Adult Bone Marrow Hematopoietic Stem Cells Research Articles

Hematopoietic Jagged1 is a fetal liver niche factor required for functional maturation and engraftment of fetal hematopoietic stem cells

Notch signaling is essential for the emergence of definitive hematopoietic stem cells (HSCs) in the embryo and their development in the fetal liver niche. However, how Notch signaling is activated and which fetal liver cell type provides the ligand for receptor activation in HSCs is unknown. Here we provide evidence that endothelial Jagged1 (Jag1) has a critical early role in fetal liver vascular development but is not required for hematopoietic function during fetal HSC expansion. We demonstrate that Jag1 is expressed in many hematopoietic cells in the fetal liver, including HSCs, and that its expression is lost in adult bone marrow HSCs. Deletion of hematopoietic Jag1 does not affect fetal liver development; however, Jag1-deficient fetal liver HSCs exhibit a significant transplantation defect. Bulk and single-cell transcriptomic analysis of HSCs during peak expansion in the fetal liver indicates that loss of hematopoietic Jag1 leads to the downregulation of critical hematopoietic factors such as GATA2, Mllt3, and HoxA7, but does not perturb Notch receptor expression. Exvivo activation of Notch signaling in Jag1-deficient fetal HSCs partially rescues the functional defect in a transplant setting. These findings indicate a new fetal-specific niche that is based on juxtracrine hematopoietic Notch signaling and reveal Jag1 as a fetal-specific niche factor essential for HSC function.

Timing and regulation of hematopoietic stem cell colonization of the human fetal bone marrow by endothelial and CAR stromal cells during pregnancy

The bone marrow (BM) contains the major reservoir of immature and maturing hematopoietic and immune cells throughout adult life while also harboring most hematopoietic stem and progenitor cells (HSPCs). BM-retained hematopoietic stem cells (HSCs) are mostly maintained in a quiescent, non-motile mode. A small fraction of BM-retained HSPC daily proliferate, differentiate, and migrate to the circulation, to replenish the blood with new immature and maturing blood and (all) immune (both myeloid and lymphoid) cells with a finite life span.1 The anti-coagulation and anti-inflammatory receptor EPCR is also functionally expressed by primitive BM-retained HSCs which are endowed with the highest competitive long-term repopulation potential (LT-HSC). Only BM-retained, quiescent EPCR-positive LT-HSCs are protected from DNA damaging insults including clinical chemotherapy and radiation treatments. Primitive EPCR-positive LT-HSC chemotherapy resistance requires the CXCL12-CXCR4 axis that also regulates HSC quiescence, cell cycle, and directional migration as well as the aPC/EPCR/PAR1 axis.2,3 The chemokine CXCL12 is highly expressed by many BM endothelial and stromal cells types including HSC niche supporting osteoprogenitor cells termed CXCL12 abundant reticular cells (CAR cells), while primitive EPCR-positive fetal liver and adult BM HSC functionally express its major receptor CXCR4.4 Most functional HSC studies involve mice experimental preclinical models as well as results obtained from clinical BM transplantation protocols. During fetal development, HSCs migrate from the fetal liver to the fetal BM and spleen for their lodgment and repopulation. Stem cell homing to the BM during development and in functional experimental transplantation assays including with human HSC in transplanted immune deficient mice is CXCL12/CXCR4-dependent.5,6 The precise timepoint of HSC colonization of the human fetal BM and spleen during pregnancy, as well as the identity of BM stromal niche cells and ligand–receptor interactions which regulate human BM HSC lodgment during fetal development are poorly understood. The group of Zheng et al7 have recently reported on the emergence of HSC in the human fetal BM and spleen and their landscape microenvironment during pregnancy by single-cell RNAseq analysis. This work revealed that functional, human fetal LT-HSC as assayed in transplanted immune deficient mice do not emerge before week 12 post conception. By careful examination of various types of fetal BM stromal and endothelial cells during weeks 10–14 post conception and their transcriptome, this study revealed that fetal BM CAR-like cells endowed with the high levels of CXCL12 production together with fetal endothelial cells which also highly express CXCL12 are the major LT-HSC niche supporting cells. Interestingly, the human fetal spleen was not repopulated with functional human LT-HSC before week 14 post conception, revealing that fetal BM is superior in providing critical signals for LT-HSC homing and lodgment over the fetal spleen. Importantly, self-renewing human CD146 positive osteoprogenitor CAR-like cells were detected in adult human BM and were found to provide a niche for adult human LT-HSC as well as in a miniature human bone implanted in immune deficient mice.8 In conclusion, this study provides evidence for the first functional fetal BM LT-HSC as well as their BM supporting microenvironment and niches.

