2.4. Coculture of MSCs-HPCs

As we previously reported [20], MSC layers at 80% confluence were incubated with 0.3 μg/mL mitomycin C to inhibit cell growth. Ten thousand cells enriched in CD34⁺CD38⁻Lin⁻ cells were seeded on MSC layers in 6-well plates (Corning Inc., Costar, New York, NY, USA) in Stem Line medium (Sigma-Aldrich, St. Louis, MO, USA) with or without the early acting cytokines thrombopoietin (TPO), Flt-3 ligand (FL), stem cell factor (SCF), and interleukin-6 (IL-6) at a concentration of 10 ng/mL (Peprotech, USA). In cultures in which MSC-HPC contact was inhibited, 0.4 μm Transwells (BD) were used. Cultures were taken on day 14, with a medium change on day 7.