Induced Pluripotent Stem Cell Generation

Lenti-X™ 293T cell line (Clontech, Cat# 632180) was maintained in DMEM medium plus10% FBS and 1% Pen Strep. Mouse embryonic fibroblasts (MEFs) from various mice strains were prepared from E13.5 embryos and maintained in DMEM medium plus 10% FBS and 1% Pen Strep. MEFs used for reprogramming were within passage three. For reprogramming, MEFs were cultured in reprogramming medium (DMEM with 15% FBS, 1% Pen Strep, Glutamax, Sodium Pyruvate, MEM Non-Essential Amino Acids, 0.1mM β-mercaptoethanol (Thermo Fisher Scientific), 1000 U ml⁻¹ LIF (Millipore Sigma, Cat# ESG1106), and 2μg ml doxycycline). Mouse embryonic stem cells (ESCs) were prepared from mouse blastocysts at embryonic day 3.5 (E3.5)^{45 (link)}. Mouse ESCs and iPSCs were cultured in reprogramming medium and grown on MEF feeder cells. For feeder-free culture, mouse ESCs and iPSCs were cultured in Knockout DMEM medium plus 20% KOSR (Thermo Fisher Scientific, Cat# 10828028), 1% Pen Strep, Glutamax, Sodium Pyruvate, MEM Non-Essential Amino Acids 0.1mM, β-mercaptoethanol, 1000 U ml⁻¹, 3 μM CHIR99021 (Selleckchem, Cat# S1263) and 1μM of PD0325901 (Selleckchem, Cat# S1036). To induce MEFs into neurons, MEFs within three passages were infected with Ascl1 lentivirus construct and cultured in N3 medium for neuronal differentiation^{33 (link)}. Cells were free of mycoplasma.