Maturation Of Hematopoietic Stem Cells Research Articles

Cis inhibition of NOTCH1 through JAGGED1 sustains embryonic hematopoietic stem cell fate

Hematopoietic stem cells (HSCs) develop from the hemogenic endothelium (HE) in the aorta- gonads-and mesonephros (AGM) region and reside within Intra-aortic hematopoietic clusters (IAHC) along with hematopoietic progenitors (HPC). The signalling mechanisms that distinguish HSCs from HPCs are unknown. Notch signaling is essential for arterial specification, IAHC formation and HSC activity, but current studies on how Notch segregates these different fates are inconsistent. We now demonstrate that Notch activity is highest in a subset of, GFI1 + , HSC-primed HE cells, and is gradually lost with HSC maturation. We uncover that the HSC phenotype is maintained due to increasing levels of NOTCH1 and JAG1 interactions on the surface of the same cell (cis) that renders the NOTCH1 receptor from being activated. Forced activation of the NOTCH1 receptor in IAHC activates a hematopoietic differentiation program. Our results indicate that NOTCH1-JAG1 cis-inhibition preserves the HSC phenotype in the hematopoietic clusters of the embryonic aorta.

Pharmacologic Reduction of Platelet Count Attenuates Whole Blood Hypofibrinolysis in a Mouse Model of Sickle Cell Disease

Increased risk of venous thrombosis is a hallmark of sickle cell disease (SCD). We previously reported that SCD dramatically alters the properties of venous clots, including resistance to tissue plasminogen activator (tPA)-mediated fibrinolysis. Using an internal fibrinolysis assay (tPA added to whole blood before clot formation), clot lysis time (CLT) was significantly (p<0.01) prolonged in clots formed ex vivo from whole blood of SCD patients (SS) (651±34 min) compared to race-matched controls (AA) (370±75). We then investigated how therapeutic RBC exchange affects CLT in SCD patients. Interestingly, despite hemoglobin A levels of approximately 50%, CLT for the pre-exchange SS clots (639±81 min) was similar to CLT recorded for blood clots from SS patients who did not undergo RBC exchange (see above). However, CLT for post-exchange SS clots was significantly shortened (240±34; p<0.05 vs pre-exchange) to the level observed for clots from AA controls. We noticed that the RBC exchange procedure not only changes the proportion of AA and SS RBCs, but also reduces both platelet counts and serum plasminogen activator inhibitor-1 (PAI-1) levels by approximately 50-60%. Importantly, exvivo restoration of the platelet number in post-exchange samples to the level observed in pre-exchange blood resulted in restoration of the hypofibrinolytic phenotype and a partial restoration of serum PAI-1 levels. Changes in platelet count after RBC exchange are quickly normalized. Therefore, in the current study, we investigated the effect of sustained reduction of platelet count on whole blood hypofibrinolysis in SCD mice. Using an external fibrinolysis assay (2.5 nM tPA added after clot formation) as described by Bonnard et al. (Sci Rep, 2017) we determined if hypofibrinolysis is also observed in Townes sickle cell mice (SS). Consistent with human SCD, the CLT was profoundly increased in ex vivo clots formed from whole blood of SS mice (n=13) compared to non-sickle (AA) control mice (n=12) (Figure 1A; first two bars). Next, we demonstrated that the cellular fraction of SS blood was responsible for resistance of the clots to tPA-mediated fibrinolysis, regardless of whether the plasma was from SS or AA mice (Figure 1A; last four bars). We then used a pharmacologic strategy to reduce platelet counts. Thrombopoietin (TPO) is a growth factor that promotes maturation of hematopoietic stem cells into megakaryocytes and thereby increases platelet production. We previously showed that silencing liver TPO expression using a liver-specific antisense oligonucleotide (TPO-ASO) resulted in a sustained 50% reduction in both plasma TPO levels and platelet counts without affecting hemostasis in both baboons and mice. In the present study, TPO-ASO (20 mg/kg subcutaneous) or vehicle control were administered to 3-month old AA and SS mice (n=3-6) on days 0, 3, 5 and 7, followed by weekly maintenance injections for the next 3 weeks. 4 weeks after initiation of treatment, mice were euthanized, and blood was collected to evaluate plasma levels of TPO and external whole blood fibrinolysis. Platelet, but no other cell count was significantly reduced in both AA (by 62%, p<0.05) and SS (by 47%, P<0.05) mice treated with TPO-ASO. Baseline plasma levels of TPO were reduced in SS mice (101±20 pg/ml) compared to AA controls (182±10; p<0.05). TPO-ASO treatment resulted in significant reduction of TPO plasma levels in both AA (21±16; p<0.01) and SS (17±9; p<0.05) mice. Interestingly, TPO-ASO-mediated reduction of platelet count did not affect CLT of ex vivo clots formed from whole blood of AA mice (Figure 1B). In contrast, TPO-ASO treatment dramatically reduced CLT in SS clots (p<0.05) (Figure 1B). In conclusion, we demonstrated that: (i) the resistance of ex vivo formed clots to tPA-mediated fibrinolysis observed in SCD patients can be reproduced in a mouse model of the disease; (i) the external fibrinolysis assay provides a robust and reliable model for studying whole blood hypofibrinolysis in SCD; (iii) hypofibrinolysis in SCD appears to be predominantly driven by the cellular fraction of blood; and (iv) platelets play an important role in the hypofibrinolytic phenotype in SCD, demonstrated by mitigation of the hypofibrinolytic phenotype in SS mice subjected to long-term reduction of platelet count. Further studies investigating the effect of platelet count reduction on venous thrombosis and vascular stasis in SS mice are currently ongoing.

