Construction of a Rodent Neural Network-skeletal Muscle Assembloid That Simulate the Postnatal Development of Spinal Cord Motor Neuronal Network

Overview

Journal Sci Rep

Date 2025 Jan 29

PMID 39880975

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

Affiliations

Soon will be listed here.

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.

References

Gomez-Pinilla F, Ying Z, Opazo P, Roy R, Edgerton V . Differential regulation by exercise of BDNF and NT-3 in rat spinal cord and skeletal muscle. Eur J Neurosci. 2001; 13(6):1078-84. DOI: 10.1046/j.0953-816x.2001.01484.x. View

Shin M, Bang J, Lee J, Tran H, Park G, Lee D . Generation of Skeletal Muscle Organoids from Human Pluripotent Stem Cells to Model Myogenesis and Muscle Regeneration. Int J Mol Sci. 2022; 23(9). PMC: 9103168. DOI: 10.3390/ijms23095108. View

Urzi A, Lahmann I, Nguyen L, Rost B, Garcia-Perez A, Lelievre N . Efficient generation of a self-organizing neuromuscular junction model from human pluripotent stem cells. Nat Commun. 2023; 14(1):8043. PMC: 10730704. DOI: 10.1038/s41467-023-43781-3. View

Lin C, Yoshida M, Li L, Ikenaka A, Oshima S, Nakagawa K . iPSC-derived functional human neuromuscular junctions model the pathophysiology of neuromuscular diseases. JCI Insight. 2019; 4(18). PMC: 6795289. DOI: 10.1172/jci.insight.124299. View

Al-Khater K, Todd A . Collateral projections of neurons in laminae I, III, and IV of rat spinal cord to thalamus, periaqueductal gray matter, and lateral parabrachial area. J Comp Neurol. 2009; 515(6):629-46. PMC: 2729698. DOI: 10.1002/cne.22081. View

Gao C, Shi Q, Pan X, Chen J, Zhang Y, Lang J . Neuromuscular organoids model spinal neuromuscular pathologies in C9orf72 amyotrophic lateral sclerosis. Cell Rep. 2024; 43(3):113892. DOI: 10.1016/j.celrep.2024.113892. View

Bucchia M, Merwin S, Re D, Kariya S . Limitations and Challenges in Modeling Diseases Involving Spinal Motor Neuron Degeneration . Front Cell Neurosci. 2018; 12:61. PMC: 5845677. DOI: 10.3389/fncel.2018.00061. View

Guo X, Smith V, Jackson M, Tran M, Thomas M, Patel A . A Human-Based Functional NMJ System for Personalized ALS Modeling and Drug Testing. Adv Ther (Weinh). 2021; 3(11). PMC: 7942691. DOI: 10.1002/adtp.202000133. View

Chambers S, Qi Y, Mica Y, Lee G, Zhang X, Niu L . Combined small-molecule inhibition accelerates developmental timing and converts human pluripotent stem cells into nociceptors. Nat Biotechnol. 2012; 30(7):715-20. PMC: 3516136. DOI: 10.1038/nbt.2249. View

10.

Horiguchi S, Takahashi J, Kishi Y, Morizane A, Okamoto Y, Koyanagi M . Neural precursor cells derived from human embryonic brain retain regional specificity. J Neurosci Res. 2004; 75(6):817-24. DOI: 10.1002/jnr.20046. View

11.

Yu H, Yang S, Li H, Wu R, Lai B, Zheng Q . Activating Endogenous Neurogenesis for Spinal Cord Injury Repair: Recent Advances and Future Prospects. Neurospine. 2023; 20(1):164-180. PMC: 10080446. DOI: 10.14245/ns.2245184.296. View

12.

Faustino Martins J, Fischer C, Urzi A, Vidal R, Kunz S, Ruffault P . Self-Organizing 3D Human Trunk Neuromuscular Organoids. Cell Stem Cell. 2020; 27(3):498. DOI: 10.1016/j.stem.2020.08.011. View

13.

Leng Y, Li X, Zheng F, Liu H, Wang C, Wang X . Advances in In Vitro Models of Neuromuscular Junction: Focusing on Organ-on-a-Chip, Organoids, and Biohybrid Robotics. Adv Mater. 2023; 35(41):e2211059. DOI: 10.1002/adma.202211059. View

14.

Kadoya K, Lu P, Nguyen K, Lee-Kubli C, Kumamaru H, Yao L . Spinal cord reconstitution with homologous neural grafts enables robust corticospinal regeneration. Nat Med. 2016; 22(5):479-87. PMC: 4860037. DOI: 10.1038/nm.4066. View

15.

Zschuntzsch J, Meyer S, Shahriyari M, Kummer K, Schmidt M, Kummer S . The Evolution of Complex Muscle Cell In Vitro Models to Study Pathomechanisms and Drug Development of Neuromuscular Disease. Cells. 2022; 11(7). PMC: 8997482. DOI: 10.3390/cells11071233. View

16.

Olmsted Z, Stigliano C, Badri A, Zhang F, Williams A, Koffas M . Fabrication of homotypic neural ribbons as a multiplex platform optimized for spinal cord delivery. Sci Rep. 2020; 10(1):12939. PMC: 7395100. DOI: 10.1038/s41598-020-69274-7. View

17.

Melamed Z, Lopez-Erauskin J, Baughn M, Zhang O, Drenner K, Sun Y . Premature polyadenylation-mediated loss of stathmin-2 is a hallmark of TDP-43-dependent neurodegeneration. Nat Neurosci. 2019; 22(2):180-190. PMC: 6348009. DOI: 10.1038/s41593-018-0293-z. View

18.

Andersen J, Revah O, Miura Y, Thom N, Amin N, Kelley K . Generation of Functional Human 3D Cortico-Motor Assembloids. Cell. 2020; 183(7):1913-1929.e26. PMC: 8711252. DOI: 10.1016/j.cell.2020.11.017. View

19.

Kim H, Kim G, Hyun S, Kim E . Advancements in 2D and 3D In Vitro Models for Studying Neuromuscular Diseases. Int J Mol Sci. 2023; 24(23). PMC: 10707046. DOI: 10.3390/ijms242317006. View

20.

Takasato M, Er P, Chiu H, Maier B, Baillie G, Ferguson C . Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis. Nature. 2015; 526(7574):564-8. DOI: 10.1038/nature15695. View