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Niches are local tissue microenvironments that maintain and regulate stem cells. Haematopoiesis provides a model for understanding mammalian stem cells and their niches, but the haematopoietic stem cell (HSC) niche remains incompletely defined and beset by competing models. Recent progress has been made in elucidating the location and cellular components of the HSC niche in the bone marrow. The niche is perivascular, created partly by mesenchymal stromal cells and endothelial cells and often, but not always, located near trabecular bone. Outstanding questions concern the cellular complexity of the niche, the role of the endosteum and functional heterogeneity among perivascular microenvironments.

A method to model expression variability in single-cell RNA-seq measurements and thus to improve subsequent data analysis. Single-cell data provide a means to dissect the composition of complex tissues and specialized cellular environments. However, the analysis of such measurements is complicated by high levels of technical noise and intrinsic biological variability. We describe a probabilistic model of expression-magnitude distortions typical of single-cell RNA-sequencing measurements, which enables detection of differential expression signatures and identification of subpopulations of cells in a way that is more tolerant of noise.

The treatment of low-risk primary prostate cancer entails active surveillance only, while high-risk disease requires multimodal treatment including surgery, radiation therapy, and hormonal therapy. Recurrence and development of metastatic disease remains a clinical problem, without a clear understanding of what drives immune escape and tumor progression. Here, we comprehensively describe the tumor microenvironment of localized prostate cancer in comparison with adjacent normal samples and healthy controls. Single-cell RNA sequencing and high-resolution spatial transcriptomic analyses reveal tumor context dependent changes in gene expression. Our data indicate that an immune suppressive tumor microenvironment associates with suppressive myeloid populations and exhausted T-cells, in addition to high stromal angiogenic activity. We infer cell-to-cell relationships from high throughput ligand-receptor interaction measurements within undissociated tissue sections. Our work thus provides a highly detailed and comprehensive resource of the prostate tumor microenvironment as well as tumor-stromal cell interactions. The immune suppressive tumour microenvironment drives recurrence and metastatic disease in prostate cancer. Here authors provide a detailed analysis of the microenvironment via single cell RNA sequencing and high-resolution spatial transcriptomics to identify tumour-dependent changes compared to healthy tissue.

Recent single-cell RNA-seq protocols based on droplet microfluidics use massively multiplexed barcoding to enable simultaneous measurements of transcriptomes for thousands of individual cells. The increasing complexity of such data creates challenges for subsequent computational processing and troubleshooting of these experiments, with few software options currently available. Here, we describe a flexible pipeline for processing droplet-based transcriptome data that implements barcode corrections, classification of cell quality, and diagnostic information about the droplet libraries. We introduce advanced methods for correcting composition bias and sequencing errors affecting cellular and molecular barcodes to provide more accurate estimates of molecular counts in individual cells.

Key Points Bone marrow cellular niches are functional compartments that regulate haematopoietic stem cell (HSC) and immune cell behaviour through direct cell contact, growth factors and cytokines, and components of the extracellular matrix. Within the bone marrow, HSCs are regulated by components of the vasculature, the trabecular bone and the surrounding stroma. In addition to its role as a primary lymphoid organ through the support of lymphoid development, the bone marrow can act as a host for naive and memory immune cells, plasma cells, regulatory T cells and myeloid immune cells. The bone marrow is heterogeneous with regard to perfusion, oxygenation and innervation. Together, studies exploring these features allow us to consider new ways in which the bone marrow cellular niches are constituted and regulated. Because the haematopoietic and immune systems need to rapidly respond and adapt to the needs of the organism, their niches should not be viewed as static entities. For example, these niches can respond to particular states of injury and inflammation. A better understanding of the dynamic interactions between HSCs, immune cells and their niches could yield significant therapeutic benefit in contexts such as chemotherapy, radiation injury or invasion of the niche by malignancies. This Review describes the dynamic interactions that occur between haematopoietic stem cells (HSCs), immune cells and their specialized niches in the bone marrow. The authors discuss how the signals provided by the niche help HSCs and immune cells to respond to injury or inflammation in peripheral tissues. Progenitor cells that are the basis for all blood cell production share the bone marrow with more mature elements of the adaptive immune system. Specialized niches within the bone marrow guide and, at times, constrain the development of haematopoietic stem and progenitor cells (HSPCs) and lineage-restricted immune progenitor cells. Specific niche components are organized into distinct domains to create a diversified landscape in which specialized cell differentiation or population expansion programmes proceed. Local cues that reflect the tissue and organismal state affect cellular interactions to alter the production of a range of cell types. Here, we review the organization of regulatory elements in the bone marrow and discuss how these elements provide a dynamic means for the host to modulate stem cell and adaptive immune cell responses to physiological challenges.

