Integrated Single-Cell Analysis Maps the Continuous Regulatory Landscape of Human Hematopoietic Differentiation.

Human hematopoiesis involves cellular differentiation of multipotent cells into progressively more lineage-restricted states. While the chromatin accessibility landscape of this process has been explored in defined populations, single-cell regulatory variation has been hidden by ensemble averaging. We collected single-cell chromatin accessibility profiles across 10 populations of immunophenotypically defined human hematopoietic cell types and constructed a chromatin accessibility landscape of human hematopoiesis to characterize differentiation trajectories. We find variation consistent with lineage bias toward different developmental branches in multipotent cell types. We observe heterogeneity within common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) and develop a strategy to partition GMPs along their differentiation trajectory. Furthermore, we integrated single-cell RNA sequencing (scRNA-seq) data to associate transcription factors to chromatin accessibility changes and regulatory elements to target genes through correlations of expression and regulatory element accessibility. Overall, this work provides a framework for integrative exploration of complex regulatory dynamics in a primary human tissue at single-cell resolution.

Plasmacytoid dendritic cells develop from Ly6D+ lymphoid progenitors distinct from the myeloid lineage.

Dendritic cells (DC) are currently classified as conventional DCs (cDCs) and plasmacytoid DCs (pDCs). Through a combination of single-cell transcriptomic analysis, mass cytometry, in vivo fate mapping and in vitro clonal assays, here we show that, at the single-cell level, the priming of mouse hematopoietic progenitor cells toward the pDC lineage occurs at the common lymphoid progenitor stage, indicative of early divergence of the pDC and cDC lineages. We found the transcriptional signature of a pDC precursor stage, defined here, in the IL-7Rα+ common lymphoid progenitor population and identified Ly6D, IL-7Rα, CD81 and CD2 as key markers of pDC differentiation, which distinguish pDC precursors from cDC precursors. In conclusion, pDCs developed in the bone marrow from a Ly6DhiCD2hi lymphoid progenitor cell and differentiated independently of the myeloid cDC lineage.

Not All Created Equal: Lineage Hard-Wiring in the Production of Blood.

The multiple cell types comprising blood have been thought to emerge from progenitors with progressively narrower lineage options. New data suggest that lineage fate may be determined earlier than thought and that myeloid progenitor populations are aggregates of individual lineage-restricted cells.

The Branching Point in Erythro-Myeloid Differentiation.

Development of mature blood cell progenies from hematopoietic stem cells involves the transition through lineage-restricted progenitors. The first branching point along this developmental process is thought to separate the erythro-myeloid and lymphoid lineage fate by yielding two intermediate progenitors, the common myeloid and the common lymphoid progenitors (CMPs and CLPs). Here, we use single-cell lineage tracing to demonstrate that so-called CMPs are highly heterogeneous with respect to cellular output, with most individual CMPs yielding either only erythrocytes or only myeloid cells after transplantation. Furthermore, based on the labeling of earlier progenitors, we show that the divergence between the myeloid and erythroid lineage develops within multipotent progenitors (MPP). These data provide evidence for a model of hematopoietic branching in which multiple distinct lineage commitments occur in parallel within the MPP pool.

Transcriptional Heterogeneity and Lineage Commitment in Myeloid Progenitors.

Within the bone marrow, stem cells differentiate and give rise to diverse blood cell types and functions. Currently, hematopoietic progenitors are defined using surface markers combined with functional assays that are not directly linked with in vivo differentiation potential or gene regulatory mechanisms. Here, we comprehensively map myeloid progenitor subpopulations by transcriptional sorting of single cells from the bone marrow. We describe multiple progenitor subgroups, showing unexpected transcriptional priming toward seven differentiation fates but no progenitors with a mixed state. Transcriptional differentiation is correlated with combinations of known and previously undefined transcription factors, suggesting that the process is tightly regulated. Histone maps and knockout assays are consistent with early transcriptional priming, while traditional transplantation experiments suggest that in vivo priming may still allow for plasticity given strong perturbations. These data establish a reference model and general framework for studying hematopoiesis at single-cell resolution.

Coexpression of CD71 and CD117 Identifies an Early Unipotent Neutrophil Progenitor Population in Human Bone Marrow.

