Hematopoietic Stem Cell Development Research Articles

Wild-type FLT3 and FLT3 ITD exhibit similar ligand-induced internalization characteristics.

Class III receptor tyrosine kinases control the development of hematopoietic stem cells. Constitutive activation of FLT3 by internal tandem duplications (ITD) in the juxtamembrane domain has been causally linked to acute myeloid leukaemia. Oncogenic FLT3 ITD is partially retained in compartments of the biosynthetic route and aberrantly activates STAT5, thereby promoting cellular transformation. The pool of FLT3 ITD molecules in the plasma membrane efficiently activates RAS and AKT, which is likewise essential for cell transformation. Little is known about features and mechanisms of FLT3 ligand (FL)‐dependent internalization of surface‐bound FLT3 or FLT3 ITD. We have addressed this issue by internalization experiments using human RS4‐11 and MV4‐11 cells with endogenous wild‐type FLT3 or FLT3 ITD expression, respectively, and surface biotinylation. Further, FLT3 wild‐type, or FLT3 ITD‐GFP hybrid proteins were stably expressed and characterized in 32D cells, and internalization and stability were assessed by flow cytometry, imaging flow cytometry, and immunoblotting. FL‐stimulated surface‐exposed FLT3 WT or FLT3 ITD protein showed similar endocytosis and degradation characteristics. Kinase inactivation by mutation or FLT3 inhibitor treatment strongly promoted FLT3 ITD surface localization, and attenuated but did not abrogate FL‐induced internalization. Experiments with the dynamin inhibitor dynasore suggest that active FLT3 as well as FLT3 ITD is largely endocytosed via clathrin‐dependent endocytosis. Internalization of kinase‐inactivated molecules occurred through a different yet unidentified mechanism. Our data demonstrate that FLT3 WT and constitutively active FLT3 ITD receptor follow, despite very different biogenesis kinetics, similar internalization and degradation routes.

Abstract P141: A Novel GWAS Locus Influences Microvascular Response to Acute Psychological Stress

Background: Excessive peripheral microvascular constriction during acute psychological stress, measured using peripheral arterial tonometry reflects similar changes in coronary blood flow and is a predictor of adverse cardiovascular outcomes. The ratio of digital pulse wave amplitude during stress compared to rest (sPAT) is used to estimate the degree of microvascular response to stress. We sought to determine if genetic factors contribute to the degree of microvascular constriction during mental stress. Methods: A total of 642 post-MI and stable CAD subjects from two prospective cohort studies underwent mental stress testing with a standardized public speaking stressor. Digital pulse wave amplitude was continuously measured using PAT and the stress/rest PAT ratio (sPAT) of pulse wave amplitude during mental stress/baseline was calculated. Genotyping was performed using Illumina’s Multi-Ethnic Genotyping Array (MEGA) platform and imputed to the 1000 Genome reference panel. Race stratified genome-wide association studies (GWAS) of sPAT were conducted using linear regression of additive genetic mode adjusted for age, sex and population stratification in the two cohorts. A trans-ethnic meta-analysis integrated the four sets GWAS results. Results: Mean age was 63±9; 65% male, 35% Black. We identified two SNPs in linkage disequilibrium on chr4:185503564 and chr4:185491706 rs13353812 (with 35% and 28% allele frequency, respectively) that were associated with greater sPAT ratio ( P = 1.42E-08). The mean sPAT ratio during mental stress for subjects with none, one and two AT insertion alleles of SNP chr4:185503564 were 0.67, 0.76 and 0.93, respectively, an average of 12% (P < 0.001), per allele. Results were similar for G insertion alleles of SNP rs13353812. The nearest gene of the sPAT-associated locus is CASP3 which encodes caspase, an essential protein for apoptosis signaling and brain and hematopoietic stem cell development. Conclusion: We have identified a genetic basis for stress-induced vasomotion. Presence of the chr4:185503564 allele is associated with less vasoconstriction during mental stress, and thus may be protective against long term cardiovascular risk. These findings need further exploration.

Identification of a norovirus outbreak on a hematopoietic stem cell transplant unit and development and implementation of a novel infection prevention algorithm for controlling transmission.

Controlling norovirus transmission in units with immunocompromised patients is challenging. We present a cluster of norovirus cases that occurred on a stem-cell transplant unit and the prevention efforts that were implemented to limit the outbreak. Protocols developed to control this cluster may provide a model for other facilities.

