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Summary Hematopoiesis serves as a paradigm for how homeostasis is maintained within hierarchically organized cell populations. However, important questions remain as to the contribution of hematopoietic stem cells (HSCs) toward maintaining steady state hematopoiesis. A number of in vivo lineage labeling and propagation studies have given rise to contradictory interpretations, leaving key properties of stem cell function unresolved. Using processed flow cytometry data coupled with a biology-driven modeling approach, we show that in vivo flux experiments that come from different laboratories can all be reconciled into a single unifying model, even though they had previously been interpreted as being contradictory. We infer from comparative analysis that different transgenic models display distinct labeling efficiencies across a heterogeneous HSC pool, which we validate by marker gene expression associated with HSC function. Finally, we show how the unified model of HSC differentiation can be used to simulate clonal expansion in the early stages of leukemogenesis., Highlights • A unifying mathematical model reconciles interpretations of three similar experiments • Model quantifies HSC heterogeneity and hematopoietic self-renewal and differentiation • Validation is performed via an independent test dataset and FACS analysis • Applicability of the model is shown via simulations of perturbed scenarios, In this article, Takahashi, Barile, Göttgens, Höfer, and colleagues demonstrate that a unifying mathematical model can be used to reconcile opposing headline statements of hematopoietic stem cell contributions to hematopoiesis. Their model is validated by both an independent test dataset and FACS analysis. Thus, their work resolves a long-running dispute about the cell kinetics that underpin native hematopoiesis.

Summary A 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., Graphical Abstract, Highlights • During hematopoiesis, Hlf is sharply downregulated upon exit from multipotency • Prolonged Hlf expression instructs GMLPs to adopt myeloid fates • Failure to downregulate Hlf is incompatible with appropriate B and T lymphopoiesis • Hlf governs molecular programs driving myelopoiesis and inhibiting lymphopoiesis, Regulators of early blood cell formation are important in both health and disease. Wahlestedt et al. identify abrupt downregulation of the transcription factor Hlf during hematopoietic differentiation. Failure to downregulate Hlf leads to a drastically skewed output of mature blood cells, positioning Hlf as a critical regulator of hematopoiesis.

Translocations involving the mixed lineage leukemia-1 are recurrent events in acute leukemia and associate with lymphoid (ALL), myeloid (AML) or mixed lineage (MLL) subtypes. Despite an association with ALL in humans, murine MLL fusion models are persistently restricted to AML. We here explored this issue using an inducible mixed lineage leukemia-eleven nineteen leukemia (MLL-ENL) mouse model. Although multiple progenitor cell types with myeloid potential are potent AML leukemia-initiating cells, also the earliest lymphoid progenitors were capable of initiating AML. This ability to evoke a latent myeloid potential in the earliest lymphoid progenitors was lost upon further lymphoid commitment. At the same time, more downstream/committed lymphoid precursors also failed to initiate lymphoid leukemia. Co-expression of MLL-ENL with a constitutively active RAS allele, the most common co-mutation in MLL fusion leukemias, could influence on both disease latency and lineage assignment of developing leukemia in what appears to be a mutation-order-dependent manner. Finally, CEBPB-mediated transdifferentation of committed and otherwise leukemia-incompetent B-cell progenitors imbued these cells with leukemic competence for AML. Therefore, apart from providing detailed insight into the differential responsiveness of candidate target cells to a first-hit MLL fusion event, our data warrants caution to therapeutic approaches based on the concept of transdifferentiation.

Morcos et al. show that there is no evidence for abrupt entry of aging hematopoietic stem cells into quiescence after four divisions as previously suggested. They examine factors confounding pulse-chase experiments in H2B-fusion protein transgenic mice that can lead to misinterpretations of HSC proliferative behavior., 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., Graphical Abstract

