devoy et al., 2017), Drosophila (Sahinet al., 2017; Watson et al., 2008; Perry et al., 2017; Xu et al., 2013),zebrafish (Shaw et al., 2018; Lissouba et al., 2018) and C. elegans(Oeda et al., 2001; Wang et al., 2009). Additionally, environmentalfactors, such as pesticides, flame retardants and military-relatedtrauma, have been correlated to ALS (Su et al., 2016). However, thesmall number of clinical cases and limited model systems makeassigning causality from environmental factors very difficult. Thenatural process of aging has also been tied to ALS, probably becauseof the aggregation of misfolded proteins and oxidative stress (Jangand Van Remmen, 2011; Turner et al., 2012). Owing, in large part,to the many causes and complex pathophysiologies of ALS, theonly therapies are the anti-glutamatergic compound riluzole and theantioxidant edaravone, both of which only assuage symptoms andextend survival for a few months (Nguyen et al., 2018).

Neuromuscular diseases can also be caused by factors external tothe motor unit. For example, myasthenia gravis (MG) is a sporadicautoimmune disease in which auto-antibodies selectively destroyacetylcholine receptors, causing a reduction in NMJ signaltransmission (Phillips and Vincent, 2016). A mouse model of MGhas been developed by injecting rat acetylcholine receptors intomice, which then triggered the development of auto-antibodies totheir own acetylcholine receptors (Granato et al., 1976). However,MG cannot be modeled in simple organisms such asDrosophila andC. elegans because they lack an adaptive immune system. Thecauses of MG are still mostly unknown and treatment is limited toacetylcholinesterase inhibitors, immunosuppressants or athymectomy (Gilhus, 2016), highlighting the need for additionalpredictive model systems to develop more targeted therapies.

Collectively, these examples highlight that neuromuscular diseasesare very diverse and are characterized by many complex genetic andnon-genetic etiologies and pathophysiologies. These complexitiesintroduce many challenges for developing comprehensive animalmodels. Thus, new disease models that are more efficient and predictiveare essential for accelerating our mechanistic knowledge of thesediseases, as well as the discovery of effective therapies. Integratingpatient-derived cells with microfabricated in vitro platforms, known asorgans-on-chips, is an emerging solution to fill the gaps of animalmodels and holds promise for patient-specific neuromuscular diseasemodeling and drug development. As discussed below, these platformsare often developed using animal cells or cell lines that are easy to scale,and that can provide important proof-of-concept and basic physiologicalinformation. To address issues of human relevance, animal cells or celllines can then be replaced with patient-derived cells, which can beacquired from a variety of sources. In the next section, we describe thecell sources for neuromuscular disease models, which can ultimately beintegrated into the two- (2D) and three-dimensional (3D) engineeredtissue platforms described in the following sections.

Cell sources for in vitro models of neuromuscular tissuesIn vitro models can mitigate many of the limitations of animalmodels described above, such as human relevance and scalability.However, the usefulness of any in vitro model is highly dependent

Box 1. Structure and physiology of the motor unitAll voluntary movements are controlled by a collection of motor units,each of which comprises a single motor neuron and all the muscle fibersthat it innervates (Fig. 1). Motor neurons have a soma that resides in themotor cortex, brain stem or spinal cord, and a single myelinated axon thatforms specialized synapses, known as neuromuscular junctions (NMJs),on muscle fibers. Muscle fibers are elongated multi-nucleated cells thatare packed with myofibrils, each of which is an interconnected chain ofcontractile sarcomere units. Multiple muscle fibers are bundled togetherand wrapped in connective tissue to form a muscle.Contraction of a motor unit begins when signals from the central nervoussystem trigger an action potential in the motor neuron, which induces theaxon to release the neurotransmitter acetylcholine into the synaptic cleftof the NMJ. Acetylcholine binds to acetylcholine receptors on themembrane of the muscle fiber, which depolarizes the membrane andinitiates an action potential. The muscle fiber then propagates this actionpotential along its length, triggering the entry of extracellular calciumthrough voltage-sensitive ion channels in the membrane andsubsequently a large release of calcium from the sarcoplasmicreticulum. This increase in cytosolic calcium enables the heads ofmyosin filaments to pull on actin filaments, shortening the sarcomere andultimately contracting the muscle fiber in an ATP-demanding process.Depending on the frequency of the action potential transmitted by themotor neuron, the muscle fiber undergoes either a singular or sustainedcontraction, referred to as twitch or tetanus, respectively. Lastly, the freeacetylcholine in the NMJ is broken down by acetylcholinesterase,cytosolic calcium is transported back into the sarcoplasmic reticulum,and the membrane potential of the muscle fiber returns to resting levels,thus causing muscle relaxation (reviewed by Hall and Hall, 2015).

