Initial seeding of the embryonic thymus by immune-restricted 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.

Loss of endothelial membrane KIT ligand affects systemic KIT ligand levels but not bone marrow hematopoietic stem cells.

A critical regulatory role of hematopoietic stem cell (HSC) vascular niches in the bone marrow has been implicated to occur through endothelial niche cell expression of KIT ligand. However, endothelial-derived KIT ligand is expressed in both a soluble and membrane-bound form and not unique to bone marrow niches, and it is also systemically distributed through the circulatory system. Here, we confirm that upon deletion of both the soluble and membrane-bound forms of endothelial-derived KIT ligand, HSCs are reduced in mouse bone marrow. However, the deletion of endothelial-derived KIT ligand was also accompanied by reduced soluble KIT ligand levels in the blood, precluding any conclusion as to whether the reduction in HSC numbers reflects reduced endothelial expression of KIT ligand within HSC niches, elsewhere in the bone marrow, and/or systemic soluble KIT ligand produced by endothelial cells outside of the bone marrow. Notably, endothelial deletion, specifically of the membrane-bound form of KIT ligand, also reduced systemic levels of soluble KIT ligand, although with no effect on stem cell numbers, implicating an HSC regulatory role primarily of soluble rather than membrane KIT ligand expression in endothelial cells. In support of a role of systemic rather than local niche expression of soluble KIT ligand, HSCs were unaffected in KIT ligand deleted bones implanted into mice with normal systemic levels of soluble KIT ligand. Our findings highlight the need for more specific tools to unravel niche-specific roles of regulatory cues expressed in hematopoietic niche cells in the bone marrow.

The earliest thymic T cell progenitors sustain B cell and myeloid lineage 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.

Hierarchically related lineage-restricted fates of multipotent haematopoietic stem cells.

Rare multipotent haematopoietic stem cells (HSCs) in adult bone marrow with extensive self-renewal potential can efficiently replenish all myeloid and lymphoid blood cells, securing long-term multilineage reconstitution after physiological and clinical challenges such as chemotherapy and haematopoietic transplantations. HSC transplantation remains the only curative treatment for many haematological malignancies, but inefficient blood-lineage replenishment remains a major cause of morbidity and mortality. Single-cell transplantation has uncovered considerable heterogeneity among reconstituting HSCs, a finding that is supported by studies of unperturbed haematopoiesis and may reflect different propensities for lineage-fate decisions by distinct myeloid-, lymphoid- and platelet-biased HSCs. 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. 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.

Canonical Notch signaling is dispensable for adult steady-state and stress myelo-erythropoiesis.

Although an essential role for canonical Notch signaling in generation of hematopoietic stem cells in the embryo and in thymic T-cell development is well established, its role in adult bone marrow (BM) myelopoiesis remains unclear. Some studies, analyzing myeloid progenitors in adult mice with inhibited Notch signaling, implicated distinct roles of canonical Notch signaling in regulation of progenitors for the megakaryocyte, erythroid, and granulocyte-macrophage cell lineages. However, these studies might also have targeted other pathways. Therefore, we specifically deleted, in adult BM, the transcription factor recombination signal-binding protein J κ (Rbpj), through which canonical signaling from all Notch receptors converges. Notably, detailed progenitor staging established that canonical Notch signaling is fully dispensable for all investigated stages of megakaryocyte, erythroid, and myeloid progenitors in steady state unperturbed hematopoiesis, after competitive BM transplantation, and in stress-induced erythropoiesis. Moreover, expression of key regulators of these hematopoietic lineages and Notch target genes were unaffected by Rbpj deficiency in BM progenitor cells.

Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy.

The blood system is maintained by a small pool of haematopoietic stem cells (HSCs), which are required and sufficient for replenishing all human blood cell lineages at millions of cells per second throughout life. Megakaryocytes in the bone marrow are responsible for the continuous production of platelets in the blood, crucial for preventing bleeding--a common and life-threatening side effect of many cancer therapies--and major efforts are focused at identifying the most suitable cellular and molecular targets to enhance platelet production after bone marrow transplantation or chemotherapy. Although it has become clear that distinct HSC subsets exist that are stably biased towards the generation of lymphoid or myeloid blood cells, we are yet to learn whether other types of lineage-biased HSC exist or understand their inter-relationships and how differently lineage-biased HSCs are generated and maintained. The functional relevance of notable phenotypic and molecular similarities between megakaryocytes and bone marrow cells with an HSC cell-surface phenotype remains unclear. Here we identify and prospectively isolate a molecularly and functionally distinct mouse HSC subset primed for platelet-specific gene expression, with enhanced propensity for short- and long-term reconstitution of platelets. Maintenance of platelet-biased HSCs crucially depends on thrombopoietin, the primary extrinsic regulator of platelet development. Platelet-primed HSCs also frequently have a long-term myeloid lineage bias, can self-renew and give rise to lymphoid-biased HSCs. These findings show that HSC subtypes can be organized into a cellular hierarchy, with platelet-primed HSCs at the apex. They also demonstrate that molecular and functional priming for platelet development initiates already in a distinct HSC population. The identification of a platelet-primed HSC population should enable the rational design of therapies enhancing platelet output.

