Fig 2: Families are highly concordant in differentiation.(A, B) Number of cell types per family in the observed data compared with the average of 250,000 permutations of the data at 24 hr (A) and 48 hr (B). Error bars indicate 95% confidence intervals based on permutations (see Materials and methods). (C) Cells from the 15 families with the largest number of cells are color-coded by family and projected onto the Uniform Manifold Approximation and Projection in Figure 3. (D) The cumulative percentage of the maximum division of offspring from ancestor SLAM-HSCs rank-ordered by their expression level (fluorescence intensity) of CD48 (blue) or Sca-1 (red) during sort. (E) The cumulative percentage of offspring, presenting a given cell type, from ancestor cells rank-ordered by increasing cell surface marker expression. * indicates a significant deviation from the diagonal (black) as determined by Jonckheere's trend test (p-values in Figure 4—source data 1). Sample sizes for all panels can be found in Figure 4—source data 2. Figure 4—source data 1.Significance values from the Jonckheere’s ?rend test between expression levels at sort and offspring maximum generation as shown in Figure 4D.Figure 4—source data 2.95% confidence intervals of Spearman r and p-values of correlations.Figure 4—source data 3.Significance values from the Jonckheere’s trend test between expression levels at sort and presence of a given cell type among offspring.Figure 4—source data 4.Summary of significant findings as shown in Figure 4E.

Fig 3: Despite population-level heterogeneity, individual hematopoietic stem and progenitor cell (HSPC) families are substantially homogeneous.Plots are fractionated by each ancestor type (SLAM-HSCs, ST-HSCs, and multi-potent progenitors [MPPs]) and cocktail (with and without IL-3 + IL-6, indicated by + and -, respectively). (A) Simultaneous visualization of family membership, generation number, and cell type of offspring from initially seeded ancestors harvested at two time points (24 and 48 hr). Each row presents the offspring from a single ancestor. Columns identify the generation number of each recovered cell, with their phenotypic cell type indicated by color coding. Rows are sorted in increasing order of the difference between maximum and minimum generations in each ancestor’s family (generational range). (B) To test the differentiation pattern of our cell phenotypes after culture, sorted cell types as shown in Figure 1 were cultured in methylcellulose cultures and evaluated for colony forming units (CFUs) formation for megakaryocytic (MK), granulocytic/monocytic (GM), erythroid burst-forming (BFU-E), or mixed colony formation of all types (CFU-MK, CFU-GM, and BFU-E) with or without MK. Late MP did not produce any colonies in methylcellulose culture (C) The percentages of MK, CFU-MK, and mixed colony formation are indicated from the culture in serum-free fibrin clot. GMP did not produce any colonies as expected. (D) Percentage of each recovered cell type. (E) Distribution of the maximum generation per family, as indicated by color coding. (F) Proportions of recovered cell types for ancestors that have not yet divided. (G) For ancestors that have divided only once and for whom two offspring are recovered, percentage having symmetric and asymmetric fates with and without differentiation. (H) Percentage of families with each generational range. The 48 hr data (red bars) is compared to a mathematical model parameterized by a single coefficient, ?, which encodes the correlation in whether cells in the same generation within a family divide or cease to divide (Materials and methods). Shown is prediction for both the maximum likelihood best-fit value of ? (value and blue/white bars) and, as a reference, a range of other values of ? (solid blue bars). For (D–H), error bars indicate 95% confidence intervals calculated via basic bootstrap (Materials and methods). Sample sizes and p-values (from permutation tests, see Materials and methods) from the panels can be found in Figure 2—source data 1 and 2. Figure 2—source data 1.Number of families per progenitor and condition.Figure 2—source data 2.Significance values from permutation testing procedures.

Fig 4: NR treatment increases lymphoid-progenitor cell frequencies in Atm-/- bone marrow.a–c Frequency analysis of early progenitor compartments in NR-treated (NR) or untreated bone marrow from Atm+/+ (WT) or Atm-/- (ATM) mice (2–3 months). CLP common lymphoid progenitors (Lin-IL-7Ra+Flk2+), LSK lineage-Sca1+c-Kit+, HSC Lin-c-Kit+Sca1-CD34-Flk2-. d Composition of the LSK compartment. e Frequencies of lineage-biased subsets of HSCs defined by Slamf1(CD150) expression. Data are represented as mean ± SEM. n: 4–6. Kruskal–Wallis test for a–c, two-way ANOVA for d and e. q value (Kruskal–Wallis test) or p value (two-way ANOVA): <0.05*, <0.01**, <0.001***.

Fig 5: Mll-AF4 maintains the progenitor B-cell compartment in the spleen of aging mice and decreases the bone marrow HSC-MPP pool. Proportions of pre-pro-B cells (B220+CD19–ckit+ IL7R+CD43+CD24–IgM–); pro-B cells (B220+CD19+ckit+IL7R+CD43+CD24+IgM–); and pre-B cells (B220+CD19+ckit+IL7R+CD43–CD24+IgM–) in the (A) bone marrow and (B) spleen of the control and Mll-AF4+VEC-Cre+ cohort at the end of study (18 months). (C) Proportions of LT-HSC (LSK CD34–FLT3–IL7R–CD48–CD150+); ST-HSCs (LSK CD34+FLT3–IL7R–CD48–CD150+); MPPs (LSK CD34+FLT3–IL7R–CD48+), LMPPs (LSK FLT3+) and LK-CLPs (Lineage–ckit+/low Sca1–/low) in the bone marrow of the control and Mll-AF4+VEC-Cre+ cohort at the end of study (18 months). Statistical differences were assessed using a nonparametric Mann–Whitney U test with a bilateral p value (*p < 0.05, **p < 0.01, ***p < 0.001).