F12 medium

CHO cells were cultured in F12 medium (Wako, Japan) supplemented with 10 % foetal bovine serum (FBS) (PAA Laboratories GmbH, Australia) and 1 % penicillin/streptomycin (Gibco, USA). Mouse A9 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10 % FBS and 1 % penicillin/streptomycin. TT2 and TT2F mES cells were hybrids of C57BL/6 and CBA strains (a gift from Dr. S. Aizawa, RIKEN, Japan). TT2 and TT2F mES cells and MI-MAC mES cell lines were cultured in KnockOut DMEM (Gibco) supplemented with 5 % FBS, 15 % KNOCKOUT SR (KSR; Gibco), 1× minimum essential medium non-essential amino acids (Gibco), 1× GlutaMAX™-1 (Gibco), 1× nucleosides (Millipore, Germany), 55 µM 2-mercaptoethanol (Gibco), 2.0 × 10⁶ units/mL ESGRO mLIF medium supplement (Millipore), and 1 % penicillin/streptomycin.

For ex vivo culture, CD34^-CD150⁺CD48^-LSK cells were isolated from femurs and tibiae of C57BL/6-CD45.2⁺ mice by flow cytometry, plated into flat-bottomed 96-well fibronectin-coated plates (Corning; 354409) at 50 cells per well in medium composed of F12 medium (Wako), 1% ITSX (Gibco), 10 mM HEPES (Gibco), 1% P/S/G (Gibco), 100 ng/ml mouse TPO (Wako), 10 ng/ml mouse SCF, 0.1% PVA (Peprotech) with or without 100 ng/ml mouse CXCL12 (Kola-Gen Pharma), and cultured at 37 °C with 5% CO₂ for 28 days. Medium changes were performed as described previously^{30 (link)}. Following ex vivo culture, 25% of the cells were prepared for flow cytometric analysis and 75% of the cells were prepared for competitive repopulation analysis from each well. Flow cytometric analysis and competitive repopulation analysis were performed as described above.