Transcriptomic characterisation of haematopoietic stem and progenitor cells from human adult bone marrow, spleen and peripheral blood
ABSTRACT: Haematopoietic stem and progenitor cells (HSPCs), the precursors of all blood cells, reside predominantly in the bone marrow (BM). Recent evidence suggests that other anatomical sites may contribute significantly to blood production, but the cellular, molecular and functional composition of extramedullary HSPC pools remains unexplored. Here, we comprehensively characterized the single-cell composition of the adult human HSPC pool within paired BM, spleen and peripheral blood (PB) from two healthy organ donors. 10x scRNA-seq of 30000 HSPCs from all three sites defined transcriptionally distinct cell clusters that correspond to precursors of all haematopoietic lineages but also stem cells (HSCs). Although most clusters were common to all organs, their proportions significantly differed between tissues. Altogether our data provides evidence that different organs sustain distinct topologies of the hematopoietic hierarchy, originating from HSC subsets with distinct lineage preferences.
This dataset is part of the Human Cell Atlas.
Project description:Constitutive egress of bone marrow (BM)-resident hematopoietic stem and progenitor cells (HSPCs) into the blood is a well-established phenomenon, but the ultimate fate and functional relevance of circulating HSPCs is largely unknown. We show that mouse thoracic duct (TD) lymph contains HSPCs that possess short- and long-term multilineage reconstitution capacity. TD-derived HSPCs originate in the BM, enter the blood, and traffic to multiple peripheral organs, where they reside for at least 36 hr before entering draining lymphatics to return to the blood and, eventually, the BM. HSPC egress from extramedullary tissues into lymph depends on sphingosine-1-phosphate receptors. Migratory HSPCs proliferate within extramedullary tissues and give rise to tissue-resident myeloid cells, preferentially dendritic cells. HSPC differentiation is amplified upon exposure to Toll-like receptor agonists. Thus, HSPCs can survey peripheral organs and can foster the local production of tissue-resident innate immune cells under both steady-state conditions and in response to inflammatory signals.
Project description:Upon tissue injury, peripheral sensory neurons release nociceptive factors (e.g. substance P [SP]), which exert local and systemic actions including the recruitment of bone marrow (BM)-derived haematopoietic stem and progenitor cells (HSPCs) endowed with paracrine pro-angiogenic properties. We herein explore whether diabetic neuropathy interferes with these phenomena.We first investigated the presence of sensory neuropathy in the BM of patients with type 2 diabetes by immunohistochemistry and morphometry analyses of nerve size and density and assessment of SP release by ELISA. We next analysed the association of sensory neuropathy with altered HSPC release under ischaemia or following direct stimulation with granulocyte colony-stimulating factor (G-CSF). BM and circulating HSPCs expressing the neurokinin 1 receptor (NK1R), which is the main SP receptor, were measured by flow cytometry. We finally assessed whether an altered modulation of SP secretion interferes with the mobilisation and homing of NK1R-HSPCs in a mouse model of type 2 diabetes after limb ischaemia (LI).Nociceptive fibres were reduced in the BM of patients and mice with type 2 diabetes. Patients with neuropathy showed a remarkable reduction in NK1R-HSPC mobilisation under ischaemia or upon G-CSF stimulation. Following LI, diabetic mice manifested an altered SP gradient between BM, peripheral blood and limb muscles, accompanied by a depressed recruitment of NK1R-HSPCs to the ischaemic site.Sensory neuropathy translates into defective liberation and homing of reparative HSPCs. Nociceptors may represent a new target for treatment of diabetic complications.
Project description:Haematopoietic stem and progenitor cells (HSPCs), the precursors of all blood cells, reside predominantly in the bone marrow. Yet, a small proportion (<1%) of phenotypic HSPCs circulates through peripheral blood (PB) at any given time. To date, the detailed characterization of steady-state circulating HSPCs in adult humans remains very poor. Here, we analyse the single-cell composition of the adult human HSPC pool within non-mobilised PB from four healthy donors. 10x scRNA-seq of 51,000 HSPCs from all six donors was paired with single-cell functional analysis using most immature haematopoietic stem cells and multipotent progenitors (HSC/MPPs). We find that long-term functional HSC/MPPs are very rare in non-mobilised PB, and that a large fraction of circulating HSPCs is biased towards the erythroid lineage. In particular, we detect the enrichment of a subset of exclusively erythroid/megakaryocyte-primed quiescent HSC-like cells within the phenotypic PB HSC/MPP compartment.
