Efficient Motor Neuron Differentiation

Motor neurons were generated using a pre-established protocol [37 (link)]. First, neuroepithelial cells were generated by culturing iPSCs in N2B27 media (50% Neurobasal™, 50% DMEM:F12, 0.5× N2 supplement, 0.5× B27™ supplement, and 1× GlutaMAX™ (all Thermo Fisher)) supplemented with 3 μM CHIR99021 (Tocris, Bristol, UK), 2 μM dorsomorphin (Tocris), and 2 μM SB431542 (Tocris) for four days. Neuroepithelial cells were expanded and differentiated into motor neuron progenitors with N2B27 media supplemented with 0.1 μM retinoic acid (RA) (Sigma-Aldrich) and 0.5 μM Purmorphamine (Tocris) for another two days. On day six, CHIR99021, dorsomorphin and SB431542 were withdrawn, and cells were cultured with RA and dorsomorphin for an additional six days. Media was transitioned to maturation media (BrainPhys™ Neuronal Culture Media (Stem Cells technologies, Vancouver, BC, Canada), 0.5× N2 supplement (Thermo Fisher), 0.5× B27™ supplement (Thermo Fisher), 10 ng/mL BDNF (PerpoTech), and 10 ng/mL GDNF (Peprotech)) including 0.1 μM Compound E (Calbiochem, San Diego, CA, USA) for the first three days to induce terminal differentiation via Notch inhibition.