Collagen

Laminin

Acetylcholine vesicle

Acetylcholine receptor

Muscle fiber

Key

Motor neuron

Neuromuscular junction

Fig. 1. Schematic of the neuromuscular junction. Multi-nucleated musclefibers are innervated by myelinated motor neurons at neuromuscular junctions(NMJs). At the NMJ, motor neurons release acetylcholine vesicles. Theneurotransmitter acetylcholine binds to acetylcholine receptors on the membraneof the muscle fiber, causing membrane depolarization and muscle contraction.

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on the source and structural and functional maturity of its cells. Thisis especially complex when modeling neuromuscular tissues, whichconsist of both muscle cells and motor neurons. In this section, wewill describe the types of muscle and motor neuron cell typesavailable today for in vitro models, and weigh up their advantagesand disadvantages.

Skeletal muscle cellsGenerating skeletal muscle tissue in vitro is amulti-step process. First,mononuclear skeletal myoblasts are seeded on a standard culturesurface that is often coated with extracellular matrix (ECM) proteins,such as collagen or laminin. The myoblasts are then expanded in ahigh-serum growthmedium until they reach confluence. Themediumis then substituted with a low-serum differentiation medium thattriggers the fusion of myoblasts into multi-nucleated myotubes, the invitro surrogate to muscle fibers (Neville et al., 1997). As illustrated inFig. 2, several different sources of myoblasts are currently available,each with distinct advantages and disadvantages that are important toconsider when engineering neuromuscular disease models.Primary myoblasts are harvested from embryonic or adult animals,

such as chicks (Urja et al., 2018; Vallette et al., 1986) or mice (Hindiet al., 2017), by excising muscle tissue and either enzymaticallydigesting it to a cell suspension or collecting the cells that migrate fromcultured tissue explants (Vaughan and Lamia, 2019). Myoblasts are

then purified using simple pre-plating steps or more sophisticatedtechniques, such as magnetic cell sorting (Sincennes et al., 2017;Spinazzola and Gussoni, 2017). Human primary myoblasts can beisolated from muscle tissue collected during a surgical procedure or byneedle biopsy (Joyce et al., 2012), and similarly processed and purified.

The structural and functional properties of myotubes differentiatedfrom primary myoblasts closely recapitulate those of native muscle,such as a high density of myofibrils and spontaneous contractilebehavior (Pimentel et al., 2017). However, primary myoblasts canonly be passaged a few times before their growth rate and myogeniccapacity decline, leading to limited supply and passage-dependentvariability. These supply and variability issues are especiallyproblematic for human primary myoblasts, which are generallyisolated from relatively small muscle biopsies. Moreover, primarymyoblasts can vary widely in purity and functional maturitydepending on the isolation and purification methods, and thecharacteristics of the subject (Cheng et al., 2014; Soriano-Arroquiaet al., 2017). This issue is further exacerbated by data indicating abeneficial role for fibroblasts in myotube function (Rao et al., 2013),raising questions about the ideal purity of primary myoblasts for invitro studies and adding further variability to the performance ofmyotubes differentiated from primary myoblasts.

Compared to primary myoblasts, immortalized myoblast cell linesare a more convenient source of cells that are relatively pure and easy

Immortalized cell lines

Pluripotent stem cell derivatives

Immortalized myoblasts

Skeletal muscle Motor neurons

Mousemotor neuron

Mouseneuroblastoma

NSC-34

Reprogrammingfactors

hiPSCs

hESCs

Spontaneousmutation or

viral transduction

Hybridization

Primary cells

Human(muscle only)

Mouse Chick

Fig. 2. Muscle and motor neuron cell sources for in vitro models. Myoblasts and motor neurons available for in vitro models fall into three categories:immortalized cell lines, primary cells and pluripotent stem cell derivatives. Immortalized cell lines recapitulate the basic properties of the original cell type and areinexpensive and easy to expand in culture. However, the immortalization process causes de-differentiation and a loss of important structural and functionalfeatures. Primary cells are usually the most mature and physiologically relevant cell source but are relatively costly and difficult to obtain, and have a limited abilityto expand in culture. Pluripotent stem cells can be widely expanded in culture and then differentiated into myoblasts or motor neurons. Induced pluripotent stemcells have the additional advantage of patient specificity. However, pluripotent stem cell derivatives are generally heterogeneous and immature. hESC, humanembryonic stem cell; hiPSC, human induced pluripotent stem cell.

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to expand. Themost commonmyoblast cell line is C2C12, which wasisolated from 2-month-old mouse muscle in 1977 (Yaffe and Saxel,1977). Another common cell line is L6, which was isolated fromnewborn rat muscle in 1968 (Yaffe, 1968). Both C2C12 and L6 cellsproliferate rapidly with a generation time of ∼24 h and fuse intomulti-nucleated contractile myotubes (McMahon et al., 1994; Öberget al., 2011). Thus, they are a convenient model system forinvestigating processes such as myotube fusion (Zhao et al., 2015)or degeneration (Menconi et al., 2008). These cells are alsocompatible with gene transfection (Balcı and Dinçer, 2009), whichis useful for establishing the functions of genetic variants in myoblastgrowth or fusion (Prinsen and Veerkamp, 1998), or introducingdisease-relevant mutations (Liang et al., 2016).Myotubes differentiated from cell lines have lower levels of

