Construction of a rodent neural network-skeletal muscle assembloid that simulate the postnatal development of spinal cord motor neuronal network

Abstract Neuromuscular diseases usually manifest as abnormalities involving motor neurons, neuromuscular junctions, and skeletal muscle (SkM) in postnatal stage. Present in vitro models of neuromuscular interactions require a long time and lack neuroglia involvement. Our study aimed to construct rod...

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Main Authors: Haiyang Yu, Shangbin Yang, Yuanfeng Chen, Chuangran Wu, Jing Xu, Yue Yang, Rongjie Wu, Yinan Guo, Zhen Chen, Ying Ding, Xiang Zeng, Ge Li, Yuanhuan Ma, Qiujian Zheng, Yuanshan Zeng, Biqin Lai
Format: Article
Language:English
Published: Nature Portfolio 2025-01-01
Series:Scientific Reports
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Online Access:https://doi.org/10.1038/s41598-025-88292-x
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author Haiyang Yu
Shangbin Yang
Yuanfeng Chen
Chuangran Wu
Jing Xu
Yue Yang
Rongjie Wu
Yinan Guo
Zhen Chen
Ying Ding
Xiang Zeng
Ge Li
Yuanhuan Ma
Qiujian Zheng
Yuanshan Zeng
Biqin Lai
author_facet Haiyang Yu
Shangbin Yang
Yuanfeng Chen
Chuangran Wu
Jing Xu
Yue Yang
Rongjie Wu
Yinan Guo
Zhen Chen
Ying Ding
Xiang Zeng
Ge Li
Yuanhuan Ma
Qiujian Zheng
Yuanshan Zeng
Biqin Lai
author_sort Haiyang Yu
collection DOAJ
description Abstract Neuromuscular diseases usually manifest as abnormalities involving motor neurons, neuromuscular junctions, and skeletal muscle (SkM) in postnatal stage. Present in vitro models of neuromuscular interactions require a long time and lack neuroglia involvement. Our study aimed to construct rodent bioengineered spinal cord neural network-skeletal muscle (NN-SkM) assembloids to elucidate the interactions between spinal cord neural stem cells (SC-NSCs) and SkM cells and their biological effects on the development and maturation of postnatal spinal cord motor neural circuits. After coculture with SkM cells, SC-NSCs developed into neural networks (NNs) and exhibited a high proportion of glutamatergic and cholinergic neurons, low proportion of neuroglia and gamma-aminobutyric acidergic neurons, and increased expression of synaptic markers. In NN-SkM assembloids, the acetylcholine receptors of SkM cells were upregulated, generating neuromuscular junction-like structures with NNs. The amplitude and frequency of SkM cell contraction in NN-SkM assembloids were increased by optogenetic and glutamate stimulation and blocked by tetrodotoxin and dizocilpine, respectively, confirming the existence of multisynaptic motor NNs. The coculture process involves the secretion of neurotrophin-3 and insulin growth factor-1 by SkM cells, which activate the related ERK-MAPK and PI3K-AKT signaling pathways in NNs. Inhibition of the ERK-MAPK and PI3K-AKT pathways significantly reduces neuronal differentiation and synaptic maturation of neural cells in NN-SkM assembloids, while also decreasing acetylcholine receptor formation on SkM cells. In brief, NN-SkM assembloids simulate the composition of spinal cord motor NNs and respond to motor regulatory signals, providing an in vitro model for studying postnatal development and maturation of spinal cord motor NNs.
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spelling doaj-art-1e785a4d852b496885750f62d9b0ed7c2025-02-02T12:18:09ZengNature PortfolioScientific Reports2045-23222025-01-0115111610.1038/s41598-025-88292-xConstruction of a rodent neural network-skeletal muscle assembloid that simulate the postnatal development of spinal cord motor neuronal networkHaiyang Yu0Shangbin Yang1Yuanfeng Chen2Chuangran Wu3Jing Xu4Yue Yang5Rongjie Wu6Yinan Guo7Zhen Chen8Ying Ding9Xiang Zeng10Ge Li11Yuanhuan Ma12Qiujian Zheng13Yuanshan Zeng14Biqin Lai15Guangdong Cardiovascular Institute, Guangdong Provincial People’s Hospital, Guangdong Academy of Medical SciencesKey Laboratory for Stem Cells and Tissue Engineering Ministry of Education, Guangdong Provincial Key Laboratory of Brain Function and Disease, Institute of Spinal Cord Injury, Department of Histology and Embryology, Zhongshan School of Medicine, Sun Yat-sen UniversityDepartment of Orthopedics, Guangdong Provincial People’s Hospital (Guangdong Academy of Medical Sciences), Southern Medical UniversityGuangdong Cardiovascular Institute, Guangdong Provincial People’s Hospital, Guangdong Academy of Medical SciencesKey Laboratory for Stem Cells and Tissue Engineering Ministry of Education, Guangdong Provincial Key Laboratory of Brain Function and Disease, Institute of Spinal Cord Injury, Department of Histology and Embryology, Zhongshan School of Medicine, Sun Yat-sen UniversityKey Laboratory for Stem Cells and Tissue Engineering Ministry of Education, Guangdong Provincial Key Laboratory of Brain Function and Disease, Institute of Spinal Cord Injury, Department of Histology and Embryology, Zhongshan School of Medicine, Sun Yat-sen UniversityDepartment of Orthopedics, Guangdong Provincial People’s Hospital (Guangdong Academy of Medical Sciences), Southern Medical UniversityKey Laboratory for Stem Cells and Tissue Engineering Ministry of Education, Guangdong Provincial Key Laboratory of Brain Function and Disease, Institute of Spinal Cord Injury, Department of Histology and Embryology, Zhongshan School of Medicine, Sun Yat-sen UniversityKey Laboratory for Stem Cells and Tissue Engineering