Longitudinal Single Cell Multiomic Analysis of a Patient with GATA2 Haploinsufficiency with ASXL1 Mutant Clonal Hematopoiesis

Introduction: GATA2 haploinsufficiency syndrome (G2HS) is an autosomal dominant germline predisposition state with broad phenotypic variability and high risk of developing myeloid neoplasms (74.3%, median age of onset 17 years; Homan CC, et al. 2021). Mechanisms resulting in myeloid transformation remain to be elucidated. To identify molecular changes and relevant drivers of gene expression prior to progression, we performed serial, single cell multiomics on a proband with G2HS and ASXL1 mutant clonal hematopoiesis (CH) over 7 years. Case report: The proband was diagnosed with G2HS at age 19 years when he presented with recurrent infections, scrotal and penile lymphedema and cytopenias. Bone marrow (BM) biopsy at diagnosis was hypocellular with minimal atypia. Next generation sequencing (NGS) identified a novel pathogenic variant in GATA2 (NM_001145661.2: c.1143+200_1198delinsC; Dorn J et al, 2017) confirmed to be germline, and a somatic pathogenic variant in ASXL1 (NM_015338.6: c.2638dupA; p.Thr880Asnfs*2) with a variant allele frequency (VAF) of 11%, indicative of CH. Methods: After IRB approval, clinical data was abstracted from the medical records. Four aliquots of BM mononuclear cells from BM biopsies obtained 6 months apart were subjected to targeted NGS, single cell RNA (scRNA-seq) and scATAC-seq. Preprocessing and poor-quality cell filtering were performed using the Cell Ranger pipeline. For scRNA-seq, filtered counts were clustered and annotated using Seurat v4.1.0 onto a CITEseq PBMC reference (Hao Y, et al. 2021). Genes were ranked within each cluster between samples based on their average log-fold change, and gene set enrichment analysis (GSEA) was performed using the fgsea package v1.16.0. In parallel, filtered scATAC-seq data was integrated with scRNA using the cluster-label transfer from Signa v1.5.0 and Seurat v4.1.0. Using MAESTRO (Wang C, et al. 2020), we predicted the transcriptional factors (TF) regulating the expression from differentially expressed genes and differential peaks between samples based on the enrichment scores from algorithms LISA (Qin Q, et al. 2020) and GIGGLE (Layer RM, et al. 2018) for RNAseq and ATACseq respectively. Results: Serial blood cell counts, blood indices and BM biopsies over 7 years remained stable (macrocytosis, monocytopenia and neutropenia), with the BM demonstrating stable hypocellularity (10-20%) with minimal atypia and with no evidence for progression to myelodysplastic syndrome (MDS). ASXL1 mutant VAF also remained stable (11 to 16% at last measurement). Quantitative flow cytometric assessments in blood revealed decreases in B and NK cells. Analysis of scRNA/scATAC-seq successfully identified monocytes, B-cells, CD4, CD8 and NK Cells yielding comparable counts to those obtained by flow cytometry. The G2HS-associated cytopenias resulted in very small numbers of monocytes and B cells being identified, and hence differential gene expression analysis was limited to CD4+, CD8+ T cells and NK cells, between time points 1 and 3 (Figure 1A). Differentially expressed genes showed aberrant enrichment of downregulated genes in pathways related to hematopoietic stem cell (HSC) maturation in all three populations, while upregulated genes were enriched in cell cycle and innate immune responses. Analysis of regulatory drivers identified BRD4 as the main TF controlling the differentially expressed genes in CD4+ T cells and NK cells, and ERG in CD8+ T cells. The expression of these TF did not change between timepoints. (Figure 1B) Discussion: We present a GATA2 haploinsufficient proband who has remained stable and MDS-free over 7 years, even with ASXL1 mutant CH. Despite the stability in his blood counts and BM, single cell analysis has identified progressive qualitative changes impacting relevant pathways including HSC maturation, cell cycle regulation and innate immune responses in T and NK cells. In addition, our scATAC-seq data predicts BRD4 and ERG to be important epigenetic readers/TF that regulate these changes. We plan to study larger cohorts of patients and additional time points to understand transcriptional and epigenetic changes induced by GATA2 haploinsufficiency and elucidate the role of ASXL1 mutant CH in disease progression. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Liver Regeneration by Hematopoietic Stem Cells: Have We Reached the End of the Road?