Cancer-associated thrombosis often lacks a clear etiology. However, it is linked to a poor prognosis and represents the second-leading cause of death in cancer patients. Recent studies have shown that chromatin released into blood, through the generation of neutrophil extracellular traps (NETs), is procoagulant and prothrombotic. Using a murine model of chronic myelogenous leukemia, we show that malignant and nonmalignant neutrophils are more prone to NET formation. This increased sensitivity toward NET generation is also observed in mammary and lung carcinoma models, suggesting that cancers, through a systemic effect on the host, can induce an increase in peripheral blood neutrophils, which are predisposed to NET formation. In addition, in the late stages of the breast carcinoma model, NETosis occurs concomitant with the appearance of venous thrombi in the lung. Moreover, simulation of a minor systemic infection in tumor-bearing, but not control, mice results in the release of large quantities of chromatin and a prothrombotic state. The increase in neutrophil count and their priming is mediated by granulocyte colony-stimulating factor (G-CSF), which accumulates in the blood of tumor-bearing mice. The prothrombotic state in cancer can be reproduced by treating mice with G-CSF combined with low-dose LPS and leads to thrombocytopenia and microthrombosis. Taken together, our results identify extracellular chromatin released through NET formation as a cause for cancer-associated thrombosis and unveil a target in the effort to decrease the incidence of thrombosis in cancer patients.

Mesenchymal stem cell (MSC) therapies demonstrate particular promise in ameliorating diseases of immune dysregulation but are hampered by short in vivo cell persistence and inconsistencies in phenotype. Here, we demonstrate that biomaterial encapsulation into alginate using a microfluidic device could substantially increase in vivo MSC persistence after intravenous (i.v.) injection. A combination of cell cluster formation and subsequent cross-linking with polylysine led to an increase in injected MSC half-life by more than an order of magnitude. These modifications extended persistence even in the presence of innate and adaptive immunity-mediated clearance. Licensing of encapsulated MSCs with inflammatory cytokine pretransplantation increased expression of immunomodulatory-associated genes, and licensed encapsulates promoted repopulation of recipient blood and bone marrow with allogeneic donor cells after sublethal irradiation by a ∼2-fold increase. The ability ofmicrogel encapsulation to sustain MSC survival and increase overall immunomodulatory capacity may be applicable for improving MSC therapies in general.

Here, using two-photon phosphorescence lifetime microscopy, the local oxygen tension in the bone marrow of live mice is found to be quite low, with spatiotemporal variations depending on the blood vessel type, distance to the endosteum, and changes in cellularity after stress. Oxygen tension in live bone marrow Low oxygen tension (hypoxia) is commonly thought to be a shared niche characteristic in maintaining quiescence in many stem cell types. However, local oxygen concentration, for example in the bone marrow, has never been measured directly. Charles Lin and colleagues have now developed a method based on two-photon microscopy to measure the absolute local oxygen tension ( p O 2 ) in the marrow of live animals. Using this method, they found that while vascular density is high throughout the bone marrow, overall oxygenation is quite low and there is heterogeneity in local p O 2 with respect to vessel type and location. For example, surprisingly, the endosteal region is not the region of the lowest p O 2 . After radiation or chemotherapy, bone marrow p O 2 becomes elevated and transplanted haematopoietic stem/progenitor cells do not seek out regions with the lowest p O 2 for homing. Characterization of how the microenvironment, or niche, regulates stem cell activity is central to understanding stem cell biology and to developing strategies for the therapeutic manipulation of stem cells 1 . Low oxygen tension (hypoxia) is commonly thought to be a shared niche characteristic in maintaining quiescence in multiple stem cell types 2 , 3 , 4 . However, support for the existence of a hypoxic niche has largely come from indirect evidence such as proteomic analysis 5 , expression of hypoxia inducible factor-1α ( Hif-1 α) and related genes 6 , and staining with surrogate hypoxic markers (for example, pimonidazole) 6 , 7 , 8 . Here we perform direct in vivo measurements of local oxygen tension ( p O 2 ) in the bone marrow of live mice. Using two-photon phosphorescence lifetime microscopy, we determined the absolute p O 2 of the bone marrow to be quite low (<32 mm Hg) despite very high vascular density. We further uncovered heterogeneities in local p O 2 , with the lowest p O 2 (∼9.9 mm Hg, or 1.3%) found in deeper peri-sinusoidal regions. The endosteal region, by contrast, is less hypoxic as it is perfused with small arteries that are often positive for the marker nestin. These p O 2 values change markedly after radiation and chemotherapy, pointing to the role of stress in altering the stem cell metabolic microenvironment.

