Spinal organoid derivation from iPSCs

Wild-type BJ fibroblast-derived iPSCs (BJ-iPS) and SMA patient-derived iPSCs (Type II SMA 1-51 N and Type I SMA 1-38 G) were cultured feeder-free on Matrigel-coated plates in MACS iPS-Brew media (Miltenyi Biotec). Routine passaging using ReLeSR (Stem Cell Technologies) is performed once every 6-7 days. Pluripotent stem cells were differentiated towards the spinal motor neuron fate following established protocols described previously^{1 (link)}. Spinal organoids were made by dissociating iPS cells into single cells, and seeded either 10,000 or 30,000 cells per well in a 96-well low-attachment plate. Eventually we used 30,000 cells per well because that resulted in better derivation of mature spinal cell types (Supplementary Figure S1). The embryoid bodies were then encapsulated in 15 μl Matrigel droplets at day 10 before transferring to spinner flasks at day 14 for neuronal maturation in the presence of growth factors BDNF and GDNF. Organoids can be maintained for at least 90 days, although in this manuscript, organoids were harvested by day 42 for analysis (Supplementary Figure S1).