Enhancing Hematopoiesis from Murine Embryonic Stem Cells through MLL1-Induced Activation of a Rac/Rho/Integrin Signaling Axis.

SummaryThe Mixed Lineage Leukemia (MLL1, KMT2A) gene is critical for development and maintenance of hematopoietic stem cells (HSCs), however, whether this protein is limiting for HSC development is unknown due to lack of physiologic model systems. Here, we develop an MLL1-inducible embryonic stem cell (ESC) system and show that induction of wild-type MLL1 during ESC differentiation selectively increases hematopoietic potential from a transitional c-Kit+/Cd41+ population in the embryoid body and also at sites of hematopoiesis in embryos. Single-cell sequencing analysis illustrates inherent heterogeneity of the c-Kit+/Cd41+ population and demonstrates that MLL1 induction shifts its composition toward multilineage hematopoietic identities. Surprisingly, this does not occur through increasing Hox or other canonical MLL1 targets but through an enhanced Rac/Rho/integrin signaling state, which increases responsiveness to Vla4 ligands and enhances hematopoietic commitment. Together, our data implicate a Rac/Rho/integrin signaling axis in the endothelial to hematopoietic transition and demonstrate that MLL1 actives this axis.

Ex Vivo Modeling of Hematopoietic Stem Cell Homing to the Fetal Liver.

Hematopoietic stem cells (HSCs) are used in the clinic to provide life-saving therapies to patients with a variety of hematological malignancies and disorders. Yet, serious deficiencies in our understanding of how HSCs develop and self-renew continue to limit our ability to make this therapy safer and more broadly available to those who have no available donor. Finding ways to expand HSCs and develop alternate sources of HSCs is an urgent priority. In the embryo, a critical transition in development of the blood system requires that newly emergent HSCs from the aorta-gonad-mesonephros (AGM) region migrate to the fetal liver where they aggressively self-renew and expand to numbers sufficient to sustain the adult long term. This process of homing to the fetal liver is orchestrated by intrinsic regulators such as epigenetic modifications to the genome, expression of transcription factors, and adhesion molecule presentation, as well as sensing of extrinsic factors like chemokines, cytokines, and other molecules. Due to technical limitations in manipulating the fetal tissue microenvironment, mechanisms mediating the homing and expansion process remain incompletely understood. Importantly, HSC development is strictly dependent upon forces created by the flow of blood, and current experimental methods make the study of biophysical cues especially challenging. In the protocol presented herein, we address these limitations by designing a biomimetic ex vivo microfluidic model of the fetal liver that enables monitoring of HSC homing to and interaction with fetal liver niches under flow and matrix elasticity conditions typical during embryonic development. This model can be easily customized for the study of key microenvironmental factors and biophysical cues that support HSC homing and expansion.

Integrated Single Cell Transcriptomics Defines an Engineered Niche Supporting Hematopoietic Stem Cell Development Ex Vivo