The liver is the organ with the highest regenerative capacity in the human body. However, various insults, including viral infections, alcohol or drug abuse, and metabolic overload, may cause chronic inflammation and fibrosis, leading to irreversible liver dysfunction. Despite advances in surgery and pharmacological treatments, liver diseases remain a leading cause of death worldwide. To address the shortage of donor liver organs for orthotopic liver transplantation, cell therapy in liver disease has emerged as a promising regenerative treatment. Sources include primary hepatocytes or functional hepatocytes generated from the reprogramming of induced pluripotent stem cells (iPSC). Different types of stem cells have also been employed for transplantation to trigger regeneration, including hematopoietic stem cells (HSCs), mesenchymal stromal cells (MSCs), endothelial progenitor cells (EPCs) as well as adult and fetal liver progenitor cells. HSCs, usually defined by the expression of CD34 and CD133, and MSCs, defined by the expression of CD105, CD73, and CD90, are attractive sources due to their autologous nature, ease of isolation and cryopreservation. The present review focuses on the use of bone marrow HSCs for liver regeneration, presenting evidence for an ongoing crosstalk between the hematopoietic and the hepatic system. This relationship commences during embryogenesis when the fetal liver emerges as the crossroads between the two systems converging the presence of different origins of cells (mesoderm and endoderm) in the same organ. Ample evidence indicates that the fetal liver supports the maturation and expansion of HSCs during development but also later on in life. Moreover, the fact that the adult liver remains one of the few sites for extramedullary hematopoiesis—albeit pathological—suggests that this relationship between the two systems is ongoing. Can, however, the hematopoietic system offer similar support to the liver? The majority of clinical studies using hematopoietic cell transplantation in patients with liver disease report favourable observations. The underlying mechanism—whether paracrine, fusion or transdifferentiation or a combination of the three—remains to be confirmed.

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

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

Definitive Hematopoietic Stem Cells Minimally Contribute to Embryonic Hematopoiesis

Definitive hematopoietic stem cells (HSCs) emerge in the embryo and sustain major adult hematopoietic lineages. Although their functional potential is detected by transplant, nascent HSC contribution during development is both unknown and difficult to address due to the overlapping emergence of HSC-independent progenitors-cells that lack the multipotency and/or longevity of HSCs but express many of the same markers. Using sorted hematopoietic stem and progenitor cells from zebrafish embryos, we performed single cell RNA sequencing to decipher HSC and HSC-independent progenitor heterogeneity during the time frame of their emergence and initial maturation. After batch correction and dimensional reduction, we identified seven distinct populations that are inferred from RNA velocity analysis to originate from pre-hemogenic endothelium and develop into three main differentiation trajectories. We also determined that HSCs can be distinguished from HSC-independent progenitors based on the temporal regulation and differential activity of the draculin (drl) promoter that was previously shown to mark adult-contributing HSCs. From these studies, we found that the drl promoter is active in HSCs and HSC-independent progenitors at 1-day post-fertilization (dpf) but becomes highly expressed primarily in HSCs by 2 dpf. We applied a drl:cre-ER T2 tamoxifen-inducible Cre-loxP lineage-tracing approach to selectively lineage trace HSCs starting at 2 dpf and track their myeloid and lymphoid contribution during larval development and adulthood. We determined that HSC-independent progenitors primarily contribute to developmental lymphomyelopoiesis with minimal HSC contribution until after 7 dpf. Consistent with this result, we demonstrated that although HSCs robustly regenerated after hematopoietic injury using a novel inducible larval HSC injury model, their depletion had almost no impact on lymphoid and myeloid cell numbers up to 7 dpf. These findings suggest that HSCs are not entirely dormant during development and that there exists an uncoupling of HSC self-renewal and differentiation in development. In conclusion, we determine that it is the HSC-independent progenitors, and not HSCs, that sustain embryonic and early larval lymphomyelopoiesis. Acquiring a greater understanding regarding developmental differences in progenitor and HSC specification and maturation will inform and improve the generation of functional HSCs from renewable pluripotent stem cells. DisclosuresNo relevant conflicts of interest to declare.

