Differentiation of iNIL Stem Cells into Motor Neurons

iNIL stem cells were generously provided by Dr Esteban Mazzoni (New York University) and maintained in the 2i‐based media (Table S3) containing leukemia inhibitory factor (ESG1106; Millipore), CHIR99021 (NC0664823; Fisher Scientific, Hanover Park, IL, USA), and PD0325901 (NC0759248; Fisher Scientific). To differentiate iNIL cells into motor neurons, cells were lifted using TrypLE (12‐604‐021; Fisher) and feeder media (Table S4), seeded to a suspension culture dish (08‐772‐32; Fisher) in AK media (Table S5) [37 ], and incubated for 2 days to allow the embryoid body formation. Doxycycline was added to the suspension culture to induce the expression of three transcription factors, neurogenin‐2, islet‐1, and lhx‐3. These three transcription factors drive neuronal specification in the embryoid body. Two days later, cells were dissociated from the embryoid body using a standard approach, and the dissociated cells were resuspended in the motor neuron media cocktail containing neurotrophic factors (GDNF, BDNF, and CNTF; Table S6). The resuspended cells were plated on poly‐l‐ornithine (PLO)‐coated plates and incubated for 2 days for motor neuron maturation. The drugs were treated to the motor neuron culture at this stage. A more detailed protocol with media recipe for creating motor neurons from iNIL stem cell was described previously [32 (link)]. The file Appendix S1 contains tables that show all culture reagents with working concentrations.