Feeder-free generation of functional dendritic cells from human pluripotent stem cells

Background: The scarcity of primary conventional dendritic cells (cDCs) and the limited functionality of monocyte-derived dendritic cells (moDCs) have long hindered mechanistic and translational studies in human dendritic cell (DC) biology and immunotherapy. Methods: We developed a feeder-free differentiation platform to generate CD1c+CD141+ human pluripotent stem cell-derived conventional dendritic cells (hPSC-cDCs) to provide a scalable source of DCs with defined properties. A Design-of-Experiments (DoE) optimization strategy was applied to refine cytokine and serum conditions, with the goal of enhancing differentiation efficiency while reducing cytokine demand. The resulting hPSC-cDCs were phenotypically, transcriptionally, and functionally characterized in comparison with primary cDC subsets and moDCs. Functional assays assessed antigen uptake, cytokine production, and the ability to prime antigen-specific CD8+ T cells. Results: The optimized protocol increased hPSC-cDC yield by more than twofold while reducing cytokine usage. hPSC-cDCs expressed canonical cDC2 markers and aligned transcriptionally with primary cDC2s. These cells exhibited efficient phagocytic activity, robust cytokine secretion in response to poly(I:C) or combined Toll-like receptors agonists, and a partially activated basal state resembling primary CD1c+CD141+ DCs in human tissues. Functionally, hPSC-cDCs induced stronger antigen-specific CD8+ T-cell proliferation, activation, and effector differentiation than moDCs. Conclusions: This feeder-free and DoE-optimized system enables reproducible, large-scale generation of functional hPSC-cDCs that phenotypically and transcriptionally resemble primary cDC2s while exhibiting stronger T-cell priming capacity than moDCs. The platform provides a defined and scalable resource for mechanistic studies, vaccine development, and ex vivo T-cell expansion for cancer immunotherapy.