Spatial mapping of human hematopoiesis at single-cell resolution reveals aging-associated topographic remodeling.

ABSTRACT: The spatial anatomy of hematopoiesis in the bone marrow (BM) has been extensively studied in mice and other preclinical models, but technical challenges have precluded a commensurate exploration in humans. Institutional pathology archives contain thousands of paraffinized BM core biopsy tissue specimens, providing a rich resource for studying the intact human BM topography in a variety of physiologic states. Thus, we developed an end-to-end pipeline involving multiparameter whole tissue staining, in situ imaging at single-cell resolution, and artificial intelligence-based digital whole slide image analysis and then applied it to a cohort of disease-free samples to survey alterations in the hematopoietic topography associated with aging. Our data indicate heterogeneity in marrow adipose tissue (MAT) content within each age group and an inverse correlation between MAT content and proportions of early myeloid and erythroid precursors, irrespective of age. We identify consistent endosteal and perivascular positioning of hematopoietic stem and progenitor cells (HSPCs) with medullary localization of more differentiated elements and, importantly, uncover new evidence of aging-associated changes in cellular and vascular morphologies, microarchitectural alterations suggestive of foci with increased lymphocytes, and diminution of a potentially active megakaryocytic niche. Overall, our findings suggest that there is topographic remodeling of human hematopoiesis associated with aging. More generally, we demonstrate the potential to deeply unravel the spatial biology of normal and pathologic human BM states using intact archival tissue specimens.

A targetable pathway to eliminate TRA-1-60+/TRA-1-81+ chemoresistant cancer cells.

Chemoresistance is a primary cause of treatment failure in pancreatic cancer. Identifying cell surface markers specifically expressed in chemoresistant cancer cells (CCCs) could facilitate targeted therapies to overcome chemoresistance. We performed an antibody-based screen and found that TRA-1-60 and TRA-1-81, two 'stemness' cell surface markers, are highly enriched in CCCs. Furthermore, TRA-1-60+/TRA-1-81+ cells are chemoresistant compared to TRA-1-60-/TRA-1-81- cells. Transcriptome profiling identified UGT1A10, shown to be both necessary and sufficient to maintain TRA-1-60/TRA-1-81 expression and chemoresistance. From a high-content chemical screen, we identified Cymarin, which downregulates UGT1A10, eliminates TRA-1-60/TRA-1-81 expression, and increases chemosensitivity both in vitro and in vivo. Finally, TRA-1-60/TRA-1-81 expression is highly specific in primary cancer tissue and positively correlated with chemoresistance and short survival, which highlights their potentiality for targeted therapy. Therefore, we discovered a novel CCC surface marker regulated by a pathway that promotes chemoresistance, as well as a leading drug candidate to target this pathway.

Transcriptional Activation of Regenerative Hematopoiesis via Vascular Niche Sensing.

Transition between activation and quiescence programs in hematopoietic stem and progenitor cells (HSC/HSPCs) is perceived to be governed intrinsically and by microenvironmental co-adaptation. However, HSC programs dictating both transition and adaptability, remain poorly defined. Single cell multiome analysis divulging differential transcriptional activity between distinct HSPC states, indicated for the exclusive absence of Fli-1 motif from quiescent HSCs. We reveal that Fli-1 activity is essential for HSCs during regenerative hematopoiesis. Fli-1 directs activation programs while manipulating cellular sensory and output machineries, enabling HSPCs co-adoptability with a stimulated vascular niche. During regenerative conditions, Fli-1 presets and enables propagation of niche-derived Notch1 signaling. Constitutively induced Notch1 signaling is sufficient to recuperate functional HSC impairments in the absence of Fli-1. Applying FLI-1 modified-mRNA transduction into lethargic adult human mobilized HSPCs, enables their vigorous niche-mediated expansion along with superior engraftment capacities. Thus, decryption of stem cell activation programs offers valuable insights for immune regenerative medicine.

Specification of fetal liver endothelial progenitors to functional zonated adult sinusoids requires c-Maf induction.