During embryonic development, hematopoietic stem cells (HSC) arise from hemogenic endothelial cells (HEC) within arterial vessels such as the aorta of the AGM (aorta-gonad-mesonephros) region, in a process referred to as the endothelial to hematopoietic transition (EHT). Although numerous signal pathways have been implicated in EHT, the precise combination of niche-derived signals required to support the generation and self-renewal of functional, long-term engrafting HSC remains poorly defined. To elucidate the niche signals regulating HSC emergence, we used single cell RNA-sequencing to simultaneously analyze the global transcriptional profiles of HEC during their transition to HSC and the AGM-derived endothelial cell stroma (AGM-EC) that supports the generation and expansion of functional HSC. Trajectory analysis of single cell transcriptomes enabled reconstruction of EHT in pseudotime, revealing dynamics of gene expression, including genes encoding cell surface receptors and downstream pathways, during the process of HSC genesis and self-renewal in vivo and in vitro. Transcriptional profiles of niche AGM-EC enabled identification of corresponding ligands which serve to activate these receptors during HSC generation. We integrated this knowledge to engineer a stromal cell-free niche for generation of engrafting HSC from hemogenic precursors in vitro. Specifically, we defined serum-free conditions combining immobilized Notch1 and Notch2-specific antibodies to activate Notch receptors, recombinant VCAM1-Fc chimera or fibronectin fragment to bind VLA-4 integrin, recombinant interleukin-3, stem cell factor, thrombopoietin, and CXCL12 to activate their respective cytokine/chemokine receptors, and small molecule inhibition of TGF-β Receptor 1. We demonstrated that this engineered niche is sufficient to support the generation of functional HSC, as measured by long-term (24 week) multilineage engraftment after transplantation to immune-competent, lethally irradiated adult recipient mice, following culture of hemogenic precursors isolated from E9.5 to E10.5 murine embryos. The observed efficiency of generating long-term engrafting HSC, particularly from precursors derived from early embryonic stages before E10, was lower in engineered conditions compared with AGM-EC stroma, suggesting additional niche signal factors remain to be defined to optimally support HSC maturation and self-renewal in the engineered niche. Single cell RNA-sequencing of hematopoietic progeny generated following culture in the engineered niche demonstrated the formation of populations with transcriptional signatures of HSC, as well as multipotent and lineage-specific progenitors, comparable to those generated following co-culture with niche AGM-EC stroma. However, we observed relative overexpression of Notch target genes promoting early T-lymphoid fate in cells generated from the engineered niche compared to those from AGM-EC stroma. Incorporating stage-specific attenuation of Notch1 receptor activation with soluble Notch1 blocking antibody during culture was sufficient to limit markers of early T-cell precursors, suggesting that temporal titration of Notch signal activation could be used to further modulate HSC and T-lymphoid output in the engineered niche. Altogether, these studies enhance our understanding of the core signal pathways necessary for the embryonic development of functional HSC, with the potential to advance in vitro engineering of therapeutically relevant pluripotent stem cell-derived HSC in stromal cell-free culture. Disclosures Bernstein: Lyell Immunopharma: Consultancy, Equity Ownership, Patents & Royalties, Research Funding; Nohla Therapeutics: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

Chromosomal Loop Editing Reveals New Epigenomic Landscape in the β-Globin Locus with Implications for Hemoglobinopathy Therapies

Human β-globin locus consists of at least six genes encoding components of the oxygen transport protein hemoglobin and an upstream locus control region (LCR) containing five DNase I hypersensitive sites. In addition, there are at least four conserved CTCF insulator elements surrounding the locus, which form dynamic chromatin interaction patterns across different cell types in the course of hematopoietic stem and progenitor cells (HSPCs) development. Chromatin conformation of the β-globin locus in normal adult CD34+ HSPCs reveals the formation of three topological associated domains (TADs), in which individual TAD boundaries are demarcated by CTCF sites (Figure 1). By comparing chromatin loop interactions between CD34+ HSPCs and its differentiated erythroid progenitors, we identify a chromatin loop that forms in erythroid progenitors and is not evident in the CD34+ HSPCs. Detailed examination of this loop shows that a DNase I hypersensitivity site, also called 3'HS1, overlaps with a CTCF site that forms a loop with another CTCF site adjacent to OR52A5 gene (Figure 2). To investigate the role of this specific chromatin loop in the regulation of hemoglobin gene expression, we knock out the 3'HS1 CTCF motif in adult CD34+ HSPCs under erythroid differentiation medium by CRISPR/Cas9-mediated gene editing. We find that deletion of 3'HS1 CTCF results in a 2-fold decrease of β-globin (HBB) and a 4-fold increase of the fetal hemoglobin gene encoded by γ-globin (HBG1/2) in erythroid colonies of edited CD34+ cells (Figure 3). Elevation of fetal hemoglobin upon 3'HS1 CTCF deletion was also confirmed in human umbilical cord blood-derived erythroid progenitor-2 cells (HUDEP-2), which results in a 12-fold increase of γ-globin expression (Figure 4). These results suggest that the 3'HS1 CTCF plays a crucial role in regulating the expression of fetal hemoglobin gene, however it remains unclear whether changes in chromatin structure is responsible for these changes. CTCF looping interactions have been described to form under convergent directionality. To validate the role of 3'HS1 CTCF in establishing chromatin interactions at the β-globin locus, we aim to invert this binding motif and evaluate how it disrupts chromatin organization and gene expression at this locus. Deciphering the underlying mechanisms will shed light on how three-dimensional chromatin structure is reorganized in differentiating erythroid cells as it undergoes nuclear condensation. Furthermore, elevation of fetal hemoglobin expression can potentially be a new therapeutic gene editing strategy to treat sickle cell disease and some cases of β-hemoglobinopathies. Disclosures No relevant conflicts of interest to declare.