Regulation of Ribosome Function by RNA Modifications in Hematopoietic Development and Leukemia: It Is Not Only a Matter of m6A.

Growth and maturation of hematopoietic stem cells (HSCs) are largely controlled at both transcriptional and post-transcriptional levels. In particular, hematopoietic development requires a tight control of protein synthesis. Furthermore, translational deregulation strongly contributes to hematopoietic malignancies. Researchers have recently identified a new layer of gene expression regulation that consists of chemical modification of RNA species, which led to the birth of the epitranscriptomics field. RNA modifications provide an additional level of control in hematopoietic development by acting as post-transcriptional regulators of lineage-specific genetic programs. Other reviews have already described the important role of the N6-methylation of adenosine (m6A) within mRNA species in regulating hematopoietic differentiation and diseases. The aim of this review is to summarize the current status of the role of RNA modifications in the regulation of ribosome function, beyond m6A. In particular, we discuss the importance of RNA modifications in tRNA and rRNA molecules. By balancing translational rate and fidelity, they play an important role in regulating normal and malignant hematopoietic development.

Integrative transcriptomic analysis of developing hematopoietic stem cells in human and mouse at single-cell resolution

Current understanding of hematopoietic stem cell (HSC) development comes from mouse models is considered to be evolutionarily conserved in human. However, the cross-species comparison of the transcriptomic profiles of developmental HSCs at single-cell level is still lacking. Here, we performed integrative transcriptomic analysis of a series of key cell populations during HSC development in human and mouse, including HSC-primed hemogenic endothelial cells and pre-HSCs in mid-gestational aorta-gonad-mesonephros (AGM) region, and mature HSCs in fetal liver and adult bone marrow. We demonstrated the general similarity of transcriptomic characteristics between corresponding cell populations of the two species. Of note, one of the previously transcriptomically defined hematopoietic stem progenitor cell (HSPC) populations with certain arterial characteristics in AGM region of human embryos showed close transcriptomic similarity to pre-HSCs in mouse embryos. On the other hand, the other two HSPC populations in human AGM region displayed molecular similarity with fetal liver HSPCs, suggesting the maturation in AGM before HSCs colonizing the fetal liver in human, which was different to that in mouse. Finally, we re-clustered cells based on the integrated dataset and illustrated the evolutionarily conserved molecular signatures of major cell populations. Our results revealed transcriptomic conservation of critical cell populations and molecular characteristics during HSC development between human and mouse, providing a resource and theoretic basis for future studies on mammalian HSC development and regeneration by using mouse models.

1616. Mechanism of Thrombocytopenia Induced by Oxazolidinones Antibiotics (Linezolid, Tedizolid): Demonstration of Impairment of Megakaryocyte Differentiation From Human Hematopoietic Stem Cells associated with Mitochondrial Toxicity

BackgroundLinezolid causes thrombocytopenia, which limits its use. In cell culture and in tissues from treated patients, linezolid impairs mitochondrial protein synthesis (due to structural similarities and common binding sites between bacterial and mitochondrial ribosomes). Recent studies have shown that mitochondria act as a key relay in the process leading from activation of the thrombopoietin receptor to megakaryocytes differentiation.MethodsValidated ex-vivo human model of hematopoietic stem cells (HSC) differentiation for (i) measuring megakaryocytes, granulocyte-monocytes, and burst-forming unit-erythroids colony formation; (ii) differentiation into megakaryocytes (conversion of CD34+ into CD41+/CD42+ cells; morphology) and proplatelets formation, (iii) mitochondrial toxicity (electron microscopy; cytochrome c-oxidase activity [partly encoded by the mitochondrial genome]).ResultsWe show that linezolid (and the recently approved tedizolid), both at concentrations corresponding to their human serum concentrations) inhibit the maturation of HSC into fully differentiated megakaryocytes (CD41 and CD42-positive cells) and the formation of proplatelets. Optic and Electron microscopy) showed an impairment of the formation of typical megakaryocytes (lack of large polylobulated nuclei and of intracellular demarcation membrane system [required for platelet formation]), together with disappearance of the internal structure of mitochondria. Biochemical studies showed a complete suppression of the activity of cytochrome c-oxidase (a key enzyme of the mitochondrial respiratory chain).ConclusionOur study provides for the first time insights in the mechanism of thrombocytopenia induced by linezolid and tedizolid, identifying mitochondria as their target and showing that the drugs will impair the differentiation of hematopoietic stem cells into mature platelets-releasing megakaryocytes. It illustrates how mitochondria dysfunction may play a key role in toxicology and diseases, while paving the way for rational approaches for the design and screening of less toxic derivatives for the benefit of future patients.Disclosures Paul M. Tulkens, MD, PhD, Bayer (Consultant, Advisor or Review Panel member, Speaker’s Bureau)Menarini (Speaker’s Bureau)Merck (Advisor or Review Panel member, Speaker’s Bureau)Trius (now part of Merck) (Advisor or Review Panel member, Research Grant or Support) Françoise Van Bambeke, PharmD, PhD, Bayer (Speaker’s Bureau)

