Fig. 2

Co-culture of human CD34⁺ HSCs with human MSCs and autologous T cells enhances granulocytic lineage development and phagocytic function. Human CD34⁺ HSCs were cultured alone or co-cultured with BM-MSCs and autologous CD3⁺ T cells at defined ratios (HSCs: 1 × 10⁵ cells, T cells: 1 × 10⁵ cells, MSCs: 2 × 10⁴ cells) with 4 × 10³ CD3/CD28 T-cell activator beads for 14 daysin vitro. Total RNA was extracted from cultured cells and analyzed by real-time PCR to quantify mRNA expression of neutrophil-associated markers, including S100A8, NOX2, MMP9, and LCN2(panels A, B). Phagocytic function was assessed by co-culturing suspension cells with 100 µg/ml pHrodo-red bioparticles for 2 h, with subsequent analysis of the CD11b⁺ MPO⁺ cell population (panel C). Phagocytic activity was determined across human peripheral blood mononuclear cells (PBMCs), freshly isolated neutrophils, and hematopoietic microenvironment (HMT)-derived CD11b⁺ MPO⁺ cells. Data are presented as means ± standard error of the mean (S.E.M.) from three independent experiments. Statistical significance was assessed using a two-sided unpaired t test (*p < 0.05, **p < 0.01, ns = not significant)