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Analysis of transplantation of single haematopoietic stem cells in mice defines stable lineage-restricted fates in long-term self-renewing multipotent stem cells, including a class of multipotent stem cells that exclusively replenishes the megakaryocyte/platelet lineage. The many fates of stem cells in the blood line Many blood disorders can be treated with haematopoietic (blood-generating) stem cell (HSC) transplants, but such treatment does not always lead to efficient replenishment of all blood lineages. Through single-cell transplantation of HSCs in mice, Sten Eirik Jacobsen and colleagues define lineage-restricted fates of long-term self-renewing cells. They identify a class of HSC that effectively replenishes the megakaryocyte and platelet lineages over other lineages, and other HSCs that are more able to participate in megakaryocyte, erythroid and myeloid lineages despite being able to sustain lymphoid potential. Genetic lineage tracing also shows that platelet-biased HSCs are able to support unperturbed adult haematopoiesis. Rare multipotent haematopoietic stem cells (HSCs) in adult bone marrow with extensive self-renewal potential can efficiently replenish all myeloid and lymphoid blood cells 1 , securing long-term multilineage reconstitution after physiological and clinical challenges such as chemotherapy and haematopoietic transplantations 2 , 3 , 4 . HSC transplantation remains the only curative treatment for many haematological malignancies, but inefficient blood-lineage replenishment remains a major cause of morbidity and mortality 5 , 6 . Single-cell transplantation has uncovered considerable heterogeneity among reconstituting HSCs 7 , 8 , 9 , 10 , 11 , a finding that is supported by studies of unperturbed haematopoiesis 2 , 3 , 4 , 12 and may reflect different propensities for lineage-fate decisions by distinct myeloid-, lymphoid- and platelet-biased HSCs 7 , 8 , 9 , 10 , 13 . Other studies suggested that such lineage bias might reflect generation of unipotent or oligopotent self-renewing progenitors within the phenotypic HSC compartment, and implicated uncoupling of the defining HSC properties of self-renewal and multipotency 11 , 14 . Here we use highly sensitive tracking of progenitors and mature cells of the megakaryocyte/platelet, erythroid, myeloid and B and T cell lineages, produced from singly transplanted HSCs, to reveal a highly organized, predictable and stable framework for lineage-restricted fates of long-term self-renewing HSCs. Most notably, a distinct class of HSCs adopts a fate towards effective and stable replenishment of a megakaryocyte/platelet-lineage tree but not of other blood cell lineages, despite sustained multipotency. No HSCs contribute exclusively to any other single blood-cell lineage. Single multipotent HSCs can also fully restrict towards simultaneous replenishment of megakaryocyte, erythroid and myeloid lineages without executing their sustained lymphoid lineage potential. Genetic lineage-tracing analysis also provides evidence for an important role of platelet-biased HSCs in unperturbed adult haematopoiesis. These findings uncover a limited repertoire of distinct HSC subsets, defined by a predictable and hierarchical propensity to adopt a fate towards replenishment of a restricted set of blood lineages, before loss of self-renewal and multipotency.

Aged haematopoietic stem cells (HSCs) generate more myeloid cells and fewer lymphoid cells compared with young HSCs, contributing to decreased adaptive immunity in aged individuals. However, it is not known how intrinsic changes to HSCs and shifts in the balance between biased HSC subsets each contribute to the altered lineage output. Here, by analysing HSC transcriptomes and HSC function at the single-cell level, we identify increased molecular platelet priming and functional platelet bias as the predominant age-dependent change to HSCs, including a significant increase in a previously unrecognized class of HSCs that exclusively produce platelets. Depletion of HSC platelet programming through loss of the FOG-1 transcription factor is accompanied by increased lymphoid output. Therefore, increased platelet bias may contribute to the age-associated decrease in lymphopoiesis. With age, haematopoietic stem cells (HSCs) produce more myeloid than lymphoid cells, affecting adaptive immunity. By combining HSC single cell transcriptomics with functional studies, Grover et al . find that platelet production is also increased in old murine HSCs and show that the FOG-1 transcription factor contributes to the age-dependent platelet bias.