The liver vascular network is patterned by sinusoidal and hepatocyte co-zonation. How intra-liver vessels acquire their hierarchical specialized functions is unknown. We study heterogeneity of hepatic vascular cells during mouse development through functional and single-cell RNA-sequencing. The acquisition of sinusoidal endothelial cell identity is initiated during early development and completed postnatally, originating from a pool of undifferentiated vascular progenitors at E12. The peri-natal induction of the transcription factor c-Maf is a critical switch for the sinusoidal identity determination. Endothelium-restricted deletion of c-Maf disrupts liver sinusoidal development, aberrantly expands postnatal liver hematopoiesis, promotes excessive postnatal sinusoidal proliferation, and aggravates liver pro-fibrotic sensitivity to chemical insult. Enforced c-Maf overexpression in generic human endothelial cells switches on a liver sinusoidal transcriptional program that maintains hepatocyte function. c-Maf represents an inducible intra-organotypic and niche-responsive molecular determinant of hepatic sinusoidal cell identity and lays the foundation for the strategies for vasculature-driven liver repair.

Engineering a niche supporting hematopoietic stem cell development using integrated single-cell transcriptomics.

Hematopoietic stem cells (HSCs) develop from hemogenic endothelium within embryonic arterial vessels such as the aorta of the aorta-gonad-mesonephros region (AGM). To identify the signals responsible for HSC formation, here we use single cell RNA-sequencing to simultaneously analyze the transcriptional profiles of AGM-derived cells transitioning from hemogenic endothelium to HSCs, and AGM-derived endothelial cells which provide signals sufficient to support HSC maturation and self-renewal. Pseudotemporal ordering reveals dynamics of gene expression during the hemogenic endothelium to HSC transition, identifying surface receptors specifically expressed on developing HSCs. Transcriptional profiling of niche endothelial cells identifies corresponding ligands, including those signaling to Notch receptors, VLA-4 integrin, and CXCR4, which, when integrated in an engineered platform, are sufficient to support the generation of engrafting HSCs. These studies provide a transcriptional map of the signaling interactions necessary for the development of HSCs and advance the goal of engineering HSCs for therapeutic applications.

Histone variant H3.3 maintains adult haematopoietic stem cell homeostasis by enforcing chromatin adaptability.

Histone variants and the associated post-translational modifications that govern the stemness of haematopoietic stem cells (HSCs) and differentiation thereof into progenitors (HSPCs) have not been well defined. H3.3 is a replication-independent H3 histone variant in mammalian systems that is enriched at both H3K4me3- and H3K27me3-marked bivalent genes as well as H3K9me3-marked endogenous retroviral repeats. Here we show that H3.3, but not its chaperone Hira, prevents premature HSC exhaustion and differentiation into granulocyte-macrophage progenitors. H3.3-null HSPCs display reduced expression of stemness and lineage-specific genes with a predominant gain of H3K27me3 marks at their promoter regions. Concomitantly, loss of H3.3 leads to a reduction of H3K9me3 marks at endogenous retroviral repeats, opening up binding sites for the interferon regulatory factor family of transcription factors, allowing the survival of rare, persisting H3.3-null HSCs. We propose a model whereby H3.3 maintains adult HSC stemness by safeguarding the delicate interplay between H3K27me3 and H3K9me3 marks, enforcing chromatin adaptability.

Developmental angiocrine diversification of endothelial cells for organotypic regeneration.

Adult organs are vascularized by specialized blood vessels. In addition to inter-organ vascular heterogeneity, each organ is arborized by structurally and functionally diversified populations of endothelial cells (ECs). The molecular pathways that are induced to orchestrate inter- and intra- organ vascular heterogeneity and zonation are shaped during development and fully specified postnatally. Notably, intra-organ specialization of ECs is associated with induction of angiocrine factors that guide cross-talk between ECs and parenchymal cells, establishing co-zonated vascular regions within each organ. In this review, we describe how microenvironmental tissue-specific biophysical, biochemical, immune, and inflammatory cues dictate the specialization of ECs with zonated functions. We delineate how physiological and biophysical stressors in the developing liver, lung, and kidney vasculature induce specialization of capillary beds. Deciphering mechanisms by which vascular microvasculature diversity is attained could set the stage for treating regenerative disorders and promote healing of organs without provoking fibrosis.

Bioengineering the Bone Marrow Vascular Niche.

