Reconciling Flux Experiments for Quantitative Modeling of Normal and Malignant Hematopoietic Stem/Progenitor Dynamics.

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

The Biology of Hematopoietic Stem Cells: transgenic approaches to Dissect Native and Perturbed Hematopoiesis

Hematopoiesis is defined as the ongoing production of blood cells. As most mature blood cells are relatively short-lived and require continuous replacement, hematopoiesis is characterized by an extraordinary turnover rate with production of trillions of new blood cells every day. To cope with the enormous proliferation required to generate sufficient numbers of blood cells to maintain homeostasis, the hematopoietic system is hierarchically organized within the bone marrow. Scarce hematopoietic stem cells (HCSs) reside at the top of this hierarchy. More abundant and increasingly developmentally restricted and proliferating progenitor cells, that massively amplify hematopoietic cell generation, reside further down in the hierarchy. 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. After transplantation of myeloablated hosts, HSCs are forced to proliferate extensively to rebuild the hematopoietic system. In sharp contrast, native HSCs display very low proliferation rates. Emerging data has highlighted fundamental differences between hematopoiesis as seen after transplantation compared to that in steady state. Therefore, analysis of native hematopoiesis in models that allows for evaluation in unperturbed settings is necessary. In article 1 we characterize HSC and progenitor proliferation dynamics in the steady state and following several types of induced stress. 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. In addition, coupling of proliferation history with gene expression analysis on single cells led to identification of subtypes of HSCs that have distinct molecular signatures and differ drastically in their reconstitution potentials.The Mx1-Cre mouse strain is the most commonly used conditional gene-knockout strain in experimental hematology. The Mx1 promoter is activated by endogenous interferon release that is induced by injection of polyinosinic:polycytidylic acid (poly I:C). However, interferon is also released as a part of the inflammatory response. In Article 2, we highlight pitfalls associated with the Mx1-Cre system. Transplantation of cells where Mx1-Cre activation is required for gene knockout resulted in high rates of spontaneous gene deletion. In addition, poly I:C administration introduced alterations to the hematopoietic stem and progenitor cell (HSPC) compartment. Collectively, this study emphasize that proper controls are crucial when modeling gene deletion with the Mx1-Cre system.A model with limited HSC contribution to native hematopoiesis has been proposed. This is in sharp contrast to the continuous contribution of HSCs to hematopoiesis after transplantation. In the work leading up to article 3 we set out to explore HSC contribution to native hematopoiesis by evaluation of blood cell generation from HSCs in native adult hematopoiesis. For this we used Fgd5-CreERT2 mediated lineage tracing, a model that can label close to 100 % of adult HSCs in a highly specific manner. We show that apart from blood cells with a known fetal origin, HSC contribution to all blood cell lineages is robust and occurs via a hierarchy of defined intermediate progenitor cells. Our experiments reveal that the time course of regeneration for distinct blood lineages varied substantially. Myeloerythroid cells were generated from HSCs more rapidly than lymphoid cells, with platelets and their corresponding progenitor cells emerging first. Therefore, adult HSCs are active contributors to all lineages of adult hematopoiesis in the steady state.In summary, we have highlighted features with experimental systems/procedures that are used in experimental hematology and have explored hematopoiesis and HSC biology in models that allows evaluation of unperturbed hematopoiesis

Mitotic History Reveals Distinct Stem Cell Populations and Their Contributions to Hematopoiesis

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

Temporal multimodal single-cell profiling of native hematopoiesis illuminates altered differentiation trajectories with age

Aging negatively affects hematopoiesis, with consequences for immunity and acquired blood cell disorders. Although impairments in hematopoietic stem cell (HSC) function contribute to this, the in vivo dynamics of such changes remain obscure. Here, we integrate extensive longitudinal functional assessments of HSC-specific lineage tracing with single-cell transcriptome and epitope profiling. In contrast to recent suggestions from single-cell RNA sequencing alone, our data favor a defined structure of HSC/progenitor differentiation that deviates substantially from HSC-derived hematopoiesis following transplantation. Native age-dependent attrition in HSC differentiation manifests as drastically reduced lymphoid output through an early lymphoid-primed progenitor (MPP Ly-I). While in vitro activation fails to rescue lymphoid differentiation from most aged HSCs, robust lymphopoiesis can be achieved by culturing elevated numbers of candidate HSCs. Therefore, our data position rare chronologically aged HSC clones, fully competent at producing lymphoid offspring, as a prime target for approaches aimed to improve lymphopoiesis in the elderly

Hematopoietic Stem Cells Are Intrinsically Protected against MLL-ENL-Mediated Transformation

Studies 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

Continuous mitotic activity of primitive hematopoietic stem cells in adult mice

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