Hematopoietic Stem And Progenitor Cells Emergence Research Articles

BF170 hydrochloride enhances the emergence of hematopoietic stem and progenitor cells

ABSTRACT Generation of hematopoietic stem and progenitor cells (HSPCs) ex vivo and in vivo, especially the generation of safe therapeutic HSPCs, still remains inefficient. In this study, we have identified compound BF170 hydrochloride as a previously unreported pro-hematopoiesis molecule, using the differentiation assays of primary zebrafish blastomere cell culture and mouse embryoid bodies (EBs), and we demonstrate that BF170 hydrochloride promoted definitive hematopoiesis in vivo. During zebrafish definitive hematopoiesis, BF170 hydrochloride increases blood flow, expands hemogenic endothelium (HE) cells and promotes HSPC emergence. Mechanistically, the primary cilia-Ca2+-Notch/NO signaling pathway, which is downstream of the blood flow, mediated the effects of BF170 hydrochloride on HSPC induction in vivo. Our findings, for the first time, reveal that BF170 hydrochloride is a compound that enhances HSPC induction and may be applied to the ex vivo expansion of HSPCs.

Nlrc3 Signaling Is Indispensable for Hematopoietic Stem Cell Emergence Via Notch Signaling in Vertebrates

Hematopoietic stem and progenitor cells (HSPCs) generate all the lineages of blood cells throughout the lifespan of vertebrates. The emergence of HSPCs occurs through the hemogenic endothelial (HE) to hematopoietic transition (EHT) process, which is finely tuned by a variety of signaling pathways. Previous studies have emerged the essential roles of pattern-recognition receptors such as Toll-like receptors and RIG-I-like receptors in EHT. However, whether the nucleotide-binding domain leucine-rich repeat (NLR) containing family members participate in vertebrate embryonic hematopoiesis remains unclear. To explore the potential pathways involved in hematopoietic ontogeny and differentiation in this stage, we screened the dynamic expression of NLR family genes and other genes related to inflammation in the published scRNA-seq data in vertebrates' embryos including zebrafish, mouse and human during HSPC development. And to verify the expression of NLR family genes in vitro by using HSC-like cells differentiation system of mouse and human embryonic stem cells (ESCs), these results suggest that NLRC3 was highly expressed during embryonic HSPC development in vivo and vitro and might play an important role in HSPC emergence in vertebrates. In order to validate this hypothesis, we synthesized probes from the full-length mRNA of nlrc3 and utilized them to observe the in-situ expression of nlrc3 in zebrafish at different developmental stages. The WISH experiment demonstrated that nlrc3 was expressed from the 1-cell stage and importantly, showed specific expression in the AGM region at the 24-28hpf and CHT region at 72hpf, which coincides with the onset and generation of HSPC generation. Then morpholino were utilized to knockdown nlrc3 in transgenic embryos Tg( runx1:EGFP/ kdrl:mCherry), Tg( cmyb:EGFP/ kdrl:mCherry) and Tg( CD41:GFP), and mutants generated by CRISPR-Cas9 were used to perform whole-mount in situ hybridization (WISH) and qPCR to detect specific markers such as runx1, cmyb(HSPCs), rag1(lymphocytes), gata1a(erythrocytes) and l-plastin(myelocytes), etc. The results showed that nlrc3 was required for HSPCs emergence, expansion and differentiation. In primitive hematopoietic wave, nlrc3 signaling may not impact primitive erythropoiesis but required for primitive neutrophils and macrophages. Mechanistically, we sorted EGFP + cells in Tg ( fli1a:EGFP) zebrafish embryos at 28 hpf to perform RNA-Seq, which was the stage where EHT and these EGFP + cells contained hemogenic endothelial cells. After bioinformatics analysis of sequencing data, we found that the effect of nlrc3 in zebrafish hematopoiesis is not based on affecting the development of arterial vessels or causing apoptosis. By using targeted inhibitor such as DAPT and JSH23, conditional rescue model like Tg ( hsp70:GAL4/ UAS:NICD), overexpression expression experiment in Tg ( tp1:EGFP/ kdrl:mCherry) and nlrc3 -/- mutants, etc, through Imaging systems and qPCR we found that NF-kB- nlrc3-Notch- hey1 axis triggers HSPCs emergence and differentiation in zebrafish. To further extend our findings in mammals, we utilized Nlrc3 knockout mice to explore whether Nlrc3 signaling was evolutionarily conserved in HSPCs emergence. Hematogenic endothelial cells (CD31 + CD41 - CD45 - TER119 -) from the embryonic AGM tissues at E10.5 were sorted and cultured in OP9 for 4 days and flow cytometry was performed to assess that c-Kit +CD45 + hematopoietic cells and CD31 +Sca-1 +CD201 + hematopoietic stem cells were notably decreased in these Nlrc3-/- embryos. Colony-forming unit-cell (CFU-C) assay and BM transplantation experiments were used to observe that fetal liver HSCs from E14.5 Nlrc3-/- embryos was decrease in the number and impaired in function in vitro and in vivo. Finally qPCR using mRNA from FL-derived LSK ( Lin-c-Kit+Sca-1+) cells further demonstrated that the NF-kB- Nlrc3-Notch- Hey1 axis orchestrated development of embryonic HSCs was highly conserved in vertebrates. Overall, by summarizing the results of the physiological and developmental roles in vivo and in vitro, we uncover an indispensable role of Nlrc3 signaling in vertebrates' HSPCs emergence, our findings provide new insights into inflammation-related hematopoietic ontogeny and Nlrc3 signal is of major importance since this investigation might be taken into consideration when recapitulation definitive hematopoiesis in vitro.

