Fig. 4: Single-cell age estimation revealed that epigenomic age determines clonal expansion potential.

a, Schematic of the experiment. CD34⁺ HSCs were used in the in vitro expansion/differentiation experiment. Cells were first expanded to D8 (CD34_500) or D14 (CD34_800) and then differentiated by SCF, IL-3 and EPO until D20. Mitochondrial mutations from the scATAC experiment were used for tracking cells derived from similar clones. Cell phenotypes were determined by the scATAC profile. b, Cells from experiments performed on D8, D14 and D20, showing a gradual transition toward terminally differentiated myeloid (my4) and erythroid (ery6) cells. c, Tracking the mean EpiTrace age of each myeloid cell clone at each timepoint. Sample numbers of independent biological clones: n = 35 (Prog D8–D20); 10 (Diff D8–D20); and 67 (Terminal D8–D20). d, Ratio of rejuvenated (clone age decrease over time) clones in all clones for the myeloid cells. The terminally differentiated cells are dominated by rejuvenated clones. e, Number of terminal myeloid cells derived from young proliferator clones (mean initial clonal EpiTrace age < 0.7) and old proliferator clones (mean initial clonal EpiTrace age ≥ 0.7) at three timepoints. f, Scatter plot of the log clonal expansion ratio on D14 (y axis) compared to the mean initial clonal EpiTrace age of the same clone (x axis). Clonal types are color-labeled. g, EpiTrace age (color) of the single cells derived from a similar clone. Three clones with different fates are shown for example. The CD34_800_42 clone was a myeloid-specific clone that generated only myeloid cells. The CD34_800_8 clone was a bipotent clone that generated both myeloid and erythroid decedents. The CD34_800_10 clone was an erythroid-specific clone that generated predominantly erythroid cells. h, Relative contribution of young proliferator clones (mean initial clonal EpiTrace < 0.7) and old proliferator clones (mean initial clonal EpiTrace age ≥ 0.7) in the terminal myeloid cell population at three timepoints. i, Scatter plot of the log clonal expansion ratio on D20 (y axis) compared to the mean initial clonal EpiTrace age of the same clone (x axis). Clonal types are color-labeled. Correlation statistics (R and P value): Pearson’s. Group statistics: t-test, two-sided. For box plots, the upper and lower bounds of boxes show 25% and 75% percentiles of the data. The median of data is shown as the horizontal line in the box. The distribution minima and maxima, defined as farthest data point distanced ≤1.5 IQR from the box bounds, are shown by the whiskers. The violin plot shows the empirically estimated density distribution of data.