Neutrophils are the most abundant peripheral immune cells and thus, are continually replenished by bone marrow-derived progenitors. Still, how newly identified neutrophil subsets fit into the bone marrow neutrophil lineage remains unclear. Here, we use mass cytometry to show that two recently defined human neutrophil progenitor populations contain a homogeneous progenitor subset we term "early neutrophil progenitors" (eNePs) (Lin-CD66b+CD117+CD71+). Surface marker- and RNA-expression analyses, together with in vitro colony formation and in vivo adoptive humanized mouse transfers, indicate that eNePs are the earliest human neutrophil progenitors. Furthermore, we identified CD71 as a marker associated with the earliest neutrophil developmental stages. Expression of CD71 marks proliferating neutrophils, which were expanded in the blood of melanoma patients and detectable in blood and tumors from lung cancer patients. In summary, we establish CD117+CD71+ eNeP as the inceptive human neutrophil progenitor and propose a refined model of the neutrophil developmental lineage in bone marrow.

Clonal analysis unveils self-renewing lineage-restricted progenitors generated directly from hematopoietic stem cells.

Consensus holds that hematopoietic stem cells (HSCs) give rise to multipotent progenitors (MPPs) of reduced self-renewal potential and that MPPs eventually produce lineage-committed progenitor cells in a stepwise manner. Using a single-cell transplantation system and marker mice, we unexpectedly found myeloid-restricted progenitors with long-term repopulating activity (MyRPs), which are lineage-committed to megakaryocytes, megakaryocyte-erythroid cells, or common myeloid cells (MkRPs, MERPs, or CMRPs, respectively) in the phenotypically defined HSC compartment together with HSCs. Paired daughter cell assays combined with transplantation revealed that HSCs can give rise to HSCs via symmetric division or directly differentiate into MyRPs via asymmetric division (yielding HSC-MkRP or HSC-CMRP pairs). These myeloid bypass pathways could be essential for fast responses to ablation stress. Our results show that loss of self-renewal and stepwise progression through specific differentiation stages are not essential for lineage commitment of HSCs and suggest a revised model of hematopoietic differentiation.

Adult Connective Tissue-Resident Mast Cells Originate from Late Erythro-Myeloid Progenitors.

Tissue-resident mast cells are associated with many inflammatory and physiological processes. Although mast cells arise from the yolk sac, the exact ontogeny of adult mast cells remains unclear. Here we have investigated the hematopoietic origin of mast cells using fate-mapping systems. We have shown that early erythro-myeloid progenitors (EMPs), late EMPs, and definitive hematopoietic stem cells (HSCs) each gave rise to mast cells in succession via an intermediate integrin ß7+ progenitor. From late embryogenesis to adult, early EMP-derived mast cells were largely replaced by late EMP-derived cells in most connective tissues except adipose and pleural cavity. Thus, mast cells with distinct origin displayed tissue-location preferences: early EMP-derived cells were limited to adipose and pleural cavity and late EMP-derived cells dominated most connective tissues, while HSC-derived cells were a main group in mucosa. Therefore, embryonic origin shapes the heterogeneity of adult mast cells, with diverse functions in immunity and development.

Deciphering human macrophage development at single-cell resolution.

Macrophages are the first cells of the nascent immune system to emerge during embryonic development. In mice, embryonic macrophages infiltrate developing organs, where they differentiate symbiotically into tissue-resident macrophages (TRMs)1. However, our understanding of the origins and specialization of macrophages in human embryos is limited. Here we isolated CD45+ haematopoietic cells from human embryos at Carnegie stages 11 to 23 and subjected them to transcriptomic profiling by single-cell RNA sequencing, followed by functional characterization of a population of CD45+CD34+CD44+ yolk sac-derived myeloid-biased progenitors (YSMPs) by single-cell culture. We also mapped macrophage heterogeneity across multiple anatomical sites and identified diverse subsets, including various types of embryonic TRM (in the head, liver, lung and skin). We further traced the specification trajectories of TRMs from either yolk sac-derived primitive macrophages or YSMP-derived embryonic liver monocytes using both transcriptomic and developmental staging information, with a focus on microglia. Finally, we evaluated the molecular similarities between embryonic TRMs and their adult counterparts. Our data represent a comprehensive characterization of the spatiotemporal dynamics of early macrophage development during human embryogenesis, providing a reference for future studies of the development and function of human TRMs.

Targeted Enhancer Activation by a Subunit of the Integrator Complex.

