Hematopoietic Stem And Progenitor Cell Formation Research Articles

Kdm6a Modulates Hematopoiesis and Leukemogenesis Via Demethylase-Dependent Epigenetic Programming

Hematopoietic stem and progenitor cells (HSPCs) maintain their differentiation by precisely regulating gene expression patterns at the transcriptional and epigenetic levels. KDM6A encodes an epigenetic regulator of the repressive chromatin mark histone 3 lysine 27 tri-methylation (H3K27me3). Growing evidence suggested the involvement of KDM6A in hematological malignancies. KDM6A mutations in leukemia frequently occur in males, reflecting that KDM6A escapes X chromosome inactivation. Therefore, female patients with single-allele loss-of-function KDM6A mutations will partially retain KDM6A expression, which is called haploinsufficiency. Furthermore, KDM6A haploinsufficiency was reported to be associated with refractory and/or relapsed leukemia, while the role of KDM6A haploinsufficiency in hematopoiesis remains unexplored. We established kdm6a-null zebrafish using CRISPR-Cas9 gene editing. We found that kdm6a loss impaired HSPCs formation. kdm6a-null embryos showed reduced expression of the hemogenic endothelial and HSPC markers runx1 at 28 hours post-fertilization (hpf). Using Tg(kdrl:mCherry;cmyb:en-GFP) embryos, we also found that the number of hemogenic endothelium and emerging HSPCs in kdm6a-null was significantly decreased. To study the importance of Kdm6a-mediated demethylase activity, we ectopically expressed kdm6a constructs (wild-type kdm6a, or catalytically-dead kdm6a) in kdm6a-null embryos. The result showed that, unlike restoring effect by wild-type kdm6a, the ectopic expression of catalytically-dead kdm6a constructs ( kdm6a H1134A) showed no rescue effect for decreased HSPC numbers. Then, we aimed to understand the potential role of kdm6a haploinsufficiency in hematopoiesis and leukemogenic mechanisms. We found that zebrafish mutants that are haploinsufficient in kdm6a ( kdm6a +/-) exhibited uncontrolled myeloid differentiation. The expression of myeloid-specific markers substantially increased in kdm6a +/- mutants, compared with WT siblings at 72 hpf. Confocal analysis showed that the ratio of myeloid-primed HSPCs was markedly increased in kdm6a +/- embryos compared to WT siblings. Consistently, a significant upregulation in transcripts encoding key myeloerythorid TFs including cebps, irf8 and gata1a was observed in kdm6a +/- embryonic HSPCs. Furthermore, kdm6a +/- adults exhibited the phenotype of monocytosis, which mimics chronic myelomonocytic leukemia, or exhibited the phenotype of increased progenitors, which mimics acute myeloid leukemia. We next sorted HSPCs from WT or kdm6a +/- at 72 hpf for RNA-sequencing and ATAC-sequencing. Enrichment analysis showed transcriptional dysregulation of genes involving “Purine metabolism”, “Cysteine and methionine metabolism”, “TGF-beta signaling pathway”, and “Cell cycle”. GSEA further uncovered positive enrichment of gene sets in kdm6a +/- embryonic HSPCs, including “Oxidative phosphorylation”, “mTORC1 signaling” and “DNA repair”. These dysregulated pathways in kdm6a +/- HSPCs are highly involved in cellular homeostasis and hallmarks of aged hematopoietic system. ATAC analysis also revealed robust changes in chromatin accessibility, especially including genes related to aged hematopoietic system. Integrated RNA-seq and ATAC-seq analyses showed that the chromatin accessibility at promoters and transcription of 63 genes were decreased in kdm6a +/- HSPCs. And we found that the negative regulator of Jak-Stat signaling- socs3a was on the list. Ectopic expression of socs3a in kdm6a +/- could partially rescue the uncontrolled myeloid differentiation of HSPCs. Consistently, abnormal myeloid differentiation from kdm6a +/- mutations was rescued by Jak or Stat3 inhibitors. We also tested the importance of Kdm6a-mediated demethylase activity. Unlike restoring effect by wild-type kdm6a, the ectopic expression of catalytically-dead kdm6a constructs ( kdm6a H1134A) showed no rescue effect for dysregulated expression of socs3a and key myeloerythorid genes including cebps. In conclusion, our data demonstrate the multiple roles of kdm6a in regulating HSPC homeostasis and provide new insight into the mechanism of kdm6a haploinsufficiency in uncontrolled myeloid differentiation through demethylase-dependent epigenetic programming.

The microbiota regulates hematopoietic stem and progenitor cell development by mediating inflammatory signals in the niche.

