B6 mESCs were cultured in ESGRO Complete Plus Serum-free Clonal Grade Medium with GSK3β inhibitor supplement (Millipore, Temecula, CA). The mESCs were transfected with a pEF1a-IRES-AcGFP vector with or without MOG gene using Lipofectamine® 2000 Transfection Reagent (Invitrogen, Carlsbad, CA) [10 (link)]. It has been reported that the transfection efficiency of this reagent in mESCs was more than 70% [12 (link)]. The mESCs were then screened in medium containing 200 μg/ml of G418 (Clontech Laboratories, Inc., Mountain View, CA) to obtain mESC lines that stably expressed the vector with MOG (MOG/mESCs) or without MOG (control mESCs).
The MOG/mESCs and control mESCs were first induced to differentiate into definitive endoderm, and then TEPs in the presence of BMP-4, FGF 7, FGF10, and EGF (BFFE), as well as rFOXN1 and rHOXA3 protein as we previously described [11 ].
The MOG/mESCs and control mESCs were first induced to differentiate into definitive endoderm, and then TEPs in the presence of BMP-4, FGF 7, FGF10, and EGF (BFFE), as well as rFOXN1 and rHOXA3 protein as we previously described [11 ].