Thymic T cell development is initiated from bone-marrow-derived multi potent thymus-seeding progenitors. During the early stages of thymocyte differentiation, progenitors become T cell restricted. However, the cellular environments supporting these critical initial stages of T cell development within the thymic cortex are not known. Here we use the dependence of early, c-Kit-expressing thymic progenitors on Kit ligand (KitL) to show that CD4 − CD8 − c-Kit + CD25 − DN1-stage progenitors associate with, and depend on, the membrane-bound form of KitL (mKitL) provided by a cortex-specific KitL-expressing vascular endothelial cell (VEC) population. In contrast, the subsequent CD4 − CD8 − c-Kit + CD25 + DN2-stage progenitors associate selectively with cortical thymic epithelial cells (cTECs) and depend on cTEC-presented mKitL. These results show that the dynamic process of early thymic progenitor differentiation is paralleled by migration-dependent change to the supporting niche, and identify VECs as a thymic niche cell, with mKitL as a critical ligand. Nerlov and colleagues show that expression of mKitL by cortical vascular endothelial cells is important for DN1 progenitor maintenance, whereas expression of mKitL by cortical thymic epithelial cells is required for maintaining DN2 progenitor cells.

Hematopoietic stem cells (HSCs) residing in specialized niches in the bone marrow are responsible for the balanced output of multiple short-lived blood cell lineages in steady-state and in response to different challenges. However, feedback mechanisms by which HSCs, through their niches, sense acute losses of specific blood cell lineages remain to be established. While all HSCs replenish platelets, previous studies have shown that a large fraction of HSCs are molecularly primed for the megakaryocyte-platelet lineage and are rapidly recruited into proliferation upon platelet depletion. Platelets normally turnover in an activation-dependent manner, herein mimicked by antibodies inducing platelet activation and depletion. Antibody-mediated platelet activation upregulates expression of Interleukin-1 (IL-1) in platelets, and in bone marrow extracellular fluid in vivo. Genetic experiments demonstrate that rather than IL-1 directly activating HSCs, activation of bone marrow Lepr + perivascular niche cells expressing IL-1 receptor is critical for the optimal activation of quiescent HSCs upon platelet activation and depletion. These findings identify a feedback mechanism by which activation-induced depletion of a mature blood cell lineage leads to a niche-dependent activation of HSCs to reinstate its homeostasis. Hematopoietic stem cells (HSCs) replenish blood cells. Here, Luis et al., identify a feedback mechanism by which IL-1 secreted by activated platelets signals through niche Lepr+ cells to activate HSCs and restore platelet homeostasis.

Precision-cut lung slices (PCLS) are used for a variety of applications. However, methods to manipulate genes in PCLS are currently limited. We developed a new method, TAT-Cre recombinase-mediated floxed allele modification in tissue slices (TReATS), to induce highly effective and temporally controlled gene deletion or activation in ex vivo PCLS. Treatment of PCLS from Rosa26-flox-stop-flox-EYFP mice with cell-permeant TAT-Cre recombinase induced ubiquitous EYFP protein expression, indicating successful Cre-mediated excision of the upstream loxP-flanked stop sequence. Quantitative real-time PCR confirmed induction of EYFP. We successfully replicated the TReATS method in PCLS from Vangl2flox/flox mice, leading to the deletion of loxP-flanked exon 4 of the Vangl2 gene. Cre-treated Vangl2flox/flox PCLS exhibited cytoskeletal abnormalities, a known phenotype caused by VANGL2 dysfunction. We report a new method that bypasses conventional Cre-Lox breeding, allowing rapid and highly effective gene manipulation in ex vivo tissue models.

Severe infections are a major stress on haematopoiesis, where the consequences for haematopoietic stem cells (HSCs) have only recently started to emerge. HSC function critically depends on the integrity of complex bone marrow (BM) niches; however, what role the BM microenvironment plays in mediating the effects of infection on HSCs remains an open question. Here, using a murine model of malaria and combining single-cell RNA sequencing, mathematical modelling, transplantation assays and intravital microscopy, we show that haematopoiesis is reprogrammed upon infection, whereby the HSC compartment turns over substantially faster than at steady-state and HSC function is drastically affected. Interferon is found to affect both haematopoietic and mesenchymal BM cells and we specifically identify a dramatic loss of osteoblasts and alterations in endothelial cell function. Osteo-active parathyroid hormone treatment abolishes infection-triggered HSC proliferation and—coupled with reactive oxygen species quenching—enables partial rescuing of HSC function.Haltalli et al. show that Plasmodium berghei infection induces interferon release, and affects haematopoietic stem cell proliferation and function, as well as osteoblasts and vascular integrity, in the bone marrow niche.