Summary Homeostasis of short-lived blood cells is dependent on rapid proliferation of immature precursors. Using a conditional histone 2B-mCherry-labeling mouse model, we characterize hematopoietic stem cell (HSC) and progenitor proliferation dynamics in steady state and following several types of induced stress. HSC proliferation following HSC transplantation into lethally irradiated mice is fundamentally different not only from native hematopoiesis but also from other stress contexts. Whereas transplantation promoted sustained, long-term proliferation of HSCs, both cytokine-induced mobilization and acute depletion of selected blood cell lineages elicited very limited recruitment of HSCs to the proliferative pool. By coupling mCherry-based analysis of proliferation history with multiplex gene expression analyses on single cells, we have found that HSCs can be stratified into four distinct subtypes. These subtypes have distinct molecular signatures and differ significantly in their reconstitution potentials, showcasing the power of tracking proliferation history when resolving functional heterogeneity of HSCs., Graphical Abstract, Highlights • Histone 2B-mCherry labeling enables cell division tracking in vivo • Stressors enforce proliferation of MPPs rather than HSCs • Transplantation enforces sustained HSC proliferation • Mitotic history allows for functional separation of candidate HSCs, Säwén et al. apply histone 2B-mCherry labeling to study proliferation dynamics in normal and perturbed hematopoiesis. Of several stressors evaluated, transplantation was unique in enforcing sustained stem cell proliferation. They also demonstrate the utility of histone 2B labeling to separate candidate stem cells into subpopulations with distinct molecular and functional characteristics.

A hallmark of adult hematopoiesis is the continuous replacement of blood cells with limited lifespans. It is well established that adult hematopoietic stem cells (HSCs) are active contributors to these processes after transplantation, yet their role in native hematopoiesis has recently been called into question. Here, we use inducible lineage tracing from genetically marked adult HSCs to explore their roles in the steady state. We show that adult HSCs contribute robustly to all lineages via intermediate progenitor cells, but with neglible production of hematopoietic cells with a known fetal origin. We further reveal that the timing for regeneration of distinct blood lineages varies substantially. Finally, HSC contribution to multilineage hematopoiesis in aged animals declines with increasing age. Therefore, while HSCs are active contributors to native adult hematopoiesis, it appears that the numerical increase of HSCs is a physiologically relevant compensatory mechanism to account for a reduced differentiation capacity with age.

A hallmark of adult hematopoiesis is the continuous replacement of blood cells with limited lifespans. While active hematopoietic stem cell (HSC) contribution to multilineage hematopoiesis is the foundation of clinical HSC transplantation, recent reports have questioned the physiological contribution of HSCs to normal/steady-state adult hematopoiesis. Here, we use inducible lineage tracing from genetically marked adult HSCs and reveal robust HSC-derived multilineage hematopoiesis. This commences via defined progenitor cells, but varies substantially in between different hematopoietic lineages. By contrast, adult HSC contribution to hematopoietic cells with proposed fetal origins is neglible. Finally, we establish that the HSC contribution to multilineage hematopoiesis declines with increasing age. Therefore, while HSCs are active contributors to native adult hematopoiesis, it appears that the numerical increase of HSCs is a physiologically relevant compensatory mechanism to account for their reduced differentiation capacity with age., eLife digest As far as we know, all adult blood cells derive from blood stem cells that are located in the bone marrow. These stem cells can produce red blood cells, white blood cells and platelets – the cells fragments that form blood clots to stop bleeding. They can also regenerate, producing more stem cells to support future blood cell production. But, our understanding of the system may be incomplete. The easiest way to study blood cell production is to watch what happens after a bone marrow transplant. Before a transplant, powerful chemotherapy kills the existing stem cells. This forces the transplanted stem cells to restore the whole system from scratch, allowing scientists to study blood cell production in fine detail. But completely replacing the bone marrow puts major stress on the body, and this may alter the way that the stem cells behave. To understand how adult stem cells keep the blood ticking over on a day-to-day basis, experiments also need to look at healthy animals. Säwén et al. now describe a method to follow bone marrow stem cells as they produce blood cells in adult mice. The technique, known as lineage tracing, leaves an indelible mark, a red glow, on the stem cells. The cells pass this mark on every time they divide, leaving a lasting trace in every blood cell that they produce. Tracking the red-glowing cells over time reveals which types of blood cells the stem cells make as well as provides estimates on the timing and extent of these processes. It has previously been suggested that a few types of specialist blood cells, like brain-specific immune cells, originate from cells other than adult blood stem cells. As expected, the adult stem cells did not produce such cells. But, just as seen in transplant experiments, the stem cells were able to produce all the other major blood cell types. They made platelets at the fastest rate, followed by certain types of white blood cells and red blood cells. As the mice got older, the stem cells started to slow down, producing fewer blood cells each. To compensate, the number of stem cells increased, helping to keep blood cell numbers up. This alternative approach to studying blood stem cells shows how the system behaves in a more natural environment. Away from the stresses of transplant, the technique revealed that blood stem cells are not immune to aging. In the future, understanding more about the system in its natural state could lead to ways to boost blood stem cells as we get older.