The commensal microbiota regulates the self-renewal and differentiation of hematopoietic stem and progenitor cells (HSPCs) in bone marrow. Whether and how the microbiota influences HSPC development during embryogenesis is unclear. Using gnotobiotic zebrafish, we show that the microbiota is necessary for HSPC development and differentiation. Individual bacterial strains differentially affect HSPC formation, independent of their effects on myeloid cells. Early-life dysbiosis in chd8-/- zebrafish impairs HSPC development. Wild-type microbiota promote HSPC development by controlling basal inflammatory cytokine expression in kidney niche, and chd8-/- commensals elicit elevated inflammatory cytokines that reduce HSPCs and enhance myeloid differentiation. We identify an Aeromonas veronii strain with immuno-modulatory activities that fails to induce HSPC development in wild-type fish but selectively inhibits kidney cytokine expression and rebalances HSPC development in chd8-/- zebrafish. Our studies highlight the important roles of a balanced microbiome during early HSPC development that ensure proper establishment of lineal precursor for adult hematopoietic system.

Splicing factor deficits render hematopoietic stem and progenitor cells sensitive to STAT3 inhibition

Hematopoietic stem and progenitor cells (HSPCs) sustain lifelong hematopoiesis. Mutations of pre-mRNA splicing machinery, especially splicing factor 3b, subunit 1 (SF3B1), are early lesions found in malignancies arising from HSPC dysfunction. However, why splicing factor deficits contribute to HSPC defects remains incompletely understood. Using zebrafish, we show that HSPC formation in sf3b1 homozygous mutants is dependent on STAT3 activation. Clinically, mutations in SF3B1 are heterozygous; thus, we explored if targeting STAT3 could be a vulnerability in these cells. We show that SF3B1 heterozygosity confers heightened sensitivity to STAT3 inhibition in zebrafish, mouse, and human HSPCs. Cells carrying mutations in other splicing factors or treated with splicing modulators are also more sensitive to STAT3 inhibition. Mechanistically, we illustrate that STAT3 inhibition exacerbates aberrant splicing in SF3B1 mutant cells. Our findings reveal a conserved vulnerability of splicing factor mutant HSPCs that could allow for their selective targeting in hematologic malignancies.

A Link between Blood Flow and the Inflammatory Networks That Govern Hematopoietic Stem Cell Specification

Hematopoietic stem and progenitor cells (HSPCs) are multipotent, self-renewing cells that maintain the blood-forming system throughout the life of all individuals. The unique properties of these cells make HSPC transplantation an effective therapy for treatment of many blood disorders. The advent of induced-pluripotent stem cells (iPSCs) led to great optimism in the field as a potentially limitless source of immunologically-matched, clinical-grade hematopoietic cells. Despite success in producing in vitro hematopoietic progenitors and cells that are capable of limited in vivo reconstitution, there are no robust, reproducible protocols that can generate long-term reconstituting HSPCs. Having a more complete understanding of the signals and molecular pathways required to develop HSPCs in their native environment will move the field forward, leading to more robust protocols and the development of novel HSPC-based therapeutics. Years of investigation have revealed that HSPCs require multiple molecular inputs for proper specification, including activity of the Notch, Wnt, FGF, and BMP signaling pathways. In addition, we have reported inflammatory signaling (Tnfa and NF-kB) as a novel group of HSPC fate modulators. However, the spatial temporal requirement of NF-kB activity has been enigmatic due to: (1) lack of tools that faithfully report the dynamic activation of NF-kB; (2) difficulty on assessing NF-kB dynamics in vivo in a mammalian system during embryonic development due to artifactual inflammatory conditions triggered by invasive techniques required for in utero experimentation. Addressing this knowledge gap will be critical for optimizing in vitro protocols for the generation of patient-specific HSPCs. To date, all existing zebrafish NF-kB reporter lines fail to report dynamic cellular activation of NF-kB in real time due to the long half-life of their fluorescent reporter proteins. To overcome this limitation, we have generated a novel NF-kB reporter zebrafish line driving a destabilized version of eGFP (D2eGFP) containing a PEST domain. This modified eGFP has a short half-life of two hours, enabling us to pinpoint the precise developmental window these signals are required. Time-lapse confocal imaging, in conjunction with flow cytometry, of double transgenic kdrl:mCherry, NF-kB:D2eGFP embryos revealed that NF-kB was activated in vivo within the hemogenic endothelium (HE) just prior to the establishment of circulation (18-24 hpf) and was deactivated soon after its initiation (26 hpf). When this window of NF-kB activity was disrupted by inhibiting the translocation of the NF-kB p65 subunit to the nucleus using Caffeic acid phenethyl ester (CAPE), HSPCs failed to specify. qPCR analysis from FACS-isolated HE with activated NF-kB kdrl:mCherry+ ,NF-kB:D2eGFP+ and the surrounding endothelium kdrl:mCherry+ ,NF-kB:D2eGFP- showed a significant enrichment of the early HSPC markers gfi1 and runx1 in the NF-kB+ endothelial population at 22 hpf. In addition, mRNAseq of kdrl:mCherry+ HE of NF-kB+ showed hematopoietic GO terms enriched, demonstrating that NF-kB is a contributing activator of the endothelial to hematopoietic transition. Blood flow is a well-known conserved regulator of HSPC formation. To investigate the correlation between NF-kB deactivation and circulation, blood flow was inhibited by morpholino knockdown of tnnt2a in Tg(kdrl:mCherry, NF-kB:D2eGFP) embryos and assessed by confocal microscopy and flow cytometry for NF-kB activity. In the absence of circulation, NF-kB displayed sustained activity throughout the HE. To determine if this prolonged NF-kB activity was contributing to the loss of HSPCs observed in tnnt2a morphants, we blocked NF-kB at the developmental time circulation would normally occur using CAPE and observed a significant rescue of HSPC numbers. These data provide evidence of a link between the extrinsic biomechanical forces of blood flow and the intrinsic molecular regulation of the inflammatory networks that govern HSPC specification. Altogether, our NF-kB reporter line provides one of the earliest markers that can be utilized to isolate HE in vivo. These data pinpoint the early NF-kB requirement to form HSPCs and could be applied in vitro to improve current protocols of hematopoietic differentiation from human PSCs.