Klotho Expression Predicts Poor Prognosis in Myelodysplastic Syndrome

Introduction: An important explanation for the occurrence of Myelodysplastic syndrome (MDS) in the elderly is related to hematopoietic stem-cell (HSC) senescence. Recent studies have identified aging-related alterations in mesenchymal stromal cells in MDS. During the aging process, the different HSC subtypes lose the ability to differentiate into lymphoid and myeloid precursor cells, causing a breakdown in the immune system balance. This process exposes HSC to important genotoxic factors and may induce genetic or epigenetic alterations because exposure to these factors increases the risk of hematological malignancies. A new biomarker for cancer is Klotho (α and β subtypes), which has been identified as an anti-aging gene that extended the lifespan in murine models when it was overexpressed. Several studies have demonstrated that αKlotho (KL) deficiency causes development of aging features, including increased proliferation, reduction in tumor cell apoptosis, and disruption of HSC development. Some studies have established an association between low expression of KL and risks of cancer (i.e. lymphoma) by inducing cell apoptosis, inhibiting tumor growth, and causing a decrease in insulin-like growth factor 1 receptor (IGF-1R) activation. In parallel to these studies, a recent study reported that KL downregulation was associated with the occurrence of anemia, the main clinical feature of MDS, in cancer patients. However, the expression profile of KL and βKlotho (KLB) genes in the bone marrow (BM) of healthy patients with different ages and the way in which the genes modulate the pathogenesis of MDS in the elderly are unknown. Methods: Total BM cell samples from 62 de novo MDS patients with a mean age of 66 ± 16 years old (10 MDS with single lineage dysplasia, 14 MDS with ring sideroblasts, 27 MDS with multilineage dysplasia, and 11 MDS with excess blasts based on World Health Organization's (WHO) 2016 classification) and from eleven healthy volunteer individuals were collected with informed consents. Healthy patients were stratified into young (n=6; mean 25 ± 6 years old) and elderly (n=5; mean 64 ± 9 years old) individuals. Pre-developed TaqMan gene expression assays for KL (Hs00934627_m1) and KLB (Hs00545621_m1) were used to quantify mRNA expression. Each sample was performed in duplicate, and the expression ratios were calculated using the 2−ΔCq method. Results: Our results demonstrate that the KL gene was expressed in the BM samples from all experimental groups. High KL expression was identified in MDS patients when compared to young (p <0.001) and elderly (p <0.001) controls (Figure 1A). In MDS, KL was downregulated in male patients (p=0.042) (Figure 1B), neutropenic MDS cases (p=0.003) (Figure 1C), MDS patients with transfusion dependence (p=0.001) (Figure 1D), and MDS with an excess of blasts (p=0.038) (Figure 1D), representing more aggressive diseases. A multiple regression was performed in order to predict KL expression from gender, absolute neutrophil count (ANC), transfusion dependence, and WHO 2016 classification. These variables significantly predicted KL expression (F [4, 27] = 3.396; p=0.023; r2=0.335). Transfusion dependence (p=0.027) and WHO 2016 classification (p=0.049) variables significantly added to the prediction. The KLB gene was only expressed in a total of 8/62 MDS patients. The feature that stood out in these patients was the presence of dyserythropoiesis (62.5%). No control sample expressed the KLB gene. Conclusions: Our results demonstrated an important expression profile of KL and KLB in BM of healthy young and old individuals and/or in MDS patients for the first time in contrast to a previous study in which it was demonstrated that human BM induces a negligible expression of these genes. Based on absence of differences between controls, our data showed that KL expression not was associated with the MDS pathogenesis-related senescence process, but its lower expression contributed to the poor prognostic MDS features, probably due to its high potential for affecting HSC number, function, and erythropoiesis. Additionally, low KLB expression in MDS patients with dyserythropoiesis suggests a compensatory response of the bone marrow environment to hematopoietic development block via clonal expansion, especially in the erythroid lineage. These approaches may define KL and KLB as new pathological markers of poor prognosis in MDS and improve future potential therapies. Disclosures No relevant conflicts of interest to declare.

