scholarly journals Compartmentalized three-dimensional human neuromuscular tissue models fabricated on a well-plate-format microdevice

2021 ◽  
Author(s):  
Kazuki Yamamoto ◽  
Nao Yamaoka ◽  
Yu Imaizumi ◽  
Takunori Nagashima ◽  
Taiki Furutani ◽  
...  
Keyword(s):  
Motor Neurons ◽  
Neuromuscular Junctions ◽  
Neuromuscular Disorders ◽  
Three Dimensional ◽  
Induced Pluripotent Stem Cell ◽  
Motor Units ◽  
Mechanical Stimuli ◽  
Tissue Models

Engineered three-dimensional models of neuromuscular tissues are promising for use in mimicking their disorder states in vitro. Although several models have been developed, it is still challenging to mimic the physically separated structures of motor neurons (MNs) and skeletal muscle (SkM) fibers in the motor units in vivo. In this study, we aimed to develop microdevices for precisely compartmentalized coculturing of MNs and engineered SkM tissues. The developed microdevices, which fit a well of 24 well plates, had a chamber for MNs and chamber for SkM tissues. The two chambers were connected by microtunnels for axons, permissive to axons but not to cell bodies. The axons from human-induced-pluripotent-stem-cell-derived MN spheroids in the MN chamber elongated in microtunnels, reached the tissue-engineered human SkM in the SkM chamber, and formed functional neuromuscular junctions with the muscle fibers. The cocultured SkM tissues with MNs on the device contracted spontaneously in response to spontaneous firing of MNs. The addition of a neurotransmitter, glutamate, into the MN chamber induced contraction of the cocultured SkM tissues. Selective addition of tetrodotoxin or vecuronium bromide into either chamber induced SkM tissue relaxation, which could be explained by the inhibitory mechanisms. We also demonstrated the application of chemical or mechanical stimuli to the middle of the axons of cocultured tissues on the device. Thus, compartmentalized neuromuscular tissue models fabricated on the device could be used for phenotypic screening to evaluate the cellular type specific efficacy of drug candidates and would be a useful tool in fundamental research and drug development for neuromuscular disorders.

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

2020 ◽  
Author(s):  
Katarina Stoklund Dittlau ◽  
Emily N. Krasnow ◽  
Laura Fumagalli ◽  
Tijs Vandoorne ◽  
Pieter Baatsen ◽  
...  
Keyword(s):  
Motor Neuron ◽  
Neurite Outgrowth ◽  
Motor Neurons ◽  
Neuromuscular Junctions ◽  
Induced Pluripotent Stem Cell ◽  
Microfluidic Devices ◽  
Motor Units ◽  
Human Motor ◽  
Induced Pluripotent

AbstractNeuromuscular 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.

scholarly journals Microfluidic device for the formation of optically excitable, three-dimensional, compartmentalized motor units

2016 ◽  
Vol 2 (8) ◽  
pp. e1501429 ◽  
Author(s):  
Sebastien G. M. Uzel ◽  
Randall J. Platt ◽  
Vidya Subramanian ◽  
Taylor M. Pearl ◽  
Christopher J. Rowlands ◽  
...  
Keyword(s):  
Motor Neurons ◽  
Neuromuscular Junctions ◽  
Three Dimensional ◽  
Embryonic Stem ◽  
Motor Units ◽  
Cell Types ◽  
In Vitro Assays ◽  
Traumatic Injuries ◽  
Muscle Innervation

