SummaryA gradual restriction in lineage potential of multipotent stem/progenitor cells is a hallmark of adult hematopoiesis, but the underlying molecular events governing these processes remain incompletely understood. Here, we identified robust expression of the leukemia-associated transcription factor hepatic leukemia factor (Hlf) in normal multipotent hematopoietic progenitors, which was rapidly downregulated upon differentiation. Interference with its normal downregulation revealed Hlf as a strong negative regulator of lymphoid development, while remaining compatible with myeloid fates. Reciprocally, we observed rapid lymphoid commitment upon reduced Hlf activity. The arising phenotypes resulted from Hlf binding to active enhancers of myeloid-competent cells, transcriptional induction of myeloid, and ablation of lymphoid gene programs, with Hlf induction of nuclear factor I C (Nfic) as a functionally relevant target gene. Thereby, our studies establish Hlf as a key regulator of the earliest lineage-commitment events at the transition from multipotency to lineage-restricted progeny, with implications for both normal and malignant hematopoiesis.

The proliferative activity of aging hematopoietic stem cells (HSCs) is controversially discussed. Inducible fluorescent histone 2B fusion protein (H2B-FP) transgenic mice are important tools for tracking the mitotic history of murine HSCs in label dilution experiments. A recent study proposed that primitive HSCs symmetrically divide only four times to then enter permanent quiescence. We observed that background fluorescence due to leaky H2B-FP expression, occurring in all H2B-FP transgenes independent of label induction, accumulated with age in HSCs with high repopulation potential. We argue that this background had been misinterpreted as stable retention of induced label. We found cell division–independent half-lives of H2B-FPs to be short, which had led to overestimation of HSC divisional activity. Our data do not support abrupt entry of HSCs into permanent quiescence or sudden loss of regeneration potential after four divisions, but show that primitive HSCs of adult mice continue to cycle rarely.

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The proliferative activity of adult hematopoietic stem cells (HSCs) is controversially discussed. Inducible fluorescent histone 2B fusion protein (H2B-FP) transgenic mice are important tools for tracking the mitotic history of murine HSCs in label dilution experiments. A recent study proposed that the most primitive HSCs divide only four times, to then enter permanent quiescence. We observed that background fluorescence due to leaky H2B-FP expression, occurring in all H2B-FP transgenes independent of label induction, accumulated with age in primitive HSCs with high repopulation potential. We argue that this background had been misinterpreted as retention of induced label and permanent quiescence. We found cell division-independent half-lives of H2B-FPs to be short, which had led to overestimation of HSC divisional activity. Our data do not support HSC mitotic memory and entry into permanent quiescence after few divisions, but show that primitive HSCs of adult mice continue to cycle rarely.In the present study, we re-evaluated H2B-FP retention. We show that in three different H2B-FP transgenic mouse strains, the most quiescent HSC sub-population inevitably accumulates high background fluorescence in an age-dependent manner. Accordingly, long-term serial repopulation activity was enriched among cells with high background. We estimated divisionindependent degradation of H2B-GFP and H2B-RFP to proceed in HSCs with half-lives of approximately 4-6 and 2 weeks, respectively, and show that neglect of H2B-FP decay leads to overestimation of HSC mitotic activity. Two sequential rounds of H2B-RFP induction and subsequent dilution revealed that HSCs do not abruptly halt mitotic activity upon ageing, arguing against permanent quiescence of HSCs after four divisions as previously hypothesized (Bernitz et al., 2016). Mathematical modeling of H2B-FP systems revealed that background label accumulates over time in the most primitive and quiescent HSCs, providing an explanation for observing seemingly label-retaining HSCs after extended chase periods.We argue that such cells were previously misinterpreted as permanently quiescent HSCs.