Endoplasmic reticulum stress induced apoptosis and caspase activation is mediated through mitochondria during megakaryocyte differentiation

Megakaryocytopoiesis involves the process of the development of hematopoietic stem cells into megakaryocytes (MKs), which are the specialized cells responsible for the production of blood platelets. Platelets are one of the crucial factors for hemostasis and thrombosis. In terminally differentiated MKs, many molecular process such as caspase activation and a massive cytoskeletal rearrangement drive the formation of cytoplasmic extensions called proplatelets. These cytoplasmic extensions packed with granules and organelles are then released from the bone marrow into the blood circulation as platelets. Classically, caspase activation is associated with apoptosis and recent reports suggest their involvement in cell differentiation and maturation. There is no clear evidence about the stimulus for caspase activation during megakaryocyte development. In the current study, we attempted to understand the importance of endoplasmic reticulum stress in the caspase activation during megakaryocyte maturation. We used human megakaryoblstic cell line (Dami cells) as an experimental model. We used PMA (Phorbol 12-myristate 13 acetate) to induce megakaryocytic differentiation to understand the involvement of ER stress and caspase activation during MK maturation. Further, we used Thapsigargin, a non-competitive inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) as a positive control to induce ER stress. We observed larger and adherent cells with the increased expression of megakaryocytic markers (CD41 and CD61) and UPR markers in PMA or Thapsigargin treated cells as compared to control. Also, Thapsigargin treatment induced increased caspase activity and PARP cleavage. The increased expression of megakaryocyte maturation markers alongside with ER stress and caspase activation suggests the importance of ER stress in caspase activation during MK maturation.

Using GrandPrix to investigate haematopoietic stem cell development by single-cell sequencing

Using GrandPrix to investigate haematopoietic stem cell development by single-cell sequencing

Targeted Integration and High-Level Transgene Expression in AAVS1 Transgenic Mice after In Vivo HSC Transduction with HDAd5/35++ Vectors

Our goal is the development of invivo hematopoietic stem cell (HSC) transduction technology with targeted integration. To achieve this, we modified helper-dependent HDAd5/35++ vectors to express a CRISPR/Cas9 specific to the "safe harbor" adeno-associated virus integration site 1 (AAVS1) locus and to provide a donor template for targeted integration through homology-dependent repair. We tested the HDAd-CRISPR+ HDAd-donor vector system in AAVS1 transgenic mice using a standard exvivo HSC gene therapy approach as well as a new invivo HSC transduction approach that involves HSC mobilization and intravenous HDAd5/35++ injections. In both settings, the majority of treated mice had transgenes (GFP or human γ-globin) integrated into the AAVS1 locus. On average, >60% of peripheral blood cells expressed the transgene after invivo selection with low-dose O6BG/bis-chloroethylnitrosourea (BCNU). Exvivo and invivo HSC transduction and selection studies with HDAd-CRISPR+ HDAd-globin-donor resulted in stable γ-globin expression at levels that were significantly higher (>20% γ-globin of adult mouse globin) than those achieved in previous studies with a SB100x-transposase-based HDAd5/35++ system that mediates random integration. The ability to achieve therapeutically relevant transgene expression levels after invivo HSC transduction and selection and targeted integration make our HDAd5/35++-based vector system a new tool in HSC gene therapy.

CONTRIBUTION OF SCLEROTOME TO THE EMBRYONIC HEMATOPOIETIC STEM CELL NICHE AND VASCULAR SMOOTH MUSCLE

Hematopoietic stem cells (HSCs) are a self-renewing population that sustains production of blood lineages throughout life. Vertebrate HSCs are first specified in the embryo, arising from hemogenic endothelium in the ventral wall of the dorsal aorta (DA). The cell types within this three-dimensional environment and the signals they provide to promote HSC development are not well defined. A better understanding of native embryonic HSC specification niche could inform efficient directed differentiation of true HSCs from induced pluripotent stem cells for clinical and research applications. We previously showed that wnt16 is required for development of zebrafish HSCs, as well as normal patterning of the ventral somite, the sclerotome. We hypothesized that normal sclerotome patterning is required for HSC specification by contributing to the cellular environment that presents inductive signals. To follow sclerotome development in living embryos, we developed transgenic reporter zebrafish where fluorophore expression is driven by regulatory elements from the sclerotome gene, pax1a. We show that sclerotome-derived cells migrate to and make contact with hemogenic endothelium of the DA shortly before initiation of the definitive hematopoietic program, demonstrating that they are present at the right time and place to present specification signals. By live imaging and profiling of sorted cells, we show these cells also give rise to arterial vascular smooth muscle cells. Loss-of-function analyses demonstrate a requirement for multiple sclerotome genes, for both patterning of sclerotome, and subsequent specification of HSCs and downstream hematopoietic lineages. Our data indicate that sclerotomal cells contribute to the HSC specification niche of the early embryo, and form a basis for defining the niche architecture and signals that drive definitive hematopoietic programming.