2009 – BNIP3LB REGULATED MITOPHAGY MAINTAINS THE EMBRYONIC POOL OF HEMATOPOIETIC STEM CELLS BY PROTECTING THEM FROM ROS INDUCED APOPTOSIS

In contrast to the established detrimental effect that elevated Reactive Oxygen Species (ROS) has on adult hematopoietic stem and progenitor cells (HSPCs), our previous work has shown that ROS promotes HSPC formation in the dorsal aorta. In order to elucidate the mechanism and developmental timing of this shift in the role of ROS in HSC biology we sought to examine the initiation and regulation of mitophagy in these cells, this being a key process by which adult HSCs regulate ROS by removing and recycling damaged mitochondria. Our recent data shows that oxidative stress begins to limit HSPC numbers immediately upon their colonization of the secondary hematopoietic niche, the caudal hematopoietic tissue (CHT, the zebrafish analog of the fetal liver), and live imaging with Tg(ubi:mitoGR), a fluorescent transgenic reporter for mitophagy, shows that this coincides with the onset of mitophagy in HSPCs. Mitophagy can be induced via multiple pathways but our scRNAseq and in situ-hybridization analyses suggest that it is induced in these cells via the bnip3lb receptor. We validated the role of bnip3lb via morpholino directed knockdown and found that mitophagy was reduced alongside HSPC marker expression in the CHT, whereas induction of mitophagy by small molecules or hsp70:ΔOTC mutants elevated detection of HSPC markers runx1 and cmyb by WISH, and CD41:GFP by flow cytometry. Mechanistically, we found that bnip3lb knockdown increases ROS levels, and that the reduction in HSPC numbers can be rescued by chemically reducing ROS. Finally, we demonstrate that the enhancement in ROS levels caused by reduced mitophagy increases apoptosis in the CHT region and alters HSPC fate. We therefore propose that bnip3lb directed developmentally programmed mitophagy is responsible for protecting embryonic HSCs from the harmful effects of oxidative stress while the HSC pool expands. In contrast to the established detrimental effect that elevated Reactive Oxygen Species (ROS) has on adult hematopoietic stem and progenitor cells (HSPCs), our previous work has shown that ROS promotes HSPC formation in the dorsal aorta. In order to elucidate the mechanism and developmental timing of this shift in the role of ROS in HSC biology we sought to examine the initiation and regulation of mitophagy in these cells, this being a key process by which adult HSCs regulate ROS by removing and recycling damaged mitochondria. Our recent data shows that oxidative stress begins to limit HSPC numbers immediately upon their colonization of the secondary hematopoietic niche, the caudal hematopoietic tissue (CHT, the zebrafish analog of the fetal liver), and live imaging with Tg(ubi:mitoGR), a fluorescent transgenic reporter for mitophagy, shows that this coincides with the onset of mitophagy in HSPCs. Mitophagy can be induced via multiple pathways but our scRNAseq and in situ-hybridization analyses suggest that it is induced in these cells via the bnip3lb receptor. We validated the role of bnip3lb via morpholino directed knockdown and found that mitophagy was reduced alongside HSPC marker expression in the CHT, whereas induction of mitophagy by small molecules or hsp70:ΔOTC mutants elevated detection of HSPC markers runx1 and cmyb by WISH, and CD41:GFP by flow cytometry. Mechanistically, we found that bnip3lb knockdown increases ROS levels, and that the reduction in HSPC numbers can be rescued by chemically reducing ROS. Finally, we demonstrate that the enhancement in ROS levels caused by reduced mitophagy increases apoptosis in the CHT region and alters HSPC fate. We therefore propose that bnip3lb directed developmentally programmed mitophagy is responsible for protecting embryonic HSCs from the harmful effects of oxidative stress while the HSC pool expands.