Capturing Endothelial-to-Hematopoietic Transition Using a Highly Efficient in Vitro Platform

Introduction De novo generation of hematopoietic stem and progenitor cells (HSPCs) for therapeutic use has been a long-standing goal of regenerative medicine. During development, the first definitive hematopoietic stem cells (HSCs) derive from a small number of specialized vascular endothelial cells (EC), named hemogenic endothelial cells (HECs), located in the Aorta-Gonado-Mesonephrose (AGM). The process of HEC conversion to HSPCs is known as endothelial-to-hematopoietic transition (EHT). During EHT, endothelial features are lost while switching on hematopoietic transcriptional programs leading to drastic functional and morphological changes that generate free-floating, round hematopoietic cells from large, flat, anchorage-dependent ECs. Key steps regulating EHT are difficult to elucidate using in vivo models because the hemogenic capacity of HECs occurs within a 2-day developmental window. Only a tiny number of cells undergo EHT in vivo. New model systems that overcome current in vivo obstacles are required so the details of EHT and the mechanisms that lead to the generation of HSPCs can be better understood. We previously reported that fully-committed ECs can be converted to functional HSPCs by ectopic expression of transcription factors Spi1, FOSB, GFI1, and Runx1 (SFGR) and an appropriate vascular niche (Sandler et al. Nature, 2014; Lis et al. Nature, 2017). But conversion using this method required several weeks and was associated with variable and low efficiency. Here, we show that our new in vitro EHT platform (ivEHT), very closely recapitulates the process of EHT in vivo and can be used to uncover heretofore unrecognized steps necessary for EC conversion to HSPCs. Methods All steps in the in vitro conversion of ECs to HSPCs were systematically analyzed and optimized. The refined model utilized new media constituents and polycistronic constructs to ensure ectopic expression of transcription factors Spi1, FOSB, GFI1 and Runx1 (SFGR)in human umbilical vein-ECs (HUVECs) in appropriate ratios and sequence. Results The new ivEHT platform yields efficient EC conversion to hematopoietic like CD45+ cells (≥80%) and high temporal reproducibility of conversion that is largely complete within 96 hrs. The ivEHT sequence of conversion closely follows the timing and sequence of EHT in vivo. Initially, transduced HUVECs are large, flat cells with CDH5-marked intercellular junctions and firm adhesion to basement membrane. Within the first 24hrs of induction, SFGR-HUVECs downregulate ECs markers CD31, KDR, CDH5 and activate the endogenous RUNX1 locus detected by the expression of nuclear GFP (GFPn) under control of the human RUNX1+24 enhancer (detected using a lentiviral reporter vector). Day 2 (D2) is characterized by complete clearance of endothelial-specific Weibel-Palade Bodies (WPBs, marked by Von Willebrand Factor, VWF) and excess mitochondria. This window also initiates morphologic changes in RUNX1-GFPn marked cells with loss of intercellular junctions and attachment to basement membrane and initial expression of hematopoietic markers such as CD45. Days 3-4 are marked by loss of cell volume and cytoplasm, cellular rounding and detachment from matrix, and increased expression of CD45 and other hematopoietic markers like cKIT, MPL, CD43. These stages of conversion are only identified in RUNX1-GFPn marked cells and by the end of D5, ~80% of cells have fully converted to CD45+ cells. Conversion is not accompanied by cell proliferation, but after D5 cell numbers expand rapidly. The ivEHT platform is scalable and highly reproducible making it amenable to higher-throughput testing in 96-well format using flow cytometry readouts. Using the ivEHT platform, we have captured and validated in AGM explants previously unobserved aspects of early EHT that is complete between D2-D3. Discussion This work promises a reproducible, faithful, and scalable platform which generates hematopoietic like cells while successfully recapitulating early steps of the EHT in the AGM. Importantly, this platform is readily accessible and is easily customized for discovery of EHT molecular details currently hidden to science, allowing significant advances in the understanding of how HSPCs emergence in vivo.

