“…Mouse embryonic stem cells (mESCs) were differentiated in serum-free medium for 7 days, as detailed by Tripathy et al 32 In short, D3 mESCs (American Type Culture Collection [ATCC] no. CRL11632; Manassas, VA) maintained in Dulbecco's modified Eagle Medium (DMEM; GibcoInvitrogen) supplemented with 15% fetal bovine serum (FBS; Hyclone, Logan, UT), 2 mM glutamine (Gibco-Invitrogen), and 1000 Units of non-essential amino acids, 100 units of penicillin and 100 mg streptomycin (Gibco-Invitrogen), 0.7% b-mercaptoethanol (MP Biomedicals, Solon, OH), and 1000 units of leukemia inhibitory factor (LIF; Millipore, Billerica, MA).…”