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Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas genome engineering has emerged as a powerful tool to modify precise genomic sequences with unparalleled accuracy and efficiency. Major advances in CRISPR technologies over the last 5 years have fueled the development of novel techniques in hematopoiesis research to interrogate the complexities of hematopoietic stem cell (HSC) biology. In particular, high throughput CRISPR based screens using various "flavors" of Cas coupled with sequencing and/or functional outputs are becoming increasingly efficient and accessible. In this review, we discuss recent achievements in CRISPR-mediated genomic engineering and how these new tools have advanced the understanding of HSC heterogeneity and function throughout life. Additionally, we highlight how these techniques can be used to answer previously inaccessible questions and the challenges to implement them. Finally, we focus on their translational potential to both model and treat hematological diseases in the clinic.

Hematopoietic stem cells (HSCs) have been studied extensively since their initial functional description in 1961 when Dr. James Till and Dr. Ernest McCulloch developed the first in vivo clonal strategy, termed the "spleen colony-forming unit" (CFU-S) assay, to assess the functional capacity of bone marrow–derived hematopoietic progenitors at the single-cell level. Through transplantation of bone marrow cells and analysis of the resulting cellular nodules in the spleen, the CFU-S assay revealed both the self-renewal and clonal differentiation capacity of hematopoietic progenitors. Further development and use of this assay have identified highly proliferative, self-renewing, and differentiating HSCs that possess clonal, multilineage differentiation. The CFU-S strategy has also been adapted to interrogating single purified hematopoietic stem and progenitor cell populations, advancing our knowledge of the hematopoietic hierarchy. In this review, we explore the major discoveries made with the CFU-S assay, consider its modern use and recent improvements, and compare it with commonly used long-term transplantation assays to determine the continued value of the CFU-S assay for understanding HSC biology and hematopoiesis.

Simple Summary E-cigarettes have gained popularity as alternatives to traditional tobacco products over the past several decades. Despite being marketed as safer, they still contain several highly toxic compounds, which pose as dangers to human health. Nicotine is one of those toxic compounds and is known to have many deleterious effects on human health and disease susceptibility. Hematopoietic stem cells (HSCs) are the stem cells that give rise to the entire immune system and therefore serve as a compelling point of interrogation for the source of altered health and disease susceptibility in exposed individuals. Here we discuss how nicotine influences HSCs and the immune cells they make, as well as highlight potential mechanisms of altered immunity for life. Abstract Hematopoiesis is a tightly regulated process orchestrated by cell-intrinsic and cell-extrinsic cues. Over the past several decades, much effort has been focused on understanding how these cues regulate hematopoietic stem cell (HSC) function. Many endogenous key regulators of hematopoiesis have been identified and extensively characterized. Less is known about the mechanisms of long-term effects of environmental toxic compounds on hematopoietic stem and progenitor cells (HSPCs) and their mature immune cell progeny. Research over the past several decades has demonstrated that tobacco products are extremely toxic and pose huge risks to human health by causing diseases like cancer, respiratory illnesses, strokes, and more. Recently, electronic cigarettes have been promoted as a safer alternative to traditional tobacco products and have become increasingly popular among younger generations. Nicotine, the highly toxic compound found in many traditional tobacco products, is also found in most electronic cigarettes, calling into question their purported “safety”. Although it is known that nicotine is toxic, the pathophysiology of disease in exposed people remains under investigation. One plausible contributor to altered disease susceptibility is altered hematopoiesis and associated immune dysfunction. In this review, we focus on research that has addressed how HSCs and mature blood cells respond to nicotine, as well as identify remaining questions.

Summary Age-related morbidity is associated with a decline in hematopoietic stem cell (HSC) function, but the mechanisms of HSC aging remain unclear. We performed heterochronic HSC transplants followed by quantitative analysis of cell reconstitution. Although young HSCs outperformed old HSCs in young recipients, young HSCs unexpectedly failed to outcompete the old HSCs of aged recipients. Interestingly, despite substantial enrichment of megakaryocyte progenitors (MkPs) in old mice in situ and reported platelet (Plt) priming with age, transplanted old HSCs were deficient in reconstitution of all lineages, including MkPs and Plts. We therefore performed functional analysis of young and old MkPs. Surprisingly, old MkPs displayed unmistakably greater regenerative capacity compared with young MkPs. Transcriptome analysis revealed putative molecular regulators of old MkP expansion. Collectively, these data demonstrated that aging affects HSCs and megakaryopoiesis in fundamentally different ways: whereas old HSCs functionally decline, MkPs gain expansion capacity upon aging., Graphical abstract, Highlights • Reconstitution deficit by old HSCs was observed by chimerism and absolute cell numbers • Young HSCs did not outcompete resident HSCs in aged recipient mice • Old MkPs display remarkable capacity to engraft, expand, and reconstitute platelets • Aging is associated with changes in MkP genome-wide expression signatures, Poscablo et al. explored age-related changes to hematopoietic stem cell and megakaryocyte progenitor (MkP) function. They found an unexpected gain of in vitro expansion and in vivo reconstitution potential of MkPs upon aging. These functional changes were accompanied by differences in transcriptome profiles between young and old MkPs, suggesting progenitor cell mechanisms contributing to hematopoietic aging.

