Novel neuromuscular junction model in 2D and 3D myotubes co-cultured with induced pluripotent stem cell-derived motor neurons

Yoshioka, Kantaro; Ito, Akira; Kawabe, Yoshinori; Kamihira, Masamichi

doi:10.1016/j.jbiosc.2019.10.004

Cited by 21 publications

(12 citation statements)

References 39 publications

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“…It is practical to build complex 3D structures comprising multiple cell types as co-cultures either with established cell lines (spheroids) or PD cells (PDO) that correspond more closely to the organ or tumor tissues of interest. The PDO models [49] can show macroscopic functional responses [119,120,140] markedly different from those of the 2D counterparts and closer to the patient responses [121,133,134] due to their ability to emulate the 3D architectures and cell-cell interactions in patient's TME.…”

Section: D Spheroids and Organoidsmentioning

confidence: 96%

“…Since then, 3D cultures and organoids have been developed for a wide range of cell types [49,108,111,112,[119][120][121][122][123][124][125]. There are many types of support matrices now available, from the widely used Matrigel (a mouse sarcoma cell extracellular matrix) to other organic polymer supports such as alginates and hydrogels [126][127][128][129][130][131][132][133][134]. Three-dimensional cultures can also be induced without matrix support by concentrating "magnetized" cells via introduction of nano magnetic beads under a magnetic field [121,135].…”

Section: D Spheroids and Organoidsmentioning

confidence: 99%

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Resolving Metabolic Heterogeneity in Experimental Models of the Tumor Microenvironment from a Stable Isotope Resolved Metabolomics Perspective

et al. 2020

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The tumor microenvironment (TME) comprises complex interactions of multiple cell types that determines cell behavior and metabolism such as nutrient competition and immune suppression. We discuss the various types of heterogeneity that exist in solid tumors, and the complications this invokes for studies of TME. As human subjects and in vivo model systems are complex and difficult to manipulate, simpler 3D model systems that are compatible with flexible experimental control are necessary for studying metabolic regulation in TME. Stable Isotope Resolved Metabolomics (SIRM) is a valuable tool for tracing metabolic networks in complex systems, but at present does not directly address heterogeneous metabolism at the individual cell level. We compare the advantages and disadvantages of different model systems for SIRM experiments, with a focus on lung cancer cells, their interactions with macrophages and T cells, and their response to modulators in the immune microenvironment. We describe the experimental set up, illustrate results from 3D cultures and co-cultures of lung cancer cells with human macrophages, and outline strategies to address the heterogeneous TME.

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Section: D Spheroids and Organoidsmentioning

confidence: 96%

Section: D Spheroids and Organoidsmentioning

confidence: 99%

Resolving Metabolic Heterogeneity in Experimental Models of the Tumor Microenvironment from a Stable Isotope Resolved Metabolomics Perspective

et al. 2020

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“…For example, human iPSC-derived co-cultures of astrocytes and motor neurons were recently used in a study showing that astrocytes exhibit non-cell autonomous effects on motor neurons in an in vitro ALS model ( Zhao et al, 2020 ). For studies on neuromuscular disease, co-cultures of iPSC-derived motor neurons with skeletal muscle cells (such as those derived from C2C12 mouse myoblasts, primary human-derived myoblasts, or differentiated from hiPSCs, with varying degrees of difficulty and availability) to simulate a simplified version of the neuromuscular system present in vivo , and allowing for assays of NMJ function ( Demestre et al, 2015 ; Picchiarelli et al, 2019 ; Lin et al, 2020 ; Yoshioka et al, 2020 ; Stoklund Dittlau et al, 2021 ). Furthermore, organoid culture systems allow for the differentiation of multiple cell types to form a miniature version and experimentally approachable model of the tissue of interest ( Vieira de Sa et al, 2021 ).…”

Section: Enhancing Cell Maturity and Exploring Cell Biological Mechanismsmentioning

confidence: 99%

An Integrated Approach to Studying Rare Neuromuscular Diseases Using Animal and Human Cell-Based Models

Hines

Lutz

Murray

et al. 2022

Front. Cell Dev. Biol.