Alcam-a and Pdgfr-α are essential for the development of sclerotome-derived stromal cells that support hematopoiesis

Mesenchymal stromal cells are essential components of hematopoietic stem and progenitor cell (HSPC) niches, regulating HSPC proliferation and fates. Their developmental origins are largely unknown. In zebrafish, we previously found that the stromal cells of the caudal hematopoietic tissue (CHT), a niche functionally homologous to the mammalian fetal liver, arise from the ventral part of caudal somites. We have now found that this ventral domain is the sclerotome, and that two markers of mammalian mesenchymal stem/stromal cells, Alcam and Pdgfr-α, are distinctively expressed there and instrumental for the emergence and migration of stromal cell progenitors, which in turn conditions the proper assembly of the vascular component of the CHT niche. Furthermore, we find that trunk somites are similarly dependent on Alcam and Pdgfr-α to produce mesenchymal cells that foster HSPC emergence from the aorta. Thus the sclerotome contributes essential stromal cells for each of the key steps of developmental hematopoiesis.

Vascular endothelial growth factor c regulates hematopoietic stem cell fate in the dorsal aorta.

Hematopoietic stem and progenitor cells (HSPCs) are multipotent cells that self-renew or differentiate to establish the entire blood hierarchy. HSPCs arise from the hemogenic endothelium of the dorsal aorta (DA) during development in a process called endothelial-to-hematopoietic transition. The factors and signals that control HSPC fate decisions from the hemogenic endothelium are not fully understood. We found that Vegfc has a role in HSPC emergence from the zebrafish DA. Using time-lapse live imaging, we show that some HSPCs in the DA of vegfc loss-of-function embryos display altered cellular behavior. Instead of typical budding from the DA, emergent HSPCs exhibit crawling behavior similar to myeloid cells. This was confirmed by increased myeloid cell marker expression in the ventral wall of the DA and the caudal hematopoietic tissue. This increase in myeloid cells corresponded with a decrease in HSPCs that persisted into larval stages. Together, our data suggest that Vegfc regulates HSPC emergence in the hemogenic endothelium, in part by suppressing a myeloid cell fate. Our study provides a potential signal for modulation of HSPC fate in stem cell differentiation protocols.

