Ex vivo expansion of adult MNCs

For each experiment, 50 × 10⁶ adult MNCs were thawed and washed twice in IMDM plus 1% BSA or HSA to remove residual DMSO. Cells were kept overnight in IMDM, 5% FBS, 4% BSA (or 9% HSA), 1% l-glutamine/PSF/β-Mpt and supplemented with SCF and IL-3 (1 ng/ml each) at 37°C in a fully humidified 5% CO₂/air atmosphere (Forma Scientific, Marietta, OH, USA). The following day (considered day 0 culture), viable MNCs were counted, washed once with IMDM plus 1% FBS (or HSA), and seeded at 10⁶ cells/ml in HEMA^ser or HEMA^def medium and stimulated with human SCF (SCF, 10 ng/ml, Amgen, Thousand Oaks, CA, USA), human EPO (3 U/ml, Neorecormon, Auckland, New Zeland), and human IL-3 (1 ng/ml, Biosource, San Jose, CA, USA). DXM and ES (both from Sigma-Aldrich) were added at 10⁻⁶ M final concentration, unless otherwise stated. Selected experiments were performed in the presence of human TPO (50 ng/ml of PEG-MGDF, Kirin Brewery, Gunma, Japan). The cells were cultured at 37°C in a fully humidified 5% CO₂/air atmosphere for 17–21 days. During the culture, the cell concentration was monitored on a regular basis and the cultures supplemented with fresh media to maintain the concentration below 10⁶ cells/ml. DXM and ES (0.5% v/v) were freshly added to the culture every 48 h.