Human thrombopoietin htpo

In cooperation with the Department of Gynecology and Prenatal Medicine (Hannover Medical School), umbilical cord blood (UCB) was collected from healthy donors after obtaining signed informed consent. The study was conducted according to the guidelines of the Declaration of Helsinki, and informed consent was obtained from all subjects involved in the study prior to UCB collection, as approved by the Hannover Medical School Ethics Committee (protocol code 1303-2012). All samples were anonymized. In short, peripheral blood mononuclear cells (PBMCs) were isolated under Ficoll density gradient centrifugation (400× g for 45 min at RT). Red cell lysis was performed for 3 min at RT (buffer: 4.15 g NH₄Cl; 2.3 g KHCO₃; 0.5 mL 0.5 M EDTA; in 500 mL H₂O), and CD34⁺ cells were enriched from PBMCs by magnetic separation using a bead-conjugated anti-CD34 antibody (Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer’s instructions. Isolated CD34⁺ cells were cultured in StemSpan medium (STEMCELL Technologies, Vancouver, BC, Canada) supplemented with 2 mM L-glutamine, 1 mM P/S (both Thermo Fisher Scientific, Waltham, MA, USA), 100 ng/mL human Stem Cell Factor (hSCF), 100 ng/mL human Fms-related Tyrosine Kinase 3 Ligand (hFLT3L), and 50 ng/mL human thrombopoietin (hTPO) (all from Peprotech, Hamburg, Germany) at 37 °C and 5% CO₂.