structural and functional maturity, and distinct metabolic propertiescompared to myotubes differentiated from primary myoblasts(Abdelmoez et al., 2019; Robinson et al., 2019), probablybecause the immortalization process causes some amount of de-differentiation and loss of myogenic properties that continue todecline with increasing passage number. Thus, although myoblastcell lines are a reproducible and cost-effective cell source comparedto primary myoblasts, myotubes differentiated from cell lines havelimited relevance to native muscle tissue. Furthermore, acommercialized human myoblast cell line does not currently exist,raising further concerns about the translatability of data collectedusing common myoblast cell lines, which are derived from rodents.However, primary human myoblasts have been immortalized by theforced expression of a telomerase subunit and cyclin dependentkinase 4, which blocks a stress pathway (Mamchaoui et al., 2011).Myotubes generated from these cell lines produce relatively maturesarcomeres (Morris et al., 2020) and form NMJs when co-culturedwith motor neurons (Saini et al., 2019), suggesting that they couldbecome a promising cell source.To overcome the limitations of primary myoblasts and myoblast

cell lines, several protocols for deriving myogenic cells from humanembryonic stem cells (hESCs) or human induced pluripotent stemcells (hiPSCs) have recently emerged (Salani et al., 2012). Thesecells have the advantages of human origin and essentially limitlesssupply, as hESCs and hiPSCs can be expanded in culture for manypassages without loss of functionality. hESCs and hiPSCs are alsocompatible with gene editing techniques, such as CRISPR/Cas9,which can be used to introduce or correct select disease-relevantmutations (Shi et al., 2018; Young et al., 2016). Because hiPSCs arereprogrammed from somatic cells, such as skin fibroblasts, hiPSC-derived myogenic cells can be used to generate patient-specificmyotubes, which makes them especially desirable for modelinginherited neuromuscular diseases.One approach for generating myogenic progenitors from hiPSCs is

to overexpress master regulators of myogenic differentiation, such asMYOD1 (Abujarour et al., 2014; Rao et al., 2018) or PAX7 (Darabiet al., 2012). A similar reprogramming process has also been used todirectly transdifferentiate other cell types, such as fibroblasts, intomyogenic progenitors (Boularaoui et al., 2018; Ito et al., 2017; Lattanziet al., 1998). A second approach, known as directed differentiation,guides hiPSCs through native-like myogenic developmental pathwaysby sequentially adding small molecules that activate or suppressspecific signaling pathways (Chal et al., 2016, 2015; Maffioletti et al.,2015; Shelton et al., 2016; van der Wal et al., 2018; Xi et al., 2017).Directed differentiation is generally slower than transdifferentiation,but the resultingmyogenic progenitors are thought to be a closer matchto native myoblasts because they follow a more natural differentiationprocess (Jiwlawat et al., 2018).

Although impressive progress has been made in derivingmyogenicprogenitors from hiPSCs, most current protocols generally suffer fromwide variability and low efficiency (Jiwlawat et al., 2018). Theseissues limit cell yield and purity. However, protocols forcryopreserving and expanding hiPSC-derived myogenic progenitorsare being developed (van der Wal et al., 2018), which helps mitigateissues with differentiation variability and throughput. Despite thesepractical limitations related to cell differentiation, hESC- and hiPSC-derived myogenic progenitors successfully fuse into myotubes thatcontain myofibrils and exhibit key functional behaviors, such ascalcium cycling and contractility (Rao et al., 2018; Skoglund et al.,2014). However, myofibrils in hESC- and hiPSC-derived myotubesstill have immature features compared to native muscle fibers ormyotubes derived from primary myoblasts (Lainé et al., 2018).

The structural and functional immaturity of myotubes probablycontributes to the stunted maturation of NMJs that form betweenhiPSC-derived myotubes and motor neurons. However, severalapproaches for maturing hiPSC-derived myotubes are underdevelopment, such as identifying small molecules that boostmaturation (Selvaraj et al., 2019) or applying other strategiesdiscussed below. Additionally, the maturation of C2C12 myotubeshas been improved by applying biophysical cues, such as mechanicalstretch (Chang et al., 2016; Heher et al., 2015) or electricalstimulation (Ito et al., 2014; Nedachi et al., 2008), which mighthave similar benefits for hiPSC-derived myotubes. Thus, althoughhiPSC-derived myotubes have significant potential as an essentiallylimitless source of patient-specific myotubes, researchers need toenhance the differentiation efficiency and maturity of these cells toimprove their throughput and relevance for modeling neuromusculardiseases in vitro.

