Paired cell cycle state and molecular analysis of individually tracked primitive human hematopoietic cells reveal an age-associated prolongation of G₁in the adult cells. (A) Experimental design to enable the in vitro serial immunofluorescence tracking of changes in the properties of individual cells isolated from donors of different ages over time. (B) Representative fluorescence images obtained from the experimental design described in panel A; scale bar = 100 μm. (C) UMAP distribution of different phenotypic properties of individually assessed CD49f⁺ and CD34⁺CD38^– cells from CB and young or aged adult BM cultured for 0, 24, 42, 50, or 64 hours and analyzed as described in panel A. Assignment of regions of the UMAP distribution to the different phases of the cell cycle by K-means clustering (bottom-right). (D) Proportion of CD49f⁺ cells in different phases of the cell cycle after different times in culture. The size of each circle is scaled to show the proportion of total cells in each cell cycle phase at the culture time point when the assessment was made (∗P < .05; ∗∗P < .01; ∗∗∗P < .001; Holm-corrected Wilcox tests). (E) Scaled intensity (Z scores) of CDK6, CDK2, Ki67, and pRb protein levels measured in the CD49f⁺ cells analyzed at each time point shown (∗P < .05; ∗∗P < .01; ∗∗∗P < .001; Holm-corrected Wilcox tests).