iPSC-derived functional human neuromuscular junctions model the pathophysiology of neuromuscular diseases

Lin, Chwan-Fwu; Yoshida, Michiko; Li, Li-Tzu; Ikenaka, Akihiro; Oshima, Shiro; Nakagawa, Kazuhiro; Sakurai, Hiroyuki; Matsui, Eriko; Nakahata, Tatsutoshi; Saito, Megumu K.

doi:10.1172/jci.insight.124299

Cited by 55 publications

(61 citation statements)

References 46 publications

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“…CC-BY-NC-ND 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted October 21, 2020. ; https://doi.org/10.1101/2020.10.21.346874 doi: bioRxiv preprint 4 functional NMJs were established (21)(22)(23)(24)(25)(26). However, such models are unable to account for the unique culture microenvironments occupied by cell-specific domains of MNs and muscle fibers (27).…”

Section: Introductionmentioning

confidence: 99%

Human motor units in microfluidic devices are impaired by FUS mutations and improved by HDAC6 inhibition

Dittlau

Krasnow

Fumagalli

et al. 2020

Preprint

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Neuromuscular junctions (NMJs) ensure proper communication between motor neurons and muscle through the release of neurotransmitters. In motor neuron disorders, such as amyotrophic lateral sclerosis (ALS), NMJs degenerate resulting in muscle atrophy, paralysis and respiratory failure. The aim of this study was to establish a versatile and reproducible in vitro model of a human motor unit to study the effect of ALS-causing mutations. Therefore, we generated a co-culture of human induced pluripotent stem cell-derived motor neurons and human primary mesoangioblast-derived myotubes in microfluidic devices. A chemotactic and volumetric gradient facilitated the growth of motor neuron neurites through microgrooves resulting in the interaction with myotubes and the formation of NMJs. We observed that ALS-causing FUS mutations resulted in a reduced neurite outgrowth and in a decreased NMJ number. Interestingly, the selective HDAC6 inhibitor, Tubastatin A, improved the neurite outgrowth and the NMJ morphology of FUS-ALS co-cultures, further prompting HDAC6 inhibition as a potential therapeutic strategy for ALS.

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Section: Introductionmentioning

confidence: 99%

Human motor units in microfluidic devices are impaired by FUS mutations and improved by HDAC6 inhibition

Dittlau

Krasnow

Fumagalli

et al. 2020

Preprint

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“…Various strategies have been published for generating in vitro NMJ of different maturation stages, but the in vitro NMJ generated in our system showed the significant maturity, as we can observe the transition of gamma to epsilon subunits of AChR during the culture 18 . Maturity is an important consideration for disease modeling with in vitro NMJ 21 .…”

Section: Discussionmentioning

confidence: 90%

“…Our The chemically triggered myotubes contraction and curaredependent inhibition confirmed that our in vitro NMJ was functional and could be potentially applied for practical assays. Spontaneous muscle contractions were observed randomly at 3-4 weeks of culture, but it could be avoided by extending the culture time to as long as two months 18 .…”

Section: Discussionmentioning

confidence: 99%

“…8 , and, in the meanwhile, the Schwann cells can also be induced 16 , 17 . Through the interactions among the myotubes, the motor neurons and the Schwann cells, the mature NMJ are formed 18 . This method can generate functional NMJ efficiently, enabling the potential study of pathological mechanisms and therapeutic compound screening.…”

Section: Introductionmentioning

confidence: 99%

“…Three cell types in the NMJ were observed in our induction system, including the myotubes, the motor neurons and the Schwann cells18 . In addition, mature morphology and function were verified.Based on our previous study, the initial cell density is a critical factor for NMJ formation, and different cell densities induce various numbers of NMJs in the culture18 . A low cell density (< 1 x 10 4 /cm 2 ) induced a low number of neurons, which is unfavorable for NMJ formation.…”

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In vitro Neuromuscular Junction Induced from Human Induced Pluripotent Stem Cells

Lin¹,

Yoshida

et al. 2020

JoVE

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The neuromuscular junction (NMJ) is a specialized synapse that transmits action potentials from the motor neuron to skeletal muscle for mechanical movement. The architecture of the NMJ structure influences the functions of the neuron, the muscle and the mutual interaction. Previous studies have reported many strategies by coculturing the motor neurons and myotubes to generate NMJ in vitro with complex induction process and long culture period but have struggled to recapitulate mature NMJ morphology and function. Our in vitro NMJ induction system is constructed by differentiating human iPSC in a single culture dish. By switching the myogenic and neurogenic induction medium for induction, the resulting NMJ contained pre-and postsynaptic components, including motor neurons, skeletal muscle and Schwann cells in the one month culture. The functional assay of NMJ also showed that the myotubes contraction can be triggered by Ca ++ then inhibited by curare, an acetylcholine receptor (AChR) inhibitor, in which the stimulating signal is transmitted through NMJ. This simple and robust approach successfully derived the complex structure of NMJ with functional connectivity. This in vitro human NMJ, with its integrated structures and function, has promising potential for studying pathological mechanisms and compound screening.

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Neuromuscular Regeneration of Volumetric Muscle Loss Injury in Response to Agrin‐Functionalized Tissue Engineered Muscle Grafts and Rehabilitative Exercise

Mihaly,

Chellu,

Iyer

et al. 2024

Adv Healthcare Materials

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Neuromuscular deficits compound the loss of contractile tissue in volumetric muscle loss (VML). Two avenues for promoting recovery are neuromuscular junction (NMJ)‐promoting substrates (e.g., agrin) and endurance exercise. Although mechanical stimulation enhances agrin‐induced NMJ formation, the two modalities have yet to be evaluated combinatorially. It is hypothesized that the implantation of human myogenic progenitor‐seeded tissue‐engineered muscle grafts (hTEMGs) in combination with agrin treatment and/or exercise will enhance neuromuscular recovery after VML. The hTEMGs alone transplant into VML defects promote significant regeneration with minimal scarring. A sex‐appropriate, low‐intensity continuous running exercise paradigm increases acetylcholine receptor (AChR) cluster density in male mice twofold relative to hTEMG alone after 7 weeks of treadmill training (p < 0.05). To further promote neuromuscular recovery, agrin is incorporated into the scaffolds via covalent tethering. In vitro, agrin increases the proliferation of hMPs, and trends toward greater myogenic maturity and AChR clustering. Upon transplantation, both hTEMGs + agrin and hTEMGs + exercise induce near 100% recovery of muscle mass and increase twitch and tetanic force output (p > 0.05). However, agrin treatment in combination with exercise produces no additional benefit. These data highlight the unprecedented regenerative potential of using hTEMGs together with either agrin or exercise supplementation to treat VML injuries.

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iPSC-derived functional human neuromuscular junctions model the pathophysiology of neuromuscular diseases

Cited by 55 publications

References 46 publications

Human motor units in microfluidic devices are impaired by FUS mutations and improved by HDAC6 inhibition

Human motor units in microfluidic devices are impaired by FUS mutations and improved by HDAC6 inhibition

In vitro Neuromuscular Junction Induced from Human Induced Pluripotent Stem Cells

Neuromuscular Regeneration of Volumetric Muscle Loss Injury in Response to Agrin‐Functionalized Tissue Engineered Muscle Grafts and Rehabilitative Exercise

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