This document describes a method for generating functional human neuromuscular junctions (NMJs) in vitro using human induced pluripotent stem cells (hiPSCs). The system recapitulates early NMJ development and maturation over time in culture. It expresses key NMJ cell types and structural features. The NMJs formed synaptic connections and responded appropriately to pharmacological agents. Additionally, the model was able to reveal developmental and functional defects of NMJ formation in spinal muscular atrophy. This in vitro NMJ system has potential for investigating NMJ physiology and modeling NMJ-related diseases.

1.  Generation of neuromuscular junction in in-vitro that mimic the early developmental NMJ
formation.
 Established functional connectivity and in-vivo synapse formation.
 Model used to reveal developmental and functional defects of NMJ formation and NMJ-dependent
muscular contraction for spinal muscular atrophy.
Highlights:

2. Neuromuscular junction (NMJ):
 NMJ is a specialized synapse that transmits signals from motor neurons (MNs) to skeletal muscle
 Requires coordinated signals from MNs, skeletal muscle, glial cells, and the extracellular matrix
 Maintaining and altering the development of NMJs or the skeletal muscle contribute to the pathology of
NMJ-related diseases

3. Purpose: Recapitulate hNMJ model in in-vitro for investigating the physiology and pathology of hNMJs
Expression construct of MYOD1
 MYOD1-myoblast determination protein 1
 Regulating muscle differentiation
 Myotube is a type of cell which will develop into a
muscle fiber

4. Purpose: Functional differentiation of myotubes, neuronal differentiation and maturation
Methods: Immunocytochemistry
Myosin heavy chain– positive
(MYH+) myotubes
Tuj1+ neurons
Acetylcholine receptors- AChRs
Neurofilament 7 synaptic vesicles
Acetylcholine receptors- AChRs
 AChRs clustering on the surface of myotubes and juxtaposed indicates the emergence of NMJ-like structures

5. Myogenic markers Motor neuronal markers
Schwann cell marker
Purpose: Functional differentiation of myotubes, neuronal differentiation and maturation
Methods: Immunocytochemistry

6. Myogenic markers
Neural & Schwann cell markers
Purpose: Functional differentiation of myotubes, neuronal differentiation and maturation
Methods: qPCR
Upregulated expressions of genes associated with pre- and postsynaptic NMJ cell types
 The ACTA1 gene encode a protein called skeletal alpha
(α)-actin
 Dystrophin a protein complex that connects the
cytoskeleton of a muscle fiber
 Sox1 Transcriptional activator for neuronal development

7. synaptic bouton capped by the terminal Schwann cell and
myelinated axon
Synaptic vesicles and mitochondria accumulated at the pre- and postsynaptic sites of NMJs
Purpose : To see the architecture of neuromuscular junction
Methods: Scanning electron microscopy (SEM) and transmission electron microscopy (TEM)
in vitro NMJs expressed major cell types and promising architecture

8. Purpose : To see the architecture of neuromuscular junction
Methods: Scanning electron microscopy (SEM) and transmission electron microscopy (TEM)
A series of hNMJ developmental stages in NMJ culture system

9. 1. Agrin administration had no significant effect on the ratio of NMJs in long term culture or vigorous myotubes and junctional folds
2. in the presence of antiagrin antibody, the area of NMJs decreased, and only remnants of myotubes and deteriorated axon terminals were
found in the culture
Purpose: investigated the contribution of the neuronal factor agrin in in vitro NMJ system
Methods: Scanning electron microscopy (SEM)
 Role is in the development of the neuromuscular
junction during embryogenesis.
 Involvement in the aggregation of acetylcholine
receptors during synaptogenesis
 Agrin-targeted autoantibodies have been
infrequently observed in myasthenia gravis
Agrin’s Function:
Observations indicate agrin plays an important role in the formation and maintenance of in vitro
hNMJs, which mimics the role of agrin in vivo

10. Purpose: Investigate weather AChR signal–dependent spontaneous synchronized contraction of skeletal muscle in early hNMJ
culture or not?
Methods: Calcium imaging
Synchronized movement with the intracellular Ca2+ concentration
Curare- inhibitor of AChRs

11. Purpose: Investigate weather AChR signal–dependent spontaneous synchronized contraction of skeletal muscle in early hNMJ
culture or not?
Methods: Calcium imaging
From the motion vector analysis, it is found the contractions were
not completely synchronized but propagated to adjacent regions
GAP27- Gap junction inhibitor
Administration of GAP27, indicating action potentials were
propagated through gap junctions
in vitro NMJs at this phase (days 15–30) recapitulate key features of early in vivo NMJ development

12. Purpose: Optogenetic neuronal excitation controls muscular contraction in mature hNMJ culture
Methods: Optogenetic
The expression of channelrhodopsin–enhanced yellow fluorescent
protein (channelrhodopsin-EYFP) driven by synapsin1 promoter
Muscle contraction was triggered only by blue light, which
confirmed the photoactivated system was established
Contractions were completely inhibited by curare administration,
confirming muscle contractions were caused by the ACh/AChR route

13. Purpose: Modeling the pathophysiology of Spinal muscular atrophy with in-vitro hNMJ system
The area of the NMJ in 201B7MYOD-SMNKD was significantly smaller than in 201B7MYOD
Bunches of myotubes and axons that were well extended and anchored to the
myotubes with enlarged axon terminals in control 201B7MYOD culture but not
in 201B7MYOD-SMNKD culture

14. Purpose: Modeling the pathophysiology of Spinal muscular atrophy with in-vitro hNMJ system
 201B7MYOD-SMNKD showed a smaller area of contractions than control 201B7MYOD
 optogenetics is also appiled to 201B7MYOD-SMNKD but could not detect muscle contraction

15. Discussion:
1. Generation of functional hNMJs derived from hiPSCs
2. hNMJs follows in vivo developmental stages and NMJs showed a fully matured appearance
in prolonged culture
3. This system can be used to development of NMJ-related diseases and have potential to
evaluate the effects of related pharmacological interventions

16. mCherry+ cells to address whether MYOD1-expressing cells could differentiate into neurons
These results suggest both MYOD1-expressing cells and non–MYOD1-expressing cells differentiate into neuronal cells.

17. Expression construct of MYOD1
Schematic illustration of in vitro hNMJ formation
The induction steps for the in vitro hNMJ formation from hiPSCs