Adult hematopoietic stem and progenitor cells (HSPCs) respond directly to inflammation and infection, causing both acute and persistent changes to quiescence, mobilization, and differentiation. Here we show that murine fetal HSPCs respond to prenatal inflammation in utero and that the fetal response shapes postnatal hematopoiesis and immune cell function. Heterogeneous fetal HSPCs show divergent responses to maternal immune activation (MIA), including changes in quiescence, expansion, and lineage-biased output. Single-cell transcriptomic analysis of fetal HSPCs in response to MIA reveals specific upregulation of inflammatory gene profiles in discrete, transient hematopoietic stem cell (HSC) populations that propagate expansion of lymphoid-biased progenitors. Beyond fetal development, MIA causes the inappropriate expansion and persistence of fetal lymphoid-biased progenitors postnatally, concomitant with increased cellularity and hyperresponsiveness of fetal-derived innate-like lymphocytes. Our investigation demonstrates how inflammation in utero can direct the output and function of fetal-derived immune cells by reshaping fetal HSC establishment.

C-X-C motif chemokine ligand 12 (CXCL12; aka SDF1α) is a major regulator of a number of cellular systems, including hematopoiesis, where it influences hematopoietic cell trafficking, proliferation, and survival during homeostasis and upon stress and disease. A variety of constitutive, temporal, ubiquitous, and cell-specific loss-of-function models have documented the functional consequences on hematopoiesis upon deletion of Cxcl12. Here, in contrast to loss-of-function experiments, we implemented a gain-of-function approach by generating a doxycycline-inducible transgenic mouse model that enables spatial and temporal overexpression of Cxcl12. We demonstrated that ubiquitous CXCL12 overexpression led to an increase in multipotent progenitors in the bone marrow and spleen. The CXCL12+ mice displayed reduced reconstitution potential as either donors or recipients in transplantation experiments. Additionally, we discovered that Cxcl12 overexpression improved hematopoietic stem and progenitor cell mobilization into the blood, and conferred radioprotection by promoting quiescence. Thus, this new CXCL12+ mouse model provided new insights into major facets of hematopoiesis and serves as a versatile resource for studying CXCL12 function in a variety of contexts.

Hematopoietic stem cell (HSC) multipotency and self-renewal are typically defined through serial transplantation experiments. Host conditioning is necessary for robust HSC engraftment, likely by reducing immune-mediated rejection and by clearing limited HSC niche space. Because irradiation of the recipient mouse is non-specific and broadly damaging, there is a need to develop alternative models to study HSC performance at steady-state and in the absence of radiation-induced stress. We have generated and characterized two new mouse models where either all hematopoietic cells or only HSCs can be specifically induced to die in vivo or in vitro. Hematopoietic-specific Vav1-mediated expression of a loxP-flanked diphtheria-toxin receptor (DTR) renders all hematopoietic cells sensitive to diphtheria toxin (DT) in “Vav-DTR” mice. Crossing these mice to Flk2-Cre mice results in “HSC-DTR” mice which exhibit HSC-selective DT sensitivity. We demonstrate robust, rapid, and highly selective cell ablation in these models. These new mouse models provide a platform to test whether HSCs are required for long-term hematopoiesis in vivo, for understanding the mechanisms regulating HSC engraftment, and interrogating in vivo hematopoietic differentiation pathways and mechanisms regulating hematopoietic homeostasis.

Tissue-resident lymphoid cells (TLCs) span the spectrum of innate-to-adaptive immune function. Unlike traditional, circulating lymphocytes that are continuously generated from hematopoietic stem cells (HSCs), many TLCs are of fetal origin and poorly generated from adult HSCs. Here, we sought to further understand murine TLC development and the roles of Flk2 and IL7Rα, two cytokine receptors with known function in traditional lymphopoiesis. Using Flk2- and Il7r-Cre lineage tracing, we found that peritoneal B1a cells, splenic marginal zone B (MZB) cells, lung ILC2s and regulatory T cells (Tregs) were highly labeled. Despite high labeling, loss of Flk2 minimally affected the generation of these cells. In contrast, loss of IL7Rα, or combined deletion of Flk2 and IL7Rα, dramatically reduced the number of B1a cells, MZBs, ILC2s and Tregs, both in situ and upon transplantation, indicating an intrinsic and essential role for IL7Rα. Surprisingly, reciprocal transplants of wild-type HSCs showed that an IL7Rα−/− environment selectively impaired reconstitution of TLCs when compared with TLC numbers in situ. Taken together, our data defined Flk2- and IL7Rα-positive TLC differentiation paths, and revealed functional roles of Flk2 and IL7Rα in TLC establishment.

