Single-cell transcriptomics analysis of MBCs, prePBs, plasmablasts and PCs during B to PC differentiation. (A) Schematic representation of the in vitro model of B to PC differentiation. MBCs from human peripheral blood were purified and cultured with activating molecules, sCD40L and oligodeoxynucleotides, and cytokines, IL-2, IL-10, and IL-21 to obtain prePBs at day 4. Cells were then cultured with IL-2, IL-6, IL-10, IL-15, and IL-21 cytokines to obtain PBs at day 7. Finally, PBs were cultured with IL-6, IL-15, and IFNα until day 10 to obtain PCs. Flow cytometry gating of CD19⁺/CD27⁺ MBCs at day 0, CD20⁻/CD38⁻ prePBs at day 4, CD20⁻/CD38⁺ PBs at day 7 and CD38⁺/CD138⁺ PCs at day 10. Schematic representation of the BD Rhapsody single-cell analysis system used in this study. MBC, prePB, PB, and PC were thawed and tagged with 4 different tags to associate, after sequencing, each read to 1 stage. The 4 populations (almost 10 000 cells) were pooled and loaded onto a cartridge composed of more than 200 000 wells. Unique barcoded beads were added in excess and after washing, each cell was associated to a unique bead, allowing the association of each read to a unique cell. Then, cells were lysed and messenger RNA (mRNA) was hybridized on the beads. To finish, beads were recovered to synthetize complementary DNA and amplify libraries prior to sequence. (B) UMAP representation of the 4 stages identified using tags and demultiplexing. (C) Number of positive differentially expressed genes identified for the 4 stages using pairwise comparisons (one stage vs all other cells). (D) mRNA expression of B-cell TFs: BACH2, BCL6 and PX5; and PC TFs: IRF4, PRDM1 and XBP1. (E) Heat map of the top 10 genes upregulated of each stage. (F) Gene ontology enrichment analysis showing both pathways enriched in upregulated and downregulated genes during transitions: from MBC to prePB, from prePB to PB and from PB to PC.