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. 2021 Jun 3;11:11695. doi: 10.1038/s41598-021-91203-5

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Number of iPSC motor neuron spheroids loaded in tissue engineered skeletal muscle determines functionality of neuromuscular tissues. (a) Schematic detailing method of iPSC motor neuron loading to generate neuromuscular tissues at different stages of myogenic development. (i) MN progenitors are loaded to spheroid microplates and matured for 14 days. (ii) Primary HDMCs are seeded in established type I collagen matrix, loaded into 3D printed mould and cultured to maturity for 2 weeks. Matured MNs are then added within optimised type I collagen hydrogel, seeded around SkM and cultured for a further 2 weeks. MN; Motor neuron, SkM; Skeletal muscle, HDMC; Human derived muscle cells, GM; Growth medium, DM; Differentiation medium, ECM; Extracellular matrix, MM; Motor neuron maintenance medium. (b) Addition of iPSC motor neurons with type I collagen perimysium at loading densities of 2, 4 or 6 spheroids per tissue evidence tetanic force and (c) concentration dependent spontaneous twitch profiles compared to skeletal muscle only tissues. (d) Quantification of functional tetanic and (e) spontaneous twitch force output with 2, 4 or 6 motor neuron spheroids. Beginning of representative tetanus and twitch contractions represents initiation of electrical field stimulation, time elapsed before this point is un-stimulated baseline recording. Individual functional data points indicative of n = 3 contraction profiles, totalling minimum of n = 9 and maximum of n = 15 per condition and presented ± standard deviation (SD). **P ≤ 0.01 and ***P ≤ 0.001.