Dynamic changes across Lin⁻CD34⁺ progenitor and CD56⁺ cell compartments over time during in vitro differentiation. (A) Schematic diagram summarizes in vitro generation of NK cells from Lin⁻CD34⁺ progenitors. (B) UMAP created from multiparameter FACS analysis of cells collected from each time point during 4-week culture, with annotated CD34⁺ and CD56⁺ clusters. Bar graphs show proportions of CD34⁺ and CD56⁺ cells across the different time points from 3 individual donors. (C) NK cell in vitro trajectory projected on UMAP embedding separated into 4 clusters based on sequential upregulation of key developmental markers: CD34 at stage1/2, CD161 and CD56 at stage 3a and 3b, and CD94 at stage 4. (D) Changes in proportions of stage1/2, stage 3a, stage 3b, and stage 4 clusters across different culture time points. (E) UMAP shows expression levels of key NK cell receptors upregulated within CD56⁺ cluster during 4-week culture.