Fig. 3

Motor neuron and myotube verification for the establishment of microfluidic coculture model. A Overview of motor neuron differentiation from hiPSCs through an EB state towards MN-NPCs and finally post-mitotic spinal motor neurons. B Representative confocal images of mature motor neurons at day 28 of differentiation stained with motor neuron markers ChAT and Islet-1 in addition to pan-neuronal markers NEFH and βIII-tubulin (Tubulin). Nuclei stained with DAPI. Scale bar: 25 μm. C Quantification of the number of cells positive for neuronal markers. Mean ± s.e.m. of 3 biological replicates (n = 15 images). Kruskal–Wallis test with Dunn’s multiple comparisons test. D Overview of myotube differentiation. Primary human myoblasts were isolated from vastus lateralis muscle, expanded and differentiated into multinucleated elongated myotubes. E Representative confocal images of myotubes stained with markers (AB⁺) MyoG, MyHC, desmin, titin and ACTN2. Nuclei stained with DAPI. Scale bar: 200 μm. F Quantification of myogenic markers in multinucleated myotubes. Mean ± s.e.m. of 3 biological replicates (n = 15 images). G Schematic overview of coculture between hiPSC-derived astrocytes and motor neurons with human primary myoblast-derived myotubes in a compartmentalized microfluidic device. A chemotactic and volumetric gradient is established to promote motor neuron neurite polarization [25, 26]. Experiments were conducted 1 week (d28) and 2 weeks (d35) after plating the mature astrocytes. Cell illustrations are modified from Smart Servier Medical Art licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/). See also Suppl. Figure 5, Additional file 2