The control of cell fate is an epigenetic process initiated by transcription factors (TFs) that recognize DNA motifs and recruit activator complexes and transcriptional machineries to chromatin. Lineage specificity is thought to be provided solely by TF-motif pairing, while the recruited activators are passive. Here, we show that INTS13, a subunit of the Integrator complex, operates as monocytic/macrophagic differentiation factor. Integrator is a general activator of transcription at coding genes and is required for eRNA maturation. Here, we show that INTS13 functions as an independent sub-module and targets enhancers through Early Growth Response (EGR1/2) TFs and their co-factor NAB2. INTS13 binds poised monocytic enhancers eliciting chromatin looping and activation. Independent depletion of INTS13, EGR1, or NAB2 impairs monocytic differentiation of cell lines and primary human progenitors. Our data demonstrate that Integrator is not functionally homogeneous and has TF-specific regulatory potential, revealing a new enhancer regulatory axis that controls myeloid differentiation.

Myelopoiesis Reloaded: Single-Cell Transcriptomics Leads the Way.

Lineage commitment during myelopoiesis has been linked to late-stage progenitors with multi-lineage potential. In a recent issue of Cell, Paul et al. (2015) now suggest a much earlier commitment toward distinct lineages even prior to the common myeloid progenitor state.

C-Myb(+) erythro-myeloid progenitor-derived fetal monocytes give rise to adult tissue-resident macrophages.

Although 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.

Erythro-myeloid progenitors contribute endothelial cells to blood vessels.

The earliest blood vessels in mammalian embryos are formed when endothelial cells differentiate from angioblasts and coalesce into tubular networks. Thereafter, the endothelium is thought to expand solely by proliferation of pre-existing endothelial cells. Here we show that a complementary source of endothelial cells is recruited into pre-existing vasculature after differentiation from the earliest precursors of erythrocytes, megakaryocytes and macrophages, the erythro-myeloid progenitors (EMPs) that are born in the yolk sac. A first wave of EMPs contributes endothelial cells to the yolk sac endothelium, and a second wave of EMPs colonizes the embryo and contributes endothelial cells to intraembryonic endothelium in multiple organs, where they persist into adulthood. By demonstrating that EMPs constitute a hitherto unrecognized source of endothelial cells, we reveal that embryonic blood vascular endothelium expands in a dual mechanism that involves both the proliferation of pre-existing endothelial cells and the incorporation of endothelial cells derived from haematopoietic precursors.

Phenotypic and functional plasticity of cells of innate immunity: macrophages, mast cells and neutrophils.

Hematopoietic cells, including lymphoid and myeloid cells, can develop into phenotypically distinct 'subpopulations' with different functions. However, evidence indicates that some of these subpopulations can manifest substantial plasticity (that is, undergo changes in their phenotype and function). Here we focus on the occurrence of phenotypically distinct subpopulations in three lineages of myeloid cells with important roles in innate and acquired immunity: macrophages, mast cells and neutrophils. Cytokine signals, epigenetic modifications and other microenvironmental factors can substantially and, in some cases, rapidly and reversibly alter the phenotype of these cells and influence their function. This suggests that regulation of the phenotype and function of differentiated hematopoietic cells by microenvironmental factors, including those generated during immune responses, represents a common mechanism for modulating innate or adaptive immunity.

Deletion of TLR2+ erythro-myeloid progenitors leads to embryonic lethality in mice.

Early embryonic hematopoiesis in mammals is defined by three successive waves of hematopoietic progenitors which exhibit a distinct hematopoietic potential and provide continuous support for the development of the embryo and adult organism. Although the functional importance of each of these waves has been analyzed, their spatio-temporal overlap and the lack of wave-specific markers hinders the accurate separation and assessment of their functional roles during early embryogenesis. We have recently shown that TLR2, in combination with c-kit, represents the earliest signature of emerging precursors of the second hematopoietic wave, erythro-myeloid precursors (EMPs). Since the onset of Tlr2 expression distinguishes EMPs from primitive progenitors which coexist in the yolk sac from E7.5, we generated a novel transgenic "knock in" mouse model, Tlr2Dtr , suitable for inducible targeted depletion of TLR2+ EMPs. In this model, the red fluorescent protein and diphtheria toxin receptor sequences are linked via a P2A sequence and inserted into the Tlr2 locus before its stop codon. We show that a timely controlled deletion of TLR2+ EMPs in Tlr2Dtr embryos results in a marked decrease in both erythroid as well as myeloid lineages and, consequently, in embryonic lethality peaking before E13.5. These findings validate the importance of EMPs in embryonic development.

Single-cell analysis of mixed-lineage states leading to a binary cell fate choice.