Motor neuronsCompared to myoblasts, fewer sources of motor neurons exist forin vitro models. Because most motor neurons stem from the spinalcord and project onto muscle fibers, it is not possible to isolate intactprimary motor neurons from humans. However, primary motorneurons can be isolated from embryonic or adult mice by extractingand digesting spinal cord tissue and using density gradientseparation to isolate motor neurons from supporting cell types,such as astrocytes and other glial cells (Beaudet et al., 2015; Gingraset al., 2007). Although rodents are a viable source of primary motorneurons, these cells are not human, which limits their relevance forneuromuscular disease modeling. Furthermore, the cell yield isrelatively low and cannot be increased with passaging becausemotor neurons are terminally differentiated and non-proliferative.

Because motor neurons do not proliferate, true motor neuron celllines do not exist. However, a hybrid mouse cell line (NSC-34) hasbeen generated by fusing neuroblastoma cells with embryonic motorneurons. NSC-34 cells retain the proliferative properties of the tumorcells while also exhibiting select neuronal properties, such asacetylcholine synthesis, neurotransmitter release and neurofilamentproteins (Cashman et al., 1992). This cell line has been used tomeasure the neurotoxicity of drugs (Maier et al., 2013) and receptortrafficking (Matusica et al., 2008), and has also been transfected tointroduce mutations relevant to ALS (Gomes et al., 2010; Pinto et al.,2017). Similar tomyoblast cell lines, the disadvantage of these cells istheir non-human origin and limited relevance to native motorneurons. For example, these cells do not replicate glutamate-mediatedexcitotoxicity (Madji Hounoum et al., 2016), questioning their abilityto replicate key features of neuromuscular diseases.

Human motor neurons can also be derived from hESCs and hiPSCsvia reprogramming or directed differentiation. hESCs and hiPSCs have

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been reprogrammed intomotor neurons byoverexpressingNGN2 (alsoknown asNEUROG2), ISL1 and LHX3 (Goto et al., 2017; Hester et al.,2011; Lee et al., 2012). Human fibroblasts have also beentransdifferentiated into motor neurons by overexpressing eight genes(Son et al., 2011). Several directed differentiation methods have alsobeen established, which entail dosing hESCs or hiPSCs with acombination of neurotrophic factors, retinoic acid, sonic hedgehog andNotch inhibitors (Du et al., 2015; Hu and Zhang, 2009; Li et al.,2008b; Qu et al., 2014; Shimojo et al., 2015). Motor neurons have alsobeen differentiated from the human fetal spinal cord stem cell line NSI-566RSC (Guo et al., 2010), which serves as another relativelyaccessible source of human motor neurons.Similar to other stem cell derivatives, stem cell-derived motor

neurons can be limited by cell heterogeneity, varying differentiationefficiency and stunted maturation (Ichida et al., 2018). Althoughmouse motor neurons derived from transdifferentiated fibroblasts ordirectly differentiated iPSCs have a transcriptome that is similar toprimary motor neurons (Ichida et al., 2018), how the structural andfunctional properties of these cells compare to their primarycounterpart is mostly unknown. Despite the limited functionalcharacterization of these cells, hiPSC-derived motor neurons havealready been shown to be a promising cell source for patient-specificmodeling of neuromuscular diseases such as ALS (Dimos et al., 2008;Sances et al., 2016; Sareen et al., 2013; Shi et al., 2018) and SMA(Fuller et al., 2016;Murdocca et al., 2016). Thus, hiPSC-derivedmotorneurons are likely to contribute to the development of new therapies forthese diseases that account for the genotype of the patient.

Engineered in vitro models of neuromuscular tissuesIn addition to cell source, another important consideration for in vitromodel development is the configuration of the cells, such that thecultured tissue is anatomically relevant and integrated with assays tomeasure functional phenotypes. Initial approaches for engineeringneuromuscular tissues in vitro entailed simply seeding dissociatedmotor neurons (Daniels et al., 2000; Das et al., 2010; Guo et al., 2011;Kengaku et al., 1991; Son et al., 2011; Umbach et al., 2012) or spinalcord explants (Askanas et al., 1987; Braun et al., 1997) on top of a 2Dlayer of myotubes attached to a conventional culture surface. Over thecourse of several days, the neurons extend axons and formNMJswiththe myotubes that successfully exhibit functional post-synapticpotentials. However, image analysis has revealed blotchycolocalization of pre- and post-synaptic markers and pooracetylcholine receptor clustering in these simple co-culturescompared to native NMJs (Das et al., 2010; Umbach et al., 2012).This limited synaptic maturity brings into question the ability of theseculture systems to accurately model disease-relevant phenotypes.The relatively stunted NMJ development in conventional co-

cultures could be attributed to many factors. First, without spatialorganization cues, myoblasts fuse into branched myotubes withrandom orientations that poorly recapitulate the architecture ofnative muscle fibers (Bettadapur et al., 2016; Denes et al., 2019),which can limit the formation of elongated myofibrils and maturesarcomeres. Second, myotubes often delaminate from conventionalculture surfaces within ∼2 weeks as they generate increasingamounts of mechanical stress (Wang et al., 2012; Sun et al., 2013).This can probably be attributed to both the high stiffness ofconventional culture substrates and the limited number of cell-adhesive molecules presented on their surfaces (Bettadapur et al.,2016). Limited culture lifetime is especially problematic forengineering neuromuscular tissues because NMJ maturationprobably requires longer than 2 weeks. Third, in situ, motorneuron soma are located in the spinal cord and only the axons of

motor neurons physically interact with muscle fibers. Thus, seedingmotor neurons on top of myotubes is not anatomically relevant andmight alter the physiology of one or both cell types.

