Single-cell transcriptomic analysis of in vitro–generated CD56 subsets uncovered diverging NK cell and ILC3 signatures highly resembling primary ILC profiles. (A) Schematic summary of experimental outline: cells generated in vitro from Lin⁻CD34⁺ progenitors after 4-week culture were column-purified for CD56⁺ cells and subjected to scRNA-seq. (B) UMAP obtained from scRNA-seq data displayed CD56⁺ cells (cluster 0-5 and cluster 8) and other non-ILC clusters. (C) Violin plots of expression levels of NK cell receptor transcripts (top) and ILC3 associated transcripts (bottom) among CD56⁺ clusters. Statistical significance was performed with Kruskal-Wallis test between pairwise comparisons cluster 4 against the remaining clusters (top panels), or cluster 5 against the remaining clusters (bottom panels); ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001; and n.s. not significant. (D) Dot plots showing percentage (as size) and expression level (as color scale) of cytotoxic genes (top) and ILC3-specific genes (bottom) among cells within CD56⁺ clusters. (E) Enrichment score for “human primary bone marrow” gene set from MSigDB database (left) and “NK cell–mediated cytotoxicity” from KEGG pathway (right) across single cells within CD56⁺ clusters. (F) Enrichment score for custom gene modules derived from bulk RNA-seq data from Collins et al,²⁸ of ex vivo CD56^dim and CD56^br circulating NK cells, and tonsil-derived ILC3 across single cells within CD56⁺ clusters. (G) UMAP obtained from scRNA-seq data displayed cell clusters generated in 2-week in vitro cultures. (H-I) Circle plots depicted cell–cell interactions from Mono-DC (H) and pDC (I) cluster toward NK-ILC cluster, in which projected chord diagrams shown top 5 pairs of ligand-receptor for these cell interactions inferred from CellChat analysis. pDC, plasmacytoid dendritic cell.