Neuromuscular Development and Disease: Learning From in vitro and in vivo Models

Abstract

The neuromuscular junction (NMJ) is a specialized cholinergic synaptic interface between a motor neuron and a skeletal muscle fiber that translates presynaptic electrical impulses into motor function. NMJ formation and maintenance require tightly regulated signaling and cellular communication among motor neurons, myogenic cells, and Schwann cells. Neuromuscular diseases (NMDs) can result in loss of NMJ function and motor input leading to paralysis or even death. Although small animal models have been instrumental in advancing our understanding of the NMJ structure and function, the complexities of studying this multi-tissue system in vivo and poor clinical outcomes of candidate therapies developed in small animal models has driven the need for in vitro models of functional human NMJ to complement animal studies. In this review, we discuss prevailing models of NMDs and highlight the current progress and ongoing challenges in developing human iPSC-derived (hiPSC) 3D cell culture models of functional NMJs. We first review in vivo development of motor neurons, skeletal muscle, Schwann cells, and the NMJ alongside current methods for directing the differentiation of relevant cell types from hiPSCs. We further compare the efficacy of modeling NMDs in animals and human cell culture systems in the context of five NMDs: amyotrophic lateral sclerosis, myasthenia gravis, Duchenne muscular dystrophy, myotonic dystrophy, and Pompe disease. Finally, we discuss further work necessary for hiPSC-derived NMJ models to function as effective personalized NMD platforms.

Keywords: disease modeling; drug development; human skeletal muscle; induced pluripotent stem cells; muscular dystrophy; neuromuscular junction; organ on a chip; tissue engineering.

FIGURE 1

Early development and hiPSC-based differentiation of motor neurons. (A) After the notochord (NC) signals inward folding of the ectoderm at the neural plate, the neural crest is brought together forming the neural tube (NT). The neural crest then forms neural crest cells (NCCs) which differentiate in the peripheral nervous system. The cells of mesoderm (MD) differentiate into somites and, eventually, the musculoskeletal system. (B) Along the rostral–caudal axis of the NT, WNT gradients dictate regionalization of the brain and RA/FGF/GDF11 gradients dictate segmentation of the spinal cord. (C) Along the dorsal–ventral axis of the NT, antithetical WNT/BMP [derived from the roof plate (RP)] and SHH gradients [derived from the floor plate (FP)] dictate patterning and the formation of the five ventral progenitor cell domains (p0, p1, p2, pMN, and p3). The pMN domain is the source of subsequent MN specification. (D) hiPSC-derivations of MNs begin with dual SMAD inhibition of TGFβ and BMP pathways to trigger neural stem cell (NSC) differentiation. WNT and RA signaling direct NSC differentiation into neural progenitor cells (NPCs) of the spinal cord region. With the addition of SHH signaling, NPCs further differentiate to Olig2-expressing MN progenitors (MNPs). Suppression of Olig2 and upregulation of Ngn2 commit MNPs to a post-mitotic MN lineage that express Hb9, Isl1, Isl2, and Lhx3. Distinct colors are used to denote approximate correspondence between stages of hiPSC differentiation in panel (D) and embryonic development in panel (C).

FIGURE 2

Early development and hiPSC-based differentiation of skeletal muscle. (A) Caudal-rostral development of SkM occurs bilaterally along the neural tube (NT) and notochord (NC). From the progenitor zone, cells migrate to the posterior presomitic mesoderm (pPSM) with a decreasing gradient of FGF and WNT signals. They then cross the determination front to enter the anterior presomitic mesoderm (aPSM). With increasing retinoic acid (RA) gradient, somite formation begins. As cells continue to travel rostrally, the dermomyotome (DT), sclerotome (ST), and myotome (MT) form, initiating primary myogenesis. (B) hiPSC differentiation to skeletal muscle begins with WNT and FGF activation, inducing a shift into neuromesodermal progenitors (NMPs) and then muscle progenitor cells (MPCs) expressing the transcription factors T, Tbx6, and Msgn1. Subsequent loss of T expression leads to formation of paraxial mesoderm cells resembling skeletal muscle progenitors of pPSM. Through RA activation, these muscle progenitors begin to express the muscle stem cell markers Pax3 and Pax7 and eventually differentiate into myoblasts expressing early myogenic markers Myf5, MyoD, and Mrf4. The myoblasts can fuse into myotubes that express the late muscle differentiation marker MyoG. Distinct colors are used to denote approximate correspondence between stages of hiPSC differentiation in panel (B) and embryonic development in panel (A).