AN MLL1-REGULATED RAC/RHO/INTEGRIN SIGNALING AXIS ENHANCES HEMATOPOIESIS FROM EMBRYONIC HEMOGENIC ENDOTHELIAL PRECURSORS

We have previously shown that the Mixed Lineage Leukemia (MLL1, KMT2A) gene is critical for the development and maintenance of transplantable hematopoietic stem cells (HSCs), however, whether overexpression of wild-type (WT) MLL1 could enhance HSC production has not been tested due to lack of suitable model systems. Here, we report the WT MLL1 inducible embryonic stem (ES) cell model and show that induction of MLL1 during ES differentiation selectively expands hematopoietic colony potential from a transitional c-Kit+/CD41+ population. Single cell sequencing analysis illustrates the inherent heterogeneity of this population and demonstrates that MLL1 induction shifts its composition from a hemogenic endothelium identity toward multilineage hematopoietic identities. Surprisingly, this does not occur through increasing Hox or other canonical MLL1 target genes, but occurs through an enhanced Rac/Rho/integrin signaling state, which increases responsiveness to VLA4 ligands and enhances hematopoietic commitment. Together, our data implicate a Rac/Rho/integrin signaling axis as critical during the endothelial to hematopoietic transition and demonstrate that WT MLL1 actives this axis.

2034 – RAB5C-MEDIATED ENDOCYTIC TRAFFICKING REGULATES HEMATOPOIETIC STEM AND PROGENITOR CELL DEVELOPMENT THROUGH NOTCH AND AKT SIGNALING

A better understanding of molecular mechanisms underlying hematopoietic stem and progenitor cell (HSPC) development may lead to improved protocols to generate functional and transplantable HSPCs ex vivo. Here we report that Rab5c is essential for HSPC specification by endocytic trafficking of Notch and AKT signaling in zebrafish embryos. Rab5c deficiency leads to defects in HSPC production. Mechanistically, Rab5c regulates hemogenic endothelium specification by endocytic trafficking of Notch ligand and receptor. We further show that the interaction between Rab5c and Appl1 in endosome is required for the survival of endothelial cells in the aorta-gonad-mesonephros region through AKT signaling. Interestingly, Rab5c over-activation can also lead to defects in HSPC production, which is attributed to excessive endolysosomal trafficking inducing Notch signaling defect. Taken together, our findings establish a previously unrecognized role of Rab5c-mediated endocytic trafficking in HSPC development, and provide new insights into how spatiotemporal signals are orchestrated to accurately execute cell fate transition.

2004 - NOVEL PRO-INFLAMMATORY AGM-ASSOCIATED MACROPHAGES ARE INVOLVED IN EMBRYONIC DEVELOPMENT OF HEMATOPOIETIC STEM CELLS

Hematopoietic stem cells (HSCs) are responsible for the life-long maintenance and regeneration of the adult vertebrate blood system. The first HSCs are generated from specialized endothelial cells of the embryonic aorta. Inflammatory factors have been implicated in regulating HSC development, but it is unclear what cells in the mouse embryonic aorta-gonad-mesonephros (AGM) microenvironment produce these factors. In the adult, macrophages play both pro- and anti-inflammatory roles and they are involved in bone marrow hematopoiesis. We sought to examine whether macrophages or other hematopoietic cells found in the embryo prior to HSC generation are involved in the AGM HSC-generative microenvironment. Our mass cytometry (CyTOF) results indicate the absence of lymphoid cells and mature neutrophils in E10.5 mouse AGM, while two abundant myeloid cell types are present – mannose-receptor positive AGM-associated macrophages (AGM-aM) and mannose-receptor negative macrophages/progenitors. We show that the appearance of macrophages in the AGM is dependent on CX3CR1 and it occurs just before the time of HSC generation. Differently from mannose-receptor positive adult macrophages, AGM-aM express a pro-inflammatory signature. They localize to the embryonic aorta and dynamically interact with nascent and emerging intra-aortic hematopoietic cells (IAHC). Importantly, upon macrophage depletion, no adult-repopulating HSCs are detected, thus implicating unique pro-inflammatory AGM-associated macrophages in regulating the embryonic development of HSCs.