Project description:The emergence of haematopoietic stem and progenitor cells (HSPCs) from hemogenic endothelium results in the formation of sizeable HSPC clusters attached to the vascular wall. We evaluate the cell cycle and proliferation of HSPCs involved in cluster formation, as well as the molecular signatures from their initial appearance to the point when cluster cells are capable of adult engraftment (definitive HSCs). We uncover a non-clonal origin of HSPC clusters with differing cell cycle, migration, and cell signaling attributes. In addition, we find that the complement cascade is highly enriched in mature HSPC clusters, possibly delineating a new role for this pathway in engraftment.
Project description:Several studies have suggested that caffeic acid phenethyl ester (CAPE) can induce the expression of hypoxia inducible factor-1? (HIF-1?) protein. We determined whether CAPE has a novel function in improving the homing and engraftment of haematopoietic stem/progenitor cells (HSPCs) by regulating HIF-1? gene expression in the bone marrow (BM) niche.For survival experiments, lethally irradiated C57BL/6 mice were injected with a low number of BM mononuclear cells (MNCs) and CAPE according to the indicated schedule. Homing efficiency analysis was conducted using flow cytometry and colony-forming unit (CFU) assays. The influence of intraperitoneal injection of CAPE on short-term and long-term engraftment of HSPCs was evaluated using competitive and non-competitive mouse transplantation models. To investigate the mechanism by which CAPE enhanced HSPC homing, we performed these experiments including Q-PCR, western blot, immunohistochemistry and CFU assays after in-vivo HIF-1? activity blockade.CAPE injection significantly increased the survival rate of recipient mice after lethal irradiation and transplantation of a low number of BM MNCs. Using HSPC homing assays, we found that CAPE notably increased donor HSPC homing to recipient BM. The subsequent short-term and long-term engraftment of transplanted HSPCs was also improved by the optimal schedule of CAPE administration. Mechanistically, we found that CAPE upregulated the expression of HIF-1?, vascular endothelial growth factor-A (VEGF-A) and stromal cell-derived factor 1? (SDF-1?). The HIF-1? inhibitor PX-478 blocked CAPE-enhanced HSPC homing, which supported the idea that HIF-1? is a key target of CAPE.Our results showed that CAPE administration facilitated HSPC homing and engraftment, and this effect was primarily dependent on HIF-1? activation and upregulation of SDF-1? and VEGF-A expression in the BM niche.
Project description:Haematopoietic stem and progenitor cells (HSPCs) give rise to all blood lineages that support the entire lifespan of vertebrates1. After HSPCs emerge from endothelial cells within the developing dorsal aorta, homing allows the nascent cells to anchor in their niches for further expansion and differentiation2-5. Unique niche microenvironments, composed of various blood vessels as units of microcirculation and other niche components such as stromal cells, regulate this process6-9. However, the detailed architecture of the microenvironment and the mechanism for the regulation of HSPC homing remain unclear. Here, using advanced live imaging and a cell-labelling system, we perform high-resolution analyses of the HSPC homing in caudal haematopoietic tissue of zebrafish (equivalent to the fetal liver in mammals), and reveal the role of the vascular architecture in the regulation of HSPC retention. We identify a VCAM-1+ macrophage-like niche cell population that patrols the inner surface of the venous plexus, interacts with HSPCs in an ITGA4-dependent manner, and directs HSPC retention. These cells, named 'usher cells', together with caudal venous capillaries and plexus, define retention hotspots within the homing microenvironment. Thus, the study provides insights into the mechanism of HSPC homing and reveals the essential role of a VCAM-1+ macrophage population with patrolling behaviour in HSPC retention.