FIGURE 3

Structural and molecular architecture of the neuromuscular junction. (A) The NMJ is comprised of three components: (1) the axonal terminal of an MN (pre-synapse), (2) the basal lamina of the synapse (synaptic cleft), and (3) the sarcolemma (membrane) of a muscle fiber (post-synapse). Following the conduction of an action potential to the axon terminal, Ca²⁺ influx occurs at the presynaptic terminal releasing ACh-containing vesicles into the synaptic cleft. Released ACh can then bind to AChRs on the sarcolemma creating an endplate potential and eventually muscle contraction. (B) AChE secreted by the muscle binds to ColQ and inactivates residual ACh within the synapse. ColQ binds to MuSK to help stabilize the synapse. The synaptogenic proteoglycan agrin secreted by MNs binds to LRP4 to facilitate formation of the NMJ. (C) AChRs are stabilized by dystrophin-associated glycan (DAG) complexes. The AChR-clustering protein rapsyn connects AChRs to the DAG complex and dystrophin anchors the complex to the SkM cytoskeleton. Lamins and glycans additionally connect the complex to the ECM while the sarcoglycan and sarcospan stabilize the DAG complex within the membrane. ACh, acetylcholine; AChE, acetylcholine esterase; ColQ, collagen Q; MuSK, muscle-specific tyrosine kinase receptor; Ag, agrin; LRP4, low density lipoprotein receptor 4; AChR, acetylcholine receptor; AChR, acetylcholine receptor; La, laminin; α/β, α/β dystroglycan; Gly, glycans; SG, sarcoglycan; SS, sarcospan; Ra, rapsyn; Cys, cysteine; Dys, dystrophin.

FIGURE 4

Engineered NMJ models. (A,B) Schematics of 2D NMJ co-culture models in mixed (A) and compartmentalized (B) configuration. (C) Mixed 3D NMJ co-culture systems incorporate MNs or neurospheres into SkM during 3D tissue formation. (D) Representative mixed 3D NMJ model with immunofluorescent staining of muscle sarcomeres (SAA), neurite extensions (SMI32), and acetylcholine receptors (BTX). Scale bar, 200 μm (Bakooshli et al., 2019; Copyright 2019, eLIFE). (E) Compartmentalized 3D NMJ co-culture systems culture MNs and SkM in separate compartments bridged by an extracellular matrix gel to facilitate axonal outreach and SkM innervation. (F) Representative compartmentalized NMJ model with immunofluorescent staining of neurite extensions (TUJ1) and myotubes [filamentous (F)-actin]. Scale bars, 100 μm (Osaki et al., 2018; Copyright 2018, Science Advances). (G) Example of optogenetic control of 3D NMJ models through blue light illumination (blue bars) of ChR2^H134R-HBG3-MN neurospheres inducing contraction in muscle (y-axis) as measured by pillar displacement within a microfluidic system. Administration of α-bungarotoxin (BTX) prevented MN-induced contractions (Uzel et al., 2016; Copyright 2016, Science Advances). (H) Example of muscle contraction induced by electrical stimulation of MNs at varying frequencies (0.5–4 Hz) (Osaki et al., 2018; Copyright 2018, Science Advances). (I) Representative recording of contractile force (y-axis) in 3D SkM-MN co-culture induced by glutamate stimulation of neurospheres (Uzel et al., 2016; Copyright 2016, Science Advances). (J) Representative recording of glutamate-induced Ca²⁺ transients in 3D SkM-MN co-culture with muscle-specific expression of GCaMP6 reporter. MN neurospheres are encircled by dashed lines (Bakooshli et al., 2019; Copyright 2019, eLIFE).

FIGURE 5

NMD-specific changes in NMJ morphology. (A) Mature human NMJs present with a characteristic “pretzel” shape and contain both synaptophysin (SY) and acetylcholine receptors (AChRs). NMJ endplates exhibit distinct membrane folds that extend into the muscle cell cytoplasm. (B) In ALS, marked decrease in synaptophysin and NMJ size occurs alongside denervation. Additionally, ALS patients present with smaller nerve terminals and flattened synaptic clefts. (C) In MG, NMJ morphology is generally maintained; however, there is synaptic accumulation of autoantibodies (Ab), decrease in AChRs, and shortening of synaptic clefts. (D) In DMD, there is marked axonal branching and NMJ fragmentation alongside shortening of the synaptic cleft. (E) In Pompe disease, there is denervation alongside NMJ fragmentation and increased rates of MN apoptosis.

FIGURE 6

Future developments of hiPSC-derived NMD models. Optimization of soluble factor treatments will be needed to improve maturity of MNs, SkM, and NMJs toward their respective in vivo-mimetic phenotypes. Incorporation of biophysical stimulation during cell culture such as electrical stimulation, mechanical stretch, and media agitation are expected to further improve functional maturity of NMJs. Biomaterial design and microfabrication/microfluidics techniques can be applied to generate biomimetic microenvironments for cell growth via controlled presentation of developmentally relevant gradients in growth factors, topography, and stiffness. As the function of native NMJs relies on complex multi-cellular interactions, the ability to incorporate Schwann cells (SCs), endothelial cells (ECs), fibro-adipogenic progenitors (FAPs), and immune system cells (ICs) would improve modeling of in vivo states and our understanding of NMJ physiology and pathology. Incorporation of additional organ compartments sharing culture media with NMJs, such as intestine, gut, and liver will better emulate human drug responses, while adding bone, ligament, cartilage, and fat compartments will allow studies of the organ-organ crosstalk in NMDs. Finally, further miniaturization and standardization of culture systems along with development of non-invasive metabolic and functional readouts will enable establishment of versatile NMD platforms for high-throughput drug discovery research.