Tsang and colleagues combined a series of targeted and unbiased analyses to uncover the drivers of transcriptomic heterogeneity in murine HSCs. Multiple analyses, including the identification of variable genes, hierarchical clustering, and principal component analysis, all pointed to fluctuations in cell-cycle state as the predominant source of cellular heterogeneity. By comparing their results with a database of stage-specific cell-cycle genes, the authors showed that they could assign individual cells to each of the four cell-cycle stages. Similar results have been reported across a variety of single-cell studies , and this finding enabled the authors to calculate the fraction of cells at each stage, a measurement reflecting the kinetics of cell-cycle progression. For example, with high reproducibility across replicate experiments, they identified that 28 % of Bcl11a
+/+ cells were assigned to a G0/early G1 phase, potentially representing a quiescent or dormant subpopulation of HSCs.
When analyzing Bcl11a-deficient cells, the authors similarly found significant heterogeneity in cell-cycle progression. Intriguingly, they observed that these cells tended to be assigned (61 %) to the S and G2/M cell-cycle phases, a more than twofold increase compared with the wild-type controls. Consistent with this finding, knockout cells exhibited reduced levels of known HSC quiescence regulators, and signature genes associated with HSC self-renewal. The authors thus proposed that Bcl11a acts as a key regulator of HSC proliferation and maintenance, and validated this finding using staining for proliferation markers, as well as donor transplantation experiments. The collection of these analyses demonstrates an important lesson that is likely to hold true in many single-cell studies: although cell-cycle variation may represent an unsurprising source of cellular heterogeneity, changes in cycling kinetics may also reflect important differences in cellular activation state and fate potential.