Clonal Analysis Reveals Functional and Transcriptional Heterogeneity of Self-Renewing Fetal Liver Hematopoietic Stem Cells

The fetal liver (FL) is a key organ supporting the expansion of hematopoietic stem cells (HSCs) during development. The ability to expand transplantable HSCs in vitro is a long-standing challenging; thus, decoding the gene pathways that regulate proliferation and self-renewal during FL-HSC development is highly relevant to engineering HSCs for therapeutic applications. Moreover, while heterogeneity in self-renewal and engraftment properties of HSCs in adult bone marrow has been extensively studied, these properties remain largely uncharacterized in FL-HSCs. To explore FL-HSC properties at the single cell level, we engineered a serum-free culture platform designed to recapitulate the murine FL endothelial niche (FL-EC) (Figure1), and sorted single FL-HSC (CD45+GR1-F4/80-SCA1highEPCRhigh) into individual wells containing FL-EC. Following co-culture, we observed emergence of immunophenotypically distinct types of hematopoietic colonies as determined by flow cytometry. Only colonies containing cells predominantly expressing high levels of HSC markers SCA1 and EPCR (Figure1, HSC colonies) provided multilineage engraftment as determined by transplantation assays, whereas colonies containing cells expressing HSC markers and more differentiated progeny (mixed colonies) were unable to provide serial multilineage engraftment. Furthermore, cellular progeny from a single HSC colony transplanted into multiple recipients demonstrated serial engraftment, indicating the FL-EC niche could amplify single FL-HSC with long-term reconstitution capacity. These studies suggest that transplantable FL-HSCs are uniquely distinguished by their propensity for symmetric self-renewal in this assay. However, not all HSC colonies provided engraftment in secondary recipients, suggesting heterogeneity in self-renewal properties of immunophenotypically defined FL-HSCs that required deeper analysis. Thus, to further assess transcriptional heterogeneity in HSC colonies, we next integrated single-cell RNA sequencing (scRNAseq) into the clonal FL-HSC assay (Figure 2). Residual cells from six HSC colonies were divided for simultaneous scRNAseq and transplantation. Consistent with experiments above, we observed heterogeneity in long-term serial engraftment from individual colonies. Remarkably, cells from the colony capable of serial engraftment were distinctly enriched in genes associated with dormant HSCs in adult marrow (Cabezas-Wallscheid et.al., Cell 2017). Conversely, cells from HSC colonies that lacked serial engraftment capacity exhibited increased expression of Myc target genes by Gene Set Enrichment Analysis (GSEA), and ribosome biogenesis and mitochondrial translation by Gene Ontogeny (GO) analysis, which are hallmarks of biosynthetically activated HSCs in adult marrow that are primed for differentiation to progenitor states. To further assess the transcriptional heterogeneity of primary FL-HSC, we next performed scRNAseq with freshly isolated FL-HSC. Unsupervised clustering and co-regulated modules of differentially expressed genes identified a rare subset of cells highly enriched for genes associated with HSC dormancy, while the other cells featured higher expression of genes involved in biosynthetic processes via GO analysis (e.g. cytoplasmic translation, rRNA processing). This data further supports transcriptional heterogeneity within the FL-HSC compartment, which includes a unique biosynthetically dormant subset. Collectively, our studies elucidate previously unrecognized heterogeneity in FL-HSCs, highlighting a state of relative biosynthetic dormancy as a key to symmetric self-renewal of serially engraftable FL-HSCs. These findings complement recent studies demonstrating distinct developmental origins for multipotent progenitors and functional engraftable HSCs in definitive hematopoiesis (Dignum et.al., Cell Reports 2021; Patel et.al., Nature 2022), suggesting functional heterogeneity in immunophenotypically defined FL-HSCs in our study may in part reflect these distinct ontogenies. Building on these studies, we are currently using CRISPR-Cas9 gene knockout to define the roles of candidate genes identified by our scRNAseq analysis that are implicated in regulating dormancy and symmetric self-renewal of FL-HSCs, which would provide essential insight for future strategies for ex vivo amplification of HSCs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Notch Ligand Jagged1 Is a Fetal Liver Niche Factor for the Function of Embryonic Hematopoietic Stem Cells