The bone marrow (BM) tissue is the main physiological site for adult hematopoiesis. In recent years, the cellular and matrix components composing the BM have been defined with unprecedent resolution, both at the molecular and structural levels. With the expansion of this knowledge, the possibility of reproducing a BM-like structure, to ectopically support and study hematopoiesis, becomes a reality. A number of experimental systems have been implemented and have displayed the feasibility of bioengineering BM tissues, supported by cells of mesenchymal origin. Despite being known as an abundant component of the BM, the vasculature has been largely disregarded for its role in regulating tissue formation, organization and determination. Recent reports have highlighted the crucial role for vascular endothelial cells in shaping tissue development and supporting steady state, emergency and malignant hematopoiesis, both pre- and postnatally. Herein, we review the field of BM-tissue bioengineering with a particular focus on vascular system implementation and integration, starting from describing a variety of applicable in vitro models, ending up with in vivo preclinical models. Additionally, we highlight the challenges of the field and discuss the clinical perspectives in terms of adoptive transfer of vascularized BM-niche grafts in patients to support recovering hematopoiesis.

Lactate released by inflammatory bone marrow neutrophils induces their mobilization via endothelial GPR81 signaling.

Neutrophils provide first line of host defense against bacterial infections utilizing glycolysis for their effector functions. How glycolysis and its major byproduct lactate are triggered in bone marrow (BM) neutrophils and their contribution to neutrophil mobilization in acute inflammation is not clear. Here we report that bacterial lipopolysaccharides (LPS) or Salmonella Typhimurium triggers lactate release by increasing glycolysis, NADPH-oxidase-mediated reactive oxygen species and HIF-1α levels in BM neutrophils. Increased release of BM lactate preferentially promotes neutrophil mobilization by reducing endothelial VE-Cadherin expression, increasing BM vascular permeability via endothelial lactate-receptor GPR81 signaling. GPR81-/- mice mobilize reduced levels of neutrophils in response to LPS, unless rescued by VE-Cadherin disrupting antibodies. Lactate administration also induces release of the BM neutrophil mobilizers G-CSF, CXCL1 and CXCL2, indicating that this metabolite drives neutrophil mobilization via multiple pathways. Our study reveals a metabolic crosstalk between lactate-producing neutrophils and BM endothelium, which controls neutrophil mobilization under bacterial infection.

Daily Onset of Light and Darkness Differentially Controls Hematopoietic Stem Cell Differentiation and Maintenance.

Hematopoietic stem and progenitor cells (HSPCs) tightly couple maintenance of the bone marrow (BM) reservoir, including undifferentiated long-term repopulating hematopoietic stem cells (LT-HSCs), with intensive daily production of mature leukocytes and blood replenishment. We found two daily peaks of BM HSPC activity that are initiated by onset of light and darkness providing this coupling. Both peaks follow transient elevation of BM norepinephrine and TNF secretion, which temporarily increase HSPC reactive oxygen species (ROS) levels. Light-induced norepinephrine and TNF secretion augments HSPC differentiation and increases vascular permeability to replenish the blood. In contrast, darkness-induced TNF increases melatonin secretion to drive renewal of HSPCs and LT-HSC potential through modulating surface CD150 and c-Kit expression, increasing COX-2/αSMA+ macrophages, diminishing vascular permeability, and reducing HSPC ROS levels. These findings reveal that light- and darkness-induced daily bursts of norepinephrine, TNF, and melatonin within the BM are essential for synchronized mature blood cell production and HSPC pool repopulation.

Open the gates: vascular neurocrine signaling mobilizes hematopoietic stem and progenitor cells.

Mobilization of hematopoietic stem and progenitor cells (HSPCs) from the bone marrow (BM) into the peripheral blood is a complex process that is enhanced dramatically under stress-induced conditions. A better understanding of how the mobilization process is regulated will likely facilitate the development of improved clinical protocols for stem cell harvesting and transplantation. In this issue of the JCI, Singh et al. (1) showed that the truncated cleaved form of neurotransmitter neuropeptide Y (NPY) actively promotes a breach of BM vascular sinusoidal portals, thereby augmenting HSPC trafficking to the circulation. The authors report a previously unrecognized axis, in which expression of the enzyme dipeptidylpeptidase-4 (DPP4)/CD26 by endothelial cells activates NPY-mediated signaling by increasing the bioavailability of the truncated form of NPY. These findings underscore the importance of and urgency to develop pharmacological therapies that target the vasculature and regulate diverse aspects of hematopoiesis, such as HSPC trafficking, in steady-state and stress-induced conditions.

Leukemic Cells "Gas Up" Leaky Bone Marrow Blood Vessels.