Hematopoietic stem cells (HSCs) undergo a developmental switch in neonatal mice hallmarked by a decrease in self-renewing divisions and entry into quiescence. Molecular determinants like dynamic changes in the chromatin landscape across the switch hold key insights into developmental changes in cell fate and function but remain poorly understood. Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) has emerged as a powerful tool to study chromatin accessibility in rare cells and in combination with genome-wide expression profiling has the potential to resolve cell specific transcriptional networks. To explore the regulation of the switch between fetal liver and adult bone marrow HSC states as well as during the first stages of hematopoietic differentiation, we here asses the changes in chromatin accessibility and genome-wide gene expression patterns in immunophenotypically defined HSCs and lymphoid primed multipotent progenitors (LMPPs). Chromatin accessibility unique to adult bone marrow HSCs were found to be enriched for putative binding sites of the megakaryocyte and erythroid lineage transcription factors Nuclear factor erythroid 2 (NFE2). In parallel, adult HSC gene expression and epigenetic profiles were highly enriched for the megakaryocyte gene signature which is strikingly absent in the E14.5 fetal liver. Taken together, our results identify a previously unrecognized hallmark of the developmental switch of the HSC compartment centered around acquisition of a megakaryocyte bias. These age specific features may have important implications in host response to environmental insults.

Summary Conditional knockout mice are commonly used to study the function of specific genes in hematopoiesis. Different promoters that drive Cre expression have been utilized, with the interferon-inducible Mx1-Cre still being the most commonly used “deleter strain” in experimental hematology. However, different pitfalls associated with this system could lead to misinterpretation in functional studies. We present here two of these issues related to the use of Mx1-Cre: first, a high spontaneous recombination rate when applying commonly used techniques in experimental hematology, and second, undesired short-term consequences of the use of polyinosinic:polycytidylic acid, including changes in cellular phenotypes that, however, resolve within days. Our studies emphasize therefore that proper controls are crucial when modeling gene deletion using the Mx1-Cre transgene., Graphical Abstract, Highlights • Common research methods induce uncontrolled activation of the Mx1-Cre system • The leakiness of the Mx1-Cre system can result in misinterpretation of results • pIpC alters the phenotype of HSPCs that resolves by day 8 • pIpC has limited effects on the proliferation of HSPCs, In this article, Cammenga, Velasco-Hernandez, and colleagues describe technical issues associated with the Mx1-Cre model in hematopoiesis, including a high level of spontaneous activation induced by multiple experimental procedures. These pitfalls can lead to misinterpretation of results so proper controls are needed to avoid these problems.

Recent studies have suggested that bone marrow cells might possess a much broader differentiation potential than previously appreciated. In most cases, the reported efficiency of such plasticity has been rather low and, at least in some instances, is a consequence of cell fusion. After myocardial infarction, however, bone marrow cells have been suggested to extensively regenerate cardiomyocytes through transdifferentiation. Although bone marrow-derived cells are already being used in clinical trials, the exact identity, longevity and fate of these cells in infarcted myocardium have yet to be investigated in detail. Here we use various approaches to induce acute myocardial injury and deliver transgenically marked bone marrow cells to the injured myocardium. We show that unfractionated bone marrow cells and a purified population of hematopoietic stem and progenitor cells efficiently engraft within the infarcted myocardium. Engraftment was transient, however, and hematopoietic in nature. In contrast, bone marrow-derived cardiomyocytes were observed outside the infarcted myocardium at a low frequency and were derived exclusively through cell fusion.