Project description:Pluripotent stem cells (PSCs) may provide a potential source of haematopoietic stem/progenitor cells (HSPCs) for transplantation; however, unknown molecular barriers prevent the self-renewal of PSC-HSPCs. Using two-step differentiation, human embryonic stem cells (hESCs) differentiated in vitro into multipotent haematopoietic cells that had the CD34(+)CD38(-/lo)CD90(+)CD45(+)GPI-80(+) fetal liver (FL) HSPC immunophenotype, but exhibited poor expansion potential and engraftment ability. Transcriptome analysis of immunophenotypic hESC-HSPCs revealed that, despite their molecular resemblance to FL-HSPCs, medial HOXA genes remained suppressed. Knockdown of HOXA7 disrupted FL-HSPC function and caused transcriptome dysregulation that resembled hESC-derived progenitors. Overexpression of medial HOXA genes prolonged FL-HSPC maintenance but was insufficient to confer self-renewal to hESC-HSPCs. Stimulation of retinoic acid signalling during endothelial-to-haematopoietic transition induced the HOXA cluster and other HSC/definitive haemogenic endothelium genes, and prolonged HSPC maintenance in culture. Thus, medial HOXA gene expression induced by retinoic acid signalling marks the establishment of the definitive HSPC fate and controls HSPC identity and function.
Project description:Haematopoietic stem/progenitor cells (HSPCs) can mobilise into blood and produce immune cell lineages following stress. However, the homeostasis and function of HSPCs after infection in teleosts are less well known. Here, we report that Listonella anguillarum infection enhances HSPC mobilisation and reduces their differentiation into myeloid cells in ayu (Plecoglossus altivelis), an aquacultured teleost in East Asia. We established a colony-forming unit culture (CFU-C) assay to measure HSPCs using conditioned medium from peripheral blood mononuclear cells stimulated with phytohaemagglutinin. The number of CFU-Cs decreased in the head kidney and increased in the blood and spleen of ayu infected with L. anguillarum. HSPC mobilisation after L. anguillarum infection was mediated by norepinephrine. Furthermore, HSPCs from ayu treated with L. anguillarum lipopolysaccharide (LPS) showed defective myeloid differentiation and could no longer rescue L. anguillarum-infected ayu. HSPC expansion was suppressed after L. anguillarum infection or its LPS treatment in vitro. These results reveal a link between HSPC regulation and pathogen infection in teleosts.
Project description:Regulation of hematopoietic stem and progenitor cell (HSPC) steady-state egress from the bone marrow (BM) to the circulation is poorly understood. While glycogen synthase kinase-3? (GSK3?) is known to participate in HSPC proliferation, we revealed an unexpected role in the preferential regulation of CXCL12-induced migration and steady-state egress of murine HSPCs, including long-term repopulating HSCs, over mature leukocytes. HSPC egress, regulated by circadian rhythms of CXCL12 and CXCR4 levels, correlated with dynamic expression of GSK3? in the BM. Nevertheless, GSK3? signaling was CXCL12/CXCR4 independent, suggesting that synchronization of both pathways is required for HSPC motility. Chemotaxis of HSPCs expressing higher levels of GSK3? compared with mature cells was selectively enhanced by stem cell factor-induced activation of GSK3?. Moreover, HSPC motility was regulated by norepinephrine and insulin-like growth factor-1 (IGF-1), which increased or reduced, respectively, GSK3? expression in BM HSPCs and their subsequent egress. Mechanistically, GSK3? signaling promoted preferential HSPC migration by regulating actin rearrangement and microtubuli turnover, including CXCL12-induced actin polarization and polymerization. Our study identifies a previously unknown role for GSK3? in physiological HSPC motility, dictating an active, rather than a passive, nature for homeostatic egress from the BM reservoir to the blood circulation.
Project description:Hematopoietic stem and progenitor cells (HSPCs) undergo self-renewal and differentiation to guarantee a constant supply of short-lived blood cells. Both intrinsic and extrinsic factors determine HSPC fate, but the underlying mechanisms remain elusive. Here, we report that Proteinase 3 (PR3), a serine protease mainly confined to granulocytes, is also expressed in HSPCs. PR3 deficiency intrinsically suppressed cleavage and activation of caspase-3, leading to expansion of the bone marrow (BM) HSPC population due to decreased apoptosis. PR3-deficient HSPCs outcompete the long-term reconstitution potential of wild-type counterparts. Collectively, our results establish PR3 as a physiological regulator of HSPC numbers. PR3 inhibition is a potential therapeutic target to accelerate and increase the efficiency of BM reconstitution during transplantation.