Embryonic hematopoietic stem cells (HSC) expand rapidly during development in the fetal liver. Notch1 is required for emergence of the definitive hematopoietic stem cells (HSCs) from the hemogenic endothelium, and is essential for survival and function of HSCs in the fetal liver. 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 analysis for all Notch signaling proteins and many known targets in E14.5 fetal HSCs and adult bone marrow HSCs as well as fetal liver endothelial cells. We determined that Jagged1 (Jag1) is highly expressed in both endothelial cells as well as in fetal HSCs but not in adult HSCs. We have performed conditional loss-of-function analysis of Jag1 in fetal endothelial cells as well as in fetal hematopoietic lineages, where both myeloid and megakaryocytic progenitors are shown to express high levels of Jag1. 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. RNA-Sequencing analysis of long-term fetal HSCs in Jag1-mutant embryos (VavCreJag f/f) revealed reduced expression of Gata2, Mllt3, Hoxa7, Angpt1 and IL-12a genes in fetal HSCs, which are well-known regulators of self-renewal and expansion. Our findings indicate that Jag1 is an essential niche factor for development of HSCs in the fetal liver and for functional potential of fetal HSCs once in the bone marrow microenvironment. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Hematopoietic Stem Cells: Nature and Niche Nurture.

Like all cells, hematopoietic stem cells (HSCs) and their offspring, the hematopoietic progenitor cells (HPCs), are highly sociable. Their capacity to interact with bone marrow niche cells and respond to environmental cytokines orchestrates the generation of the different types of blood and immune cells. The starting point for engineering hematopoiesis ex vivo is the nature of HSCs, and a longstanding premise is that they are a homogeneous population of cells. However, recent findings have shown that adult bone marrow HSCs are really a mixture of cells, with many having lineage affiliations. A second key consideration is: Do HSCs “choose” a lineage in a random and cell-intrinsic manner, or are they instructed by cytokines? Since their discovery, the hematopoietic cytokines have been viewed as survival and proliferation factors for lineage committed HPCs. Some are now known to also instruct cell lineage choice. These fundamental changes to our understanding of hematopoiesis are important for placing niche support in the right context and for fabricating an ex vivo environment to support HSC development.

Unexpected redundancy of Gpr56 and Gpr97 during hematopoietic cell development and differentiation

AbstractIntegrated molecular signals regulate cell fate decisions in the embryonic aortic endothelium to drive hematopoietic stem cell (HSC) generation during development. The G-protein–coupled receptor 56 (Gpr56, also called Adgrg1) is the most highly upregulated receptor gene in cells that take on hematopoietic fate and is expressed by adult bone marrow HSCs. Despite the requirement for Gpr56 in hematopoietic stem/progenitor cell (HS/PC) generation in zebrafish embryos and the highly upregulated expression of GPR56 in treatment-resistant leukemic patients, its function in normal mammalian hematopoiesis remains unclear. Here, we examine the role of Gpr56 in HS/PC development in Gpr56 conditional knockout (cKO) mouse embryos and Gpr knockout (KO) embryonic stem cell (ESC) hematopoietic differentiation cultures. Our results show a bias toward myeloid differentiation of Gpr56 cKO fetal liver HSCs and an increased definitive myeloid progenitor cell frequency in Gpr56KO ESC differentiation cultures. Surprisingly, we find that mouse Gpr97 can rescue Gpr56 morphant zebrafish hematopoietic generation, and that Gpr97 expression is upregulated in mouse Gpr56 deletion models. When both Gpr56 and Gpr97 are deleted in ESCs, no or few hematopoietic PCs (HPCs) are generated upon ESC differentiation. Together, our results reveal novel and redundant functions for these 2 G-protein coupled receptors in normal mammalian hematopoietic cell development and differentiation.