3175 – HEMATOPOIETIC STEM CELL-DEPENDENT NOTCH TRANSCRIPTION IS MEDIATED BY P53 THROUGH THE HISTONE CHAPERONE SUPT16

Hematopoietic stem cells (HSCs) are rare cells that can self-renew and differentiate into all blood cell lineages for life. HSCs have long been the focus of developmental and regenerative studies, yet our understanding of the signaling events regulating their specification remains incomplete. In order to identify novel genes and transcription factors involved in hematopoietic specification, we performed a forward genetic screen to identify zebrafish mutants defective in HSC formation. Through large-scale whole mount in situ hybridization based screens followed by RNA-sequencing-based linkage mapping, we identified that supt16h, a component of the FAcilitates Chromatin Transcription (FACT) complex, is required for HSC formation. Zebrafish supt16h mutants express reduced levels of Notch signaling components, genes essential for HSC development, due to abrogated transcription. Although cellular functions of Supt16h is generally known for regulating transcription and reorganizing nucleosomes to alter chromatin accessibility, global chromatin accessibility in the zebrafish supt16h mutants is unaffected. However, we observe a specific increase in accessibility at the p53 locus leading to an accumulation of P53 protein in the supt16h mutants and abrogation of increased p53 levels in supt16h mutants rescues both loss of Notch and HSC phenotypes. We further demonstrate that P53 levels directly influence expression of the Polycomb Group protein, Phc1, which functions as a transcriptional repressor of Notch genes. Suppression of phc1 or its upstream regulator, p53, rescues both loss of Notch and loss of HSC phenotypes in supt16h mutants. Taken together, our results highlight a previously uncharacterized relationship between supt16h, p53, and phc1 to specify HSCs via modulation of Notch signaling.

1035 - DECODING THE MOLECULAR CUES THAT REGULATE HSC SPECIFICATION.

Hematopoietic stem cells (HSCs) underlie the continued production of blood and immune cell lineages for the lifetime of an organism. In all vertebrate embryos examined, HSCs arise from the unique transdifferentiation of hemogenic endothelium comprising the floor of the dorsal aorta during a brief developmental window. To date, this process has not been replicated in vitro from pluripotent precursors, both because the full complement of required signaling inputs remains to be determined and because the lineage relationships among each subtype of arterial endothelium remain to be elucidated. Our recent efforts to elucidate the signaling pathways required to generate HSC fate have demonstrated that Notch signaling is needed iteratively, with both environmental and intrinsic requirements. The earliest known requirement is present within the somite to generate the sclerotome, the ventromedial domain of each developing somite. The sclerotome, in turn, is necessary to present Notch ligand to the shared vascular precursors of HSCs as they migrate across the ventral face of the somites. These studies provide the earliest known molecular cue required for HSC development. We have followed upon this work to demonstrate additional roles of the somite in patterning HSC formation and function. Wnt9a is produced from the somites and received via Frizzled9b / EGF receptors present upon HSC precursors, which regulates the first proliferative events within nascent HSCs. Finally, we have also shown that the somite produces endothelial cells that migrate and incorporate into the developing dorsal aorta. Thus, it appears that three different classes of endothelial cells are present at the time of HSC emergence – hemogenic endothelium, arterial endothelium, and somite-derived endothelial cells. What regulates production of each subset, and how each contributes to HSC generation are topics of our ongoing studies.

Interferon gamma inhibits the differentiation of mouse adult liver and bone marrow hematopoietic stem cells by inhibiting the activation of notch signaling

BackgroundThe paradigm of hematopoietic stem and progenitor cells (HSPCs) has become accepted ever since the discovery of adult mouse liver hematopoietic stem cells and their multipotent characteristics that give rise to all blood cells. However, differences between bone marrow (BM) and liver hematopoietic stem cells and the hematopoietic microenvironment remain poorly understood. In addition, the regulation of the liver hematopoietic system remains unknown.MethodsClone formation assays were used to confirm that the proliferation of adult mouse liver and bone marrow HSPCs. Model mice with different interferon gamma (IFN-γ) levels and a co-culture system were used to detect the differentiation of liver HSPCs. The γ-secretase inhibitor (GSI) and the JAK/STAT inhibitor ruxolitinib and cell culture assays were used to explore the molecular mechanism by which IFN-γ impairs HSPC proliferation and differentiation.ResultsThe colony-forming activity of liver and bone marrow HSPCs was inhibited by IFN-γ. Model mice with different IFN-γ levels showed that the differentiation of liver HSPCs was impaired by IFN-γ. Using a co-culture system comprising liver HSPCs, we found that IFN-γ inhibited the development of liver hematopoietic stem cells into γδT cells. We then demonstrated that IFN-γ might impair liver HSPC differentiation by inhibiting the activation of the notch signaling via the JAK/STAT signaling pathway.ConclusionsIFN-γ inhibited the proliferation of liver-derived HSPCs. IFN-γ also impaired the differentiation of long-term hematopoietic stem cells (LT-HSCs) into short-term hematopoietic stem cells (ST-HSCs) and multipotent progenitors (MPPs) and the process from LSK (Lineage−Sca-1+c-Kit+) cells to γδT cells. Importantly, we proposed that IFN-γ might inhibit the activation of notch signaling through the JAK/STAT signaling pathway and thus impair the differentiation process of mouse adult liver and BM hematopoietic stem cells.