Hematopoietic Jagged1 Is Required for the Transition of Hematopoietic Stem Cells from the Fetal Liver to the Adult Bone Marrow Niche

Notch signaling is known to play important roles in hematopoietic development and differentiation. Notch1 is required for emergence of the definitive hematopoietic stem cells (HSCs) from the hemogenic endothelium, and we have previously shown that Notch signaling is essential for survival and function of HSCs in the fetal liver. Activation of canonical Notch signaling requires direct cellular contact; thus, the identity of the ligand and the ligand-presenting cell during hematopoietic development would provide valuable information of the Notch signaling mechanism in HSCs as well as the identity of key niche cells that drive the expansion and cell fate decisions of embryonic HSCs. In the present study, we have taken a comprehensive approach to determine the ligands and cells that initiate Notch signaling in the mouse fetal liver. To this end, we have performed single-cell PCR analysis for all Notch signaling proteins in E14.5 fetal HSCs and compared the findings to the adult bone marrow HSCs. We also have analyzed fetal liver endothelial cells for surface expression of all Notch ligands. We determined that Jagged1 (Jag1) is highly expressed in both endothelial cells as well as in fetal HSCs but not adult HSCs. We have performed conditional loss-of-function analysis of Jag1 in fetal endothelial cells using inducible Ve-cadherinCreERT2 as well as in fetal hematopoietic lineages using constitutive VavCre. Our results indicate that while loss of endothelial Jag1 has severe effects in embryonic vascular development, loss of hematopoietic Jag1 allows for normal fetal morphology, yet severely impedes the functional ability of fetal liver HSCs to expand and differentiate both in vitro and in vivo. Fetal to adult transplantation of VavCre+Jag1f/f HSCs indicated a defect in reconstitution potential of fetal HSCs that lack Jag1 expression. Our findings indicate that hematopoietic Jag1 is essential for maturation of HSCs in the fetal liver and for homing and reconstitution potential of HSCs into the post-natal bone marrow microenvironment. Disclosures No relevant conflicts of interest to declare.

3004 – IDENTIFICATION AND CHARACTERIZATION OF NOVEL FUNCTIONAL MARKERS DURING THE HEMATOPOIETIC STEM CELL SPECIFICATION PROCESS

Endothelial to hematopoietic transition (EHT) is a crucial step in the formation of definitive hematopoietic stem cells during embryonic development. Holding such a critical developmental role not many details regarding molecular changes and cell surface marker (CSM) expression on these transitional cells are known. We think that sub-segregating the process of EHT holds great therapeutic potential by giving new indications to understand the hematopoietic stem cell maturation. We generated embryonic stem (ES) cell lines from Hoxb4-YFP reporter mice (Hills, 2011), in which YFP expression marks functional HSCs in adult and embryonic mice. When these cell lines were subject to embryoid body differentiation assays, we observed a transient Hoxb4/YFP+ cell population, which corresponded to the emergence of hemogenic endothelial cells in culture. We then analyzed the gene expression profile of the Hoxb4+ cells and compared it to immediate precursor (Hoxb4-Flk1+) and daughter (Hoxb4-CD41+) cell populations. As well as documenting the expression of numerous molecular markers previously associated with EHT, we also observed an elevated inflammatory gene expression signature that has previously been characterized as a mediator of hematopoietic specification in vivo. We were additionally able to identify 45 novel cell surface markers that could potentially be used to prospectively isolate and sub segregate cells undergoing EHT. 26 of these targets were subsequently verified using MRM mass spectrometry. To establish if any of these markers were functionally relevant, we generated knockout (KO) cell lines using CRISPR/Cas9. Upon EB differentiation, we observed a profound block in hematopoietic differentiation for ES cells that were KO for the membrane proteins Evi2a and Lyve1. This block was manifest at the EHT stage, as verified by time-lapse video imaging, and was equivalent in magnitude to Runx1 KO, suggesting a crucial role of Evi2a and Lyve1 during the EHT stage of hematopoietic development. Notably, this defect could be rescued by genetic replacement of the deleted gene. When comparing our data to human fetal liver data sets Evi2a is highly expressed in fetal liver, as well as in HSCs isolated from murine AGM and fetal liver.

A non-linear reverse-engineering method for inferring genetic regulatory networks.

