Human scf

CD34⁺ cells were purified from mononuclear cells of UCB by using immunomagnetic cell separation (Dynabeads; Invitrogen, https://www.thermofisher.com). Cells were cultured in DMEM supplemented with each batches of fetal bovine serum (FBS) in the presence of 100ng/ml human Flt-3 ligand (Prospec Tany, Rehovot, Israel), 100ng/ml human SCF (Prospec Tany), 40ng/ml human IL-6 (R&D Systems), 40ng/ml human IL-3 (R&D Systems) and 40ng/ml human G-CSF (Prospec Tany) supplemented with 10⁻⁶ M hydrocortisone sodium hemisuccinate (Sigma). For co-culture, MSCs were irradiated (1500 cGy) before use and co-cultured with CD34⁺ cells in similar medium conditions for 5 days as described [13 (link)]. For colony forming assay of hematopoietic progenitors, hematopoietic cells were plated for 14 days in semi-solid methylcellulose media (MethoCult; StemCell Technologies) containing cytokines, and analyzed for colony numbers and lineages as described[13 (link)]. For long-term culture-initiating cell (LTC-IC) analysis, CD34⁺ cells were co-cultured with normal MSCs for 5 days, transferred to a 6-week long-term culture, and subjected to a colony-forming assay in semi-solid medium.

For co-culture assays, MSCs were irradiated with 15 Gy of ¹³⁷Cs γ-rays (Gammacell 1000; MDS Nordion, Ottawa, ON, Canada) 18–24 h prior to co-culture with CD34⁺ cells. Purified CD34⁺ cells co-cultured with MSCs for 4 days in DMEM supplemented with FBS in the presence of a cytokine mixture (20 ng/mL human SCF; 20 ng/mL human Flt3L (ProSpec-Tany TechnoGene Ltd.), 4 ng/mL human IL3, IL6 (R&D Systems, Minneapolis, MN), and 4 ng/mL human G-CSF (ProSpec) supplemented with 10⁻⁶ M hydrocortisone (Sigma-Aldrich, St Louis, MO). Controls were performed by seeding CD34⁺ cells in suspension culture without MSCs.