Summary Hematopoietic stem cell (HSC) transplantation is a curative treatment for a variety of blood and immune disorders. Currently available methods to obtain donor HSCs are suboptimal, and the limited supply of donor HSCs hampers the success and availability of HSC transplantation therapies. We recently showed that manipulation of vascular integrity can be employed to induce HSC mobilization from the bone marrow to the blood stream, facilitating non-invasive collection of HSCs. Here, we tested whether FDA-approved vasodilators are capable of mobilizing HSCs. We found that a rapid, 2-h regimen of a single oral dose of Viagra (sildenafil citrate) combined with a single injection of the CXCR4 antagonist AMD3100 leads to efficient HSC mobilization at levels rivaling the standard-of-care 5-day regimen of granulocyte-colony stimulating factor (G-CSF/Filgrastim/Neupogen). Our findings solidify vascular integrity as an essential regulator of HSC trafficking and provide an attractive, single-day regimen for HSC mobilization using already FDA-approved drugs., Graphical Abstract, Highlights • Viagra enhances AMD3100-mediated HSC mobilization • Extremely rapid and efficient HSC mobilization with two FDA-approved drugs • Vascular integrity regulates HSC trafficking, In this article, Forsberg and colleagues demonstrate that Viagra, a widely used vasodilator, enhances Plerixafor-mediated hematopoietic stem cell (HSC) mobilization from the bone marrow to the blood stream. These findings solidify vascular integrity as an essential regulator of HSC trafficking and provide an attractive, single-day regimen for HSC mobilization using already FDA-approved drugs.

Summary Hematopoiesis is arguably one of the best understood stem cell systems; however, significant challenges remain to reach a consensus understanding of the lineage potential, heterogeneity, and relationships of hematopoietic stem and progenitor cell populations. To gain new insights, we performed quantitative analyses of mature cell production from hematopoietic stem cells (HSCs) and multiple hematopoietic progenitor populations. Assessment of the absolute numbers of mature cell types produced by each progenitor cell revealed a striking erythroid dominance of all myeloid-competent progenitors assessed, accompanied by strong platelet reconstitution. All populations with myeloid potential also produced robust numbers of red blood cells and platelets in vivo. Clonal analysis by single-cell transplantation and by spleen colony assays revealed that a significant fraction of HSCs and multipotent progenitors have multilineage potential at the single-cell level. These new insights prompt an erythroid-focused model of hematopoietic differentiation., Graphical Abstract, Highlights • RBCs are the predominant cell type produced by multipotent hematopoietic progenitors • All cell types with myeloid potential also produced RBCs and platelets in vivo • Single HSCs and MPPF cells are capable of multilineage hematopoietic reconstitution • Erythroid cell production emerges as a default hematopoietic fate, In this article, Forsberg and colleagues use quantitative reconstitution assays to demonstrate that all myeloid-competent progenitor cells also contribute to erythroid and platelet production. Single-cell in vivo analyses showed that hematopoietic stem cells and multipotent progenitors are clonally multipotent. These results suggest that erythropoiesis is a default hematopoietic fate and challenges current views on hematopoietic lineage potential and fate decisions.

Hematopoietic stem cells (HSCs) have the capacity to differentiate into vastly different types of mature blood cells. The epigenetic mechanisms regulating the multilineage ability, or multipotency of HSCs are not well understood. To test the hypothesis thatcisregulatory elements that control fate decisions for all lineages are primed in HSCs, we used ATAC-seq to compare chromatin accessibility of HSCs with five unipotent cell types. We observed the highest similarity in accessibility profiles between Megakaryocyte Progenitors and HSCs, whereas B cells had the greatest number of regions withde novogain in accessibility during differentiation. Despite these differences, we identifiedcisregulatory elements from all lineages that displayed epigenetic priming in HSCs. These findings provide new insights into the regulation of stem cell multipotency, as well as a resource to identify functional drivers of lineage fate.HIGHLIGHTSHSCs have higher global chromatin accessibility than any unilineage progenyMegakaryocyte Progenitors are the most closely related unipotent cell type to HSCsB cell commitment involvesde novochromatin accessibilityEvidence ofciselement priming of lineage-specific genes in HSCs

Platelets provide life-saving functions by halting external and internal bleeding. There is also a dark side to platelet biology, however. Recent reports provide evidence for increased platelet reactivity during aging of mice and humans, making platelets main suspects in the most prevalent aging-related human pathologies, including cardiovascular diseases, stroke, and cancer. What drives this platelet hyperreactivity during aging? Here, we discuss how hematopoietic stem cell differentiation pathways into the platelet lineage offer avenues to understand the fundamental differences between young and old platelets. Recent advances begin to unravel how the cellular and molecular regulation of the parent hematopoietic stem and progenitor cells likely imbue aging characteristics on the resulting Plt progeny. The resulting mechanistic insights into intrinsic platelet reactivity will provide strategies for selectively targeting age-related pathways. This brief viewpoint focuses on current concepts on aging hematopoiesis and the implications for platelet hyperactivity during aging.