Sry-related High Mobility Group Box 17 Functions as a Tumor Suppressor by Antagonizing the Wingless-related Integration Site Pathway.

A transcription factor Sry-related high mobility group box (Sox) 17 is involved in developmental processes including spermatogenesis, cardiovascular system, endoderm formation, and so on. In this article, we firstly review the studies on the relation between the Sox17 expression and tumor malignancy. Although Sox17 positively promotes various tissue development, most of the cancers associated with Sox17 show decreased expression levels of Sox17, and an inverse correlation between Sox17 expression and malignancy is revealed. We briefly discuss the mechanism of such Sox17 down-regulation by focusing on DNA methylation of CpG sites located in the Sox17 gene promoter. Next, we overview the function of Sox17 in the fetal hematopoiesis, particularly in the dorsal aorta in midgestation mouse embryos. The Sox17 expression in hematopoietic stem cell (HSC)-containing intra-aortic hematopoietic cell cluster (IAHCs) is important for the cluster formation with the hematopoietic ability. The sustained expression of Sox17 in adult bone marrow HSCs and the cells in IAHCs of the dorsal aorta indicate abnormalities that are low lymphocyte chimerism and the aberrant proliferation of common myeloid progenitors in transplantation experiments. We then summarize the perspectives of Sox17 research in cancer control.

Transcriptional regulatory network controlling the ontogeny of hematopoietic stem cells

Hematopoietic stem cell (HSC) ontogeny is accompanied by dynamic changes in gene regulatory networks. We performed RNA-seq and histone mark ChIP-seq to define the transcriptomes and epigenomes of cells representing key developmental stages of HSC ontogeny in mice. The five populations analyzed were embryonic day 10.5 (E10.5) endothelium and hemogenic endothelium from the major arteries, an enriched population of prehematopoietic stem cells (pre-HSCs), fetal liver HSCs, and adult bone marrow HSCs. Using epigenetic signatures, we identified enhancers for each developmental stage. Only 12% of enhancers are primed, and 78% are active, suggesting the vast majority of enhancers are established de novo without prior priming in earlier stages. We constructed developmental stage-specific transcriptional regulatory networks by linking enhancers and predicted bound transcription factors to their target promoters using a novel computational algorithm, target inference via physical connection (TIPC). TIPC predicted known transcriptional regulators for the endothelial-to-hematopoietic transition, validating our overall approach, and identified putative novel transcription factors, including the broadly expressed transcription factors SP3 and MAZ. Finally, we validated a role for SP3 and MAZ in the formation of hemogenic endothelium. Our data and computational analyses provide a useful resource for uncovering regulators of HSC formation.

3167 – DEVELOPMENTAL CHANGES IN THE CHROMATIN ACCESSIBILITY OF HEMATOPOIETIC STEM CELLS ARE ASSOCIATED WITH DISTINCT GENETIC PROGRAMS

Hematopoietic stem cells (HSCs) undergo a developmental switch in neonatal mice hallmarked by a decrease in self-renewing divisions and entry into quiescence. Molecular determinants like dynamic changes in the chromatin landscape across the switch hold key insights into developmental changes in cell fate and function but remain poorly understood. Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) has emerged as a powerful tool to study chromatin accessibility in rare cells and in combination with genome-wide expression profiling has the potential to resolve cell specific transcriptional networks. To explore the regulation of the switch between fetal liver and adult bone marrow HSC states as well as during the first stages of hematopoietic differentiation, we here asses the changes in chromatin accessibility and genome-wide gene expression patterns in immunophenotypically defined HSCs and lymphoid primed multipotent progenitors (LMPPs). Chromatin accessibility unique to adult bone marrow HSCs were found to be enriched for putative binding sites of the megakaryocyte and erythroid lineage transcription factors Nuclear factor erythroid 2 (NFE2). In parallel, adult HSC gene expression and epigenetic profiles were highly enriched for the megakaryocyte gene signature which is strikingly absent in the E14.5 fetal liver. Taken together, our results identify a previously unrecognized hallmark of the developmental switch of the HSC compartment centered around acquisition of a megakaryocyte bias. These age specific features may have important implications in host response to environmental insults.