Simple mixed co-cultures also suffer from technical problemsthat limit data collection. For example, measuring forces generatedby cells is not possible on most, if not all, conventional culturesubstrates, precluding quantitative assessment of muscle forceproduction due to motor neuron stimulation. This is a key functionalreadout in animal models, with high relevance to the severity ofneuromuscular disease (Bonetto et al., 2015). A second limitation isthat mixed co-cultures afford minimal independent control oranalysis of each cell type, as the cells are cultured in the samemedium and any electrical stimulation or drug treatment reachesboth cell types simultaneously. Similarly, isolating material fromeach cell type independently to measure changes in gene or proteinexpression, which is often important for establishing diseasemechanisms as well as drug effects, is challenging.

To overcome these diverse biological and technical challenges,researchers have developed several types of tunable culture surfacesand microfabricated devices to engineer more sophisticatedneuromuscular tissues in vitro. These surfaces and devices areusually also integrated with assays for quantifying structural andfunctional tissue phenotypes. In particular, when coupled with thehiPSC-derived cell sources described above, these platforms, knownas organs-on-chips or microphysiological systems, have immensepotential for advancing neuromuscular disease modeling and drugdevelopment. In this section, we describe both 2D and 3D engineeredtissue models.

Engineered 2D models of neuromuscular tissuesEngineering a neuromuscular tissue in vitro depends on first culturingmature and stable myotubes. In native skeletal muscle fibers, theECM plays a key role in tissue development and physiology bybinding to integrin receptors and providing biomechanical support asthe muscle fibers contract (Gillies and Lieber, 2011). The ECM isalso a rich source of biochemical cues that regulate behaviors such asadhesion, proliferation and differentiation. Furthermore, the ECMplays an active role in skeletal muscle disease, injury and aging, withfibrosis and subsequent tissue stiffening contributing to diminishedmuscle function (Mann et al., 2011). Owing to the documentedimportance of the ECM in native muscle fibers, several types oftunable culture substrates that mimic aspects of native muscle ECMhave been developed, as described below. However, anotherimportant feature of in vitro models of neuromuscular tissues is theability to measure muscle contractility in response to motor neuronstimulation. Thus, we also describe later in this section howengineered substrates have been integrated with contractility assays.

Natural biomaterial substratesHydrogels synthesized from natural polymers are popular culturesubstrates due to their biocompatibility, although they can suffer frombatch-to-batch variability. Collagen hydrogels are routinely used asculture substrates for myoblasts and myotubes due to their intrinsicbioactivity (Palade et al., 2019), as we also discuss above. Alignedmyotubes have been fabricated on collagen hydrogels by embeddingtopographical features into the hydrogel (Kim et al., 2017) or usingultrasound to pattern the cells acoustically (Armstrong et al., 2018).Gelatin, a partially hydrolyzed form of collagen, is also crosslinkedinto thermostable hydrogels by either mixing gelatin polymers withenzymatic crosslinking agents (Bettadapur et al., 2016; Denes et al.,2019; Suh et al., 2017) or methacrylating gelatin polymers such thatthey are compatible with photopolymerization techniques (Hosseini

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et al., 2012; Sun et al., 2018). Crosslinking increases the stiffness ofthe hydrogel and reduces its degradability (Sun et al., 2018), whichcan be advantageous for in vitro neuromuscular models that need tobe stable for several weeks.To promote myotube alignment on gelatin hydrogels, the surface

can be micromolded with polydimethylsiloxane (PDMS) (a siliconeelastomer) stamps with ridges several micrometers in size(Bettadapur et al., 2016; Chal et al., 2016; Denes et al., 2019;Hosseini et al., 2012). PDMS stamps are fabricated by casting PDMSon silicon wafer templates made using photolithography, which cangenerate feature sizes of ∼1 µm (Suh et al., 2017). Probably due totheir enhanced bioactivity, micromolded gelatin hydrogels can extendthe culture lifetime and maturation of C2C12 myotubes compared tosynthetic culture surfaces (Bettadapur et al., 2016; Denes et al., 2019).Carbon nanotubes have also been embedded into methacrylatedgelatin hydrogels to enhance myotube maturation by increasingelectrical conductivity (Ahadian et al., 2015; Ramón-Azcón et al.,2013). To better mimic the basement membrane of muscle,micromolded gelatin hydrogels have also been crosslinked with alayer of laminin, which improves the adherence, morphology andelectrophysiology of myotubes and neural cells (Besser et al., 2020).