Hematopoietic stem cell (HSC) trafficking is regulated by a number of complex mechanisms. Among them are the transmembrane protein Robo4 and the vascular cell adhesion molecule, VCAM1. Endothelial VCAM1 is a well-known regulator of hematopoietic cell trafficking, and our previous studies revealed that germline deletion of Robo4 led to impaired HSC trafficking, with an increase in vascular endothelial cell (VEC) numbers and downregulation of VCAM1 protein on sinusoidal VECs. Here, we utilized two Robo4 conditional deletion models in parallel with Robo4 germline knockout mice (R4KO) to evaluate the effects of acute and endothelial cell-specific Robo4 deletion on HSC trafficking. Strikingly similar to the R4KO, the acute deletion of Robo4 resulted in altered HSC distribution between the bone marrow and blood compartments, despite normal numbers of VECs and wild-type levels of VCAM1 cell surface protein on sinusoidal VECs. Additionally, consistent with the R4KO mice, acute loss of Robo4 in the host perturbed long-term engraftment of donor wild-type HSCs and improved HSC mobilization to the peripheral blood. These data demonstrate the significant role that endothelial Robo4 plays in directional HSC trafficking, independent of alterations in VEC numbers and VCAM1 expression.

Hematopoietic stem and progenitor cell (HSPC) transplantation is the paradigm for stem cell therapies. The protocol described here enables quantitative assessment of the body-wide HSPC reconstitution of different mature hematopoietic cells in mice based on their presence in circulating blood. The method determines donor-derived mature cell populations per mouse, over time, by quantitatively obtaining their absolute numbers in the peripheral blood and utilizing previously assessed tissue-distribution factors. A Markov-based birth/death computational model accounts for the drastic differences in mature cell half-lives. By quantifying the number of cells produced and eliminating host variability, the protocol can be used to directly compare the lineage output of different types of HSPCs on a per cell basis, thereby clarifying the lineage potential and expansion capacity of different cell populations. These protocols were developed for hematopoiesis, but can readily be extended to other contexts by simply replacing the cell types and distributions.HighlightsQuantitative assessment of stem and progenitor cell reconstitution capacityElimination of cell-specific recipient variability for accurate donor cell potentialDirectly comparable lineage output within and between stem and progenitor cellsBlood-based absolute quantification of whole-body repopulation over timeMarkov modelling-based consideration of differential mature cell half-lives

Respiratory diseases are a leading cause of death worldwide, with highly varied vulnerability to disease between individuals. The underlying reasons of disease susceptibility are unknown, but often include a variable immune response in lungs. Recently, we identified a surprising novel role of the interleukin 7 receptor (IL7R), a primarily lymphoid-associated regulator, in fetal-specified, lung-resident macrophage development. Here, we report that traditional, hematopoietic stem cell-derived myeloid cells in the adult lung, peripheral blood, and bone marrow also depend on IL7R expression. Using single and double germline knockout models, we found that eosinophil numbers were reduced upon deletion of IL7Rα. We then employed two Cre recombinase models in lineage tracing experiments to test whether these cells developed through an IL7Rα+ pathway. Despite the impact of IL7Rα deletion, IL7R-Cre labeled only a minimal fraction of eosinophils. We therefore examined the intrinsic versus extrinsic requirement for IL7R in the production of eosinophils using reciprocal hematopoietic stem cell transplantation assays. These assays revealed that extrinsic, but not eosinophil-intrinsic, IL7R is required for eosinophil reconstitution by HSCs in the adult lung. To determine which external factors may be influencing eosinophil development and survival, we performed a cytokine array analysis between wild-type and IL7Rα-deficient mice and found several differentially regulated proteins. These findings expand upon our previous publication that IL7R is required not only for proper lymphoid cell development and homeostasis, but also for myeloid cell homeostasis in tissues.

Hematopoietic stem cells (HSCs) have long been considered the continuous source of all hematopoietic cells for the life of an individual. Recent findings have questioned multiple aspects of this view, including the ability of lifelong HSCs to contribute to tissue-resident immune cells. Here we discuss the most recent findings on the source of B1a cells, innatelike lymphocytes that primarily reside in serous cavities. Powerful experimental approaches including bar coding, single cell transplantation, in vivo lineage tracing, and HSC-specific pulse-chase labeling have provided novel insights on B1a-cell generation during ontogeny. We evaluate the evidence for fetal vs adult B1a-cell production capacity and the identity of putative cells of origin. Integrating these most recent findings with previous work, we propose a working model that encapsulates our current understanding of waves of immune development.

Hematopoiesis is the process by which mature blood and immune cells are produced from hematopoietic stem and progenitor cells (HSCs and HSPCs). The last several decades of research have shed light on the origin of HSCs, as well as the heterogeneous pools of fetal progenitors that contribute to lifelong hematopoiesis. The overarching concept that hematopoiesis occurs in dynamic, overlapping waves throughout development, with each wave contributing to both continuous and developmentally limited cell types, has been solidified over the years. However, recent advances in our ability to track the production of hematopoietic cells in vivo have challenged several long-held dogmas on the origin and persistence of distinct hematopoietic cell types. In this review, we highlight emerging concepts in hematopoietic development and identify unanswered questions.