Delineating hierarchical cellular states, including rare intermediates and the networks of regulatory genes that orchestrate cell-type specification, are continuing challenges for developmental biology. Single-cell RNA sequencing is greatly accelerating such research, given its power to provide comprehensive descriptions of genomic states and their presumptive regulators. Haematopoietic multipotential progenitor cells, as well as bipotential intermediates, manifest mixed-lineage patterns of gene expression at a single-cell level. Such mixed-lineage states may reflect the molecular priming of different developmental potentials by co-expressed alternative-lineage determinants, namely transcription factors. Although a bistable gene regulatory network has been proposed to regulate the specification of either neutrophils or macrophages, the nature of the transition states manifested in vivo, and the underlying dynamics of the cell-fate determinants, have remained elusive. Here we use single-cell RNA sequencing coupled with a new analytic tool, iterative clustering and guide-gene selection, and clonogenic assays to delineate hierarchical genomic and regulatory states that culminate in neutrophil or macrophage specification in mice. We show that this analysis captured prevalent mixed-lineage intermediates that manifested concurrent expression of haematopoietic stem cell/progenitor and myeloid progenitor cell genes. It also revealed rare metastable intermediates that had collapsed the haematopoietic stem cell/progenitor gene expression programme, instead expressing low levels of the myeloid determinants, Irf8 and Gfi1 (refs 9, 10, 11, 12, 13). Genetic perturbations and chromatin immunoprecipitation followed by sequencing revealed Irf8 and Gfi1 as key components of counteracting myeloid-gene-regulatory networks. Combined loss of these two determinants 'trapped' the metastable intermediate. We propose that mixed-lineage states are obligatory during cell-fate specification, manifest differing frequencies because of their dynamic instability and are dictated by counteracting gene-regulatory networks.

Single-cell RNA sequencing reveals developmental heterogeneity among early lymphoid progenitors.

Single-cell RNA sequencing is a powerful technology for assessing heterogeneity within defined cell populations. Here, we describe the heterogeneity of a B220+CD117intCD19-NK1.1- uncommitted hematopoietic progenitor having combined lymphoid and myeloid potential. Phenotypic and functional assays revealed four subpopulations within the progenitor with distinct lineage developmental potentials. Among them, the Ly6D+SiglecH-CD11c- fraction was lymphoid-restricted exhibiting strong B-cell potential, whereas the Ly6D-SiglecH-CD11c- fraction showed mixed lympho-myeloid potential. Single-cell RNA sequencing of these subsets revealed that the latter population comprised a mixture of cells with distinct lymphoid and myeloid transcriptional signatures and identified a subgroup as the potential precursor of Ly6D+SiglecH-CD11c- Subsequent functional assays confirmed that B220+CD117intCD19-NK1.1- single cells are, with rare exceptions, not bipotent for lymphoid and myeloid lineages. A B-cell priming gradient was observed within the Ly6D+SiglecH-CD11c- subset and we propose a herein newly identified subgroup as the direct precursor of the first B-cell committed stage. Therefore, the apparent multipotency of B220+CD117intCD19-NK1.1- progenitors results from underlying heterogeneity at the single-cell level and highlights the validity of single-cell transcriptomics for resolving cellular heterogeneity and developmental relationships among hematopoietic progenitors.

A novel prospective isolation of murine fetal liver progenitors to study in utero hematopoietic defects.

In recent years, highly detailed characterization of adult bone marrow (BM) myeloid progenitors has been achieved and, as a result, the impact of somatic defects on different hematopoietic lineage fate decisions can be precisely determined. Fetal liver (FL) hematopoietic progenitor cells (HPCs) are poorly characterized in comparison, potentially hindering the study of the impact of genetic alterations on midgestation hematopoiesis. Numerous disorders, for example infant acute leukemias, have in utero origins and their study would therefore benefit from the ability to isolate highly purified progenitor subsets. We previously demonstrated that a Runx1 distal promoter (P1)-GFP::proximal promoter (P2)-hCD4 dual-reporter mouse (Mus musculus) model can be used to identify adult BM progenitor subsets with distinct lineage preferences. In this study, we undertook the characterization of the expression of Runx1-P1-GFP and P2-hCD4 in FL. Expression of P2-hCD4 in the FL immunophenotypic Megakaryocyte-Erythroid Progenitor (MEP) and Common Myeloid Progenitor (CMP) compartments corresponded to increased granulocytic/monocytic/megakaryocytic and decreased erythroid specification. Moreover, Runx1-P2-hCD4 expression correlated with several endogenous cell surface markers' expression, including CD31 and CD45, providing a new strategy for prospective identification of highly purified fetal myeloid progenitors in transgenic mouse models. We utilized this methodology to compare the impact of the deletion of either total RUNX1 or RUNX1C alone and to determine the fetal HPCs lineages most substantially affected. This new prospective identification of FL progenitors therefore raises the prospect of identifying the underlying gene networks responsible with greater precision than previously possible.