Synthetic biomaterial substratesSynthetic biomaterials are advantageous culture substrates becausetheir mechanical and biochemical properties are highly controllableand reproducible. For example, polyethylene glycol andpolyacrylamide (PA) are both biologically inert hydrophilicpolymers that can be crosslinked into hydrogels with elastic modulituned to match the developing, healthy or fibrotic muscle tissuematrices (Engler et al., 2004). Another synthetic biomaterial that isimplemented as a culture surface is the aforementioned PDMS. Theelasticity of PDMS can be easily tuned to physiological or pathologicalvalues by altering the ratio of base to crosslinker (Wang et al., 2014) orblending different formulations of PDMS (Palchesko et al., 2012).To achieve consistent cell adhesion, researchers must functionalize

the synthetic substratewith ECMproteins. This is generally consideredan advantage because ECM ligand type and concentration can bespecified. Because collagen accounts for up to 10% of the dry weightof muscle (Gillies and Lieber, 2011), several studies have fabricatedsubstrates for C2C12 cultures by transferring collagen onto PDMS(Duffy et al., 2016) or PA hydrogels (Engler et al., 2004; Li et al.,2008a). Because the basement membrane of muscle fibers is enrichedin laminin and fibronectin, synthetic substrates functionalized witheither of these glycoproteins also promote myoblast adhesion andfusion into myotubes (Duffy et al., 2016; Gilbert et al., 2010;Palchesko et al., 2012; Ziemkiewicz et al., 2018).Synthetic biomaterials are also compatible with many

micropatterning techniques that can be used to introduce microscalefeatures on the surface to spatially control cell adhesion and alignment(Falconnet et al., 2006), as shown in Fig. 3. For example, PDMSstamps generated using the same photolithography techniquesdescribed above can be used to transfer ECM proteins onto a surfacein a process known as microcontact printing (Qin et al., 2010). Thisprocess has been used to prescribe myotube alignment on Petri dishes(Bajaj et al., 2011), PDMS-coated surfaces (Bettadapur et al., 2016;Jiwlawat et al., 2019; Nesmith et al., 2016; Palchesko et al., 2012; Sunet al., 2013) and PA hydrogels (Li et al., 2008a). Photolithography hasalso been used to selectively expose strips of a PA hydrogel to UVlight, which activates only the exposed regions for collagen bindingand thus myoblast adhesion (Engler et al., 2004). Myotubes have alsobeen aligned on substrates with nanoscale ridges fabricated usingelectron beam lithography, in which electrons are scanned in a defined

pattern on awafer coatedwith a light-sensitive photoresist (Wang et al.,2012). Another alternative is solvent-assisted capillary forcelithography, in which a polymer solution is molded on a siliconwafer with features at the hundreds of nanometers scale (Yang et al.,2014).

Integrated contractility assaysMicropatterned synthetic culture substrates are especially compatiblewith assays that quantify myotube contractility because themechanical properties of the substrate are well defined, andmyotube architecture can be controlled to increase the magnitudeand reproducibility of contractile force production. MicropatternedPA gels are widely used as a substrate for traction force microscopy, atechnique that quantifies forces generated by cells by tracking thedisplacement of fluorescent beads embedded in the hydrogel.Although traction force microscopy is more commonly used forcardiac myocytes (Ariyasinghe et al., 2017; McCain et al., 2012;Pasqualini et al., 2018; Ribeiro et al., 2015), it has also been used toquantify forces generated by micropatterned C2C12 myotubes (Liet al., 2008a).

Contractility can also be quantified by culturing myotubes onflexible cantilevers, as in the muscular thin film (MTF) assay. TheMTF assay entails first spin coating a glass coverslip with a layer ofpoly(N-isopropylacrylamide) (PNIPAAm), a temperature-sensitivepolymer, followed by a layer of PDMS (Feinberg et al., 2007). ThePDMS is then laser-cut into arrays of cantilevers with dimensionsranging from 1 mm to 5 mm (Agarwal et al., 2013), microcontactprinted with lines of fibronectin, and used to culture myotubes.After the desired culture period, the muscle-PDMS cantilevers,referred to as MTFs, are released by reducing the temperature from37°C to 25°C to solubilize the PNIPAAm. Electrodes are then usedto stimulate myotube contraction, which causes cantilever bending.Contractile stress is calculated based on the radius of curvature ofeach MTF (Grosberg et al., 2011). The MTF assay has beensuccessfully used to measure twitch and tetanus forces generated byC2C12 myotubes (Sun et al., 2013) and primary human myotubes(Nesmith et al., 2016). Microfabricated silicon cantilevers withdimensions of <1 mm have also been used as a culture substrate forprimary rat myotubes, and the contractile stresses in this system aremeasured based on the myotube-induced deflection of laser light(Smith et al., 2014a; Wilson et al., 2010). These compact lasersystems are advantageous for multiplexing, which increases testingthroughput and scalability for drug screening applications (Smithet al., 2014b).