Bone marrow endothelial cells (BMECs) form a network of blood vessels that regulate both leukocyte trafficking and haematopoietic stem and progenitor cell (HSPC) maintenance. However, it is not clear how BMECs balance these dual roles, and whether these events occur at the same vascular site. We found that mammalian bone marrow stem cell maintenance and leukocyte trafficking are regulated by distinct blood vessel types with different permeability properties. Less permeable arterial blood vessels maintain haematopoietic stem cells in a low reactive oxygen species (ROS) state, whereas the more permeable sinusoids promote HSPC activation and are the exclusive site for immature and mature leukocyte trafficking to and from the bone marrow. A functional consequence of high permeability of blood vessels is that exposure to blood plasma increases bone marrow HSPC ROS levels, augmenting their migration and differentiation, while compromising their long-term repopulation and survival. These findings may have relevance for clinical haematopoietic stem cell transplantation and mobilization protocols. Bone marrow endothelial cells have dual roles in the regulation of haematopoietic stem cell maintenance and in the trafficking of blood cells between the bone marrow and the blood circulatory system; this study shows that these different functions are regulated by distinct types of endothelial blood vessels with different permeability properties, affecting the metabolic state of their neighbouring stem cells. Bone marrow blood vessel specialization Endothelial cells of the bone marrow modulate both haematopoietic stem cell (HSC) maintenance and the trafficking of blood cells out of the bone marrow. Tsvee Lapidot and colleagues find that these two aspects are controlled by two distinct types of blood vessels in the bone marrow, with different permeability properties and reactive oxygen species (ROS) levels. Less permeable arteries surrounded by pericytes maintain HSCs in a low reactive oxygen species (ROS) state, whereas the more permeable smaller sinusoids promote HSC activation and allow trafficking of immature and mature leukocytes. The authors also show that in conditions that allow for expansion of HSCs, endothelial integrity is increased, with fewer blood cells moving in and out. Disruption of the endothelial barrier has the reverse effects. Elsewhere in this issue ( page 380 ), Anjali Kusumbe et al . demonstrate that Notch signalling in endothelial cells of bone marrow induces change in the capillaries and mesenchymal stem cells of the environment to support HSC amplification.

Mutations in the protein tyrosine phosphatase SHP2 affect cells in the bone marrow environment, which leads to aberrant activation of resident haematopoietic stem cells and thereby contributes to the development of leukaemia. A mutated microenvironment in leukaemia Hereditary mutations in the tyrosine phosphatase SHP2 (encoded by PTPN11 ), part of the Ras signalling pathway, have been linked to a syndrome leading to an increased risk of developing leukaemia. Previous studies in mouse models have shown that the function of haematopoietic stem cells carrying these mutations is defective, which suggests a cell-autonomous effect. Cheng-Kui Qu and colleagues find that the mutations also affect cells in the bone marrow environment, blocking their normal control on haematopoietic stem cells and thereby promoting the development of leukaemia. Administration of CCL3 receptor antagonists effectively reversed oncogenesis driven by the Ptpn11 -mutated bone marrow microenvironment. Germline activating mutations of the protein tyrosine phosphatase SHP2 (encoded by PTPN11 ), a positive regulator of the RAS signalling pathway 1 , are found in 50% of patients with Noonan syndrome 2 . These patients have an increased risk of developing leukaemia 3 , especially juvenile myelomonocytic leukaemia (JMML), a childhood myeloproliferative neoplasm (MPN). Previous studies have demonstrated that mutations in Ptpn11 induce a JMML-like MPN through cell-autonomous mechanisms that are dependent on Shp2 catalytic activity 4 , 5 , 6 , 7 . However, the effect of these mutations in the bone marrow microenvironment remains unclear. Here we report that Ptpn11 activating mutations in the mouse bone marrow microenvironment promote the development and progression of MPN through profound detrimental effects on haematopoietic stem cells (HSCs). Ptpn11 mutations in mesenchymal stem/progenitor cells and osteoprogenitors, but not in differentiated osteoblasts or endothelial cells, cause excessive production of the CC chemokine CCL3 (also known as MIP-1α), which recruits monocytes to the area in which HSCs also reside. Consequently, HSCs are hyperactivated by interleukin-1β and possibly other proinflammatory cytokines produced by monocytes, leading to exacerbated MPN and to donor-cell-derived MPN following stem cell transplantation. Remarkably, administration of CCL3 receptor antagonists effectively reverses MPN development induced by the Ptpn11 -mutated bone marrow microenvironment. This study reveals the critical contribution of Ptpn11 mutations in the bone marrow microenvironment to leukaemogenesis and identifies CCL3 as a potential therapeutic target for controlling leukaemic progression in Noonan syndrome and for improving stem cell transplantation therapy in Noonan-syndrome-associated leukaemias.