Ing4 Suppresses Quiescence and Inflammation in Hematopoietic Stem Cells

Hematopoietic stem and progenitor cell (HSPC) development and maintenance is regulated through a complex regulatory network. In a screen of epigenetic regulators of hematopoiesis in zebrafish, we identified a requirement for the tumor suppressor protein, Inhibitor of growth 4 (Ing4) in HSPC specification. Ing4 acts to regulate transcription through interactions with transcription factors, including HIF, NF-kB, and p53. It is often mis-expressed in many human cancers and has been shown to promote stem cell-like characteristics in malignant cells, in part, due to the inhibitory role of Ing4 in the NF-kB signaling pathway. The transcription factor NF-kB is a regulator of inflammatory response and serves an important role in embryonic HSPC emergence, survival, differentiation and proliferation. The Ing4 protein binds to the p65/RelA subunit of NF-kB, inhibiting DNA binding and suppressing NF-kB cytokines and inflammatory pathways. In the absence of Ing4 there is an overexpression of NF-kB target genes that have inhibitory effects on hematopoietic programming. Given the regulatory role of Ing4 in both hematopoiesis and cancer, it is likely critical to the regulation of stem cell self-renewal, maintenance and specialization. To better define the role of Ing4 on hematopoiesis we use two Ing4 loss-of-function models: zebrafish and mouse. For the zebrafish model of Ing4 deficiency, Ing4-deficient zebrafish embryos lose >90% of runx1+/c-myb+ cells in the aorta, gonad, mesonephros (AGM) region of the developing zebrafish embryo, demonstrating a lack of HSPC specification. 36 hours post fertilization (hpf) Ing4 morphants display increased expression of NF-kB target genes when Ing4 is absent. Genetic epistasis experiments performed to block translation of RelA, IL-1b, and additional NF-kB target gene mRNA revealed recovered HSC marker expression in the aorta. To discover small molecule inhibitors that would mimic these effects, we conducted an in vivo chemical screen of NF-kB pathway inhibitors assessing their ability to rescue HSC specification in Ing4 morphant zebrafish. Ing4 morphants treated with NF-kB inhibitors had reduced NF-kB cytokine expression, as well as a dose-dependent rescue of HSC marker expression in the aorta. These results suggest that NF-kB inhibition could remediate the effects of Ing4 loss on hematopoiesis. To more thoroughly profile the effects of Ing4 loss on HSC specification and the bone marrow niche, an Ing4-/-mouse model was used. These mice are developmentally normal but are hypersensitive to stimulation with LPS due to increased inflammatory signaling. Peripheral blood analysis reveals an increase in Mac-1 cells in the Ing4-/- mouse. Ing4-/- bone marrow progenitors are skewed toward granulocyte-myocyte progenitor cells (GMPs) lending to the shift in cell populations present in the peripheral blood. Ing4 loss further disrupts the mouse hematopoietic program resulting in a dramatic increase in the number of short term-HSCs (ST-HSC) (WT: 11.4%, Null: 31.7%), a modest increase in long term-HSCs (LT-HSC) (WT: 2.4%, Null: 5.52%), and a dramatic decrease in multipotent progenitors (MPPs) (WT: 47.9%, Null: 19.3%). We also found significant alterations in stress hematopoiesis following competitive HSC transplant where sorted Ing4-/- LT-HSCs failed to engraft. Following myeloablative insult, we found no significant change in Ing4-/- LT-HSC (-1.18%) when compared with ST-HSC (-14.43%) indicating reduced sensitivity to 5-FU ablation in the Ing4-/- LT-HSC group. Cell cycle analysis identified 92.9% of Ing4-/- LT-HSCs are in G0 compared to 76.2% of wildtype LT-HSCs. ST-HSCs were also more quiescent with 27% of Ing4-/- ST-HSCs in G0 compared to 11.1% of wildtype ST-HSCs. Previously published work reports hyper proliferative HSCs that exhibit loss of quiescence as a result of proinflammatory NF-kB signaling. We believe that the interaction between Ing4 and the HIF-1a pathway may play a role in the observed phenotype of Ing4-/- LT-HSCs resulting in increased quiescence and disruption of the balance between self-renewal and differentiation critical to reconstitution of the hematopoietic compartment. Overall, our findings suggest that the regulatory effects of Ing4 play a crucial role in hematopoiesis and provides key tools for further identification and characterization of Ing4 pathways and functions. Disclosures Zon: CAMP4 Therapeutics: Current equity holder in private company, Other: Founder; Fate Therapeutics: Current equity holder in publicly-traded company, Other: Founder; Scholar Rock: Current equity holder in publicly-traded company, Other: Founder; Amagma Therapeutics: Current equity holder in private company, Other: Founder; Cellarity: Consultancy; Celularity: Consultancy.

Repetitive Elements Trigger RIG-I-like Receptor Signaling that Regulates the Emergence of Hematopoietic Stem and Progenitor Cells

Inflammatory signaling is required for hematopoietic stem and progenitor cell (HSPC) development. Here, we studied the involvement of RIG-I-like receptors (RLRs) in HSPC formation. Rig-I or Mda5 deficiency impaired, while Lgp2 deficiency enhanced, HSPC emergence in zebrafish embryos. Rig-I or Mda5 deficiency reduced HSPC numbers by inhibiting inflammatory signals that were in turn enhanced in Lgp2 deficient embryos. Simultaneous reduction of Lgp2 and either Rig-I or Mda5 rescued inflammatory signals and HSPC numbers. Modulating the expression of the signaling mediator Traf6 in RLR deficient embryos restored HSPC numbers. Repetitive element transcripts could be detected in hemogenic endothelial cells and HSPCs, suggesting a role as RLR ligands. Indeed, ectopic expression of repetitive elements enhanced HSPC formation in wild-type, but not in Rig-I or Mda5 deficient embryos. Manipulation of RLR expression in mouse fetal liver HSPCs indicated functional conservation among species. Thus, repetitive elements transcribed during development drive RLR-mediated inflammatory signals that regulate HSPC formation.