Engineered co-cultures of skeletal muscle and motor neuronsMicrofabricated surfaces have also been developed to improvemixed co-cultures of myotubes and motor neurons. For example,photopolymerization techniques have been used to fabricate PAhydrogels with alternating soft and stiff stripes that mimic therigidity of nervous tissue and muscle tissue, respectively (Happeet al., 2017). On these surfaces, myoblasts preferentially migrateonto the stiffer stripes and fuse into aligned myotubes. When co-cultured with motor neurons, myotubes on mechanically patternedhydrogels exhibited increased acetylcholine receptor clusteringcompared to myotubes co-cultured on uniform hydrogels (Happeet al., 2017).

NMJ maturation has also been achieved in mixed co-cultures byapplying electrical stimulation using a bioreactor (Charoensooket al., 2017). This approach could be further refined by transfectingone or both cell types with channelrhodopsin, a membrane channelthat is activated by blue light (Fig. 3). Because chronic optogenetic

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Microfabricated surfaces for controllingECM rigidity and myotube alignment

Optogenetic stimulationof myotubes and/or

motor neurons

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myotubes and motor neurons

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Fig. 3. Engineered 2D neuromuscular tissues. Conventional approaches for engineering neuromuscular tissues in vitro entailed mixed co-cultures (center).New advances to improve the architecture and assaying capabilities of 2D neuromuscular tissues include microfabricated surfaces (top), compartmentalizedculture devices (right), and integration of optogenetics (left). ChR2, channelrhodopsin-2; ECM, extracellular matrix; PA, polyacrylamide; PDMS,polydimethylsiloxane; UV, ultraviolet.

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stimulation of myotubes can improve maturity (Rangarajan et al.,2014), a similar strategy applied to co-cultures could be a relativelynon-invasive approach for maturation. Transfecting motor neuronswith channelrhodopsin is also a powerful experimental tool formixed co-cultures because it enables users to stimulate only motorneurons and therefore more clearly identify responses in the musclethat are driven specifically by motor neurons (Lin et al., 2019;Steinbeck et al., 2016).Compartmentalized culture devices have also beenmicrofabricated

to physically isolate motor neurons and myotubes into separatechambers (Fig. 3). These chambers are connected by microchannelsthat are permissive to axons, but not cell bodies, to allow thecontrolled formation of NMJs in the myotube chamber (Santhanamet al., 2018; Taylor et al., 2003). Because the chambers are chemicallyisolated, these devices allow each cell type to be cultured in its ownmedium, which may boost viability. Furthermore, drugs or othersmall molecules can be selectively added to one or both chambers

(Santhanam et al., 2018), which can be useful for establishing drugmechanisms. Structural and functional analyses are also easier inthese devices compared to mixed co-cultures because NMJs form inrelatively prescribed locations and cells in each chamber can beelectrically stimulated independently.

Engineered 3D models of neuromuscular tissuesAlthough engineered 2D neuromuscular tissues have manyadvantages from an assay perspective, they fundamentally lackthe bundle-like architecture and cell-ECM interactions of nativemuscle fibers. To address this, researchers have developed severalapproaches to engineer miniature 3Dmuscle bundles (Fig. 4). Thesetypes of approaches were first reported in the late 1990s and entailedinjecting primary myoblasts mixed in an ECM pre-polymer solutioninto a rectangular chamber with patches of stainless-steel screening(Shansky et al., 1997) or Velcro (Powell et al., 1999) at itslongitudinal ends. As the myoblasts fused into myotubes, they

Matrigel

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Fig. 4. Engineered 3D neuromuscular tissues. (A) Aligned 3Dmuscle bundles are engineered bymixing myoblasts in an ECM pre-polymer solution of Matrigel,thrombin and fibrinogen, and casting it into a microfabricated support structure, such as a Velcro frame. Scale bars: 50 mm (left image); 50 µm (right image).Adapted from Madden et al. (2015). (B) Compartmentalized fluidic devices have been microfabricated to controllably co-culture 3D muscle bundles and motorneuron spheroids, and generated NMJs after 14 days in culture. D7, day 7; D14, day 14; DAPI, 4′,6-diamidino-2-phenylindole; nAChR, nicotinic acetylcholinereceptor; SAA, sarcomeric alpha-actinin; Tuj1, neuron-specific class III β-tubulin. Scale bars: 2 mm (left image); 10 µm (right image). Adapted with permissionfrom Osaki et al. (2018) and Uzel et al. (2016). The images in this figure are not published under the terms of the CC-BY license of this article. For permission toreuse, please see Madden et al. (2015), Osaki et al. (2018) and Uzel et al. (2016).