Motor units are the fundamental elements responsible for muscle movement. They are formed by lower motor neurons and their muscle targets, synapsed via neuromuscular junctions (NMJs). The loss of NMJs in neurodegenerative disorders (such as amyotrophic lateral sclerosis or spinal muscle atrophy) or as a result of traumatic injuries affects millions of lives each year. Developing in vitro assays that closely recapitulate the physiology of neuromuscular tissues is crucial to understand the formation and maturation of NMJs, as well as to help unravel the mechanisms leading to their degeneration and repair. We present a microfluidic platform designed to coculture myoblast-derived muscle strips and motor neurons differentiated from mouse embryonic stem cells (ESCs) within a three-dimensional (3D) hydrogel. The device geometry mimics the spinal cord–limb physical separation by compartmentalizing the two cell types, which also facilitates the observation of 3D neurite outgrowth and remote muscle innervation. Moreover, the use of compliant pillars as anchors for muscle strips provides a quantitative functional readout of force generation. Finally, photosensitizing the ESC provides a pool of source cells that can be differentiated into optically excitable motor neurons, allowing for spatiodynamic, versatile, and noninvasive in vitro control of the motor units.

scholarly journals C9orf72-derived arginine-containing dipeptide repeats associate with axonal transport machinery and impede microtubule-based motility

2019 ◽  
Author(s):  
Laura Fumagalli ◽  
Florence L. Young ◽  
Steven Boeynaems ◽  
Mathias De Decker ◽  
Arpan R. Mehta ◽  
...  
Keyword(s):  
Axonal Transport ◽  
Motor Neurons ◽  
Single Molecule ◽  
Induced Pluripotent Stem Cell ◽  
Cytoplasmic Dynein ◽  
Inhibitory Interaction ◽  
Hexanucleotide Repeat ◽  
Repeat Expansions

ABSTRACTHexanucleotide repeat expansions in the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). How this mutation leads to these neurodegenerative diseases remains unclear. Here, we use human induced pluripotent stem cell-derived motor neurons to show that C9orf72 repeat expansions impair microtubule-based transport of mitochondria, a process critical for maintenance of neuronal function. Cargo transport defects are recapitulated by treating healthy neurons with the arginine-rich dipeptide repeat proteins (DPRs) that are produced by the hexanucleotide repeat expansions. Single-molecule imaging shows that these DPRs perturb motility of purified kinesin-1 and cytoplasmic dynein-1 motors along microtubules in vitro. Additional in vitro and in vivo data indicate that the DPRs impair transport by interacting with both microtubules and the motor complexes. We also show that kinesin-1 is enriched in DPR inclusions in patient brains and that increasing the level of this motor strongly suppresses the toxic effects of arginine-rich DPR expression in a Drosophila model. Collectively, our study implicates an inhibitory interaction of arginine-rich DPRs with the axonal transport machinery in C9orf72-associated ALS/FTD and thereby points to novel potential therapeutic strategies.

An improved method for culturing myotubes on laminins for the robust clustering of postsynaptic machinery

2019 ◽  
Author(s):  
Marcin Pęziński ◽  
Patrycja Daszczuk ◽  
Bhola Shankar Pradhan ◽  
Hanns Lochmüller ◽  
Tomasz J. Prószyński
Keyword(s):  
Motor Neurons ◽  
High Throughput Screening ◽  
Acetylcholine Receptors ◽  
Neuromuscular Junctions ◽  
Neuromuscular Disorders ◽  
Cluster Assembly ◽  
Achr Cluster ◽  
Robust Clustering ◽  
Coated Surfaces

AbstractMotor neurons form specialized synapses with skeletal muscle fibers, called neuromuscular junctions (NMJs). Cultured myotubes are used as a simplified in vitro system to study the postsynaptic specialization of muscles. The stimulation of myotubes with the glycoprotein agrin or laminin-111 induces the clustering of postsynaptic machinery that contains acetylcholine receptors (AChRs). When myotubes are grown on laminin-coated surfaces, AChR clusters undergo developmental remodeling to form topologically complex structures that resemble mature NMJs. Needing further exploration are the molecular processes that govern AChR cluster assembly and its developmental maturation. Here, we describe an improved protocol for culturing muscle cells to promote the formation of complex AChR clusters. We screened various laminin isoforms and showed that laminin-221 was the most potent for inducing AChR clusters, whereas laminin-121, laminin-211, and laminin-221 afforded the highest percentages of topologically complex assemblies. Human primary myotubes that were formed by myoblasts obtained from patient biopsies also assembled AChR clusters that underwent remodeling in vitro. Collectively, these results demonstrate an advancement of culturing myotubes that can facilitate high-throughput screening for potential therapeutic targets for neuromuscular disorders.