Selective Targeting of Splicing Factor Mutant Hematopoietic Stem and Progenitor Cells Via STAT3 Inhibition

Myelodysplastic syndrome (MDS) is a bone marrow failure disorder driven by dysfunction of hematopoietic stem and progenitor cells (HSPCs). Patient sequencing studies over the last decade have revealed that mutations in splicing machinery predominate in MDS, thus selective targeting of these cells is therapeutically attractive. STAT3 inhibition has been explored previously as a means to eradicate HSPCs in MDS. While efficacy was demonstrated in a subset of samples, the underlying mechanism for this selectivity remains unknown. We examined RNAseq of MDS CD34+ HSPCs with splicing factor mutations versus wildtype, finding alternative splicing and differential expression of STAT3 pathway components. Functionally, we explored if STAT3 signaling represents a novel vulnerability in SF3B1 mutant HSPCs using a multi-model approach of in vivo zebrafish and mouse systems, and in vitro assays of CRISPR-engineered human leukemia K562 cells and primary MDS samples. Utilizing the small molecule STAT3 inhibitor STATTIC, we found that human cells carrying MDS-associated SF3B1 point mutations had heightened sensitivity to STAT3 inhibition compared to wildtype controls. To evaluate the activity of STAT3 inhibition in vivo, we utilized an Mx1-cre conditional knock-in mouse model of mutant SF3B1 (Sf3b1+/K700E). We demonstrated that in vivo STATTIC treatment selectively depleted Sf3b1 mutant cells over wildtype in vivo. RNAseq of sf3b1 homozygous mutantzebrafish cells revealed conserved dysregulation of STAT3 pathway splicing and target expression. Diminishing Stat3 (via morpholino knockdown, stable mutants, or STATTIC treatment) decreased HSPCs in sf3b1 heterozygotes but not wildtype embryos, demonstrating synthetic lethality between Sf3b1 and Stat3. Our data indicate that SF3B1 heterozygosity, regardless of the type of mutation, confers a heightened sensitivity to STAT3 inhibition in zebrafish, mouse, and human HSPCs. Critically, our data indicate that SF3B1-mutant cells can be selectively killed in vivo while sparing wildtype cells. We sought to rescue HSPCs in sf3b1 homozygous mutant zebrafish, however overexpression of ligands Osm and Il6 or wildtype Stat3 was insufficient. Instead, overexpression of constitutively-active Stat3 partially restored HSPCs, indicating that functional Stat3 signaling downstream of Sf3b1 is critical for HSPC formation. To investigate the specificity of the synthetic lethality for SF3B1, we assessed the STAT3 synthetic lethal interaction with other mutated splicing factors in MDS. Similar to SF3B1, we demonstrated STAT3 synthetic lethality with U2AF1 and SRSF2 heterozygosity in zebrafish and human cells. RNA-sequencing analysis of STATTIC-treated K562 cells revealed an exacerbation of splicing alterations upon STAT3 inhibition that was more pronounced in SF3B1+/K666N cells compared to wildtype. Even more strikingly, we demonstrated that constitutive activation of STAT3 could partially reverse defective splicing in zebrafish sf3b1 homozygous mutant cells. Mechanistically, these data strongly support coordinated splicing dysfunction as the underlying cause for STAT3-SF3B1 synthetic lethality. Together, we demonstrated a conserved and selective synthetic lethal interaction between STAT3 function and splicing factor defects that represents a novel liability for mutant HSPCs with important implications for MDS treatment. DisclosuresShastri: Guidepoint: Consultancy; Kymera Therapeutics: Research Funding; Onclive: Honoraria; GLC: Consultancy. Verma: Curis: Research Funding; BMS: Research Funding; Stelexis: Consultancy, Current equity holder in publicly-traded company; Eli Lilly: Research Funding; Medpacto: Research Funding; Incyte: Research Funding; GSK: Research Funding; Novartis: Consultancy; Acceleron: Consultancy; Celgene: Consultancy; Stelexis: Current equity holder in publicly-traded company; Throws Exception: Current equity holder in publicly-traded company.

Efficient hemogenic endothelial cell specification by RUNX1 is dependent on baseline chromatin accessibility of RUNX1-regulated TGFβ target genes.

Hematopoietic stem and progenitor cells (HSPCs) are generated de novo in the embryo from hemogenic endothelial cells (HECs) via an endothelial-to-hematopoietic transition (EHT) that requires the transcription factor RUNX1. Ectopic expression of RUNX1 alone can efficiently promote EHT and HSPC formation from embryonic endothelial cells (ECs), but less efficiently from fetal or adult ECs. Efficiency correlated with baseline accessibility of TGFβ-related genes associated with endothelial-to-mesenchymal transition (EndoMT) and participation of AP-1 and SMAD2/3 to initiate further chromatin remodeling along with RUNX1 at these sites. Activation of TGFβ signaling improved the efficiency with which RUNX1 specified fetal ECs as HECs. Thus, the ability of RUNX1 to promote EHT depends on its ability to recruit the TGFβ signaling effectors AP-1 and SMAD2/3, which in turn is determined by the changing chromatin landscape in embryonic versus fetal ECs. This work provides insight into regulation of EndoMT and EHT that will guide reprogramming efforts for clinical applications.

Ddx41 loss R-loops in cGAS to fuel inflammatory HSPC production

In this issue of Developmental Cell, Weinreb etal. reveal that loss of the DEAD-box helicase Ddx41 unexpectedly triggers an R-loop-mediated sterile inflammatory cascade which drives HSPC production during embryonic development. Human studies suggest mechanistic conservation for inflammation in DDX41-associated hematologic disease, uncovering a potential route for future therapeutic intervention.