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detached from the bottom surface but remained embedded in theECM and anchored by the screening or Velcro, forming anelongated 3D muscle bundle with aligned myotubes.Over the past two decades, approaches for engineering 3Dmuscle

bundles have been advanced and refined. Several different types ofculture chambers with anchor points have been fabricated(Costantini et al., 2017a; Smith et al., 2016), including Velcro andnylon frames (Davis et al., 2017, 2019; Madden et al., 2015; Raoet al., 2018; Smith et al., 2016; Zhang et al., 2018) andmicrofabricated chambers with pillars (Osaki et al., 2018, 2020;Uzel et al., 2016). Testing of multiple ECM solutions has alsorevealed that fibrin hydrogels are optimal for encapsulatingmyotubes due to their strength (Hinds et al., 2011; Pollot et al.,2018), although these hydrogel compositions are not necessarilyphysiological. To improve assay capabilities, contractile forces havebeen measured in 3D muscle bundles with custom force transducers(Davis et al., 2019; Madden et al., 2015; Rao et al., 2018) or bytracking the displacement of pillars (Osaki et al., 2018; Uzel et al.,2016). Similar to 2D tissues, biophysical cues, such as mechanicalstretch (Powell et al., 2002) and optogenetic stimulation (Mills et al.,2019), or addition of fibroblasts (Dennis et al., 2001) have also beenshown to mature 3D muscle bundles.Microfluidic devices have also been fabricated to engineer and

maintain 3D muscle bundles (Agrawal et al., 2017; Shimizu et al.,2015). These systems are advantageous because they continuouslyperfuse freshmedia to the engineered tissues, which probably improvesviability compared to static culture. Furthermore, microfluidic devicescan be used to screen drugs at a higher throughput and can be linked toother microfluidic organ-on-chip systems to capture organ-organinteractions, and mimic organism-level responses (Novak et al., 2020).One common approach to innervate 3D muscle bundles is to

directly seed them with spheroids of motor neurons (Afshar Bakooshliet al., 2019; Morimoto et al., 2013; Smith et al., 2016). These systemshave demonstrated that the resulting NMJs are functional but still haverelatively diffuse acetylcholine receptor clustering (Morimoto et al.,2013). Acetylcholine clustering in 3D muscle bundles has beenadvanced by adding the basement membrane components agrin andlaminin (Wang et al., 2013), which could help improveNMJ formationin these co-cultures. Despite their limited maturity, these 3Dneuromuscular tissues show reduced contractility in response to serafrom MG patients (Morimoto et al., 2013), recapitulating thepathological response in MG and demonstrating their promise formodeling complex neuromuscular diseases.Similar to 2D models, compartmentalized microdevices have

been developed to culture motor neuron spheroids and engineeredmuscle bundles in separate compartments connected by axon-permissive channels (Fig. 4) (Osaki et al., 2018, 2020; Uzel et al.,2016). In these studies, the muscle bundles were attached to flexiblepillars and the motor neurons were optogenetically modified. Withthis combination of technologies, the users could quantify musclecontractility as a function of motor neuron stimulation, a key readoutof NMJ function. This type of device was also used to capture NMJdegeneration in tissues generated using hiPSC-derived motorneurons from an ALS patient. Importantly, the application of twoALS drug candidates, bosutinib and rapamycin, to this modelreduced muscle atrophy and dysfunction (Osaki et al., 2018),demonstrating how this type of approach has the potential forpatient-specific disease modeling and drug screening.

ConclusionsRecently developed approaches to model healthy and diseasedneuromuscular tissues on a chip have the potential to capture the

vastly heterogeneous genotypes and phenotypes of individuals witha variety of neuromuscular disorders. Newer technologies, such as3D bioprinting, which is a form of additive manufacturing that usescells and other biomaterials as ‘inks’ to print living structures (Choiet al., 2016; Costantini et al., 2017b; Kang et al., 2016; Kim et al.,2020), will probably further advance these models. However,in vitro models of neuromuscular tissues are far from achievingadult-like maturity, especially when based on hiPSC-derivedmuscle cells and motor neurons. Furthermore, in vitro models ofneuromuscular tissues lack the supporting cells known to beimportant regulators of NMJs in health and disease, such asSchwann cells (Santosa et al., 2018). Most in vitro models alsocurrently lack immune cells, despite the established role ofneuroinflammation in many neuromuscular diseases (Mäureret al., 2002; Thonhoff et al., 2018). However, researchers havebegun developing models that integrate immune cells, such asmacrophages (Juhas et al., 2018), to probe the role of the immunesystem in muscle injury and repair. Given these limitations, in vitromodels are most powerful when implemented hand-in-hand withanimal models, which have less human relevance but moreadvanced motor unit structure and physiology. Together, thesecomplementary model systems are likely to pave the way for moreeffective and personalized therapies for these debilitating diseases.

Competing interestsThe authors declare no competing or financial interests.

FundingThis project was supported by the University of Southern California Viterbi School ofEngineering, Women in Science and Engineering, University of Southern California,an Amyotrophic Lateral Sclerosis Association Starter Grant (18-IIA-401 toM.L.M.), aRose Hills Foundation Innovator Grant to M.L.M., and a National ScienceFoundation Graduate Research Fellowship Grant (DGE 1418060 to J.W.S.).

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