3131 – THE ROLE OF VASCULAR ENDOTHELIAL GROWTH FACTOR - C IN DEVELOPMENTAL HEMATOPOIESIS

Hematopoietic stem and progenitor cells (HSPCs) are multipotent cells that self-renew or differentiate to establish and replenish the entire blood hierarchy. HSPCs arise from the hemogenic endothelium of the dorsal aorta (DA) in the embryo. We use zebrafish to study endothelial-to-hematopoietic transition (EHT) because the embryos are transparent and develop externally, allowing direct observation of in vivo cellular events. My long-term goal is to identify factors that impact HSPC differentiation during EHT. One candidate, VEGFC, is expressed in the DA and specifically in long-term HSPCs of both mouse and zebrafish. VEGFC has been shown to play a role in fetal liver erythropoiesis, but its developmental role in definitive hematopoiesis has not been explored. I hypothesize that VEGFC maintains HSPC identity by negatively regulating alternative hemogenic endothelium fates. Preliminarily, we have shown in zebrafish that morpholino knock-down of vegfc results in decreased HSPC emergence and altered cell behavior during EHT. During the peak of HSPC emergence from the DA, vegfc knock-down embryos show decreased HSPC emergence, this lack of HSPCs persists into lymphoid development in the thymus. HSPCs in vegfc knock-down embryos also display altered cell behavior, they crawl similar to myeloid cells, instead of the typical and well described budding from the DA. vegfc knock-down embryos and genetic mutants also show an increase in myeloid cells in the caudal hematopoietic tissue (CHT), the zebrafish equivalent of the mammalian fetal liver. We plan to further investigate the role of VEGFC in a mammalian system using human pluripotent stem cell differentiation protocols and aorta-gonad mesonephros explants. We hope to use the results of this research to improve our ability to create HSPCs in vitro to be used in clinical stem cell transplants.

Cooperative Transcription Factor Induction Mediates Hemogenic Reprogramming

SUMMARYDuring development, hematopoietic stem and progenitor cells (HSPCs) arise from specialized endothelial cells by a process termed endothelial-to-hematopoietic transition (EHT). The genetic program driving human HSPC emergence remains largely unknown. We previously reported that the generation of hemogenic precursor cells from mouse fibroblasts recapitulates developmental hematopoiesis. Here, we demonstrate that human fibroblasts can be reprogrammed into hemogenic cells by the same transcription factors. Induced cells display dynamic EHT transcriptional programs, generate hematopoietic progeny, possess HSPC cell surface phenotype, and repopulate immunodeficient mice for 3 months. Mechanistically, GATA2 and GFI1B interact and co-occupy a cohort of targets. This cooperative binding is reflected by engagement of open enhancers and promoters, initiating silencing of fibroblast genes and activating the hemogenic program. However, GATA2 displays dominant and independent targeting activity during the early phases of reprogramming. These findings shed light on the processes controlling human HSC specification and support generation of reprogrammed HSCs for clinical applications.

A Zebrafish Model of Fetal Bone Marrow Provides a Dynamic View of Hematopoietic Stem Cell Niche Colonization

The entire blood system is supported throughout life by a small number of hematopoietic stem and progenitor cells (HSPCs) that are produced exclusively during embryonic development. These stem cells are generated from the hemogenic endothelium of the dorsal aorta, then migrate to the fetal liver where they expand, before making a final migration to the fetal bone marrow. After seeding the bone marrow, the stem cell pool stabilizes and the total number of HSPCs remains relatively constant. Very little is known about this early stage of bone marrow niche colonization. A better understanding of native stem cell pool establishment will likely lead to improved clinical HSPC transplantation that depends on repopulation of the bone marrow niche. Currently, imaging technology does not allow direct visualization of the bone marrow niche during colonization because it occurs in utero in the long bones of the fetus. The zebrafish is an advantageous model because the embryos develop externally and are transparent. To quantify the early stem cell pool, we employed long term fate mapping with clonal analysis using the multicolor Zebrabow system, which imparts a unique fluorescent hue to stem cells and their progeny. Our findings reveal that 21 HSPC clones exist prior to HSPC emergence (24 hours post fertilization) and 30 clones are present during peak production from the aorta (48 hours post fertilization). Seeding of the presumptive adult marrow niche in zebrafish begins 4-5 days post fertilization, versus 16.5 days in the mouse. We previously described a transgenic zebrafish line (Runx:mCherry) that marks long term repopulating HSPCs throughout development and into adulthood. HSPC-specific expression is driven by the well-characterized Runx1 +23 kb mouse enhancer element. We used this line to directly observe the earliest immigration events of HSPCs as they arrive in the marrow. To achieve this, we immobilized the zebrafish by injection of the snake venom protein alpha-bungarotoxin directly into circulation. This allowed long term live imaging of the niche (~16 hours) so we could quantify the dynamics of HSPC colonization and expansion. To rapidly acquire high resolution imaging data for this deep tissue we applied lightsheet microscopy. By simultaneously illuminating the sample in the X plane, while taking images in the Z plane, hundreds of optical sections can be captured in seconds. The high pixel and temporal resolution of lightsheet microscopy in a large volume of tissue provides a highly dynamic view of the entire marrow niche. We could assess the localization of HSPCs in relation to other cell types within the niche. For example, HSPCs were closely associated with endothelial cells in a perivascular niche, similar to what has been described in mammalian bone marrow. Furthermore, we could quantify single Runx+ nuclei over time on one side of the bilateral kidney marrow. During this early stage of niche colonization, we found the number of HSPCs per side was ~50 (so ~100 total) and that remained relatively constant. This was in fact a dynamic equilibrium achieved by ingress and egress of cells, as well as occasional cell divisions. This cell number was independently validated using another transgenic zebrafish line, cd41:GFP, that also marks HSPCs. This quantification, combined with our data from earlier development, suggests that HSPCs undergo around two population doublings between emergence from the aorta and engraftment in the marrow. This unique platform for the quantification of a total stem cell pool will allow further functional and mechanistic studies using both genetics and chemical biology. Our goal is to gain insights into the establishment of the stem cell pool within the niche microenvironment and how this could improve clinical transplantation outcomes. DisclosuresZon:Marauder Therapeutics: Equity Ownership, Other: Founder; Scholar Rock: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Fate, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.

