Mouse ESC and iPSC Maintenance and Differentiation

Mouse ESCs and iPSCs were maintained in mouse ESC maintenance medium (DMEM, 15% fetal bovine serum [FBS; Gemini Bio], 0.1 mM non-essential amino acid [Life Technologies], β-mercaptoethanol [Sigma-Aldrich], and 1,000 U/ml embryonic stem cell growth medium [ESGRO, Millipore]) on gelatin-coated plates as described previously (Kim et al., 2010 (link)). For EB differentiation, trypsinized wild-type or mutant iPSCs were suspended in Costar ultra-low-attachment cell culture plates (Corning) at a density of 1 × 10⁵ cells/ml in differentiation medium (ESC maintenance medium without ESGRO). EB samples were collected on the indicated days for total RNA extraction. For neuronal differentiation, EBs (day 4) were plated onto tissue culture plates and cultured in N2 medium (DMEM/F12 and N2 supplement [Gemini Bio]) for up to 25 days. All fibroblasts were cultured in D10 medium (DMEM and 10% FBS). NSCs were cultured in Mouse Neural Stem Cell Expansion medium (EMD Millipore) on tissue culture plates coated with polyornithine (Sigma-Aldrich) and laminin (EMD Millipore).