Missense mutations and other chromosomal perturbations underlie the heterogeneity of acute leukemia. While Mixed lineage leukemia-1 (MLL1/KMT2A) translocations are recurrent in human acute leukemia, most if not all MLL fusions are permissive for either acute myeloid (AML), acute lymphoid (ALL) or mixed/biphenotypic leukemia (MLL). At the same time, previous work have suggested that MLL rearranged leukemias require few additional mutations to induce transformation(1, 2). The MLL-ENL translocation is apart from being associated with AML also found in human acute T cell leukemia (T-ALL). Despite this, but consistent with most previous observations in mice, we failed to observe development of ALL even when inducing MLL-ENL in multiple early hematopoietic progenitors(3). We hypothesized that this could be caused by a compromised T cell generation induced by the MLL-ENL translocation product. Culturing bone marrow Granulocyte-Monocyte-Lymphoid Progenitor (GMLPs) on OP9-DL1 stromal cells revealed that MLL-ENL blocked differentiation at a stage that phenotypically corresponds to a primitive DN1 stage. In vivo, short-term induction of MLL-ENL led to deregulation of T cell differentiation, with an evident block at the DN2/3 stage. As such a compromise in T cell generation could omit T-ALL development from immature T cell progenitors in the bone marrow, we next assessed the leukemic capacity of defined lymphoid progenitor cells at different stages of development. The latent myeloid potential of DN1 cells and early B lymphoid progenitors (BLPs), a property lost upon further differentiation of these cells, was sufficient to confer potent leukemia initiating activity for AML. By contrast, mice transplanted with of later stages of T cell and B cell progenitors failed to associate with development of disease. The discrepancy of lineage assignment between MLL translocations in established human leukemia and attempts to mimic these in the mouse might be due to a requirement of necessary co-mutations. Although MLL fusions typically associate with few secondary mutations, previous studies have established that activating somatic mutations in the RAS pathway are frequently co-occurring with MLL rearranged leukemias. Therefore, we next investigated whether mutation order might influence on the developing murine leukemia. Co-expression of KRASG12D in GMLPs led to a significant reduction in disease latency compared to MLL-ENL alone. However, disease was still restricted to the myeloid lineage. In strikingly contrast, but in agreement with previous reports, mice receiving KRASG12D expressing GMLPs in the absence of MLL-ENL expression developed T-ALL with a median latency of 143 days and with roughly a 30% penetrance. The onset of MLL-ENL expression 5 weeks post transplantation in KRASG12D expressing GMLPs also gave rise to T-ALL, but with a shorter mean latency (111 days) and higher (50%) penetrance. The sequence of acquisition of somatic mutations therefore can influence not only on disease latency, but also on the lineage assignment of the developing leukemia. Finally, to identify novel somatic mutations occurring during MLL-ENL induced leukemogenesis, we generated paired leukemic samples by transplanting leukemia initiating cells from individual donor mice into separate cohorts of recipients, followed by whole genome and exome sequencing upon development of AML. While MLL-ENL induced AML associated with very few secondary mutations, targeted re-sequencing of a panel of variants validated a few single nucleotide variants from leukemic samples, including amongst others driver mutations in PTPN11 and RAS known from studies of human leukemia. Further studies are now undertaken to obtain functional insights into the mode of action of the identified somatic mutations. References 1. Andersson AK, Ma J, Wang J, Chen X, Gedman AL, Dang J, et al. The landscape of somatic mutations in infant MLL-rearranged acute lymphoblastic leukemias. Nature genetics. 2015;47(4):330-7. 2. Cancer Genome Atlas Research N. Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia. The New England journal of medicine. 2013;368(22):2059-74. 3. Ugale A, Norddahl GL, Wahlestedt M, Sawen P, Jaako P, Pronk CJ, et al. Hematopoietic stem cells are intrinsically protected against MLL-ENL-mediated transformation. Cell reports. 2014;9(4):1246-55. Disclosures No relevant conflicts of interest to declare.