scholarly journals Human induced pluripotent stem cell-derived three-dimensional cardiomyocyte tissues ameliorate the rat ischemic myocardium by remodeling the extracellular matrix and cardiac protein phenotype

PLoS ONE ◽  
2021 ◽  
Vol 16 (3) ◽  
pp. e0245571
Author(s):  
Junya Yokoyama ◽  
Shigeru Miyagawa ◽  
Takami Akagi ◽  
Mitsuru Akashi ◽  
Yoshiki Sawa
Keyword(s):  
Myocardial Infarction ◽  
Extracellular Matrix ◽  
Pluripotent Stem Cell ◽  
Heart Function ◽  
Three Dimensional ◽  
Induced Pluripotent Stem Cell ◽  
Left Ventricular ◽  
Induced Pluripotent

The extracellular matrix (ECM) plays a key role in the viability and survival of implanted human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). We hypothesized that coating of three-dimensional (3D) cardiac tissue-derived hiPSC-CMs with the ECM protein fibronectin (FN) would improve the survival of transplanted cells in the heart and improve heart function in a rat model of ischemic heart failure. To test this hypothesis, we first explored the tolerance of FN-coated hiPSC-CMs to hypoxia in an in vitro study. For in vivo assessments, we constructed 3D-hiPSC cardiac tissues (3D-hiPSC-CTs) using a layer-by-layer technique, and then the cells were implanted in the hearts of a myocardial infarction rat model (3D-hiPSC-CTs, n = 10; sham surgery control group (without implant), n = 10). Heart function and histology were analyzed 4 weeks after transplantation. In the in vitro assessment, cell viability and lactate dehydrogenase assays showed that FN-coated hiPSC-CMs had improved tolerance to hypoxia compared with the control cells. In vivo, the left ventricular ejection fraction of hearts implanted with 3D-hiPSC-CT was significantly better than that of the sham control hearts. Histological analysis showed clear expression of collagen type IV and plasma membrane markers such as desmin and dystrophin in vivo after implantation of 3D-hiPSC-CT, which were not detected in 3D-hiPSC-CMs in vitro. Overall, these results indicated that FN-coated 3D-hiPSC-CT could improve distressed heart function in a rat myocardial infarction model with a well-expressed cytoskeletal or basement membrane matrix. Therefore, FN-coated 3D-hiPSC-CT may serve as a promising replacement for heart transplantation and left ventricular assist devices and has the potential to improve survivability and therapeutic efficacy in cases of ischemic heart disease.

scholarly journals Dynamic neuromuscular remodeling precedes motor-unit loss in a mouse model of ALS

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Éric Martineau ◽  
Adriana Di Polo ◽  
Christine Vande Velde ◽  
Richard Robitaille
Keyword(s):  
Motor Neuron ◽  
Motor Neurons ◽  
Axonal Degeneration ◽  
Synapse Formation ◽  
Neuromuscular Junctions ◽  
Motor Units ◽  
Early Event ◽  
Motor Neuron Degeneration ◽  
Single Motor Units

Despite being an early event in ALS, it remains unclear whether the denervation of neuromuscular junctions (NMJ) is simply the first manifestation of a globally degenerating motor neuron. Using in vivo imaging of single axons and their NMJs over a three-month period, we identify that single motor-units are dismantled asynchronously in SOD1G37R mice. We reveal that weeks prior to complete axonal degeneration, the dismantling of axonal branches is accompanied by contemporaneous new axonal sprouting resulting in synapse formation onto nearby NMJs. Denervation events tend to propagate from the first lost NMJ, consistent with a contribution of neuromuscular factors extrinsic to motor neurons, with distal branches being more susceptible. These results show that NMJ denervation in ALS is a complex and dynamic process of continuous denervation and new innervation rather than a manifestation of sudden global motor neuron degeneration.