A Transcriptional Switch Point During Hematopoietic Stem and Progenitor Cell Ontogeny.

During mammalian embryogenesis, hematopoietic stem and progenitor cells (HSPCs) originate from mesoderm-derived endothelial cells in the aorta-gonad-mesonephros (AGM) region and placenta (PL). Later, HSPCs expand in fetal liver (FL) and migrate to bone marrow (BM) shortly before birth. Understanding global transcriptional regulation governing HSPC emergence from embryonic stem/induced pluripotent stem cells is necessary to devise clinical applications, such as novel transplantation approaches. In this study, to assess transcriptional dynamics during development, we performed cap analysis of gene expression on 10 developmental murine HSPC populations isolated from the AGM region, PL, FL, and BM and identified 15,681 transcripts across HSPC ontogeny. We performed microarray analysis of AGM-derived HSPCs at 9.5 and 10.5 days postcoitum (dpc) and identified 40 differentially expressed genes, 23 confirmed as significantly changed by real-time polymerase chain reaction. We conclude that a transcriptional switch point occurs in HSPC ontogeny between 9.5 and 10.5 dpc in the AGM region.

Hypoxic Stress-Induced Serotonergic Stimulation Controls Hematopoietic Stem Cell Production Via the Hypothalamic-Pituitary-Interrenal Axis and Glucocorticoid Signaling

There is increasing evidence that neuronal signals contribute to adult hematopoietic stem cell (HSC) production and maintenance; however, the mechanisms by which the central nervous system regulates HSCs and/or the HSC niche and its functional necessity during vertebrate HSC development remains unclear. Serotonin (5HT), a neurotransmitter in the brain and a hormone peripherally, was identified in a screen for HSC modulators in zebrafish. Exposure to 5HT or fluoxetine (5HT reuptake inhibitor) increased hematopoietic stem and progenitor cell (HSPC) formation in the zebrafish aorta-gonad-mesonephros (AGM) region as shown by runx1/cmyb in situ hybridization, and confirmed by FACS and qRT-PCR analysis (p<0.05). The stimulatory effect of 5HT was seen both during HSPC niche formation, as well as when AGM HSPCs emerge and expand. Peripheral and neuronal 5HT are synthesized by tryptophan hydroxylase 1 (TPH1) and TPH2, respectively. Chemical inhibition of these rate-limiting enzymes decreased HSC numbers during HSPC emergence (36 hpf). Using morpholino-mediated knockdown, only the effects of selective tph2 reduction on HSCs, and not that of tph1, were found to persist by 48 hpf (p<0.05), which could be rescued by exogenous 5HT; this finding was confirmed using an inducible tph2 -ablation model. Although the sympathetic nervous system (SNS) can contribute to HSC emergence, the effect of 5HT was independent of SNS function as shown using 6-hydroxydopamine (6-OHDA) to induce sympathetic nerve lesions. 5HT can activate the hypothalamic-pituitary-adrenal (HPA) axis, leading to cortisol production and activation of glucocorticoid receptor (GR) to mediate peripheral responses. Indeed, mRNA levels of corticotropin-releasing hormone receptor, pro-opiomelanocortin (pomc) and nr3c1 (GR), as well as cortisol levels were increased in 5HT-treated embryos (p<0.05). In contrast, cortisol production was decreased in tph2 morphants (p<0.05). Consistent with a role for neuronal 5HT in regulating HSCs through the Hypothalamic-Pituitary-Interrenal (HPI) axis (teleost HPA axis counterpart) and peripheral cortisol production, exposure to GR agonists hydrocortisone and dexamethasone increased embryonic HSPC production, while MO-knockdown of GR or analysis of GR mutants revealed drastic reductions in HSPCs and rag2+ lymphoid progenitors. Further, epistasis analysis revealed the impact of 5HT or GR agonist on HSPCs was abrogated upon MO knockdown of GR, whereas GR agonist treatment rescued HSPC numbers in corticotropin-releasing hormone and pomc morphants (p<0.05). The HPA/I axis is well recognized as a "stress response" pathway; we, and others, previously showed that local hypoxia and hypoxic stress-induced Hypoxia Inducible Factor 1a (HIF1a) activity play a pivotal role during embryonic HSPC development. We therefore examined whether hypoxic stimulation can initiate serotonergic and HPI Axis-mediated regulation of HSPC production. Consistent with previous reports, HIF1a activation by cobalt (II) chloride (CoCl2) increased runx1 + HSPCs in the AGM; CoCl2 exposure also elevated tph2 (but not tph1) and HPI axis gene expression and total cortisol production (p<0.05). Furthermore, the effect of CoCl2 stimulation on AGM HSPCs is blocked in tph2 morphants (p<0.05), suggesting that HIF1a activates TPH2-mediated 5HT production to impact HSPC number. Collectively, our data highlight a novel regulatory mechanism controlling embryonic HSPC induction and expansion that exploits the hypoxic stress response to induce serotonergic stimulation of the HPI axis and glucocorticoid signaling. DisclosuresNo relevant conflicts of interest to declare.

