Clonally tracked B + NM-progenitor outputs reveal initial slow divisions are associated with delayed differentiation. (A) Experimental design used to track clonal changes in the surface expression of CD34, CD45RA, and CLEC12A. (B) Brightfield and immunofluorescence images of representative clones examined on day 12. Scale bar, 100 μm. (C) Percentages of clonal output types of early B and NM lineage cells (see detailed phenotypes defined in supplemental Table 6). Each bar shows the mean ± SEM of values pooled from 3 experiments with 944 total single cells examined. (D) Percentages of clones that maintained an exclusive CD34⁺RA^–C^– output phenotype (black line) and RA⁺C^–/C⁺ bilineage outputs (gray line) out of all bilineage clones that eventually produced RA⁺C^– and C⁺ outputs. Each point shows the mean ± SEM of values pooled from 3 experiments. The dotted line represents the time when the loss of CD34⁺RA⁺C^– clonal output reaches 50%. (E) Cumulative growth kinetics (locally estimated scatterplot smoothing [LOESS]-smoothed) of clones initiated from CD34⁺RA^–C^– cells (n = 201) according to the time of the first appearance of RA⁺C^– or C⁺ cells in them. (F) Growth curves of the geometric means of the clone sizes analyzed in panel E (points) and generated using a logistic model. (G) Growth parameters fitted to individual clones analyzed in panels D-E. Each data point represents a clone (center line, median; box limits, first and third quartiles; whiskers, 1.5× interquartile range). (H-I) Growth kinetics and fitted logistic models of clones with NM-restricted outputs (n = 350 clones pooled from 3 experiments). (J-K) Growth kinetics and fitted logistic models of clones with B-restricted outputs (n = 60 clones pooled from 3 experiments). (L) Cumulative size distribution of blast cell colonies on days 4, 8, 12, and 16 after plating from the ensemble of colonies that show the first lineage marker expression at days 2, 4, 6, and 8, respectively. Points show experimental data, and lines show a lognormal dependence: $erfc((lnn−μ)/2σ)/2$⁠, in which $μ=⟨lnn⟩$ denotes the measured average logarithm of colony size and $σ2=⟨(lnn−μ)2⟩$ represents the corresponding variance (for details, see supplemental Mathematical Model). (M) Schematic showing the model of blast cell colony expansion. In this model, blast cells have a defined proliferative capacity at the time of plating. Blast cells expand at a rate $λB$ over a time $τ$⁠, where $τ$ varies between blast colonies. At time $τ$⁠, blast cells transition near synchronously into lineage-restricted progenitor states, which have a strictly limited proliferative capacity that varies both within and between lineages. The colony growth characteristics and size are consistent with a model in which progenitor cells expand over a fixed period $T$⁠, giving rise to subclones of size $eN$⁠, with $N$ drawn at random from a Gaussian distribution with fixed mean and variance, with the capacity of B-cell progenitors an order of magnitude smaller than NM progenitors (for details of the model and its fit to the data, see supplemental Mathematical Model).