Neurogenin 2-Driven Stem Cell Differentiation

Human excitatory neurons differentiated from pluripotent stem cells by over-expression of lineage-specific transcription factor-Neurogenin 2 (Ngn2) as described before^{36 (link)}. In summary, one day before conversion, we dissociated stem cells into single cells with Accutase (Innovative Cell Technologies) and seeded at ~ 40 K cells into one 24 well plate pre-coated with Matrigel (BD Biosciences) in a medium supplemented with Thiazovivin (5 µM) (STEMCELL Technologies) and doxycycline (2 mg/ml, Clontech). After 6 h, we infected the cells with lentivirus containing Ngn2, RTTA, and Cre recombinase or ΔCre (truncated form of Cre, which is not functional and is used as control). The next day, we replaced the medium with neuronal medium N2/DMEM/F12/NEAA (Invitrogen) containing doxycycline (2 mg/ml, Clontech). We kept the cells in this medium for five days, and on day six, we added ~ 10 K mouse glia cells into each 24 well and replaced the culture medium with a serum-containing medium. We analyzed the cultures approximately 3–5 weeks after induction. To generate homozygous knockout neurons, we infected the neurons with LV- Cre or ΔCre one day after induction of the Ngn2 transcription factor.