In this issue of Cancer Cell, Passaro et al. demonstrate how leukemia through aberrant induction of reactive oxygen species and nitric oxide production trigger marrow vessel leakiness, instigating pro-leukemic function. Disrupted tumor blood vessels promote exhaustion of non-malignant stem and progenitor cells and may facilitate leukemia relapse following chemotherapeutic treatment.

Regulation of Hematopoiesis and Osteogenesis by Blood Vessel-Derived Signals.

In addition to their conventional role as a versatile transport system, blood vessels provide signals controlling organ development, regeneration, and stem cell behavior. In the skeletal system, certain capillaries support perivascular osteoprogenitor cells and thereby control bone formation. Blood vessels are also a critical component of niche microenvironments for hematopoietic stem cells. Here we discuss key pathways and factors controlling endothelial cell behavior in bone, the role of vessels in osteogenesis, and the nature of vascular stem cell niches in bone marrow.

Age-dependent modulation of vascular niches for haematopoietic stem cells.

Blood vessels define local microenvironments in the skeletal system, play crucial roles in osteogenesis and provide niches for haematopoietic stem cells. The properties of niche-forming vessels and their changes in the ageing organism remain incompletely understood. Here we show that Notch signalling in endothelial cells leads to the expansion of haematopoietic stem cell niches in bone, which involves increases in CD31-positive capillaries and platelet-derived growth factor receptor-ß (PDGFRß)-positive perivascular cells, arteriole formation and elevated levels of cellular stem cell factor. Although endothelial hypoxia-inducible factor signalling promotes some of these changes, it fails to enhance vascular niche function because of a lack of arterialization and expansion of PDGFRß-positive cells. In ageing mice, niche-forming vessels in the skeletal system are strongly reduced but can be restored by activation of endothelial Notch signalling. These findings indicate that vascular niches for haematopoietic stem cells are part of complex, age-dependent microenvironments involving multiple cell populations and vessel subtypes.

Distinct bone marrow blood vessels differentially regulate haematopoiesis.

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.

PAR1 signaling regulates the retention and recruitment of EPCR-expressing bone marrow hematopoietic stem cells.

Retention of long-term repopulating hematopoietic stem cells (LT-HSCs) in the bone marrow is essential for hematopoiesis and for protection from myelotoxic injury. We report that signaling cascades that are traditionally viewed as coagulation related also control retention of endothelial protein C receptor-positive (EPCR(+)) LT-HSCs in the bone marrow and their recruitment to the blood via two pathways mediated by protease activated receptor 1 (PAR1). Thrombin-PAR1 signaling induces nitric oxide (NO) production, leading to EPCR shedding mediated by tumor necrosis factor-α-converting enzyme (TACE), enhanced CXCL12-CXCR4-induced motility and rapid stem and progenitor cell mobilization. Conversely, bone marrow blood vessels provide a microenvironment enriched with activated protein C (aPC) that retains EPCR(+) LT-HSCs by limiting NO generation, reducing Cdc42 activity and enhancing integrin VLA4 affinity and adhesion. Inhibition of NO production by aPC-EPCR-PAR1 signaling reduces progenitor cell egress from the bone marrow, increases retention of bone marrow NO(low) EPCR(+) LT-HSCs and protects mice from chemotherapy-induced hematological failure and death. Our study reveals new roles for PAR1 and EPCR in controlling NO production to balance maintenance and recruitment of bone marrow EPCR(+) LT-HSCs, with potential clinical relevance for stem cell transplantation.

Synaptojanin 2 is a druggable mediator of metastasis and the gene is overexpressed and amplified in breast cancer.

Amplified HER2, which encodes a member of the epidermal growth factor receptor (EGFR) family, is a target of effective therapies against breast cancer. In search for similarly targetable genomic aberrations, we identified copy number gains in SYNJ2, which encodes the 5'-inositol lipid phosphatase synaptojanin 2, as well as overexpression in a small fraction of human breast tumors. Copy gain and overexpression correlated with shorter patient survival and a low abundance of the tumor suppressor microRNA miR-31. SYNJ2 promoted cell migration and invasion in culture and lung metastasis of breast tumor xenografts in mice. Knocking down SYNJ2 impaired the endocytic recycling of EGFR and the formation of cellular lamellipodia and invadopodia. Screening compound libraries identified SYNJ2-specific inhibitors that prevented cell migration but did not affect the related neural protein SYNJ1, suggesting that SYNJ2 is a potentially druggable target to block cancer cell migration.