Summary Epigenetic regulation serves as the basis for stem cell differentiation into distinct cell types, but it is unclear how global epigenetic changes are regulated during this process. Here, we tested the hypothesis that global chromatin organization affects the lineage potential of stem cells and that manipulation of chromatin dynamics influences stem cell function. Using nuclease sensitivity assays, we found a progressive decrease in chromatin digestion among pluripotent embryonic stem cells (ESCs), multipotent hematopoietic stem cells (HSCs), and mature hematopoietic cells. Quantitative high-resolution microscopy revealed that ESCs contain significantly more euchromatin than HSCs, with a further reduction in mature cells. Increased cellular maturation also led to heterochromatin localization to the nuclear periphery. Functionally, prevention of heterochromatin formation by inhibition of the histone methyltransferase G9A resulted in delayed HSC differentiation. Our results demonstrate global chromatin rearrangements during stem cell differentiation and that heterochromatin formation by H3K9 methylation regulates HSC differentiation., Graphical Abstract, Highlights • Nuclease sensitivity decreases progressively with stem cell differentiation • Distinct chromatin architecture is apparent in cells of different lineage potential • Stem cell differentiation leads to perinuclear heterochromatin localization • G9a-mediated heterochromatin formation facilitates stem cell differentiation, Forsberg and colleagues use a variety of approaches to investigate global chromatin structure and architecture in pluripotent, multipotent, and differentiated cells. They show that progressive changes in nuclease sensitivity, chromatin condensation, and heterochromatin localization correlate with the lineage potential of embryonic and adult stem cells and committed cells. Functionally, inhibition of heterochromatin formation delayed hematopoietic stem cell differentiation. These data provide new insights on the epigenetic regulation of stem cell lineage potential and differentiation.

In February 2015, over 200 scientists gathered for the Keystone Hematopoiesis meeting, which was held at the scenic Keystone Resort in Keystone, Colorado, USA. The meeting organizers, Patricia Ernst, Hanna Mikkola and Timm Schroeder, put together an exciting program, during which field leaders and new investigators presented discoveries that spanned developmental and adult hematopoiesis within both physiologic and pathologic contexts. Collectively, the program highlighted the increasing pace of new discoveries and the substantial progress made in the hematopoiesis field since the last Keystone meeting two years ago. In this Meeting Review, we highlight the main concepts discussed at the conference, with an emphasis on topics relevant to developmental biology.

SummaryAlthough classified as hematopoietic cells, tissue-resident macrophages (MFs) arise from embryonic precursors that seed the tissues prior to birth to generate a self-renewing population, which is maintained independently of adult hematopoiesis. Here we reveal the identity of these embryonic precursors using an in utero MF-depletion strategy and fate-mapping of yolk sac (YS) and fetal liver (FL) hematopoiesis. We show that YS MFs are the main precursors of microglia, while most other MFs derive from fetal monocytes (MOs). Both YS MFs and fetal MOs arise from erythro-myeloid progenitors (EMPs) generated in the YS. In the YS, EMPs gave rise to MFs without monocytic intermediates, while EMP seeding the FL upon the establishment of blood circulation acquired c-Myb expression and gave rise to fetal MOs that then seeded embryonic tissues and differentiated into MFs. Thus, adult tissue-resident MFs established from hematopoietic stem cell-independent embryonic precursors arise from two distinct developmental programs.

Haematopoietic stem cell (HSC) niches provide an environment essential for life-long HSC function. Intense investigation of HSC niches both feed off and drive technology development to increase our capability to assay functionally defined cells with high resolution. A major driving force behind the desire to understand the basic biology of HSC niches is the clear implications for clinical therapies. Here, with particular emphasis on cell type-specific deletion of SCL and CXCL12, we focus on unresolved issues on HSC niches, framed around some very recent advances and novel discoveries on the extrinsic regulation of HSC maintenance. We also provide ideas for possible paths forward, some of which are clearly within reach while others will require both novel tools and vision.

Selective labeling of specific cell types by expression of green fluorescent protein (GFP) within the hematopoietic system would have great utility in identifying, localizing, and tracking different cell populations in flow cytometry, microscopy, lineage tracing, and transplantation assays. In this report, we describe the generation and characterization of a new transgenic mouse line with specific GFP labeling of all nucleated hematopoietic cells, as well as platelets. This new “Vav-GFP” mouse line labels the vast majority of hematopoietic cells with GFP during both embryonic development and adulthood, with particularly high expression in hematopoietic stem and progenitor cells. With the exception of transient labeling of fetal endothelial cells, GFP expression is highly selective for hematopoietic cells, and persists in donor-derived progeny after transplantation of hematopoietic progenitor cells. Finally, we also demonstrate that the loxP-flanked reporter allows for specific GFP labeling of different hematopoietic cell subsets when crossed to various Cre reporter lines. Notably, by crossing Vav-GFP mice to Flk2-Cre mice, we obtained robust and highly selective GFP expression in HSCs. Together, these data describe a new mouse model capable of directing GFP labeling exclusively of hematopoietic cells or exclusively of HSCs.

