B6 embryonic stem cells were derived from E1.5-E3.5 C57BL/6 mouse blastocysts.
Two-cell-stage embryos were flushed from the oviducts of C57BL/6 mice into phosphate-buffered saline (PBS) supplemented with fetal calf serum (FCS, 10%) and cultured for 3 days in HEPES-buffered potassium simplex-optimized medium (KSOM) or KSOM supplemented with 4-hydroxytamoxifen. The zona pellucida was removed, and the blastocysts were treated with rabbit anti-mouse red blood cell antibody for 30 minutes. The blastocysts were then washed three times with KSOM and treated with guinea pig complement diluted in KSOM, to promote lysis of the trophoectoderm. The inner cell mass (ICM) was then isolated and used for derivation of the ES cell lines. The isolated ICMs were seeded onto mouse embryonic fibroblasts (MEFs) and cultured for 6 days in Glasgow minimal essential medium (GMEM) supplemented with fetal calf serum (FCS, 10%), MEM non-essential amino acids (1%), GlutaMax (2mM), β-mercaptoethanol (0.1mM), and leukemia inhibitory factor (LIF, 100 units/ml). For serum-free culture, cells were cultured in N2B27 medium supplemented with CHIR99021 (3 mM ) and PD0325901 (1 μM). For feeder-free culture, cells are cultured in N2B27 supplemented with CHIR99021, PD0325901, and LIF.