Human iPSCs culture and amplification
The WTSli020 hiPSC line from fibroblasts of dermis of a healthy female that was provided by EBiSC (European Bank for induced pluripotent Stem Cells) was cultured in feeder-free conditions using Vitronectin-coated culture vessels (VTN-N; Thermo Fisher Scientific, Waltham, MA, USA) and Essential 8 Flex medium (Gibco, Grand-Island, NY, USA) supplemented with Penicillin/Streptomycin (1:1000 PenStrep; Gibco). Another hiPSC line derived from a healthy female was also used as previously described . Briefly, cells were thawed and manually expanded over five supplementary passages. For manual passaging, StemPro EZPassage tool (Thermo Fisher Scientific) was used. The automated cell culture system CompacT SelecT (Sartorius, Gottingen, Germany) was then used to generate a working cell bank using 0.25 mM EDTA (Thermo Fisher Scientific) in Phosphate-Buffered Saline (PBS; Gibco) without calcium or magnesium for cell passaging. Finally, cells were dispensed into cryovials using the automated system Fill-It (Sartorius) and frozen using CryoMed Controlled-Rate Freezer (Thermo Fisher Scientific). Quality controls (mycoplasma detection, pluripotency marker expression, genomic integrity) were performed before and after amplification.
Cells were detached using TrypLE (Thermo Fisher Scientific) and resuspended at 100 000 cells per 50 µL in staining buffer containing PBS with 2 mM EDTA and Bovine Serum Albumin (0.5% BSA; Gibco). Antibodies, TRA1-81-AF647 (BioLegend, San Diego, CA, USA) and SSEA4-PE (Miltenyi Biotech, Bergisch Gladbach, Germany) were added at appropriate concentrations (according to the manufacturer’s instructions) and incubated for 30 min at 4 °C in the dark. Cells were analyzed on a MACSQuant flow cytometer (Miltenyi Biotec) with FlowJo software (Tree Star, San Carlos, CA, USA). A total of 20,000 events were recorded for each sample.
Before passaging the cells, the cell culture supernatants of hiPSCs WTSli020-B cultures were collected and transferred directly into a safe-lock tube. The culture medium must have been in contact with the cells for at least 24 h. The supernatant samples containing cells were processed by Stemgenomics (St Eloi University Hospital Center, Montpellier, France) and analyzed using iCS-digital PCS test which detects by digital PCR more than 90% of recurrent genomic abnormalities in hPSCs supernatant (iCS-digital PSC 24-probes kit).
Differentiation of hMNs progenitors from hiPSCs
Human iPSC embryoid body-based differentiation was performed as previously described by Maury et al. . hiPSCs were dissociated with Accutase (Thermo Fisher Scientific) for 5 min at 37 °C and resuspended in basal medium which is a mix between DMEM-F12 Glutamax/Neurobasal (1:1 ratio; Gibco), N2 supplement/B27 no vitamin A supplement (1:2 ratio; Gibco), β-mercaptoethanol (0.1% β-ME; Gibco) and PenStrep (0.1%; Gibco), supplemented with small molecules including ascorbic acid (0.5 µM; Sigma-Aldrich), SB431542 (20 µM; TOCRIS-BioTechne, Minneapolis, MN, USA), LDN193189 (0.2 µM; Miltenyi Biotec), CHIR99021 (3 µM; Miltenyi Biotec) and Y-27632 (10 µM; STEMCELLS Technologies, Vancouver, Canada). Cells were seeded in suspension into T25 flask (Dutscher, Bernolsheim, France) to form embryoid bodies (EBs). During the entire culture process, small molecules were added at different timepoints including retinoic acid (0.1 µM RA; Sigma-Aldrich), Smoothened Agonist (0.5 µM SAG; STEMCELLS Technologies), Brain-Derived Neurotrophic Factor (10 ng/mL BDNF; PreproTech, Rocky Hill, NJ, USA) and γ-secretase inhibitor (10 µM DAPT; STEMCELLS Technologies). Then, EBs were dissociated at DIV 10 (days in vitro) and hMNs progenitors were generated. Cells were finally dispensed into cryovials and freezed using CryoMed Controlled-Rate Freezer (Thermo Fisher Scientific). The differentiation proceeded according to the schema presented among the figures.