Hematopoiesis is a highly complex developmental process that produces various types of blood cells. This process is regulated by different genetic networks that control the proliferation, differentiation, and maturation of hematopoietic stem cells (HSCs). Although substantial progress has been made for understanding hematopoiesis, the detailed regulatory mechanisms for the fate determination of HSCs are still unraveled. In this study, we propose a novel approach to infer the detailed regulatory mechanisms. This work is designed to develop a mathematical framework that is able to realize nonlinear gene expression dynamics accurately. In particular, we intended to investigate the effect of possible protein heterodimers and/or synergistic effect in genetic regulation. This approach includes the Extended Forward Search Algorithm to infer network structure (top-down approach) and a non-linear mathematical model to infer dynamical property (bottom-up approach). Based on the published experimental data, we study two regulatory networks of 11 genes for regulating the erythrocyte differentiation pathway and the neutrophil differentiation pathway. The proposed algorithm is first applied to predict the network topologies among 11 genes and 55 non-linear terms which may be for heterodimers and/or synergistic effect. Then, the unknown model parameters are estimated by fitting simulations to the expression data of two different differentiation pathways. In addition, the edge deletion test is conducted to remove possible insignificant regulations from the inferred networks. Furthermore, the robustness property of the mathematical model is employed as an additional criterion to choose better network reconstruction results. Our simulation results successfully realized experimental data for two different differentiation pathways, which suggests that the proposed approach is an effective method to infer the topological structure and dynamic property of genetic regulations.

Transcriptome Dynamics of Hematopoietic Stem Cell Formation Revealed Using a Combinatorial Runx1 and Ly6a Reporter System.

SummaryStudies of hematopoietic stem cell (HSC) development from pre-HSC-producing hemogenic endothelial cells (HECs) are hampered by the rarity of these cells and the presence of other cell types with overlapping marker expression profiles. We generated a Tg(Runx1-mKO2; Ly6a-GFP) dual reporter mouse to visualize hematopoietic commitment and study pre-HSC emergence and maturation. Runx1-mKO2 marked all intra-arterial HECs and hematopoietic cluster cells (HCCs), including pre-HSCs, myeloid- and lymphoid progenitors, and HSCs themselves. However, HSC and lymphoid potential were almost exclusively found in reporter double-positive (DP) cells. Robust HSC activity was first detected in DP cells of the placenta, reflecting the importance of this niche for (pre-)HSC maturation and expansion before the fetal liver stage. A time course analysis by single-cell RNA sequencing revealed that as pre-HSCs mature into fetal liver stage HSCs, they show signs of interferon exposure, exhibit signatures of multi-lineage differentiation gene expression, and develop a prolonged cell cycle reminiscent of quiescent adult HSCs.

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.

Embryonic lineage tracing with Procr-CreER marks balanced hematopoietic stem cell fate during entire mouse lifespan

The functional heterogeneity of hematopoietic stem cells (HSCs) has been comprehensively investigated by single-cell transplantation assay. However, the heterogeneity regarding their physiological contribution remains an open question, especially for those with life-long hematopoietic fate of rigorous self-renewing and balanced differentiation capacities. In this study, we revealed that Procr expression was detected principally in phenotypical vascular endothelium co-expressing Dll4 and CD44 in the mid-gestation mouse embryos, and could enrich all the HSCs of the embryonic day 11.5 (E11.5) aorta-gonad-mesonephros (AGM) region. We then used a temporally restricted genetic tracing strategy to irreversibly label the Procr-expressing cells at E9.5. Interestingly, most labeled mature HSCs in multiple sites (such as AGM) around E11.5 were functionally categorized as lymphomyeloid-balanced HSCs assessed by direct transplantation. Furthermore, the labeled cells contributed to an average of 7.8% of immunophenotypically defined HSCs in E14.5 fetal liver (FL) and 6.9% of leukocytes in peripheral blood (PB) during one-year follow-up. Surprisingly, in aged mice of 24 months, the embryonically tagged cells displayed constant contribution to leukocytes with no bias to myeloid or lymphoid lineages. Altogether, we demonstrated, for the first time, the existence of a subtype of physiologically long-lived balanced HSCs as hypothesized, whose precise embryonic origin and molecular identity await further characterization.

NK Cell Precursors in Human Bone Marrow in Health and Inflammation.

