Large-Scale Human Dendritic Cell Differentiation Revealing Notch-Dependent Lineage Bifurcation and Heterogeneity

Summary: The ability to generate large numbers of distinct types of human dendritic cells (DCs) in vitro is critical for accelerating our understanding of DC biology and harnessing them clinically. We developed a DC differentiation method from human CD34+ precursors leading to high yields of plasmacytoid DCs (pDCs) and both types of conventional DCs (cDC1s and cDC2s). The identity of the cells generated in vitro and their strong homology to their blood counterparts were demonstrated by phenotypic, functional, and single-cell RNA-sequencing analyses. This culture system revealed a critical role of Notch signaling and GM-CSF for promoting cDC1 generation. Moreover, we discovered a pre-terminal differentiation state for each DC type, characterized by high expression of cell-cycle genes and lack of XCR1 in the case of cDC1. Our culture system will greatly facilitate the simultaneous and comprehensive study of primary, otherwise rare human DC types, including their mutual interactions. : Balan et al. report a protocol to simultaneously generate large numbers of human pDCs, cDC1s, and cDC2s from cord blood and non-mobilized CD34+ progenitors. This culture system will enable experimental testing of mechanisms controlling the differentiation or functions of human DC types and their translational application to treat cancer. Keywords: dendritic cell types, dendritic cell differentiation, plasmacytoid dendritic cells, XCR1, CLEC9A, CLEC10A, NOTCH, hematopoiesis, adjuvant, immunotherap