Ministry of Education, Guangdong Provincial Key Laboratory of Brain Function and Disease, Institute of Spinal Cord Injury, Department of Histology and Embryology, Zhongshan School of Medicine, Sun Yat-sen UniversityKey Laboratory for Stem Cells and Tissue Engineering Ministry of Education, Guangdong Provincial Key Laboratory of Brain Function and Disease, Institute of Spinal Cord Injury, Department of Histology and Embryology, Zhongshan School of Medicine, Sun Yat-sen UniversityKey Laboratory for Stem Cells and Tissue Engineering Ministry of Education, Guangdong Provincial Key Laboratory of Brain Function and Disease, Institute of Spinal Cord Injury, Department of Histology and Embryology, Zhongshan School of Medicine, Sun Yat-sen UniversityGuangdong Cardiovascular Institute, Guangdong Provincial People’s Hospital, Guangdong Academy of Medical SciencesKey Laboratory for Stem Cells and Tissue Engineering Ministry of Education, Guangdong Provincial Key Laboratory of Brain Function and Disease, Institute of Spinal Cord Injury, Department of Histology and Embryology, Zhongshan School of Medicine, Sun Yat-sen UniversityGuangdong Cardiovascular Institute, Guangdong Provincial People’s Hospital, Guangdong Academy of Medical SciencesKey Laboratory for Stem Cells and Tissue Engineering Ministry of Education, Guangdong Provincial Key Laboratory of Brain Function and Disease, Institute of Spinal Cord Injury, Department of Histology and Embryology, Zhongshan School of Medicine, Sun Yat-sen UniversityKey Laboratory for Stem Cells and Tissue Engineering Ministry of Education, Guangdong Provincial Key Laboratory of Brain Function and Disease, Institute of Spinal Cord Injury, Department of Histology and Embryology, Zhongshan School of Medicine, Sun Yat-sen UniversityAbstract Neuromuscular diseases usually manifest as abnormalities involving motor neurons, neuromuscular junctions, and skeletal muscle (SkM) in postnatal stage. Present in vitro models of neuromuscular interactions require a long time and lack neuroglia involvement. Our study aimed to construct rodent bioengineered spinal cord neural network-skeletal muscle (NN-SkM) assembloids to elucidate the interactions between spinal cord neural stem cells (SC-NSCs) and SkM cells and their biological effects on the development and maturation of postnatal spinal cord motor neural circuits. After coculture with SkM cells, SC-NSCs developed into neural networks (NNs) and exhibited a high proportion of glutamatergic and cholinergic neurons, low proportion of neuroglia and gamma-aminobutyric acidergic neurons, and increased expression of synaptic markers. In NN-SkM assembloids, the acetylcholine receptors of SkM cells were upregulated, generating neuromuscular junction-like structures with NNs. The amplitude and frequency of SkM cell contraction in NN-SkM assembloids were increased by optogenetic and glutamate stimulation and blocked by tetrodotoxin and dizocilpine, respectively, confirming the existence of multisynaptic motor NNs. The coculture process involves the secretion of neurotrophin-3 and insulin growth factor-1 by SkM cells, which activate the related ERK-MAPK and PI3K-AKT signaling pathways in NNs. Inhibition of the ERK-MAPK and PI3K-AKT pathways significantly reduces neuronal differentiation and synaptic maturation of neural cells in NN-SkM assembloids, while also decreasing acetylcholine receptor formation on SkM cells. In brief, NN-SkM assembloids simulate the composition of spinal cord motor NNs and respond to motor regulatory signals, providing an in vitro model for studying postnatal development and maturation of spinal cord motor NNs.https://doi.org/10.1038/s41598-025-88292-xNeuromuscular interactionTissue engineeringMotor neural networkSkeletal muscleSpinal cord neural stem cells
spellingShingle Haiyang Yu
Shangbin Yang
Yuanfeng Chen
Chuangran Wu
Jing Xu
Yue Yang
Rongjie Wu
Yinan Guo
Zhen Chen
Ying Ding
Xiang Zeng
Ge Li
Yuanhuan Ma
Qiujian Zheng
Yuanshan Zeng
Biqin Lai
Construction of a rodent neural network-skeletal muscle assembloid that simulate the postnatal development of spinal cord motor neuronal network
Scientific Reports
Neuromuscular interaction
Tissue engineering
Motor neural network
Skeletal muscle
Spinal cord neural stem cells
title Construction of a rodent neural network-skeletal muscle assembloid that simulate the postnatal development of spinal cord motor neuronal network
title_full Construction of a rodent neural network-skeletal muscle assembloid that simulate the postnatal development of spinal cord motor neuronal network
title_fullStr Construction of a rodent neural network-skeletal muscle assembloid that simulate the postnatal development of spinal cord motor neuronal network
title_full_unstemmed Construction of a rodent neural network-skeletal muscle assembloid that simulate the postnatal development of spinal cord motor neuronal network
title_short Construction of a rodent neural network-skeletal muscle assembloid that simulate the postnatal development of spinal cord motor neuronal network
title_sort construction of a rodent neural network skeletal muscle assembloid that simulate the postnatal development of spinal cord motor neuronal network
topic Neuromuscular interaction
Tissue engineering
Motor neural network
Skeletal muscle
Spinal cord neural stem cells
url https://doi.org/10.1038/s41598-025-88292-x
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