Bioengineered optogenetic model of human neuromuscular junction

Vila, Olaia F.; Chavez, Miguel; Stephen, P.; Yeager, Keith; Zholudeva, Lyandysha V.; Colón-Mercado, Jennifer M.; Qu, Yihuai; Nash, Trevor R.; Lai, Carmen; Feliciano, Carissa M.; Carter, Matthew; Kamm, Roger D.; Judge, Luke M.; Conklin, Bruce R.; Ward, Michael E.; McDevitt, Todd C.; Vunjak‐Novakovic, Gordana

doi:10.1016/j.biomaterials.2021.121033

Cited by 21 publications

(30 citation statements)

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“…The group recapitulated diseased NMJ pathology by subjecting their NMJ model to serum derived from MG patients and observing diminished NMJ function, as skeletal muscle cells failed to contract upon optical stimulation of MNs. Building upon this platform, the same group incorporated automated video-processing algorithms to create a diagnostic tool that automatically quantifies NMJ function and MG severity in a high-throughput manner based on sera from MG donors (Vila et al 2021 ). Another opto-genetically engineered NMJ model was recently developed by Solomon et al to test the functionality of NMJ in response to vecuronium, a competitive inhibitor of acetylcholine receptors (AChR) (Solomon et al 2021 ).…”

Section: State-of-the-art Of In Vitro Tissue Modelsmentioning

confidence: 99%

3D bioprinting of complex tissues in vitro: state-of-the-art and future perspectives

et al. 2022

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The pharmacology and toxicology of a broad variety of therapies and chemicals have significantly improved with the aid of the increasing in vitro models of complex human tissues. Offering versatile and precise control over the cell population, extracellular matrix (ECM) deposition, dynamic microenvironment, and sophisticated microarchitecture, which is desired for the in vitro modeling of complex tissues, 3D bio-printing is a rapidly growing technology to be employed in the field. In this review, we will discuss the recent advancement of printing techniques and bio-ink sources, which have been spurred on by the increasing demand for modeling tactics and have facilitated the development of the refined tissue models as well as the modeling strategies, followed by a state-of-the-art update on the specialized work on cancer, heart, muscle and liver. In the end, the toxicological modeling strategies, substantial challenges, and future perspectives for 3D printed tissue models were explored.

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Section: State-of-the-art Of In Vitro Tissue Modelsmentioning

confidence: 99%

3D bioprinting of complex tissues in vitro: state-of-the-art and future perspectives

et al. 2022

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“…Interestingly, functional innervation was achieved following 2 weeks of culture within 3D, but not in comparable 2D NMJ co-cultures, and expression of the mature AChR ε-subunit was observed only in 3D NMJ co-cultures ( Bakooshli et al, 2019 ). Beyond mixed 3D NMJ models, compartmentalized microdevices ( Figures 4E,F ) have been developed to spatially separate MN spheroids and engineered SkM and connect them via axon-permissive channels to more appropriately mimic in vivo muscle innervation ( Uzel et al, 2016 ; Osaki et al, 2018 ; Vila et al, 2021 ). Through this compartmentalization, visualization of 3D neurite outgrowth and engineered SkM innervation is greatly simplified, similar to studies in 2D compartmentalized co-cultures.…”

Section: Current Models For Studies Of Neuromuscular Junction Function and Diseasementioning

confidence: 99%

“…Motor neuron activation within 3D NMJ models has been achieved through addition of the neurotransmitter glutamate ( Osaki et al, 2018 ; Bakooshli et al, 2019 ) or its mimic N -Methyl-D-aspartate (NMDA), optogenetic control ( Osaki et al, 2018 ; Vila et al, 2019 , 2021 ), or direct electrical stimulation ( Osaki et al, 2018 ; Rimington et al, 2021 ). Glutamate stimulates MNs ( Figure 4I ) through binding to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA), kainic acid (KA), and NMDA receptors while NMDA specifically targets NMDA receptors ( Newcomer et al, 2000 ).…”