G protein-coupled receptor 183 facilitates endothelial-to-hematopoietic transition via Notch1 inhibition.

In vertebrates, embryonic hematopoietic stem and progenitor cells (HSPCs) are derived from a subset of endothelial cells, the hemogenic endothelium (HE), through the endothelial-to-hematopoietic transition (EHT). Notch signaling is essential for HSPC development during embryogenesis across vertebrates. However, whether and how it regulates EHT remains unclear. Here, we show that G protein-coupled receptor 183 (Gpr183) signaling serves as an indispensable switch for HSPC emergence by repressing Notch signaling before the onset of EHT. Inhibition of Gpr183 significantly upregulates Notch signaling and abolishes HSPC emergence. Upon activation by its ligand 7α-25-OHC, Gpr183 recruits β-arrestin1 and the E3 ligase Nedd4 to degrade Notch1 in specified HE cells and then facilitates the subsequent EHT. Importantly, 7α-25-OHC stimulation promotes HSPC emergence in vivo and in vitro, providing an attractive strategy for enhancing the in vitro generation of functional HSPCs.

Myocardial Infarction Activates CCR2+ Hematopoietic Stem and Progenitor Cells

Following myocardial infarction (MI), myeloid cells derived from the hematopoietic system drive a sharp increase in systemic leukocyte levels that correlates closely with mortality. The origin of these myeloid cells, and the response of hematopoietic stem and progenitor cells (HSPCs) to MI, however, is unclear. Here, we identify a CCR2(+)CD150(+)CD48(-) LSK hematopoietic subset as the most upstream contributor to emergency myelopoiesis after ischemic organ injury. This subset has 4-fold higher proliferation ratesthan CCR2(-)CD150(+)CD48(-) LSK cells, displays a myeloid differentiation bias, and dominates the migratory HSPC population. We further demonstrate that the myeloid translocation gene 16 (Mtg16) regulates CCR2(+) HSPC emergence. Mtg16(-/-) mice have decreased levels of systemic monocytes and infarct-associated macrophages and display compromised tissue healing and post-MI heart failure. Together, these data provide insights into regulation of emergency hematopoiesis after ischemic injury and identify potential therapeutic targets to modulate leukocyte output after MI.

Adenosine signaling promotes hematopoietic stem and progenitor cell emergence

Hematopoietic stem cells (HSCs) emerge from aortic endothelium via the endothelial-to-hematopoietic transition (EHT). The molecular mechanisms that initiate and regulate EHT remain poorly understood. Here, we show that adenosine signaling regulates hematopoietic stem and progenitor cell (HSPC) development in zebrafish embryos. The adenosine receptor A2b is expressed in the vascular endothelium before HSPC emergence. Elevated adenosine levels increased runx1(+)/cmyb(+) HSPCs in the dorsal aorta, whereas blocking the adenosine pathway decreased HSPCs. Knockdown of A2b adenosine receptor disrupted scl(+) hemogenic vascular endothelium and the subsequent EHT process. A2b adenosine receptor activation induced CXCL8 via cAMP-protein kinase A (PKA) and mediated hematopoiesis. We further show that adenosine increased multipotent progenitors in a mouse embryonic stem cell colony-forming assay and in embryonic day 10.5 aorta-gonad-mesonephros explants. Our results demonstrate that adenosine signaling plays an evolutionary conserved role in the first steps of HSPC formation in vertebrates.