NK cells are generated from hematopoietic stem cells (HSC) residing in the bone marrow (BM), similar to other blood cells. Development toward mature NK cells occurs largely outside the BM through travel of CD34+ and other progenitor intermediates toward secondary lymphoid organs. The BM harbors multipotent CD34+ common lymphoid progenitors (CLPs) that generate T, B, NK, and Dendritic Cells and are devoid of erythroid, myeloid, and megakaryocytic potential. Over recent years, there has been a quest for single-lineage progenitors predominantly with the objective of manipulation and intervention in mind, which has led to the identification of unipotent NK cell progenitors devoid of other lymphoid lineage potential. Research efforts for the study of lymphopoiesis have almost exclusively concentrated on healthy donor tissues and on repopulation/transplant models. This has led to the widely accepted assumption that lymphopoiesis during disease states reflects the findings of these models. However, compelling evidences in animal models show that inflammation plays a fundamental role in the regulation of HSC maturation and release in the BM niches through several mechanisms including modulation of the CXCL12-CXCR4 expression. Indeed, recent findings during systemic inflammation in patients provide evidence that a so-far overlooked CLP exists in the BM (Lin−CD34+DNAM-1brightCXCR4+) and that it overwhelmingly exits the BM during systemic inflammation. These “inflammatory” precursors have a developmental trajectory toward surprisingly functional NK and T cells as reviewed here and mirror the steady state maintenance of the NK cell pool by CD34+DNAM-1−CXCR4− precursors. Our understanding of NK cell precursor development may benefit from including a distinct “inflammatory” progenitor modeling of lymphoid precursors, allowing rapid deployment of specialized Lin−CD34+DNAM-1brightCXCR4+ -derived resources from the BM.

Metabolic Regulations in Hematopoietic Stem Cells.

One of the bottlenecks of the treatments for malignant hematopoietic disorders is the unavailability of sufficient amount of hematopoietic stem cells (HSCs). HSCs areconsidered to be originated from the aorta-gonad-mesonephros and gradually migrates into fetal liver and resides in a unique microenvironment/niche of bone marrow. Although many intrinsic and extrinsic factors (niche components) are reported to be involved in the origination, maturation, migration, and localization of HSCs at different developmental stages, the detailed molecular mechanisms still remain largely unknown. Previous studies have shown that intrinsic metabolic networks may be critical for the cell fate determinations of HSCs. For example, HSCs mainly utilize glycolysis as the main energy sources; oxidative phosphorylation is required for the homeostasis of HSCs; lipid or amino acid metabolisms may also sustain HSC stemness. Mechanistically, lots of regulatory pathways, such as MEIS1/HIF1A and PI3K/AKT/mTOR signaling, are found to fine-tune the different nutrient metabolisms and cell fate commitments of HSCs. However, more efforts are required for the optimization and establishment of precise metabolic techniques specific for the HSCs with relatively rare cell frequency and understanding of the basic metabolic properties and their underlying regulatory mechanisms of different nutrients (such as glucose) during the different developmental stages of HSCs.

The VWRPY Domain Is Essential for RUNX1 Function in Hematopoietic Progenitor Cell Maturation and Megakaryocyte Differentiation