Section: Current Models For Studies Of Neuromuscular Junction Function and Diseasementioning

confidence: 99%

“…To assess NMJ functionality, recordings of calcium transients have been used as an indicator of MN-induced muscle excitation and gCaMP6 ( Bakooshli et al, 2019 ), a genetically encoded calcium indicator, has been used to visualize calcium flow through muscles ( Figure 4J ). Furthermore, MN-innervated engineered SkM tissues can be cultured on microfabricated pillars, displacement of which can be imaged to assess muscle contractions induced via glutamate or light-stimulated MN activity ( Uzel et al, 2016 ; Vila et al, 2019 , 2021 ; Afshar et al, 2020 ). In addition to indirect functional measurements by video recordings, contractile force generation in mixed 3D NMJ co-cultures can be directly measured by a force transducer ( Martin et al, 2015 ; Rizzuto et al, 2017 ; Rimington et al, 2021 ), which allows for assessment of the muscle force-length relationship and could be used for detailed functional studies in compartmentalized 3D NMJ models, similar to those performed in native nerve-muscle preparations ( Martin et al, 2015 ; Rizzuto et al, 2017 ).…”

Section: Current Models For Studies Of Neuromuscular Junction Function and Diseasementioning

confidence: 99%

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

Fralish

Lotz

Chavez

et al. 2021

Front. Cell Dev. Biol.

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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.

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“…Indeed, the latest technological advances in tissue engineering such as 3D bioprinting or the development of novel biomaterials and culture platforms ( Capel et al, 2019 ; Denes et al, 2019 ; Costantini et al, 2021 ; Gupta et al, 2021 ; Peper et al, 2021 ) are facilitating the development of better in vitro skeletal muscle constructs. To this end, researchers have recapitulated many native skeletal muscle features, including contraction ( Madden et al, 2015 ; Urciuolo et al, 2020 ; Akiyama et al, 2021 ; Alave Reyes-Furrer et al, 2021 ), vascularization ( van der Schaft et al, 2011 ; Bersini et al, 2018 ; Osaki et al, 2018a ; Gilbert-Honick et al, 2018 ), mechanical tension ( Okano and Matsuda, 1998 ; Vandenburgh and Karlisch, 1989 ), neuromuscular junction (NMJ) ( Afshar Bakooshli et al, 2019 ; Yoshioka et al, 2020 ; Vila et al, 2021 ), and muscle stiffness ( Urciuolo et al, 2020 ). These features, represented in Figure 1 (top), are considered key requirements for generating functional and mature skeletal muscle constructs.…”

Section: Introductionmentioning

confidence: 99%

Contractile force assessment methods for in vitro skeletal muscle tissues

Vesga-Castro

Aldazábal

Vallejo-Illarramendi

et al. 2022

eLife

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Over the last few years, there has been growing interest in measuring the contractile force (CF) of engineered muscle tissues to evaluate their functionality. However, there are still no standards available for selecting the most suitable experimental platform, measuring system, culture protocol, or stimulation patterns. Consequently, the high variability of published data hinders any comparison between different studies. We have identified that cantilever deflection, post deflection, and force transducers are the most commonly used configurations for CF assessment in 2D and 3D models. Additionally, we have discussed the most relevant emerging technologies that would greatly complement CF evaluation with intracellular and localized analysis. This review provides a comprehensive analysis of the most significant advances in CF evaluation and its critical parameters. In order to compare contractile performance across experimental platforms, we have used the specific force (sF, kN/m2), CF normalized to the calculated cross-sectional area (CSA). However, this parameter presents a high variability throughout the different studies, which indicates the need to identify additional parameters and complementary analysis suitable for proper comparison. We propose that future contractility studies in skeletal muscle constructs report detailed information about construct size, contractile area, maturity level, sarcomere length, and, ideally, the tetanus-to-twitch ratio. These studies will hopefully shed light on the relative impact of these variables on muscle force performance of engineered muscle constructs. Prospective advances in muscle tissue engineering, particularly in muscle disease models, will require a joint effort to develop standardized methodologies for assessing CF of engineered muscle tissues.

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Bioengineered optogenetic model of human neuromuscular junction

Cited by 21 publications

References 50 publications

3D bioprinting of complex tissues in vitro: state-of-the-art and future perspectives

3D bioprinting of complex tissues in vitro: state-of-the-art and future perspectives

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

Contractile force assessment methods for in vitro skeletal muscle tissues

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