The role of the microbiota in acute stress-induced myeloid immune cell trafficking.

There has been a growing recognition of the involvement of the gastrointestinal microbiota in the development of stress-related disorders. Acute stress leads to activation of neuroendocrine systems, which in turn orchestrate a large-scale redistribution of innate immune cells. Both these response systems are independently known to be primed by the microbiota, even though much is still unclear about the role of the gastrointestinal microbiota in acute stress-induced immune activation. In this study, we investigated whether the microbiota influences acute stress-induced changes in innate immunity using conventionally colonised mice, mice devoid of any microbiota (i.e. germ-free, GF), and colonised GF mice (CGF). We also explored the kinetics of stress-induced immune cell mobilisation in the blood, the spleen and mesenteric lymph nodes (MLNs). Mice were either euthanised prior to stress or underwent restraint stress and were then euthanised at various time points (i.e. 0, 45- and 240-minutes) post-stress. Plasma adrenaline and noradrenaline levels were analysed using ELISA and immune cell levels were quantified using flow cytometry. GF mice had increased baseline levels of adrenaline and noradrenaline, of which adrenaline was normalised in CGF mice. In tandem, GF mice had decreased circulating levels of LY6Chi and LY6Cmid, CCR2+ monocytes, and granulocytes, but not LY6C-, CX3CR1+ monocytes. These deficits were normalised in CGF mice. Acute stress decreased blood LY6Chi and LY6Cmid, CCR2+ monocytes while increasing granulocyte levels in all groups 45 min post-stress. However, only GF mice showed stress-induced changes in LY6Chi monocytes and granulocytes 240 min post-stress, indicating impairments in the recovery from acute stress-induced changes in levels of specific innate immune cell types. LY6C-, CX3CR1+ monocytes remained unaffected by stress, indicating that acute stress impacts systemic innate immunity in a cell-type-specific manner. Overall, these data reveal novel cell-type-specific changes in the innate immune system in response to acute stress, which in turn are impacted by the microbiota. In conclusion, the microbiota influences the priming and recovery of the innate immune system to an acute stressor and may inform future microbiota-targeted therapeutics aimed at modulating stress-induced immune activation in stress-related disorders.

Bone Marrow-Derived Proangiogenic Cells Mediate Pulmonary Arteriole Stiffening via Serotonin 2B Receptor Dependent Mechanism.

RATIONALE: Pulmonary arterial hypertension is a deadly disease of the pulmonary vasculature for which no disease-modifying therapies exist. Small-vessel stiffening and remodeling are fundamental pathological features of pulmonary arterial hypertension that occur early and drive further endovascular cell dysfunction. Bone marrow (BM)-derived proangiogenic cells (PACs), a specialized heterogeneous subpopulation of myeloid lineage cells, are thought to play an important role in pathogenesis. OBJECTIVE: To determine whether BM-derived PACs directly contributed to experimental pulmonary hypertension (PH) by promoting small-vessel stiffening through 5-HT2B (serotonin 2B receptor)-mediated signaling. METHODS AND RESULTS: We performed BM transplants using transgenic donor animals expressing diphtheria toxin secondary to activation of an endothelial-specific tamoxifen-inducible Cre and induced experimental PH using hypoxia with SU5416 to enhance endovascular injury and ablated BM-derived PACs, after which we measured right ventricular systolic pressures in a closed-chest procedure. BM-derived PAC lineage tracing was accomplished by transplanting BM from transgenic donor animals with fluorescently labeled hematopoietic cells and treating mice with a 5-HT2B antagonist. BM-derived PAC ablation both prevented and reversed experimental PH with SU5416-enhanced endovascular injury, reducing the number of muscularized pulmonary arterioles and normalizing arteriole stiffness as measured by atomic force microscopy. Similarly, treatment with a pharmacological antagonist of 5-HT2B also prevented experimental PH, reducing the number and stiffness of muscularized pulmonary arterioles. PACs accelerated pulmonary microvascular endothelial cell injury response in vitro, and the presence of BM-derived PACs significantly correlated with stiffer pulmonary arterioles in pulmonary arterial hypertension patients and mice with experimental PH. RNA sequencing of BM-derived PACs showed that 5-HT2B antagonism significantly altered biologic pathways regulating cell proliferation, locomotion and migration, and cytokine production and response to cytokine stimulus. CONCLUSIONS: Together, our findings illustrate that BM-derived PACs directly contribute to experimental PH with SU5416-enhanced endovascular injury by mediating small-vessel stiffening and remodeling in a 5-HT2B signaling-dependent manner.