A Novel Method for Floxed Gene Manipulation Using TAT-Cre Recombinase in Ex Vivo Precision-Cut Lung Slices (PCLS).

Precision-cut lung slices (PCLS), ex vivo 3D lung tissue models, have been widely used for various applications in lung research. PCLS serve as an excellent intermediary between in vitro and in vivo models because they retain all resident cell types within their natural niche while preserving the extracellular matrix environment. This protocol describes the TReATS (TAT-Cre recombinase-mediated floxed allele modification in tissue slices) method that enables rapid and efficient gene modification in PCLS derived from adult floxed animals. Here, we present detailed protocols for the TReATS method, consisting of two simple steps: PCLS generation and incubation in a TAT-Cre recombinase solution. Subsequent validation of gene modification involves live staining and imaging of PCLS, quantitative real-time PCR, and cell viability assessment. This four-day protocol eliminates the need for complex Cre-breeding, circumvents issues with premature lethality related to gene mutation, and significantly reduces the use of animals. The TReATS method offers a simple and reproducible solution for gene modification in complex ex vivo tissue-based models, accelerating the study of gene function, disease mechanisms, and the discovery of drug targets. Key features • Achieve permanent ex vivo gene modifications in complex tissue-based models within four days. • Highly adaptable gene modification method that can be applied to induce gene deletion or activation. • Allows simple Cre dosage testing in a controlled ex vivo setting with the advantage of using PCLS generated from the same animal as true controls. • With optimisation, this method can be applied to precision-cut tissue slices of other organs.

A method for TAT-Cre recombinase-mediated floxed allele modification in ex vivo tissue slices.

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.

TLR7 activation at epithelial barriers promotes emergency myelopoiesis and lung antiviral immunity.

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.

Perivascular niche cells sense thrombocytopenia and activate hematopoietic stem cells in an IL-1 dependent manner.

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.

Wnt3a deficiency irreversibly impairs hematopoietic stem cell self-renewal and leads to defects in progenitor cell differentiation.

Canonical Wnt signaling has been implicated in various aspects of hematopoiesis. Its role is controversial due to different outcomes between various inducible Wnt-signaling loss-of-function models and also compared with gain-of-function systems. We therefore studied a mouse deficient for a Wnt gene that seemed to play a nonredundant role in hematopoiesis. Mice lacking Wnt3a die prenatally around embryonic day (E) 12.5, allowing fetal hematopoiesis to be studied using in vitro assays and transplantation into irradiated recipient mice. Here we show that Wnt3a deficiency leads to a reduction in the numbers of hematopoietic stem cells (HSCs) and progenitor cells in the fetal liver (FL) and to severely reduced reconstitution capacity as measured in secondary transplantation assays. This deficiency is irreversible and cannot be restored by transplantation into Wnt3a competent mice. The impaired long-term repopulation capacity of Wnt3a(-/-) HSCs could not be explained by altered cell cycle or survival of primitive progenitors. Moreover, Wnt3a deficiency affected myeloid but not B-lymphoid development at the progenitor level, and affected immature thymocyte differentiation. Our results show that Wnt3a signaling not only provides proliferative stimuli, such as for immature thymocytes, but also regulates cell fate decisions of HSC during hematopoiesis.

Wnt signaling in hematopoiesis: crucial factors for self-renewal, proliferation, and cell fate decisions.

A large number of studies from many different laboratories have implicated the Wnt signaling pathway in regulation of hematopoiesis. However, different inducible gain- and loss-of-function approaches yielded controversial and some times contradictory results. In this prospect we will review the current ideas on Wnt signaling in hematopoiesis and early lymphopoiesis. Reviewing this large body of knowledge let us to hypothesize that different levels of activation of the pathway, dosages of Wnt signaling required and the interference by other signals in the context of Wnt activation collectively explain these controversies. Besides differences in dosage, differences in biological function of Wnt proteins in various blood cell types also is a major factor to take into account. Our own work has shown that while in the thymus Wnt signaling provides cytokine-like, proliferative stimuli to developing thymocytes, canonical Wnt signaling in HSC regulates cell fate decisions, in particular self-renewal versus differentiation.

