Lysosome abundance and inheritance predict CD33 upregulation. (A) Experimental design. (B) CD49fHSC real-time differentiation landscapes by Uniform Manifold Approximation and Projection (UMAP). Localization of CD49fHSCs and offspring until generation 3 in landscapes, and CD201, LysoBrite, CD49c, and CD33 levels before divisions. n = 4 independent experiments, 1994 cells analyzed. (C,D) Identification of high-dimensional cell states (= clusters) in real-time differentiation landscape. Frequency of cell states in different cell generations. (E) Average frequency of cell fates (= clusters, colors as in panel C) in offspring generations of Lyso^Low, Lyso^Mid, and Lyso^High CD49fHSCs. Lyso^Low CD49fHSC offspring more frequently acquire clusters with higher levels of CD33 than Lyso^High offspring. Two-way ANOVA, Sidak multiple comparison corrected. n = 3 independent experiments mean ± SEM; 1994 cells analyzed. (F) Quantification of CD33 levels (left) and LysoBriteNIR (right) before division in generation 3 offspring of Lyso^Low, Lyso^Mid, and Lyso^High CD49fHSCs. CD33 levels higher in Lyso^Low offspring. Box-plot elements: center line, median; box limits, upper and lower quartiles; Tukey’s 1.5× interquartile range; points, outlier. Two-tailed Mann-Whitney test. (G) LysoBrite levels inherited by CD49fHSC daughter cells after division and absolute difference in LysoBrite levels between sister cells. Lyso^Low and Lyso^High daughters can distribute lysosomes symmetrically (quadrants 1 and 2), whereas Lyso^High cells show strongest asymmetric inheritance of absolute LysoBrite levels (quadrant 4). A total of 139, 55, and 33 CD49fHSC divisions in quadrants 1, 2, and 4, respectively. (H) CD33 levels before division in generation 3 offspring after a-/symmetric LysoBrite inheritance in Lyso^Low and Lyso^High CD49fHSCs as depicted in panel G. Symmetric LysoBrite inheritance of Lyso^Low and Lyso^High CD49fHSCs gives rise to symmetrically high and low CD33 expression in generation 3 offspring, respectively. CD33 levels in offspring of Lyso^Low daughters is higher than Lyso^High daughters after asymmetric LysoBrite inheritance. n = 3 independent experiments. Mean ± SEM. Two-tailed paired t test. (I) Average frequency of cell fates (= clusters, colors as in panel C) in offspring (generation 3) after a-/symmetric LysoBrite inheritance in Lyso^Low and Lyso^High CD49fHSC as depicted in panel G. Sister offspring clusters are comparable after symmetric inheritance, but differ after asymmetric inheritance. Asymmetric Lyso^Low sisters more frequently acquire fates (clusters) with higher levels of CD33 than their Lyso^High sisters. χ² test, Sidak multiple comparison corrected. n = 3 independent experiments, mean ± SEM; 1994 cells analyzed.