Bone marrow hematopoietic stem cells (HSCs) are crucial to maintain lifelong production of all blood cells. Although HSCs divide infrequently, it is thought that the entire HSC pool turns over every few weeks, suggesting that HSCs regularly enter and exit cell cycle. Here, we combine flow cytometry with label-retaining assays (BrdU and histone H2B-GFP) to identify a population of dormant mouse HSCs (d-HSCs) within the lin(-)Sca1+cKit+CD150+CD48(-)CD34(-) population. Computational modeling suggests that d-HSCs divide about every 145 days, or five times per lifetime. d-HSCs harbor the vast majority of multilineage long-term self-renewal activity. While they form a silent reservoir of the most potent HSCs during homeostasis, they are efficiently activated to self-renew in response to bone marrow injury or G-CSF stimulation. After re-establishment of homeostasis, activated HSCs return to dormancy, suggesting that HSCs are not stochastically entering the cell cycle but reversibly switch from dormancy to self-renewal under conditions of hematopoietic stress.

Classically, blood arises from stem cells through a series of oligopotent progenitors that become increasingly restricted to unipotent progenitors, each slotted into a hierarchical layer based on their differentiation potential. The presence of oligopotent cells is critical to the standard model of blood differentiation as they define the path from stem cells to unipotent progenitors. We developed a new cell-sorting scheme to resolve myeloid (My), erythroid (Er) and megakaryocytic (Mk) fates from single CD34+ cells and then mapped the progenitor hierarchy across human development. Fetal liver contained large numbers of distinct oligopotent progenitors with entangled My, Er and Mk fates. Unexpectedly in adult bone marrow, few oligopotent progenitor intermediates were present with multipotent and unipotent progenitors predominating, and now Er-Mk lineages emerged from multipotent cells. The developmental shift to an adult 'two-tier' hierarchy challenges current dogma and provides a new framework to understand normal and disease states of human hematopoiesis. ‡

Intratumoral heterogeneity arises through the evolution of genetically diverse subclones during tumor progression. However, it remains unknown whether cells within single genetic clones are functionally equivalent. By combining DNA copy number alteration (CNA) profiling, sequencing, and lentiviral lineage tracking, we followed the repopulation dynamics of 150 single lentivirus-marked lineages from 10 human colorectal cancers through serial xenograft passages in mice. CNA and mutational analysis distinguished individual clones and showed that clones remained stable upon serial transplantation. Despite this stability, the proliferation, persistence, and chemotherapy tolerance of lentivirally marked lineages were variable within each clone. Chemotherapy promoted the dominance of previously minor or dormant lineages. Thus, apart from genetic diversity, tumor cells display inherent functional variability in tumor propagation potential, which contributes to both cancer growth and therapy tolerance.

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SummaryRegulated blood production is achieved through the hierarchical organization of dormant hematopoietic stem cell (HSC) subsets that differ in self-renewal potential and division frequency, with long-term (LT)-HSCs dividing the least. The molecular mechanisms underlying this variability in HSC division kinetics are unknown. We report here that quiescence exit kinetics are differentially regulated within human HSC subsets through the expression level of CDK6. LT-HSCs lack CDK6 protein. Short-term (ST)-HSCs are also quiescent but contain high CDK6 protein levels that permit rapid cell cycle entry upon mitogenic stimulation. Enforced CDK6 expression in LT-HSCs shortens quiescence exit and confers competitive advantage without impacting function. Computational modeling suggests that this independent control of quiescence exit kinetics inherently limits LT-HSC divisions and preserves the HSC pool to ensure lifelong hematopoiesis. Thus, differential expression of CDK6 underlies heterogeneity in stem cell quiescence states that functionally regulates this highly regenerative system.