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.

The chromatin remodeler Brg1 is required for formation and maintenance of hematopoietic stem cells.

Hematopoietic stem and progenitor cells (HSPCs) have the ability to self-renew and differentiate into various blood cells, thus playing an important role in maintenance of lifelong hematopoiesis. Brahma-related gene 1 (BRG1), which acts as the ATP subunit of mammalian SWI-SNF-related chromatin remodeling complexes, is involved in human acute myeloid leukemia and highly expresses in short-term HSPCs. But its role and regulatory mechanism for HSPC development have not yet been well established. Here, we generated a brg1 knockout zebrafish model using TALEN technology. We found that in brg1-/- embryo, the primitive hematopoiesis remained well, while definitive hematopoiesis formation was significantly impaired. The number of hemogenic endothelial cells was decreased, further affecting definitive hematopoiesis with reduced myeloid and lymphoid cells. During embryogenesis, the nitric oxide (NO) microenvironment in brg1-/- embryo was seriously damaged and the reduction of HSPCs could be partially rescued by a NO donor. Chromatin immunoprecipitation (ChIP) assays showed that BRG1 could bind to the promoter of KLF2 and trigger its transcriptional activity of NO synthase. Our findings show that Brg1 promotes klf2a expression in hemogenic endothelium and highlight a novel mechanism for HSPC formation and maintenance.

YAP Regulates Hematopoietic Stem Cell Formation in Response to the Biomechanical Forces of Blood Flow.

Hematopoietic stem and progenitor cells (HSPCs), first specified from hemogenic endothelium (HE) in the ventral dorsal aorta (VDA), support lifelong hematopoiesis. Their de novo production promises significant therapeutic value; however, current invitro approaches cannot efficiently generate multipotent long-lived HSPCs. Presuming this reflects a lack of extrinsic cues normally impacting the VDA, we devised a human dorsal aorta-on-a-chip platform that identified Yes-activated protein (YAP) as a cyclic stretch-induced regulator of HSPC formation. In the zebrafish VDA, inducible Yap overexpression significantly increased runx1 expression invivo and the number of CD41+ HSPCs downstream of HE specification. Endogenous Yap activation by lats1/2 knockdown or Rho-GTPase stimulation mimicked Yap overexpression and induced HSPCs in embryos lacking blood flow. Notably, in static human induced pluripotent stem cell (iPSC)-derived HE culture, compound-mediated YAP activation enhanced RUNX1 levels and hematopoietic colony-forming potential. Together, our findings reveal a potent impact of hemodynamic Rho-YAP mechanotransduction on HE fate, relevant to denovo human HSPC production.

Developmental Biology of the Blood System

Hematopoietic stem and progenitor cells (HSPCs) in the bone marrow are derived from a small population of hemogenic endothelial (HE) cells located in the yolk sac and major caudal arteries of the mammalian embryo. HE cells undergo an endothelial to hematopoietic cell transition, giving rise to HSPCs that accumulate in intra-arterial clusters before colonizing the fetal liver. To examine the molecular transitions between endothelial cells, HE, and intra-arterial cluster cells, and the heterogeneity of HSPCs within the intra-arterial clusters, we profiled ~40,000 cells from the caudal arteries (dorsal aorta, umbilical, vitelline) of embryonic day 9.5 to 11.5 mouse embryos by single-cell RNA sequencing (scRNA-seq) and single-cell chromatin accessibility sequencing (scATAC-Seq). A continuous developmental trajectory leads from endothelial cells to intra-arterial cluster cells, with identifiable intermediate stages between endothelial cells and HE. The intermediate endothelial stages most proximal to HE are characterized by elevated expression of genes regulated by GATA and SOX transcription factors. Developmental bottlenecks separate endothelial cells from HE cells, with the efficiency of transit through one of the last bottleneck regulated by RUNX1 dosage. Distinct developmental trajectories within intra-arterial cluster cells result in two populations of CD45+HSPCs; an initial wave of multi-lineage committed progenitors followed by precursors of hematopoietic stem cells (pre-HSCs). These and other insights gained from single cell analyses of HSPC formation from arterial endothelium will be presented. Disclosures No relevant conflicts of interest to declare.