Divide and Rule: Mitochondrial Fission Regulates Quiescence in Hematopoietic Stem Cells.

Hematopoietic stem cells (HSCs) remain quiescent to preserve long-term integrity. In this issue of Cell Stem Cell, Hinge et al. (2020) and Liang et al. (2020) demonstrate that HSCs achieve this by regulating mitochondrial fission and lysosomal activity, suppressing glucose uptake, and maintaining healthy punctate mitochondria with low metabolic activity.

Single-cell lineage tracing approaches in hematology research: technical considerations.

The coordinated differentiation of hematopoietic stem and progenitor cells (HSPCs) into the various mature blood cell types is responsible for sustaining blood and immune system homeostasis. The cell fate decisions underlying this important biological process are made at the level of single cells. Methods to trace the fate of single cells are therefore essential for understanding hematopoietic system activity in health and disease and have had a major impact on how we understand and represent hematopoiesis. Here, we discuss the basic methodologies and technical considerations for three important clonal assays: single-cell transplantation, lentiviral barcoding, and Sleeping Beauty barcoding. This perspective is a synthesis of presentations and discussions from the 2019 International Society for Experimental Hematology (ISEH) Annual Meeting New Investigator Technology Session and the 2019 ISEH Winter Webinar.

Manipulating niche composition limits damage to haematopoietic stem cells during Plasmodium infection.

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.

Biological implications of clonal hematopoiesis.

Adult hematological malignancies, such as acute myeloid leukemia, are thought to arise through the gradual acquisition of oncogenic mutations within long-lived hematopoietic stem cells (HSCs). Genomic analysis of peripheral blood DNA has recently identified leukemia-associated genetic mutations within otherwise healthy individuals, an observation that is strongly associated with age. These genetic mutations are often found at high frequency, suggesting dominance of a mutant HSC clone. Expansion of clones carrying other mutations not associated with leukemia or larger chromosomal deletions was also observed. This clinical observation has been termed clonal hematopoiesis, a condition associated with increased risk of both hematological malignancy and cardiovascular disease. Here, we discuss the identification of clonal hematopoiesis and its implications on human health, based on the May 2019 International Society for Experimental Hematology New Investigator Committee Webinar.

In Vivo Labeling by CD73 Marks Multipotent Stromal Cells and Highlights Endothelial Heterogeneity in the Bone Marrow Niche.

Despite much work studying ex vivo multipotent stromal cells (MSCs), the identity and characteristics of MSCs in vivo are not well defined. Here, we generated a CD73-EGFP reporter mouse to address these questions and found EGFP+ MSCs in various organs. In vivo, EGFP+ mesenchymal cells were observed in fetal and adult bones at proliferative ossification sites, while in solid organs EGFP+ cells exhibited a perivascular distribution pattern. EGFP+ cells from the bone compartment could be clonally expanded ex vivo from single cells and displayed trilineage differentiation potential. Moreover, in the central bone marrow CD73-EGFP+ specifically labeled sinusoidal endothelial cells, thought to be a critical component of the hematopoietic stem cell niche. Purification and molecular characterization of this CD73-EGFP+ population revealed an endothelial subtype that also displays a mesenchymal signature, highlighting endothelial cell heterogeneity in the marrow. Thus, the CD73-EGFP mouse is a powerful tool for studying MSCs and sinusoidal endothelium.

The evolving view of the hematopoietic stem cell niche.

Hematopoietic stem cells (HSCs) reside in specialized microenvironments known as niches. The niche is essential to support HSC function and to maintain a correct balance between self-renewal and differentiation. Recent advances in defining different mesenchymal and endothelial bone marrow cell populations, as well as hematopoietic stem and progenitor cells, greatly enhanced our understanding of these niches and of the molecular mechanisms by which they regulate HSC function. In addition to the role in maintaining HSC homeostasis, the niche has also been implicated in the pathogenesis of blood disorders including hematological malignancies. Characterizing the extrinsic regulators and the cellular context in which the niches interact with HSCs will be crucial to define new strategies to enhance blood regeneration. Furthermore, a better understanding of the role of the niche in leukemia development will open new possibilities for the treatment of these disorders by using therapies aiming to target the leukemic niche specifically. To update on recent findings on this topic, the International Society for Experimental Hematology (ISEH) organized a webinar, presented by Prof. Sean J. Morrison and Dr. Simón Méndez-Ferrer and moderated by Dr. Cristina Lo Celso, entitled "The evolving view of the hematopoietic stem cell niche," which we summarize here.