Ex vivo Expansion of Hematopoietic Stem Cells

For ex vivo culture, CD34^-CD150⁺CD48^-LSK cells were isolated from femurs and tibiae of C57BL/6-CD45.2⁺ mice by flow cytometry, plated into flat-bottomed 96-well fibronectin-coated plates (Corning; 354409) at 50 cells per well in medium composed of F12 medium (Wako), 1% ITSX (Gibco), 10 mM HEPES (Gibco), 1% P/S/G (Gibco), 100 ng/ml mouse TPO (Wako), 10 ng/ml mouse SCF, 0.1% PVA (Peprotech) with or without 100 ng/ml mouse CXCL12 (Kola-Gen Pharma), and cultured at 37 °C with 5% CO₂ for 28 days. Medium changes were performed as described previously^{30 (link)}. Following ex vivo culture, 25% of the cells were prepared for flow cytometric analysis and 75% of the cells were prepared for competitive repopulation analysis from each well. Flow cytometric analysis and competitive repopulation analysis were performed as described above.