INFLAMMASOME-MEDIATED EXPANSION OF THE HEMATOPOIETIC SYSTEM IN THE VERTEBRATE EMBRYO

Embryonic hematopoietic stem and progenitor cells (HSPCs) have the capacity to rapidly expand while maintaining multilineage potential. As efforts to derive HSPCs in vitro are suboptimal, a better understanding of mechanisms regulating embryonic HSPC formation and expansion is needed. We previously reported that sterile inflammation stimulates embryonic HSPC production, yet it remains unclear how the embryo initiates inflammatory cues. The NLRP3 inflammasome cleaves and activates interleukin-1-beta (IL-1β) in response to sterile stimuli, including reactive oxygen species and metabolic flux. Building on previous findings that glucose exposure expands embryonic HSPCs, we hypothesized that these effects rely on inflammasome activation and IL-1β release. Glucose exposure promoted il1b expression; il1b knockdown suppressed the inductive effects of glucose on numbers of CD41+ HSPCs. Conversely, overexpression of the active form of il1b or pharmacologic inflammasome stimulation increased runx1/cmyb expression and numbers of Flk1+cMyb+ HSPCs. Inflammasome activation required NF-kB priming, and upregulated IL-1β targets IL-6 and IL-8, which are known to expand embryonic HSPCs. Loss of inflammasome components reduced HSPCs, and prevented their expansion in response to glucose stimulation. Furthermore, knockdown of pu.1/spi1l and cell ablation studies indicate that macrophages serve an important role in mediating the effects of inflammasome stimulation on HSPCs. Significantly, inflammasome stimulation of human iPSC-derived hemogenic precursors also increased the frequency of multilineage hematopoietic colony-forming units formed in culture. This work identifies the inflammasome as a conserved metabolic sensor that expands HSPC production in vivo and in vitro, which may ultimately promote human HSPC production for cell therapy.

Inflammasome-Mediated Regulation of Hematopoiesis in the Vertebrate Embryo

Hematopoietic stem and progenitor cells (HSPCs) arise in the embryonic dorsal aorta (DA) through a Runx1-dependent process of endothelial-to-hematopoietic transition (EHT). Subsequently, HSPCs colonize secondary niches to proliferate, differentiate and maintain lifelong hematopoiesis. We previously reported that elevated glucose metabolism and sterile inflammatory signaling each stimulate zebrafish HSPC production from hemogenic endothelium. Consistent with the hypothesis that transient metabolic activation induces an inflammatory response to influence HSPC formation, we found that glucose stimulation from 12-36 hours post fertilization (hpf) increased expression of pro-inflammatory cytokines and receptors by quantitative PCR. In particular, morpholino-mediated knockdown of il1b blunted the inductive effects of glucose metabolism on runx1 expression in the DA and numbers of CD41+ HSPCs in the caudal hematopoietic tissue (CHT). In contrast, overexpression of mature il1b increased HSPC numbers as assessed by flow cytometry and runx1/cmyb in situ hybridization. As IL-1β is cleaved and activated by the inflammasome, which is a well-known sensor of metabolic stimuli, we treated embryos with validated inflammasome activators, such as nigericin, and found that they also increased runx1/cmyb expression and Flk1+cMyb+ HSPCs. Inflammasome stimulation required NF-κB activity, and upregulated canonical IL-1β targets, including IL-6 and IL-8, which have been previously shown by our laboratory to play significant roles in embryonic HSPC formation. Importantly, loss of the inflammasome adapter pycard or effector caspa reduced HSPC numbers, and prevented expansion of the HSPC pool in response to glucose stimulation. In further support of a role for sterile inflammatory activity in regulating HSPC number downstream of metabolic activation, we found that inflammasome-mediated IL-1β action bypassed the suppressive effect of antioxidant treatment on runx1/cmyb expression in the DA. Conversely, inflammasome inhibition partially blocked effects of metabolism-associated HIF1α activation on runx1/cmyb expression, indicating that the inflammasome acts downstream of reactive-oxygen-species (ROS) generation and HIF1α activation to promote HSPC production and/or expansion from hemogenic endothelium. Inflammasome components are highly expressed in myeloid cells; targeted knockdown and cell ablation studies indicate that macrophages are necessary to mediate the effects of inflammasome stimulation on HSPC numbers in the CHT at 72hpf. Interestingly, prolonged inflammasome stimulation also expands myeloid and lymphoid progenitors, as cmyb, rag1 and mpo expression were each elevated at 120hpf; increased numbers of Rag2+ and Mpo+ cells were confirmed by flow cytometry. To determine whether the effects of inflammasome activation were conserved in higher vertebrates, we induced inflammasome activation during differentiation of human induced pluripotent stem cells (iPSC) into hematopoietic progenitors. In this system, nigericin treatment of iPSC-derived hemogenic endothelial cells yielded an increased frequency of functional colony-forming units by day 7 of EHT culture. Taken together, inflammasome-mediated IL-1β action appears to serve as an integrator of metabolic activity, downstream of ROS/ HIF1α, to promote HSPC formation and development of myeloid and lymphoid lineages in vivo and in vitro. These studies identify the inflammasome as a promising target to promote human HSPC production from iPSCs for therapeutic purposes. DisclosuresNo relevant conflicts of interest to declare.

The Role of Runx1 in Embryonic Blood Cell Formation.

The de novo generation of hematopoietic stem and progenitor cells (HSPC) occurs solely during embryogenesis from a population of epithelial cells called hemogenic endothelium (HE). During midgestation HE cells in multiple intra- and extraembryonic vascular beds leave the vessel wall as they transition into HSPCs in a process termed the endothelial to hematopoietic transition (EHT). Runx1 expression in HE cells orchestrates the transcriptional switch necessary for the transdifferentiation of endothelial cells into functional HSPCs. Runx1 is widely considered the master regulator of developmental hematopoiesis because it plays an essential function during specification of the hematopoietic lineage during embryogenesis. Here we review the role of Runx1 in embryonic HSPC formation, with a particular focus on its role in hemogenic endothelium.