MEGAKARYOCYTE-LINEAGE BIAS AND RESTRICTION OF HEMATOPOIETIC STEM AND PROGENITOR CELLS

Hematopoietic stem cells (HSCs), defined by long-term self-renewal and ability to replenish all blood lineages, are molecularly and functionally heterogeneous. This heterogeneity might have important implications in both steady-state and emergency hematopoiesis, as well as aging and hematological malignancies. We previously uncovered a highly organized framework of lineage-biased and lineage-restricted HSC fates through sensitive tracking of post-transplantation contribution of hundreds of single HSCs from adult murine bone marrow (BM) (Carrelha et al. Nature 2018). Most notably, we identified HSCs that in vivo contribute stably, effectively, and exclusively towards the megakaryocyte (Mk) branch of the hematopoietic tree, despite possessing multipotency. No long-term self-renewing HSCs contribute exclusively to any other lineage upon transplantation. We also identified Mk/erythroid/myeloid and Mk/erythroid/myeloid/B-cell restricted outputs. Contrary to previous studies, we found no evidence for long-term adult BM HSCs with sustained lymphoid bias. Recently we also performed single-cell transplantations of HSCs from late gestation mouse embryos (E19.5). We found that these fetal liver and fetal BM HSCs undergo almost exclusively multilineage fates, rarely producing lineage-restricted outputs, which suggests that the lineage biases and restrictions of adult HSCs are established in the BM postnatally. Through fate-mapping analysis with an inducible Vwf-Cre reporter we demonstrated that Mk-biased HSCs play an important role in unperturbed adult BM hematopoiesis. We have further observed that Mk-biased HSCs play a more prominent role in the rapid replenishment of platelets following an acute insult with 5-fluorouracil. In agreement with this data, single transplanted HSCs with Mk-restricted fate replenish platelets more rapidly than HSCs undergoing a multilineage fate.

YY1 Controls Hematopoietic Stem Cell Quiescence By Repressing Cohesin Expression

Hematopoietic stem cells (HSCs) are undifferentiated, self-renewing, pluripotent cells that have the capacity to differentiate into all mature lineage-specific cells in adult blood. Adult HSCs can remain in a quiescent state for a prolonged time, and quiescence is a fundamental characteristic of HSCs in adult bone marrow. Thus, the cell cycle must be precisely regulated. Yin Yang 1 (YY1) is a multifunctional transcription factor and Polycomb Group Protein (PcG) that is important for embryonic development, adult hematopoiesis, cell proliferation and maintaining higher-order chromosomal structure. We have generated YY1 conditional knockout mice (Yy1f/f Mx1-Cre) and showed that Yy1 deficient HSCs fail to self-renewal and had disrupted HSCs quiescence. Stem cell factor (SCF)/c-Kit signaling, a critical regulatory pathway in HSC development, is significantly downregulated in Yy1-/- HSCs. Interestingly, YY1 regulation of HSCs self-renewal and quiescence is independent on its PcG domain/function. Instead, YY1 occupied at Smc3 promoter area and repressed Smc3 expression. In Yy1-/-HSCs, Smc3 expression was upregulated. SMC3 is a core component of cohesin protein complex and plays critical roles in HSC self-renewal, myeloid differentiation and leukemogenesis. To further dissect the underlying mechanisms by which YY1 regulates SMC3 expression in HSCs, we have generated conditional knockout mice with YY1 homozygous deletion and SMC3 heterozygous deletion (Yy1f/f Smc3f/+Mx1-Cre). In Yy1-/- Smc3+/- bone marrow cells, SMC3 expression was normalized to the wild-type level. In adult bone marrow cells, YY1 physically interacted with cohesin complex proteins through its zinc finger domain. By analyzing the YY1, SMC1A and SMC3 ChIP-Seq database, our study showed that YY1 and cohesin co-occupied at promoter areas of genes that are critical for cell metabolism. Evidence from previous study showed that impaired metabolism, including increased reactive oxygen species (ROS) and decreased mitochondrial function, can cause defect in stem cell self-renewal and quiescence. In Yy1-/- Smc3+/-HSCs, cell quiescence was restored although HSC self-renewal was still impaired, indicates that YY1 and SMC3 may control HSC cell quiescence via regulating genes critical for cell metabolism. Our study identified YY1 as the first transcription factor that regulates expression of cohesin complex component SMC3. We are currently further dissecting underlying mechanisms and functional significances of metabolic pathways regulated by YY1-SMC3 axis in HSCs. DisclosuresLevine:Imago: Equity Ownership; Isoplexis: Equity Ownership; Janssen: Consultancy, Honoraria; Gilead: Honoraria; Epizyme: Patents & Royalties; Loxo: Consultancy, Equity Ownership; C4 Therapeutics: Equity Ownership; Roche: Consultancy, Research Funding; Prelude: Research Funding; Qiagen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Novartis: Consultancy.

