Fig. 1: Geometric engineering and generation of the human motor assembloids-on-a-chip.

a Schematic of the geometric confinement, assembly processes, and mechanical anisotropy mechanisms for human motor assembloids-on-a-chip. b Heatmap of the temporal expression patterns of developmental markers over 24 days during the generation of hMNS. n = 3 independent experiments. c Whole-cell current-clamp recording of action potential induction from a neuron within hMNS in response to depolarizing currents. d and e Anchoring and geometric configurations of motor assembloids. Representative images showing models of failure and success as defined in our research (d), along with quantification of these events (e). Unmodified: n = 4 independent experiments, Surface modification: n = 5 independent experiments. f Schematic representation of ECM alignment and tissue sculpting progression over 14 days. g and h Representative immunofluorescence of motor assembloids showing muscle fiber and axon orientation in isotropic and anisotropic models, stained for myosin heavy chain (MHC; gray), β-III tubulin (TUJ1; red), and nuclei (blue). i and j Dominant directions of axon (i) and myofiber (j) orientation in isotropic and anisotropic models. n = 3 assembloids. k Displacement trace of hSkM contractions in response to electrical stimulation at 1 Hz and 10 Hz. l Immunofluorescence analysis of the motor assembloid reveals hMNS projecting TUJ1 + axons (red) into the organized striated muscle region marked by sarcomeric α-actinin (SAA; green). (i) co-localization of an axon terminal with an AChR cluster labeled by α-bungarotoxin (α-BTX; magenta); (ii) distribution of AChR clusters surrounding hMNS; (iii) localization of AChR clusters on the striated muscle fibers. Three independent experiments was repeated independently with similar results. Source data are provided as a Source Data file.