Hematopoietic stem cell (HSC) function is typically evaluated using transplantation experiments, which offers quantitative and qualitative information on their self-renewal and multilineage differentiation potential. In this setting, potent long-term multilineage contribution can be observed from even single HSCs. However, emerging data has highlighted fundamental differences between hematopoiesis as seen after transplantation compared to that in steady state, with surprisingly little evidence of robust lymphomyeloid contribution from HSCs in native adult hematopoiesis (Busch et al., 2015; Sun et al., 2014). On the other hand, multilineage capacity has most often been defined as the ability to produce mature B-, T- and myeloid lineage cells, with evaluation of the capacity to generate erythroid cells and thrombocytes often neglected. Thus, the bulk amount of hematopoietic cells in need of continuous regeneration has been omitted from such analyses. Here, we evaluated the kinetics of blood cell generation in adult hematopoiesis using Fgd5-CreERT2 mediated cell tracing (Gazit et al., 2014), a model that can approach close to 100% label of adult HSCs in a highly specific manner. In agreement with that seen using an alternative model for HSC lineage tracing (Busch et al., 2015), HSC derived peripheral B and T lymphoid cells emerged with very slow kinetics (detection beginning at week 5 for B cells and at week 8 for T cells), while HSC derived granulocytes and monocytes emerged earlier (detectable by 3 weeks). NK cells were generated from HSCs with kinetics more resembling the kinetics for the myeloid lineages (detectable by 4 weeks), indicative of an intermediate developmental relationship of NK cells to myeloid and lymphoid cells. Finally, our ability to trace peripheral platelets revealed that this lineage was generated from HSCs not only in a highly robust manner, but also with the fastest kinetics (detectable by 2 weeks). Consistent with the mature blood cell labeling kinetics, detailed kinetic evaluations of defined myeloerythroid progenitors in the bone marrow revealed that megakaryocyte progenitors acquire label earlier than other hematopoietic progenitor subsets while thymocytes acquired label slowly. Evaluation of label progression from HSCs to other subfractions of the immature Lineage negative, c-kit positive and Sca-1 (LSK) positive compartment revealed that a CD150+CD48+ subset acquired label faster than other LSK fractions, pointing to a close temporal relationship of cells with this phenotype and HSCs. The demonstration that HSCs are potent and relatively rapid contributors to the megakaryocytic/platelet lineage in the settings evaluated strongly argues for their physiological importance not only upon transplantation, but also in steady state. Disclosures Rossi: Magenta Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Intellia Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Moderna Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Studies on the developmental pathways of hematopoietic stem cells (HSCs) have led to roadmaps of differentiation and resulted in key information concerning lineage relationships and restriction points in the blood system. This knowledge is also central to understand the etiology of acute myeloid leukemia (AML), where recent work has proposed that the heterogeneity and aggressiveness of AML can associate with the developmental stage of transformation. Balanced chromosomal translocations that result in fusion proteins with aberrant transcriptional regulatory activities are frequent initiating events in acute myeloid leukemia, and a prototype family of such chimeric transcription factors is represented by fusions involving the mixed lineage leukemia-1 (MLL1) gene. Previous work using mouse models have suggested that at some stage of normal differentiation there is a loss of competence to induce AML. However discrepancies exists between these mouse models concerning the target cells of MLL fusion genes. While it is clear that cells can lose competence for leukemic transformation as part of their normal differentiation, the question remains whether the most primitive HSCs are always imbued with leukemogenic competency as part of their normal biology. To address this, we developed a Doxycycline inducible transgenic mouse model of the human chimeric transcription factor Mixed Lineage Leukemia-Eleven Nineteen Leukemia (MLL-ENL). Prospective isolations of candidate leukemia-initiating cells followed by adoptive transfers allowed us to detail leukemia-initiation and competence throughout the hematopoietic hierarchy. We show that AML can origin from multiple HPC subsets with intrinsic granulocytic/monocytic potential. Closely related myeloid progenitors displayed distinct leukemic- and functional capacity in response to physiological levels of MLL-ENL, highlighting the importance of a careful prospective isolation of progenitor populations. AML could also develop efficiently from common lymphoid progenitors, supporting a latent myeloid potential of these cells. By contrast, early commitment to the megakaryocytic/erythroid lineages was incompatible with leukemic development. By contrast, disease failed to arise from the most primitive progenitor subsets, including HSCs. Investigations of the immediate transcriptional responses to MLL-ENL showed evidence for a block in differentiation in both myeloid progenitors and HSCs, while MLL-ENL restricted cell cycle progression uniquely in HSCs. Our study highlights how an oncogene can exert unique functions depending on the developmental position of its cellular targets and demonstrate the existence of a mechanism, operational at the level of immature HSCs/progenitors, which act to prevent leukemic development. Figure 1 Graphical abstract Figure 1. Graphical abstract Disclosures No relevant conflicts of interest to declare.

SummaryStudies of developmental pathways of hematopoietic stem cells (HSCs) have defined lineage relationships throughout the blood system. This is relevant to acute myeloid leukemia (AML), where aggressiveness and therapeutic responsiveness can be influenced by the initial stage of transformation. To address this, we generated a mouse model in which the mixed-lineage leukemia/eleven-nineteen-leukemia (MLL-ENL) transcription factor can be conditionally activated in any cell type. We show that AML can originate from multiple hematopoietic progenitor subsets with granulocytic and monocytic potential, and that the normal developmental position of leukemia-initiating cells influences leukemic development. However, disease failed to arise from HSCs. Although it maintained or upregulated the expression of target genes associated with leukemic development, MLL-ENL dysregulated the proliferative and repopulating capacity of HSCs. Therefore, the permissiveness for development of AML may be associated with a narrower window of differentiation than was previously appreciated, and hijacking the self-renewal capacity of HSCs by a potent oncogene is insufficient for leukemic development.