Genetic fate-mapping approaches provide a unique opportunity to assess differentiation pathways under physiological conditions. We have recently employed a lineage tracing approach to define hematopoietic differentiation pathways in relation to expression of the tyrosine kinase receptor Flk2.1 Based on our examination of reporter activity across all stem, progenitor and mature populations in our Flk2-Cre lineage model, we concluded that all mature blood lineages are derived through a Flk2+ intermediate, both at steady-state and under stress conditions. Here, we re-examine in depth our initial conclusions and perform additional experiments to test alternative options of lineage specification. Our data unequivocally support the conclusion that onset of Flk2 expression results in loss of self-renewal but preservation of multilineage differentiation potential. We discuss the implications of these data for defining stem cell identity and lineage potential among hematopoietic populations.

Tobacco use is associated with an increase in the white blood cell (WBC) count. This association has been attributed to bronchopulmonary inflammation and/or infection. It is not known if nicotine itself may play a role. The objective of this study was to determine whether nicotine itself could affect the WBC count, and to determine whether this was due to a direct effect on hematopoietic stem cells (HSC). C57Bl6J mice received nicotine orally, and measurements of the WBC count, bone marrow and spleen cellularity, and HSC count were made. To determine the functionality of HSCs, irradiated animals received bone marrow transplants from vehicle or nicotine-treated mice. Nicotine increased leukocytes in the peripheral blood, bone marrow and spleen. The peripheral red cell and platelet count were unaffected. Nicotine increased the frequency of HSC in the bone marrow. Isolated long-term HSCs from nicotine-treated mice transplanted into irradiated mice regenerated all hematopoietic cell lineages, demonstrating the functional competence of those HSCs. HSCs expressed nicotinic acetylcholine receptors (nAChRs), as documented by FITC-conjugated alpha-bungarotoxin binding. Nicotine increased soluble Kit ligand, consistent with stem cell activation. In conclusion, the data suggest a new mechanism for the increased WBC associated with tobacco use. The effect of nicotine to activate hematopoiesis may contribute to tobacco-related diseases.

Hematopoietic stem cells (HSC) produce all blood cell lineages by virtue of their capacity to self-renew and differentiate into progenitors with decreasing cellular potential. Recent studies suggest that epigenetic mechanisms play an important role in controlling stem cell potency and cell fate decisions. To investigate this hypothesis in HSC, we have modified the conventional chromatin immunoprecipitation assay allowing for the analysis of 50,000 prospectively purified stem and progenitor cells. Together with bisulfite sequencing analysis, we found that methylated H3K4 and AcH3 and unmethylated CpG dinucleotides colocalize across defined regulatory regions of lineage-affiliated genes in HSC. These active epigenetic histone modifications either accumulated or were replaced by increased DNA methylation and H3K27 trimethylation in committed progenitors consistent with gene expression. We also observed bivalent histone modifications at a lymphoid-affiliated gene in HSC and downstream transit-amplifying progenitors. Together, these data support a model in which epigenetic modifications serve as an important mechanism to control HSC multipotency.

Haematopoietic stem cells (HSCs) self-renew for life, thereby making them one of the few blood cells that truly age(1,2). Paradoxically, although HSCs numerically expand with age, their functional activity declines over time, resulting in degraded blood production and impaired engraftment following transplantation(2). While many drivers of HSC ageing have been proposed(2-5), the reason why HSC function degrades with age remains unknown. Here we show that cycling old HSCs in mice have heightened levels of replication stress associated with cell cycle defects and chromosome gaps or breaks, which are due to decreased expression of mini-chromosome maintenance (MCM) helicase components and altered dynamics of DNA replication forks. Nonetheless, old HSCs survive replication unless confronted with a strong replication challenge, such as transplantation. Moreover, once old HSCs re-establish quiescence, residual replication stress on ribosomal DNA (rDNA) genes leads to the formation of nucleolar-associated gamma H2AX signals, which persist owing to ineffective H2AX dephosphorylation by mislocalized PP4c phosphatase rather than ongoing DNA damage. Persistent nucleolar gamma H2AX also acts as a histone modification marking the transcriptional silencing of rDNA genes and decreased ribosome biogenesis in quiescent old HSCs. Our results identify replication stress as a potent driver of functional decline in old HSCs, and highlight the MCM DNA helicase as a potential molecular target for rejuvenation therapies.