RUNX1 is a transcription factor essential during definitive hematopoiesis. Germline mutations in RUNX1 results in a disorder called Familial Platelet Disorder with Associated Myeloid Malignancy (FPDMM). FPDMM patients have abnormal bleeding due to reduced platelet count and/or function. Importantly, 20-60% of the FPDMM patients develop hematological malignancies, which are mainly myeloid. Reported RUNX1 mutations in FPDMM families are mostly clustered in the N-terminal runt domain and the C-terminal transactivation domain. Recently, three mutations have been reported at or near the end of the C-terminal repression domain, the VWRPY motif. However, the mechanism behind the VWRPY motif involvement in the FPDMM pathogenesis has not been studied. Interestingly, these VWRPY-mutated RUNX1 proteins still have intact runt and transactivation domains, but patients still show FPDMM phenotype. Here, we evaluate the functional defects of a RUNX1 mutation, L472fsX, which was reported in a FPDMM family, using three different experimental models. Our study is aimed to unravel the significance of the VWRPY motif in FPDMM pathogenesis.The RUNX1 L472fsX mutation is caused by a GC insertion upstream of the VWRPY motif. The mutation results in a frameshift and a run on protein for an additional 123 amino acids. The frameshift abolishes the VWRPY motif, which is responsible for the binding between RUNX1 and a co-repressor protein, TLE1. As expected, from both FRET and co-IP assays, the mutated RUNX1 lost binding with TLE1. Interestingly, we observed increased binding between the mutated RUNX1 and its co-factor CBFβ in the FRET assay, as compared to the wildtype RUNX1. Furthermore, in reporter assays we found that TLE1 failed to repress the expression of a RUNX1 target, M-CSFR promoter, when co-transfected with the mutated RUNX1, which is contrary to what has been seen with wildtype RUNX1. Consistent with increased binding between the mutated RUNX1 and CBFβ in the FRET assay, co-transfecting mutant RUNX1 and CBFβ resulted in a significant increase of M-CSFR promoter expression as compared to wildtype RUNX1 with CBFβ. For another RUNX1 target, Hmga2, wildtype RUNX1 and CBFβ decreased Hmga2 expression, which could be restored by adding TLE1. Transfected mutant RUNX1 and CBFβ also decreased Hmga2 expression, but TLE1 could not restore Hmga2 expression when co-transfected with the mutant RUNX1. These findings suggest that the VWRPY-disrupting L472fsX mutation leads to the loss of binding between mutant RUNX1 and TLE1, which in turn resulted in defective repression of the RUNX1 activity by TLE1.To assess for hematopoietic defects in the FPDMM patients with the L472fsX mutation, blood cells from two family members were reprogrammed to induced pluripotent stem cells (iPSCs). Similar to previous studies, iPSCs from these patients gave rise to fewer megakaryocyte progenitors and mature megakaryocytes during in vitro differentiation. In addition, these FPDMM iPSCs showed decrease in hematopoietic stem cell (HSCs) maturation and differentiation to progenitors. The L472fsX mutation in the iPSCs was then corrected by genome editing using zinc finger nuclease. Importantly, the hematopoietic defects of the FPDMM iPSCs mentioned above were rescued after mutation correction. Overall, the findings in these iPSCs differentiation assays showed that the VWRPY motif is essential for RUNX1 activity in megakaryocytes differentiation. In addition, the VWRPY motif is important for HSCs maturation and differentiation to progenitors.To evaluate the impact of this VWRPY-deletion mutation on hematopoiesis in an in vivo model, CRISPR-mediated genome editing was used to generate mice with frameshift mutations that remove the VWRPY domain. Preliminary observations showed that the mutant mice have minor defects in the peripheral blood. More data on this mouse model will be presented at the meeting.In conclusion, we present a novel RUNX1 mutation (L472fsX) with unique hematopoietic defect that has not been reported previously in FPDMM. Our findings imply the significance of the VWRPY motif in megakaryopoiesis, as well as HSCs maturation and differentiation. DisclosuresNo relevant conflicts of interest to declare.

Development of Human Mast Cells from Hematopoietic Stem Cells within a 3D Collagen Matrix: Effect of Stem Cell Media on Mast Cell Generation.

Mast cells (MCs) arise from hematopoietic stem cells (HSCs) that mature within vascularized tissues. Fibroblasts and endothelial cells (ECs) play a role in the maturation of HSCs in the tissues. Due to difficulties in isolating MCs from tissues, large numbers of committed MC precursors can be generated in 2D culture systems with the use of differentiation factors. Since MCs are tissue-resident cells, the development of a 3D tissue-engineered model with ancillary cells that more closely mimics the 3D in vivo microenvironment has greater relevance for MC studies. The goals of this study were to show that MCs can be derived from HSCs within a 3D matrix and to determine a media to support MCs, fibroblasts, and ECs. The results show that HSCs within a collagen matrix cultured in StemSpan media with serum added at the last week yielded a greater number of c-kit+ cells and a greater amount of histamine granules compared to other media tested. Media supplemented with serum were necessary for EC survival, while fibroblasts survived irrespective of serum with higher cell yields in StemSpan. This work demonstrates the development of functional MCs within a 3D collagen matrix using a stem cell media that supports fibroblast and ECs.

Epirubicin induces apoptosis in osteoblasts through death-receptor and mitochondrial pathways.

Epirubicin is an anthracycline and is widely used in tumor treatment, but has toxic and undesirable side effects on wide range of cells and hematopoietic stem cells (HSC). Osteoblasts play important roles in bone development and in supporting HSC differentiation and maturation. It remains unknown whether epirubicin-induced bone loss and hematological toxicity are associated with its effect on osteoblasts. In primary osteoblast cell cultures, epirubicin inhibited cell growth and decreased mineralization. Moreover, epirubicin arrested osteoblasts in the G2/M phase, and this arrest was followed by apoptosis in which both the extrinsic (death receptor-mediated) and intrinsic (mitochondrial-mediated) apoptotic pathways were evoked. The factors involved in the extrinsic apoptotic pathway were increased FasL and FADD as well as activated caspase-8. Those involved in the intrinsic apoptotic pathway were decreased Bcl-2; increased reactive oxygen species, Bax, cytochrome c; and activated caspase-9 and caspase-3. These results demonstrate that epirubicin induced osteoblast apoptosis through the extrinsic and intrinsic apoptotic pathways, leading to the destruction of osteoblasts and consequent lessening of their functions in maintaining bone density and supporting hematopoietic stem cell differentiation and maturation.