scholarly journals Culturing Keratinocytes on Biomimetic Substrates Facilitates Improved Epidermal Assembly In Vitro

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1177
Author(s):  
Eve Hunter-Featherstone ◽  
Natalie Young ◽  
Kathryn Chamberlain ◽  
Pablo Cubillas ◽  
Ben Hulette ◽  
...  
Keyword(s):  
Cell Biology ◽  
Three Dimensional ◽  
Epidermal Keratinocytes ◽  
Mechanical Stimuli ◽  
Linc Complex ◽  
Mechanical Conditioning ◽  
Keratinocyte Cell ◽  
The Impact

Mechanotransduction is defined as the ability of cells to sense mechanical stimuli from their surroundings and translate them into biochemical signals. Epidermal keratinocytes respond to mechanical cues by altering their proliferation, migration, and differentiation. In vitro cell culture, however, utilises tissue culture plastic, which is significantly stiffer than the in vivo environment. Current epidermal models fail to consider the effects of culturing keratinocytes on plastic prior to setting up three-dimensional cultures, so the impact of this non-physiological exposure on epidermal assembly is largely overlooked. In this study, primary keratinocytes cultured on plastic were compared with those grown on 4, 8, and 50 kPa stiff biomimetic hydrogels that have similar mechanical properties to skin. Our data show that keratinocytes cultured on biomimetic hydrogels exhibited major changes in cellular architecture, cell density, nuclear biomechanics, and mechanoprotein expression, such as specific Linker of Nucleoskeleton and Cytoskeleton (LINC) complex constituents. Mechanical conditioning of keratinocytes on 50 kPa biomimetic hydrogels improved the thickness and organisation of 3D epidermal models. In summary, the current study demonstrates that the effects of extracellular mechanics on keratinocyte cell biology are significant and therefore should be harnessed in skin research to ensure the successful production of physiologically relevant skin models.

scholarly journals Interaction between a Novel Oligopeptide Fragment of the Human Neurotrophin Receptor TrkB Ectodomain D5 and the C-Terminal Fragment of Tetanus Neurotoxin

Molecules ◽  
2021 ◽  
Vol 26 (13) ◽  
pp. 3988
Author(s):  
Ana Candalija ◽  
Thomas Scior ◽  
Hans-Richard Rackwitz ◽  
Jordan E. Ruiz-Castelan ◽  
Ygnacio Martinez-Laguna ◽  
...  
Keyword(s):  
Motor Neurons ◽  
Neuromuscular Junctions ◽  
Hot Spot ◽  
Potential Interaction ◽  
Neurotrophin Receptor ◽  
Downstream Signaling ◽  
Cellular Models ◽  
Tetanus Neurotoxin

This article presents experimental evidence and computed molecular models of a potential interaction between receptor domain D5 of TrkB with the carboxyl-terminal domain of tetanus neurotoxin (Hc-TeNT). Computational simulations of a novel small cyclic oligopeptide are designed, synthesized, and tested for possible tetanus neurotoxin-D5 interaction. A hot spot of this protein-protein interaction is identified in analogy to the hitherto known crystal structures of the complex between neurotrophin and D5. Hc-TeNT activates the neurotrophin receptors, as well as its downstream signaling pathways, inducing neuroprotection in different stress cellular models. Based on these premises, we propose the Trk receptor family as potential proteic affinity receptors for TeNT. In vitro, Hc-TeNT binds to a synthetic TrkB-derived peptide and acts similar to an agonist ligand for TrkB, resulting in phosphorylation of the receptor. These properties are weakened by the mutagenesis of three residues of the predicted interaction region in Hc-TeNT. It also competes with Brain-derived neurotrophic factor, a native binder to human TrkB, for the binding to neural membranes, and for uptake in TrkB-positive vesicles. In addition, both molecules are located together In Vivo at neuromuscular junctions and in motor neurons.

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