Defining differentiation pathways is central to understanding the pathogenesis of hematopoietic disorders, including leukemia. The function of the receptor tyrosine kinase Flk2 (Flt3) in promoting myeloid development remains poorly defined, despite being commonly mutated in acute myeloid leukemia. We investigated the effect of Flk2 deficiency on myelopoiesis, focusing on specification of progenitors between HSC and mature cells. We provide evidence that Flk2 is critical for proliferative expansion of multipotent progenitors that are common precursors for all lymphoid and myeloid lineages, including megakaryocyte/erythroid (MegE) cells. Flk2 deficiency impaired the generation of both lymphoid and myeloid progenitors by abrogating propagation of their common upstream precursor. At steady state, downstream compensatory mechanisms masked the effect of Flk2 deficiency on mature myeloid output, whereas transplantation of purified progenitors revealed impaired generation of all mature lineages. Flk2 deficiency did not affect lineage choice, thus dissociating the role of Flk2 in promoting cell expansion and regulating cell fate. Surprisingly, despite impairing myeloid development, Flk2 deficiency afforded protection against myeloablative insult. This survival advantage was attributed to reduced cell cycling and proliferation of progenitors in Flk2-deficient mice. Our data support the existence of a common Flk2(+) intermediate for all hematopoietic lineages and provide insight into how activating Flk2 mutations promote hematopoietic malignancy by non-Flk2-expressing myeloid cells.

The bone marrow (BM) niche is essential for lifelong hematopoietic stem cell (HSC) maintenance, proliferation and differentiation. Several BM cell types, including osteoblast lineage cells (OBC), mesenchymal stem cells (MSC) and endothelial cells (EC) have been implicated in supporting HSC location and function, but the relative importance of these cell types and their secreted ligands remain controversial. We recently found that the cell surface receptors Robo4 and CXCR4 cooperate to localize HSC to BM niches. We hypothesized that Slit2, a putative ligand for Robo4, cooperates with the CXCR4 ligand SDF1 to direct HSC to specific BM niche sites. Here, we have isolated OBC, MSC and EC by flow cytometry and determined their frequency within the bone marrow and the relative mRNA levels of Slit2, SDF1 and Robo4. We found that expression of Slit2 and SDF1 were dynamically regulated in MSC and OBC-like populations following radiation, while Robo4 expression was restricted to EC. Radiation also significantly affected the cellularity and frequency of both the non-adherent and adherent cells within the BM stroma. These data support a physiological role for Slit2 in regulating the dynamic function of Robo-expressing cells within BM niches at steady state and following radiation.

The stemness hypothesis states that all stem cells use common mechanisms to regulate self-renewal and multi-lineage potential. However, gene expression meta-analyses at the single gene level have failed to identify a significant number of genes selectively expressed by a broad range of stem cell types. We hypothesized that stemness may be regulated by modules of homologs. While the expression of any single gene within a module may vary from one stem cell type to the next, it is possible that the expression of the module as a whole is required so that the expression of different, yet functionally-synonymous, homologs is needed in different stem cells. Thus, we developed a computational method to test for stem cell-specific gene expression patterns from a comprehensive collection of 49 murine datasets covering 12 different stem cell types. We identified 40 individual genes and 224 stemness modules with reproducible and specific up-regulation across multiple stem cell types. The stemness modules included families regulating chromatin remodeling, DNA repair, and Wnt signaling. Strikingly, the majority of modules represent evolutionarily related homologs. Moreover, a score based on the discovered modules could accurately distinguish stem cell-like populations from other cell types in both normal and cancer tissues. This scoring system revealed that both mouse and human metastatic populations exhibit higher stemness indices than non-metastatic populations, providing further evidence for a stem cell-driven component underlying the transformation to metastatic disease.

Hematopoietic stem cells (HSC) are rare, multipotent cells capable of generating all specialized cells of the blood system. Appropriate regulation of HSC quiescence is thought to be crucial to maintain their lifelong function; however, the molecular pathways controlling stem cell quiescence remain poorly characterized. Likewise, the molecular events driving leukemogenesis remain elusive. In this study, we compare the gene expression profiles of steady-state bone marrow HSC to non-self-renewing multipotent progenitors; to HSC treated with mobilizing drugs that expand the HSC pool and induce egress from the marrow; and to leukemic HSC in a mouse model of chronic myelogenous leukemia. By intersecting the resulting lists of differentially regulated genes we identify a subset of molecules that are downregulated in all three circumstances, and thus may be particularly important for the maintenance and function of normal, quiescent HSC. These results identify potential key regulators of HSC and give insights into the clinically important processes of HSC mobilization for transplantation and leukemic development from cancer stem cells.

Hematopoietic stem cells (HSCs) have been highly enriched using combinations of 12–14 surface markers. Genes specifically expressed by HSCs as compared with other multipotent progenitors may yield new stem cell enrichment markers, as well as elucidate self-renewal and differentiation mechanisms. We previously reported that multiple cell surface molecules are enriched on mouse HSCs compared with more differentiated progeny. Here, we present a definitive expression profile of the cell adhesion molecule endothelial cell-selective adhesion molecule (Esam1) in hematopoietic cells using reverse transcription-quantitative polymerase chain reaction and flow cytometry studies. We found Esam1 to be highly and selectively expressed by HSCs from mouse bone marrow (BM). Esam1 was also a viable positive HSC marker in fetal, young, and aged mice, as well as in mice of several different strains. In addition, we found robust levels of Esam1 transcripts in purified human HSCs. Esam1−/− mice do not exhibit severe hematopoietic defects; however, Esam1−/− BM has a greater frequency of HSCs and fewer T cells. HSCs from Esam1−/− mice give rise to more granulocyte/monocytes in culture and a higher T cell:B cell ratio upon transplantation into congenic mice. These studies identify Esam1 as a novel, widely applicable HSC-selective marker and suggest that Esam1 may play roles in both HSC proliferation and lineage decisions.

