Differentiation of hiPSCs into Motor Neurons

Healthy hiPSCs were procured from Coriell (ND03719, ND05280, ND00184, ND03231) and differentiated into motor neurons following published protocols.^12,85 (link) Briefly, hiPSCs were treated with small molecule cocktails that included CHIR99021, DMH1, and SB431542 to differentiate into OLIG2-positive motor neuron progenitors. These progenitors were removed from the culture surface with Accutase and seeded into non-tissue culture treated polystyrene dishes to form spheroidal aggregates. Cultures were further enriched through Notch inhibition (Compound E) and Hedgehog activation (purmorphamine). Motor neuron spheroids were then broken apart into small aggregates by slowly dissociating with a P1000 pipette until no chunks remained visible by eye. A homogenous sample of these aggregates was taken and further dissociated to obtain a reliable cell count. Finally, motor neurons were seeded dropwise at a density of roughly 750 000 neurons per substrate on top of myotubes undergoing differentiation for two to three days. All co-cultures were maintained in chick differentiation media supplemented with 10 μm Rho-associated protein kinase inhibitor (Selleck, S1049), which was removed after one day, and 10 ng/ml BDNF, CNTF, and GDNF, which were removed after one week.^24,85 (link) Media was refreshed every other day.