Human immortalized myoblasts culture
The human immortalized myoblasts cell line (AB1167c4, from fascia lata muscle of a healthy 20 years old male) was obtained from the MyoBank, Institute of Myology (Paris, France) . Cells were seeded onto 96-well plate precoated with Collagen I (Thermo Fisher Scientific) at a concentration of 70,000 cells/cm2 in the myogenic induction medium composed of a mix between Medium 199/Dulbecco’s modified Eagle’s medium (DMEM) high glucose GlutaMAX (1:4 ratio; Gibco), Fetal Bovine Serum (20% FBS; Sigma-Aldrich, Saint-Louis, MO, USA), fetuin (25 µg/mL; Sigma-Aldrich), insulin (5 µg/mL; Gibco), Dexamethasone (0.2 µg/mL DXT; Sigma-Aldrich), Fibroblast Growth Factor (0.5 ng/mL FGF; STEMCELL Technologies), Epidermal Growth Factor (5 ng/mL EGF; STEMCELL Technologies) and gentamicin (0.1%; Gibco).
Coculture of human immortalized myoblasts and hMNs
Human immortalized myoblasts were seeded onto 96-well plate and incubated over 24 h at 37 °C within a 5% CO2 environment in 96-well plate. The myogenic induction medium was replaced with coculture medium which was a mix between 1:3 of myogenic differentiation medium and 2:3 of hMNs growth medium. The myogenic differentiation medium was composed of DMEM high glucose GlutaMAX (Gibco), gentamicin (1%; Gibco) and insulin (10 µg/mL; Gibco). The hMNs growth medium was composed of a basal medium which is a mix between DMEM-F12 Glutamax/Neurobasal (1:1 ratio; Gibco), N2 supplement/B27 no vitamin A supplement (1:2 ratio; Gibco), β-mercaptoethanol (0.1% β-ME; Gibco) and Penicillin/Streptomycin (0.1% PenStrep; Gibco), supplemented with small molecules including ascorbic acid (0.5 µM; Sigma-Aldrich), retinoic acid (0.1 µM; Sigma-Aldrich), Smoothened Agonist (0.5 µM SAG; STEMCELLS Technologies), Brain-Derived Neurotrophic Factor (10 ng/mL BDNF; PreproTech, Rocky Hill, NJ, USA), Glial-Derived Neurotrophic Factor (10 ng/mL GDNF; PreproTech), γ-secretase inhibitor (10 nM DAPT; STEMCELLS Technologies). Y-27632 (10 µM; STEMCELLS Technologies) was used only for the thawing. hMNs progenitors generated in 10 days by following the protocol previously described in the section “Differentiation of hMNs progenitors from hiPSCs” were present at a concentration of 90 000 cells/cm2 and plated directly over the myoblasts and incubated at 37 °C with 5% CO2 for up to 15 days to mature and differentiate. Half of coculture medium was changed every 5 days.
Cells were fixed with 4% paraformaldehyde for 15 min and further permeabilized and blocked with Triton X-100 (0.1%; Sigma-Aldrich) and BSA (2%; Gibco) in PBS (Gibco) for 30 min. Primary antibodies were then added and were incubated at 4 °C overnight in PBS/BSA/Triton solution. Human iPSCs were labelled with Nanog (1:1000; Abcam, Cambridge, UK), Oct4 (1:400; Thermo Fisher Scientific), Sox2 (1:200; Thermo Fisher Scientific) and TRA1-60 (1:500; Abcam). Neurons were labelled with Tuj1 (1:1000; BioLegend), Islet1 (1:200; Neuromics, Edina, MN, USA), Synaptophysin (1:100 SYN; Abcam), Neurofilament H Non-Phosphorylated (1:200 SMI32; Abcam) and SNAP25 (1:1000; Sigma-Aldrich) antibodies. Myotubes were labelled with Rhodamine-Phalloidin (1:200; Invitrogen, Carlsbad, CA, USA), Myosin-Alexa Fluor 488 (1:500 MF20; Invitrogen) and Sarcomeric Alpha Actinin (1:200 SAA; Sigma-Aldrich) antibodies. The clusters of acetylcholine receptors were labelled with AChR antibody (1:100; DSHB, Iowa City, IA, USA). Appropriate Alexa Fluor 488/594/647-conjugated secondary antibodies (1:1000; Invitrogen) were used with 4′,6-diamidino-2-phenylindole (DAPI) nuclear counterstain (1:1000; Invitrogen) for 2 h at room temperature. Staining was visualized and imaged on an Evolve EMCCD camera (Zeiss, Oberkocen, Germany) coupled to a Spinning Disk system (Nipkow, CSU-X1M 5000; Zeiss) with Metamorph (Molecular Devices, San José, CA, USA). Images were processed with Photoshop 2020 (Adobe, Mountain View, CA, USA) and ImageJ version 1.52 (NIH, Bethesda, MD, USA). To quantify Islet1+ hMNs, cells were counted with the aid of the Cell Counter plugin for ImageJ. To quantify myotubes fusion index, the ratio of the nuclei number in MF20+ myotubes with ≥ 3 nuclei versus the total number of nuclei was calculated with the aid of an ImageJ plug-in determining the number of nuclei at which another staining of interest is colocalized.
