Mobilized HSPC Isolation and Culture

The mobilized peripheral blood was collected from four healthy donors, after informed consent and approval by the local ethics committee (Ethikkommission an der TUD, Ethic board no. EK201092004). Mobilization was achieved by subcutaneous injection of granulocyte colony-stimulating factor (7.5 µg/kg per day; Granocyte, Chugai Pharma) [14 (link)]. CD34⁺ HSPCs were isolated directly after leukapheresis by magnetic-activated cell sorting (MACS) (#130-046-702, Miltenyi Biotec, Bergisch Gladbach, Germany) technology based on CD34, as we described previously [14 (link), 20 (link), 37 (link)]. CD34⁺ HSPCs were cultured in serum-free HSPC medium (CellGenix® GMP SCGM, CellGenix GmbH, Freiburg, Germany) supplemented with early acting cytokines (50 ng/mL stem cell factor, (CellGenix), 50 ng/mL fms-related tyrosine kinase-3 ligand (PeproTech, Cranbury, NJ) and 15 ng/mL interleukin-3 (R&D Systems, MN)) at a density of 7.5 × 10⁴/cm² of surface area for 1–2 days on confluent MSCs in a humidified 5% CO₂ atmosphere at 37 °C. Afterward, they were collected and cultured on sub-confluent MSCs (see below).