Insights into blood cell formation from hemogenic endothelium in lesser-known anatomic sites.

Hematopoietic stem and progenitor cells (HSPCs) are generated de novo in the embryo in a process termed the endothelial to hematopoietic transition (EHT). EHT is most extensively studied in the yolk sac and dorsal aorta. Recently new sites of hematopoiesis have been described, including the heart, somites, head, and venous plexus of the yolk sac. We examined sites of HSPC formation in well-studied and in less well-known sites by mapping the expression of the key EHT factor Runx1 along with several other markers by means of confocal microscopy. We identified sites of HSPC formation in the head, heart and somites. We also identified sites of HSPC formation in both the arterial and venous plexuses of the yolk sac, and show that progenitors with lymphoid potential are enriched in hematopoietic clusters in close proximity to arteries. Furthermore, we demonstrate that many of the cells in hematopoietic clusters resemble monocytes or granulocytes based on nuclear shape. We identified sites of HSPC formation in the head, heart, and somites, confirming that embryonic hematopoiesis is less spatially restricted than previously thought. Furthermore, we show that HSPCs in the yolk sac with lymphoid potential are located in closer proximity to arteries than to veins. Developmental Dynamics 245:1011-1028, 2016. © 2016 Wiley Periodicals, Inc.

The Central Nervous System Regulates Embryonic HSPC Production via Stress-Responsive Glucocorticoid Receptor Signaling

Hematopoietic stem and progenitor cell (HSPC) specification is regulated by numerous defined factors acting locally within the hemogenic niche; however, it is unclear whether production can adaptto fluctuating systemic needs. Here we show that the CNS controls embryonic HSPC numbers via the hypothalamic-pituitary-adrenal/interrenal (HPA/I) stress response axis. Exposure to serotonin or the reuptake inhibitor fluoxetine increased runx1 expression and Flk1(+)/cMyb(+) HSPCs independent of peripheral innervation. Inhibition of neuronal, but not peripheral, tryptophan hydroxlyase (Tph) persistently reduced HSPC number. Consistent with central HPA/I axis induction and glucocorticoid receptor (GR) activation, GR agonists enhanced, whereas GRloss diminished, HSPC formation. Significantly, developmental hypoxia, as indicated by Hif1α function, induced the HPA/I axis and cortisol production. Furthermore, Hif1α-stimulated HSPC enhancement was attenuated by neuronal tph or GR loss. Our data establish that embryonic HSC production responds to physiologic stress via CNS-derived serotonin synthesis and central feedback regulation to control HSC numbers.

The Spliceosomal Component Sf3b1 Is Essential for Hematopoietic Differentiation

Myelodysplastic syndrome (MDS) is a disorder arising from hematopoietic stem and progenitor cell (HSPC) dysfunction resulting in ineffective hematopoiesis. A multitude of recurrent somatic mutations in spliceosomal components were recently identified in MDS that likely contribute to the pathogenesis of the disease. The lack of in vivo models to study cell-type specific effects of spliceosomal mutations limits our understanding of why such mutations lead to hematopoietic abnormalities. Using a zebrafish with a loss-of-function mutation in sf3b1 (sf3b1hi3394), an essential member of the spliceosome, we demonstrate hematopoietic cell differentiation and hematopoietic stem and progenitor cell (HSPC) specification are processes sensitive to spliceosomal malfunction. Primitive erythropoiesis initiates normally in sf3b1 mutants as evidenced by expression of scl in the posterior lateral plate mesoderm at 14 hours post fertilization (hpf) as well as gata1 and beta-globin at 24 hpf. Flow cytometry quantification of gata1:gfp positive erythrocytes showed sf3b1 mutants have 25% more cells at 24 hpf, but greater than 3-fold fewer cells at 36 and 48 hpf, time points when wild type erythroblasts are expanding and differentiating. At 48 hpf, we also observed decreased levels of o-dianisidine positive erythrocytes, low numbers of morphologically mature erythroblasts, and higher levels of immature erythroblasts in sf3b1 mutants. Similarly, we observed normal initiation of primitive myelopoiesis marked by stem cell leukemia (scl) expression in the anterior lateral plate mesoderm at 14 hpf, but diminished expression of more differentiated markers, l-plastin and myeloperoxidase at 24 and 28 hpf in sf3b1 mutants. Quantification of lysozyme C:dsred positivemyeloid cells using flow cytometry also showed 24-fold fewer mature myeloid cells in sf3b1 mutants at 36 hpf. Our data on primitive erythropoiesis and myelopoiesis indicate sf3b1 is required for hematopoietic cell differentiation. Additionally, sf3b1 mutants have diminished expression of the definitive HSPC marker runx1 within the aorta at 28 hpf. In contrast, we observed normal expression of the pan-endothelial marker kinase insert domain receptor-like (kdrl) and aorta-specific markers notch1b and notch3 at 24 hpf. Flow cytometry quantification of kdrl:gfp endothelial cells at 24 hpf shows no difference in the frequency of endothelial cells in sf3b1 mutants. Moreover, we observed fewer cmyb:gfp; kdrl:dsred double positive HSPCs along the dorsal aorta. The data suggest that decreased HSPC formation in sf3b1 mutants is due to a failure in hemogenic induction. From these studies, we show sf3b1 is required at specific stages of hematopoietic cell development. These results provide novel insight into the role of splicing in blood cell development, and can afford a deeper understanding of the mechanism of splicing regulation on the origins of MDS. DisclosuresNo relevant conflicts of interest to declare.

