Isolation and Expansion of Mouse Trophoblast Stem Cells

Cell culture was performed as previously described with modifications [37 (link), 40 (link), 41 (link)]. Placentas were harvested from pregnancies of mice at E18.5. Placentas were chopped into small pieces after trimming off ~ 1 mm of the maternal portion and dissociated in an enzyme buffer containing collagenase I (1 mg/ml) and collagenase II (2.5 mg/ml, Worthington Biochemical) in PBS. The cells were expanded in DMEM/F12 (Lonza) supplemented with 20% fetal bovine serum (FBS; GE Healthcare), 100 mM nonessential amino acids (Cellgro), 55 mM beta-mercaptoethanol (Gibco), 1 mM sodium pyruvate (Cellgro), 10 ng/ml LIF (Millipore), 20 ng/ml murine basic fibroblast growth factor (bFGF; PeproTech), and 1% penicillin/streptomycin/l-glutamine (Corning). Culture medium was changed every other day. CD117⁺ TSCs were isolated using anti-mouse CD117 MicroBeads (Miltenyi Biotec, Cat. 130-091-224, 20 μl per 10⁷ cells) [37 (link)].