RNA extraction and quantitative reverse transcriptase chain reaction (RT-qPCR)
hMNs were plated onto 24-well plate. Total RNA from two wells was isolated with the PicoPure RNA Isolation Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. Human total adult Spinal Cord RNA (BioChain Institute Inc., Newark, CA, USA) was used as positive control. cDNA was generated from 0.5 μg of RNA with High Capacity cDNA Reverse Transcription kit (Applied Biosystems). Quantitative real time-polymerase chain reactions (QRT-PCRs) were performed with the TaqMan Universal PCR Master Mix (Applied Biosystems) and the following TaqMan Human Gene Expression Assays: ISLET1 (Hs00158126_m1); ACHE (Hs01085739_g1); CHAT (Hs00252848_m1); SYP (Hs00300531_m1); SNAP25 (Hs00938962_m1); VAMP1 (Hs01042310_m1); VAMP2 (Hs00360269_m1); VAMP3 (Hs00922166_m1); SV2A (Hs01059458_m1); SV2B (Hs00208178_m1); SV2C (Hs00392676_m1); SYT1 (Hs00194572_m1); SYT2 (Hs00980604_m1); and GAPDH (Hs03929097_g1). Quantification was performed at a threshold detection line (Ct value). The Ct of each target gene was normalized to GAPDH housekeeping gene.
Botulinum neurotoxins treatment
Cells were exposed to several doses of recombinant BoNT/A (rBoNT/A) produced from Escherichia Coli as previously described  or modified BoNT (mBoNT). Both toxins were engineered and purified to more than 90% by IPSEN (Milton Park, UK). Each dose of BoNTs was tested in triplicate and a negative control (toxin-free medium) was always included. For Western blotting experiment, after 24 h the medium containing the toxin was removed, cells were washed with PBS and lysed.
SNAP25 cleavage assay by Western blot
Cells protein lysis and Western blot were performed as previously described . Bands were visualized on an ImageQuant LAS 4000 (GE Healthcare Life Sciences, Marlborough, MA, USA) and processed with Image Studio Lite (LI-COR Biosciences, Lincoln, NE, USA). For analysis, the half maximal effective concentration (EC50) was calculated with GraphPad Prism version 8.3 (GraphPad Software).