Abstract 5193: Conditional knockout of histone chaperone FACT in mice: Consequences for normal tissues

Abstract Histone chaperone FAcilitates Chromatin Transcription (FACT) is, a complex of SSRP1 and SPT16, involved in chromatin remodeling during transcription, replication, and DNA repair. FACT is selectively expressed during early stages of development and later exclusively present in the stem cell niches in adult normal tissues. It is expressed in undifferentiated aggressive tumors with poor prognosis.¹ FACT inhibition is not tolerated by most tumor, unlike normal cells. This vulnerability of tumor cells upon FACT inactivation suggests evaluating FACT inhibition as an antitumor therapeutic approach.¹ Since FACT knockout (KO) is embryonically lethal (E3.5),² study of FACT’s function in different non-tumor cells of mammalian organisms was not possible and was mostly studied in single cell organisms and in tumor cell lines. Therefore, to assess the safety of FACT inhibition as an anticancer approach, by understanding its role in normal physiology, we generated a conditional KO of Ssrp1 mouse model using CreERT2-LoxP system.³ We harvested stem cells of different lineages to better understand the role of FACT in normal mouse physiology. Using the hematopoietic stem cells (HSCs) from the bone marrow (BM), we performed BM colony formation assay. We found that FACT inactivation significantly decreased the number of colonies of HSCs. Furthermore, inhibiting FACT significantly increased caspase 3 expression in mouse adipose stem cells (ASCs), resulting in apoptosis and decreased survival. These results prompted us to comprehensively evaluate roles of FACT in stem cell development and survival. To this end, fibroblasts in a conditional knockout mouse were reprogrammed by retroviral vector encoding four pluripotency transcription factors (Oct3/4, Klf4, Sox2 and c-Myc). We have found successful generation of induced pluripotent stem (iPS) cells 14 days after reprogramming. In conclusion, FACT associates with development and/or survival of mouse HSCs and ASCs. Further evaluation using iPS cells is expected to delineate roles of FACT in various stages of stem cell development and thus normal physiology. 1. Garcia H, et al. Cell Rep, 2013 4(1) 2. Cao S, et al. Mol Cel Bio. 2003 23(15) 3. Sandlesh P et al. PLOS ONE. 2018 13(6) Citation Format: Masaki A. Ito, Poorva Sandlesh, Imon Goswami, Laura A. Prendergast, Katerina Gurova. Conditional knockout of histone chaperone FACT in mice: Consequences for normal tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5193.

Pro-inflammatory Aorta-Associated Macrophages Are Involved in Embryonic Development of Hematopoietic Stem Cells

SummaryHematopoietic stem cells (HSCs) are generated from specialized endothelial cells of the embryonic aorta. Inflammatory factors are implicated in regulating mouse HSC development, but which cells in the aorta-gonad-mesonephros (AGM) microenvironment produce these factors is unknown. In the adult, macrophages play both pro- and anti-inflammatory roles. We sought to examine whether macrophages or other hematopoietic cells found in the embryo prior to HSC generation were involved in the AGM HSC-generative microenvironment. CyTOF analysis of CD45+ AGM cells revealed predominance of two hematopoietic cell types, mannose-receptor positive macrophages and mannose-receptor negative myeloid cells. We show here that macrophage appearance in the AGM was dependent on the chemokine receptor Cx3cr1. These macrophages expressed a pro-inflammatory signature, localized to the aorta, and dynamically interacted with nascent and emerging intra-aortic hematopoietic cells (IAHCs). Importantly, upon macrophage depletion, no adult-repopulating HSCs were detected, thus implicating a role for pro-inflammatory AGM-associated macrophages in regulating the development of HSCs.