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 Embryonic Hematopoietic Stem and Lymphoid Progenitor Cell Formation

Hematopoietic stem cells (HSCs) are defined as cells that can self-renew and provide robust, long-term multilineage engraftment of immunocompromised adults. Only one HSC is sufficient to reconstitute the entire hematopoietic system of an adult mouse, thus blood should theoretically be a simple organ system to generate from embryonic stem cells or induced pluripotent stem cells. Yet, despite two decades of effort, no one has been able to produce HSCs ex vivo from these sources. In the vertebrate embryo, transient waves of hematopoietic cells are produced prior to the formation of HSCs. While the role of erythrocyte production is self-evident, the necessity of myeloid production in the midgestation embryo is less clear. HSCs differentiate from hemogenic endothelium, and in mammalian embryos briefly accumulate in the aorta/gonad/mesonephros (AGM) region within intra-aortic hematopoietic clusters, although the vast majority of cluster cells are not functional HSCs. Approximately 20% of cells in hematopoietic clusters express a Ly6a-GFP transgene (Ly6a encodes Sca-1), and all lymphoid progenitors, pre-HSCs, and HSCs at embryonic day (E)11.5 are found within the Ly6a-GFP+ population. Genetic profiling of Ly6a-GFP+ and Ly6a-GFP- hematopoietic cluster cells from the AGM region revealed enrichment in gene ontology (GO) terms associated with innate immunity and inflammatory response in the Ly6a-GFP+ population, suggesting inflammatory signaling may regulate the formation of lymphoid progenitors and HSCs. To identify inflammatory cytokines that may be involved in lymphoid progenitor/HSC formation, we performed explant cultures of E9.5 embryos and added inflammatory factors known to induce Sca-1 expression in the adult mouse. Both type I and type II interferons (IFN-α4 and IFN-γ), and to a lesser extent TNFα, induced Ly6a-GFP expression in hematopoietic cells of explanted embryos. IFN-γ receptor 1 (IFN-γR1) was expressed on >80% of Ly6a-GFP+ hematopoietic cluster cells in the AGM, and Stat1 was phosphorylated in response to the addition of either IFN-γ or IFN-α4 to AGM explant cultures, indicating that embryonic hematopoietic stem and progenitor cells (HSPCs) are wired to respond to interferon stimulation. Mouse embryos deficient for IFN-g, the IFN-gR1, or the IFN-a4 receptor had decreased numbers of progenitors with lymphoid potential in the AGM region, as measured by limiting dilution culture on OP9 stromal cells, and IFN-gR1 deficient embryos had fewer HSCs, as determined by transplantation into adult mice. The role of IFN-γ signaling in HSC production in the AGM is evolutionarily conserved, as morpholino (MO) knockdown of IFN-g and its receptor likewise reduced HSPC numbers in the dorsal aorta and caudal hematopoietic tissue of zebrafish embryos, and the subsequent population of rag2 expressing lymphoid cells in the thymus. ChIP-Seq demonstrated that interferon regulatory factor 2 (IRF2) occupied genes in human fetal liver CD34+ HSPCs, with GO analysis indicating innate immunity and interferon signaling as significantly enriched processes, further demonstrating that interferon signaling is active during vertebrate development. IRF2 is a negative regulator of interferon signaling in the adult, and consistent with this, MO-knockdown of IRF2 in the zebrafish embryo enhanced the production of HSPCs, whereas knockdown of the positive effector IRF1 mimicked the phenotype of IFN-g loss. Myeloid cells have previously been shown to produce IFNs, and primitive F4/80+ macrophages were observed in close proximity to hematopoietic cluster cells in the murine AGM region. To determine if the presence of primitive myeloid cells impacted HSPC formation, macrophage specific as well as pan-myeloid knockdown was employed in zebrafish; macrophage depletion by IRF8-MO significantly reduced runx1 expression in the AGM and HSPC production. Significantly, MO-mediated inhibition of PU.1 function, which depletes the majority of myeloid cells, caused an even more striking loss, suggesting IFN-γ is not the only inflammatory mediator, nor primitive macrophages the only myeloid cells, involved in controlling embryonic HSPC production. Together these data demonstrate that myeloid-mediated inflammatory signaling is active in the developing vertebrate embryo, and in the absence of pathogenic challenge function to regulate the production of lymphoid progenitors and definitive HSCs. DisclosuresNo relevant conflicts of interest to declare.