Energy Producing Metabolic Pathways in Functional Regulation of the Hematopoietic Stem Cells.

The hematopoietic system has a very well-studied hierarchy with the long-term (LT) hematopoietic stem cells (HSCs) taking the top position. The pool of quiescent adult LT-HSCs generated during the fetal and early postnatal life acts as a reservoir to supply all the blood cells. Therefore, the maintenance of this stem cell pool is pivotal to maintaining homeostasis in hematopoietic system. It has long been known that external cues, along with the internal genetic factors influence the status of HSCs in the bone marrow (BM). Hypoxia is one such factor that regulates the vascular as well as hematopoietic ontogeny from a very early time point in development. The metabolic outcomes of a hypoxic microenvironment play important roles in functional regulation of HSCs, especially in case of adult BM HSCs. Anaerobic metabolic pathways therefore perform prominent role in meeting energy demands. Increased oxidative pathways on the other hand result in loss of stemness. Recent studies have attributed the functional differences in HSCs across different life stages to their metabolic phenotypes regulated by respective niches. Indicating thus, that various energy production pathways could play distinct role in regulating HSC function at different developmental/physiological states. Here, we review the current status of our understanding over the role that energy production pathways play in regulating HSC stemness. © 2018 IUBMB Life, 70(7):612-624, 2018.

The Wave2 scaffold Hem-1 is required for transition of fetal liver hematopoiesis to bone marrow

The transition of hematopoiesis from the fetal liver (FL) to the bone marrow (BM) is incompletely characterized. We demonstrate that the Wiskott–Aldrich syndrome verprolin-homologous protein (WAVE) complex 2 is required for this transition, as complex degradation via deletion of its scaffold Hem-1 causes the premature exhaustion of neonatal BM hematopoietic stem cells (HSCs). This exhaustion of BM HSC is due to the failure of BM engraftment of Hem-1−/− FL HSCs, causing early death. The Hem-1−/− FL HSC engraftment defect is not due to the lack of the canonical function of the WAVE2 complex, the regulation of actin polymerization, because FL HSCs from Hem-1−/− mice exhibit no defects in chemotaxis, BM homing, or adhesion. Rather, the failure of Hem-1−/− FL HSC engraftment in the marrow is due to the loss of c-Abl survival signaling from degradation of the WAVE2 complex. However, c-Abl activity is dispensable for the engraftment of adult BM HSCs into the BM. These findings reveal a novel function of the WAVE2 complex and define a mechanism for FL HSC fitness in the embryonic BM niche.