The hematopoietic system is an invaluable model both for understanding basic developmental biology and for developing clinically relevant cell therapies. Using highly purified cells and rigorous microarray analysis we have compared the expression pattern of three of the most primitive hematopoietic subpopulations in adult mouse bone marrow: long-term hematopoietic stem cells (HSC), short-term HSC, and multipotent progenitors. All three populations are capable of differentiating into a spectrum of mature blood cells, but differ in their self-renewal and proliferative capacity. We identified numerous novel potential regulators of HSC self-renewal and proliferation that were differentially expressed between these closely related cell populations. Many of the differentially expressed transcripts fit into pathways and protein complexes not previously identified in HSC, providing evidence for new HSC regulatory units. Extending these observations to the protein level, we demonstrate expression of several of the corresponding proteins, which provide novel surface markers for HSC. We discuss the implications of our findings for HSC biology. In particular, our data suggest that cell–cell and cell–matrix interactions are major regulators of long-term HSC, and that HSC themselves play important roles in regulating their immediate microenvironment., Synopsis Hematopoietic, or blood-forming, stem cells (HSC) are responsible for the continual replenishment of all blood cells throughout life. This ability to both renew themselves and give rise to expanded populations of differentiating and mature cells is a hallmark of stem cells and is therefore an area of intense research. The rarity of HSC as well as their location in the bone marrow environment has made it difficult to identify the genes that regulate these properties. The earliest stages of blood development begins with the long-term (LT) repopulating HSC that then differentiate into short-term (ST) repopulating HSC and non-self renewing multipotent progenitors (MPP). The authors investigated the gene expression differences in these highly purified populations that differ mainly in their capacity to self renew, and identified a number of genes specific to each of these populations. Intriguingly, many of these genes code for proteins that are involved in cell–cell and cell–matrix interactions that were not previously identified on these populations. These novel discoveries will, together with future experiments, enhance our understanding of the basic biology of stem cells and their clinical uses.

This review focuses on the genomics of mouse hematopoiesis, but also draws parallels to other systems and discusses issues common to the analysis of rare populations such as stem cells. As examples from the mouse blood forming system are used to illustrate several points, the authors first give a brief introduction to mouse hematopoiesis as a model system. We review the multiple microarray analyses that have been performed on various mouse hematopoietic subpopulations and comment on both technical and biological aspects of such experiments. The concept of stemness is discussed, and the importance of biological function of gene products, protein-protein interactions and molecular pathways highlighted. Finally, the authors discuss some major unresolved issues in hematopoiesis and discuss the potential uses of future microarray analysis as well as other genomic and functional approaches that might prove useful to further our understanding of hematopoiesis and other stem cell systems.

SummarySpecific bone marrow (BM) niches are critical for hematopoietic stem cell (HSC) function during both normal hematopoiesis and in stem cell transplantation therapy. We demonstrate that the guidance molecule Robo4 functions to specifically anchor HSCs to BM niches. Robo4-deficient HSCs displayed poor localization to BM niches and drastically reduced long-term reconstitution capability while retaining multilineage potential. Cxcr4, a critical regulator of HSC location, is upregulated in Robo4−/− HSCs to compensate for Robo4 loss. Robo4 deletion led to altered HSC mobilization efficiency, revealing that inhibition of both Cxcr4- and Robo4-mediated niche interactions are necessary for efficient HSC mobilization. Surprisingly, we found that WT HSCs express very low levels of Cxcr4 and respond poorly to Cxcr4 manipulation relative to other hematopoietic cells. We conclude that Robo4 cooperates with Cxcr4 to endow HSCs with competitive access to limited stem cell niches, and we propose Robo4 as a therapeutic target in HSC transplantation therapy.

SummaryLoss of the JunB/AP-1 transcription factor induces a myeloproliferative disease (MPD) arising from the hematopoietic stem cell (HSC) compartment. Here, we show that junB inactivation deregulates the cell-cycle machinery and increases the proliferation of long-term repopulating HSCs (LT-HSCs) without impairing their self-renewal or regenerative potential in vivo. We found that JunB loss destabilizes a complex network of genes and pathways that normally limit myeloid differentiation, leading to impaired responsiveness to both Notch and TGF-β signaling due in part to transcriptional deregulation of the Hes1 gene. These results demonstrate that LT-HSC proliferation and differentiation are uncoupled from self-renewal and establish some of the mechanisms by which JunB normally limits the production of myeloid progenitors, hence preventing initiation of myeloid malignancies.