Inflammatory signaling regulates hematopoietic stem and progenitor cell emergence in vertebrates

AbstractInflammatory signaling has been shown to be essential for stress hematopoiesis in adult bone marrow, either through increasing proliferation or by directing differentiation of hematopoietic stem and progenitor cells (HSPCs) toward myeloid or lymphoid lineages. However, its role in embryonic normal hematopoiesis has been unknown. Here, we demonstrate that in both zebrafish and mouse embryos, inflammatory signaling is necessary and sufficient for HSPC emergence, in the absence of infection or pathological inflammation. Mechanistically, inflammatory signaling regulates hemogenic endothelium-derived HSPC development through a conserved Toll-like receptor 4 (TLR4)–nuclear factor κ-light-chain enhancer of activated B core (NF-κB) signaling, which then promotes Notch activity, a well-known signal required for HSPC specification in vertebrates. Our findings establish a previously unrecognized link between inflammatory signaling and HSPC emergence, and provide new insights into regenerative medicine and novel therapies to treat innate immune-related diseases.

Interactions between the chromatin remodeller CHD1 and the spliceosome are critical for hematopoietic stem and progenitor cell emergence

Defects in hematopoietic stem cells (HSCs) lead to hematological diseases, such as myelodysplastic syndromes (MDS) and leukemia. Recurrent mutations in spliceosomal components were recently identified in these diseases, suggesting an underappreciated role for splicing regulation in HSC biology. Based on this hypothesis, we explored the importance of splicing on hematopoietic stem and progenitor cell (HSPC) specification during zebrafish development. Zebrafish mutants in U2 snRNP components, specifically sf3a3hi1950 and sf3b1hi3394, have diminished runx1 expression within their aorta by 28 hours post fertilization (hpf), while expression of the endothelial marker flk1/kdrl is mostly unaffected, indicating a defect at the level of HSPC formation. Using these mutants, we defined chd1 as a genetic interaction partner with the spliceosome that is critical for HSPC emergence. CHD1 (Chromodomain Helicase DNA Binding Protein 1) is a chromatin remodeler that interacts with SF3A and SF3B spliceosomal complexes in human cells and is needed for their recruitment to actively transcribed genes. Transient morpholino-mediated knockdown of chd1 in zebrafish embryos has no effect on HSPC development. Similarly, embryos heterozygous for sf3a3hi1950 have no noticeable hematopoietic phenotype, however, in combination, diminished chd1 levels in sf3a3hi1950 heterozygotes leads to a significant decline in HSPC levels. These results demonstrate a clear in vivo connection between splicing and epigenetic regulation within HSPCs. As these two processes are the most frequently mutated in MDS, our data might also have implications for understanding the origins of MDS at the HSC level. Defects in hematopoietic stem cells (HSCs) lead to hematological diseases, such as myelodysplastic syndromes (MDS) and leukemia. Recurrent mutations in spliceosomal components were recently identified in these diseases, suggesting an underappreciated role for splicing regulation in HSC biology. Based on this hypothesis, we explored the importance of splicing on hematopoietic stem and progenitor cell (HSPC) specification during zebrafish development. Zebrafish mutants in U2 snRNP components, specifically sf3a3hi1950 and sf3b1hi3394, have diminished runx1 expression within their aorta by 28 hours post fertilization (hpf), while expression of the endothelial marker flk1/kdrl is mostly unaffected, indicating a defect at the level of HSPC formation. Using these mutants, we defined chd1 as a genetic interaction partner with the spliceosome that is critical for HSPC emergence. CHD1 (Chromodomain Helicase DNA Binding Protein 1) is a chromatin remodeler that interacts with SF3A and SF3B spliceosomal complexes in human cells and is needed for their recruitment to actively transcribed genes. Transient morpholino-mediated knockdown of chd1 in zebrafish embryos has no effect on HSPC development. Similarly, embryos heterozygous for sf3a3hi1950 have no noticeable hematopoietic phenotype, however, in combination, diminished chd1 levels in sf3a3hi1950 heterozygotes leads to a significant decline in HSPC levels. These results demonstrate a clear in vivo connection between splicing and epigenetic regulation within HSPCs. As these two processes are the most frequently mutated in MDS, our data might also have implications for understanding the origins of MDS at the HSC level.