Differentiation of Mouse ESCs into Spinal Motor Neurons

HBG3 Hb9::EGFP mouse ESCs (a gift from H. Wichterle, Columbia University) and newly generated Hb9::GFP and Hb9::Foxp1; Hb9::GFP mouse ESCs were maintained and differentiated into MNs as previously described^{16 ,52 (link)}. Briefly, mouse ESCs were first plated on gelatin to remove MEFs prior to differentiation, and then plated in 60mm bacterial petri dishes in core MN medium to induce embryoid body (EB) formation. Core MN medium consisted of a 1:1 mixture of Dulbecco’s Modified Eagle’s Medium/F12 (DMEM/F12) and Neurobasal Medium supplemented with 10% Knockout Serum Replacement, 1% Glutamax, 2-mercaptoethanol (560 nM), 1% Penicillin/Streptomycin, and Primocin (50 µg ml⁻¹; Invivogen). Except as noted, media components were obtained from Invitrogen. 2 days later, N2 supplement (1x, Invitrogen), Retinoic Acid (RA; 1 µM; Sigma), and Smoothened agonist (SAG; 1 µM; Calbiochem) were added to the EBs. After 5–6 days, the EBs were either dissociated and plated on matrigel-coated coverslips for immunostaining or used for transplantation and muscle assays. For culturing the MNs past day 6, RA and SAG were removed from the medium and replaced with Glia-Derived Neurotrophic Factor (10 ng ml⁻¹; Peprotech), Brain-Derived Neurotrophic Factor (10 ng ml⁻¹; Peprotech), and Ciliary Neurotrophic Factor (10 ng ml⁻¹; Peprotech).