Monocytes are heterogeneous innate effector leukocytes generated in the bone marrow and released into circulation in a CCR2-dependent manner. During infection or inflammation, myelopoiesis is modulated to rapidly meet the demand for more effector cells. Danger signals from peripheral tissues can influence this process. Herein we demonstrate that repetitive TLR7 stimulation via the epithelial barriers drove a potent emergency bone marrow monocyte response in mice. This process was unique to TLR7 activation and occurred independently of the canonical CCR2 and CX3CR1 axes or prototypical cytokines. The monocytes egressing the bone marrow had an immature Ly6C-high profile and differentiated into vascular Ly6C-low monocytes and tissue macrophages in multiple organs. They displayed a blunted cytokine response to further TLR7 stimulation and reduced lung viral load after RSV and influenza virus infection. These data provide insights into the emergency myelopoiesis likely to occur in response to the encounter of single-stranded RNA viruses at barrier sites.

The identity and lineage potential of the embryonic thymus-seeding progenitors that first seed the embryonic thymic rudiment is unclear. Jacobsen and colleagues find that these cells do not include multipotent stem cells or T cell–restricted progenitors but instead are lympho-myeloid progenitors. The final stages of restriction to the T cell lineage occur in the thymus after the entry of thymus-seeding progenitors (TSPs). The identity and lineage potential of TSPs remains unclear. Because the first embryonic TSPs enter a non-vascularized thymic rudiment, we were able to directly image and establish the functional and molecular properties of embryonic thymopoiesis-initiating progenitors (T-IPs) before their entry into the thymus and activation of Notch signaling. T-IPs did not include multipotent stem cells or molecular evidence of T cell–restricted progenitors. Instead, single-cell molecular and functional analysis demonstrated that most fetal T-IPs expressed genes of and had the potential to develop into lymphoid as well as myeloid components of the immune system. Moreover, studies of embryos deficient in the transcriptional regulator RBPJ demonstrated that canonical Notch signaling was not involved in pre-thymic restriction to the T cell lineage or the migration of T-IPs.

Key Points There are different WNT signalling pathways: the canonical WNT pathway, which involves β-catenin and members of the T-cell factor (TCF)/lymphocyte-enhancer-binding factor (LEF) family, the planar cell polarity (PCP) pathway and the WNT–Ca 2+ pathway. Most studies of immune and blood cells concern canonical WNT signalling. WNT signalling probably controls aspects of haematopoietic stem cell (HSC) self-renewal, although there are some controversies surrounding this topic. The level of WNT signalling seems to be important. WNT signalling is required for T-cell development in the thymus and might also be involved in developing B cells in the bone marrow. WNT signalling regulates aspects of peripheral T-cell activation and migration. WNT signalling is also involved in dendritic cell (DC) maturation, and activation of WNT signalling increases the survival of regulatory T cells. Dysregulated WNT signalling can cause leukaemia. This Review covers the recently discovered roles of WNT proteins in the regulation of haematopoietic stem-cell fate, T- and B-cell development and activation, and dendritic-cell maturation. These new immunohaematological functions of WNT proteins have implications for the development of haematological malignancies. WNT proteins are secreted morphogens that are required for basic developmental processes, such as cell-fate specification, progenitor-cell proliferation and the control of asymmetric cell division, in many different species and organs. In blood and immune cells, WNT signalling controls the proliferation of progenitor cells and might also affect the cell-fate decisions of stem cells. Recent studies indicate that WNT proteins also regulate effector T-cell development, regulatory T-cell activation and dendritic-cell maturation. WNT signalling seems to function as a universal mechanism in leukocytes to establish a pool of undifferentiated cells for further selection, effector-cell maturation and terminal differentiation. WNT signalling is therefore subject to strict molecular control, and dysregulated WNT signalling is implicated in the development of haematological malignancies.

The commitment stage at which progenitors seed the thymus remains unclear. Jacobsen and colleagues show that the earliest progenitors in the neonatal thymus have combined myeloid, T lymphocyte and B lymphocyte potential but not megakaryocyte-erythroid potential. The stepwise commitment from hematopoietic stem cells in the bone marrow to T lymphocyte–restricted progenitors in the thymus represents a paradigm for understanding the requirement for distinct extrinsic cues during different stages of lineage restriction from multipotent to lineage-restricted progenitors. However, the commitment stage at which progenitors migrate from the bone marrow to the thymus remains unclear. Here we provide functional and molecular evidence at the single-cell level that the earliest progenitors in the neonatal thymus had combined granulocyte-monocyte, T lymphocyte and B lymphocyte lineage potential but not megakaryocyte-erythroid lineage potential. These potentials were identical to those of candidate thymus-seeding progenitors in the bone marrow, which were closely related at the molecular level. Our findings establish the distinct lineage-restriction stage at which the T cell lineage–commitment process transits from the bone marrow to the remote thymus.