Muscle cells contractions and Ca2+ transient measurements
Cocultures at DIV 15 were placed in 96-well plate in a live imaging system with an Evolve EMCCD camera coupled to a Spinning Disk system (Zeiss), and timelapse imaging (1 min film with time interval of 300 ms for each recording) was performed under physiological conditions (37 °C and 5% CO2). Recording was done in a same myofiber for each condition. Recording was done in basal condition before and after any drug addition, i.e., 5 µM tetrodotoxin (TTX; Tocris Bioscience, Bristol, UK), 150 µM tubocurarine (Sigma-Aldrich), rBoNT/A (IPSEN). For Ca2+, measurements cells were loaded for 15 min with 2 µM Cal520 (Abcam, Cambridge, UK), which is a Ca2+ indicator, dissolved in loading buffer, then washed 3 times with recording buffer. The loading buffer was composed of a mix between 10X Hanks' Balanced Salt Solution (HBSS) calcium magnesium/distilled water (1:8 ratio; Gibco), 1 M hepes (20 mM; Gibco) and sodium hydroxide (2 mM NaOH; Sigma-Aldrich). The recording buffer was composed of a mix between 10X HBSS/distilled water (1:8 ratio; Gibco), 1 M hepes (20 mM; Gibco), sodium hydroxide (2 mM NaOH; Sigma-Aldrich) and calcium chloride (2 mM CaCl2; Sigma-Aldrich). For each recording (contractions and Ca2+), we used three myofibers/well and three wells/condition. Myotube contraction analyses were performed with an open-source software tool MUSCLEMOTION following the instructions from the software . This software quantifies movement by subtracting the summed, absolute changes in pixel intensity between a reference frame and the frame of interest. Ca2+ oscillation analyses were performed with the semi-automated open-source Ca2+ imaging analyzer CALIMA to detect Ca2+ activity in myotubes by following the instructions from the software 
Adeno-associated virus transduction
The photoactivated domain ReaChR was obtained from Addgene (Watertown, MA, USA) under human Synapsin 1 (hSyn1) promoter (Plasmid #50954) as well as the plasmid containing the calcium sensor GCaMP6f under Human cytomegalovirus (CMV) promoter (Plasmid #40755). Both plasmids were packaged into Adeno-associated virus serotype 2 (AAV2) with a functional titer of 1013 GC/mL by Vigene Biosciences (Rockville, MD, USA). To define the best MOI for ReaChR and GCaMP6f transduction, muscle cells and hMNs were exposed to viral doses ranging in MOI of control vectors AAV2-CMV-GFP (Applied Biological Materials, Richmond, Canada) for muscle cells and AAV2-hSyn1-GFP (Vigene Biosciences) for hMNs. To quantified GFP+ control signal in hMNs and in muscle cells, colocalization between GFP+/Phalloidin+ muscle cells and GFP+/Islet1+ hMNs was counted using the ImageXpress Micro Confocal High-Content Imaging System (Molecular Devices, San José, CA, USA). After selecting the appropriate MOI, muscle cells were transduced at DIV 1 post-seeding with AAV2-CMV-GCaMP6f-WPRE-SV40pA construct with 5000 MOI, and hMNs were transduced with AAV2-hSyn1-ReaChR-citrine construct at DIV 8 post-seeding hMNs over muscle cells with 10 000 MOI.
Optogenetic stimulation procedure and Ca2+ measurements
Optogenetic imaging experiments were performed in specific recording buffer where detailed in the section “Muscle cells contractions and Ca2+ transient measurements” above. Red light stimulation of cells was triggered with a Fiber-Coupled LED 590 nm (Thorlabs, Newton, NJ, USA) connected to a DC4100 4-Channel LED Driver (Thorlabs) in live imaging system with a Spinning Disk (Zeiss) under physiological conditions. ReaChR+ hMNs were activated at 20 mW/cm2 intensity with the optical fiber by 20 pulses of light, each pulse having a duration of 20 ms. Recordings were acquired with Metamorph (Molecular Devices). The addition of drugs was performed for optogenetics assay: 150 µM tubocurarine (Sigma-Aldrich), 10–50 µM range of glutamate (Sigma-Aldrich), BoNTs (IPSEN). For BoNTs assay, recordings were performed before addition and 4 h/8 h after exposure. For all recording, we used three myofibers/well and three wells/condition. Ca2+ oscillation analysis was performed using ImageJ version 1.52 (NIH) with Region of Interest (ROI) manager and using CALIMA software . Fluorescence changes in GCaMP6f+ myotubes were expressed as the ratio F590/F0 normalized to basal values (ΔF/F0). Subsequently, the transmission of emission light intensity was quantified with a thermophile UNO laser power meter (Gentec Electro-Optics, Québec, Canada).
All statistical analysis was performed using GraphPad Prism version 8.3 (GraphPad Software). One-way ANOVA with Sidak’s post hoc tests was used to determine statistical significance (*,#p < 0.05; **,##p < 0.01; ***,###p < 0.001; ****,####p < 0.0001; ns, not significant).