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As sequencing technology improves, the identification of new disease-associated genes and new alleles of known genes is rapidly increasing our understanding of the genetic underpinnings of rare diseases, including neuromuscular diseases. However, precisely because these disorders are rare and often heterogeneous, they are difficult to study in patient populations. In parallel, our ability to engineer the genomes of model organisms, such as mice or rats, has gotten increasingly efficient through techniques such as CRISPR/Cas9 genome editing, allowing the creation of precision human disease models. Such in vivo model systems provide an efficient means for exploring disease mechanisms and identifying therapeutic strategies. Furthermore, animal models provide a platform for preclinical studies to test the efficacy of those strategies. Determining whether the same mechanisms are involved in the human disease and confirming relevant parameters for treatment ideally involves a human experimental system. One system currently being used is induced pluripotent stem cells (iPSCs), which can then be differentiated into the relevant cell type(s) for in vitro confirmation of disease mechanisms and variables such as target engagement. Here we provide a demonstration of these approaches using the example of tRNA-synthetase-associated inherited peripheral neuropathies, rare forms of Charcot-Marie-Tooth disease (CMT). Mouse models have led to a better understanding of both the genetic and cellular mechanisms underlying the disease. To determine if the mechanisms are similar in human cells, we will use genetically engineered iPSC-based models. This will allow comparisons of different CMT-associated GARS alleles in the same genetic background, reducing the variability found between patient samples and simplifying the availability of cell-based models for a rare disease. The necessity of integrating mouse and human models, strategies for accomplishing this integration, and the challenges of doing it at scale are discussed using recently published work detailing the cellular mechanisms underlying GARS-associated CMT as a framework.

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“…Immunostaining of cells was performed according to our previous study with slight modifications [19]. Briefly, cell samples washed three times with phosphate buffered saline (PBS) were fixed with 4% paraformaldehyde (Nara Pathology Laboratory, Nara, Japan).…”

Section: Immunostainingmentioning

confidence: 99%

Contractile Activity of Myotubes Derived from Human Induced Pluripotent Stem Cells: A Model of Duchenne Muscular Dystrophy

Yoshioka

Ito

Horie

et al. 2021

Cells

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Duchenne muscular dystrophy (DMD) is a genetic disorder that results from deficiency of the dystrophin protein. In recent years, DMD pathological models have been created using induced pluripotent stem (iPS) cells derived from DMD patients. In addition, gene therapy using CRISPR-Cas9 technology to repair the dystrophin gene has been proposed as a new treatment method for DMD. However, it is not known whether the contractile function of myotubes derived from gene-repaired iPS cells can be restored. We therefore investigated the maturation of myotubes in electrical pulse stimulation culture and examined the effect of gene repair by observing the contractile behaviour of myotubes. The contraction activity of myotubes derived from dystrophin-gene repaired iPS cells was improved by electrical pulse stimulation culture. The iPS cell method used in this study for evaluating muscle contractile activity is a useful technique for analysing the mechanism of hereditary muscular disease pathogenesis and for evaluating the efficacy of new drugs and gene therapy.

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Novel neuromuscular junction model in 2D and 3D myotubes co-cultured with induced pluripotent stem cell-derived motor neurons

Cited by 21 publications

References 39 publications

Resolving Metabolic Heterogeneity in Experimental Models of the Tumor Microenvironment from a Stable Isotope Resolved Metabolomics Perspective

Resolving Metabolic Heterogeneity in Experimental Models of the Tumor Microenvironment from a Stable Isotope Resolved Metabolomics Perspective

An Integrated Approach to Studying Rare Neuromuscular Diseases Using Animal and Human Cell-Based Models

Contractile Activity of Myotubes Derived from Human Induced Pluripotent Stem Cells: A Model of Duchenne Muscular Dystrophy

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