A three-step serum-free protocol for the robust generation of monocyte-derived dendritic cells from human hematopoietic stem/progenitor cells.

Dendritic cells (DCs) and their monocyte precursors are rare in peripheral blood, which limits their use in immunotherapy. Although hematopoietic stem/progenitor cells (HSPCs) provide an alternative source, they are even less abundant in vivo. To address this limitation and compensate for apoptosis during the generation of DCs from monocytes, we developed a three-step serum-free protocol (culture system) that combines HSPC expansion with sequential differentiation. The protocol comprises a 7-day HSPC expansion phase, a 14-day monocyte induction phase, and a 7-day DC generation phase-5 days of differentiation followed by 2 days of maturation with either tumor necrosis factor-α or lipopolysaccharide. Cell quantification revealed that the system generated approximately 5,000 monocytes and 50 DCs from a single HSPC. The generated DCs expressed the key surface markers CD40 and CD209 and exhibited antigen uptake capacity comparable to that of native CD14⁺ monocyte-derived DCs. Overall, this system provides a reproducible and scalable platform for studying DC biology and supports applications in preclinical and translational immunotherapy research.