Regulation of Fetal Liver Hematopoietic Stem Cell Function By the Dpy30 Subunit of Set1/Mll Complexes

While stem cells undergo phenotypic and functional changes in development, the capacity of self-renewal and differentiation remains the defining property of stem cells throughout life, indicating certain fundamental regulatory mechanisms underlying these cardinal features of stem cells. A profound transition occurs to hematopoietic stem cells (HSCs) from embryonic to adult hematopoiesis, resulting in pronounced distinctions between fetal liver (FL) and adult bone marrow (BM) HSCs in many aspects. While many studies have documented a different dependence of fetal versus adult HSC function on epigenetic modulators including several Polycomb proteins, little is known about if Trithorax proteins play a similar or different role in fetal versus adult HSC function. More specifically, despite being a prominent epigenetic mark associated with gene activation, the role of H3K4 methylation (an activity of many Trithorax proteins) in different stages of HSCs remains unclear.As the major H3K4 methylases in mammals, the Set1/Mll family complexes play important roles in development and stem cell function, and are extensively associated with diseases including blood cancers. We have previously established a direct role of Dpy30, a core subunit in all Set1/Mll complexes, in facilitating genome-wide H3K4 methylation, and this allows an effective interrogation of the functional role of efficient H3K4 methylation through genetic studies of Dpy30. While dispensable for the self-renewal of embryonic stem cells (ESCs), Dpy30 is crucial for efficient differentiation of ESCs by facilitating the induction of many bivalently marked developmental genes (Jiang et al., Cell, 2011). We have then generated a Dpy30 conditional knockout mouse, and shown that Dpy30 plays a crucial role in the long term maintenance and differentiation of adult BM HSCs, and preferentially controls H3K4 methylation and expression of many hematopoiesis-associated genes in adult BM cells (Yang et al., J Exp Med, accepted). However, the role of Dpy30 and efficient H3K4 methylation in fetal HSCs is still unknown.To study the role of efficient H3K4 methylation in fetal HSCs, we deleted Dpy30 in fetal hematopoietic cells using VavCre line. VavCre; Dpy30F/- fetuses are anemic at E14.5 and E15.5, with reduced H3K4 methylation but significantly increased numbers of FL HSCs. However, these FL HSCs were functionally defective in colony formation and blood reconstitution following transplantation. Proliferation of the progenitors, but not HSCs, was significantly (but modestly) reduced. These results suggest a role of Dpy30 in differentiation of HSCs and progenitor proliferation in FL. We also competitively transplanted Mx1Cre; Dpy30F/- FL and deleted Dpy30 after stable engraftment. Our analysis at an early time point after deletion showed little effect on donor contribution to HSCs, but significant reduction of oligopotent progenitors. Analysis at a later time point after deletion, however, showed marked reduction of all hematopoietic cells including HSCs. These results support a cell-autonomous role of Dpy30 in the differentiation and long term maintenance of FL HSCs.The phenotypes of FL HSCs are largely similar to those of BM HSCs following Dpy30 loss, suggesting that Dpy30 and certain Dpy30 targets are fundamentally important in regulating HSCs regardless of the developmental stages. To identify these targets, we performed RNA-seq analyses for purified FL HSCs from VavCre; Dpy30F/- versus VavCre; Dpy30F/+ littermates. Among hundreds of genes that were significantly changed in FL HSCs, however, only a handful of genes were found to be co-downregulated in both FL and BM HSCs following Dpy30 loss, suggesting that Dpy30 may have different functional targets in different stages of HSCs. To identify Dpy30 targets fundamentally important to HSC regulation, we are now selectively investigating the function of a few common Dpy30 targets in HSCs by colony formation and potentially transplantation assays following their stable knockdown.The similar requirement of Dpy30 in both fetal and adult HSC differentiation as well as long-term maintenance underscores the fundamental importance of this epigenetic modulator in the central properties of stem cells, and studies of the common Dpy30 targets may identify new regulatory genes controlled by this modulator in fetal and adult HSC function. DisclosuresNo relevant conflicts of interest to declare.

Terminal differentiation induction as DNA damage response in hematopoietic stem cells by GADD45A.

Hematopoietic stem cells (HSCs) sustain lifelong blood cell regeneration by balancing their ability for self-renewal with their ability to differentiate into all blood cell types. To prevent organ exhaustion and malignant transformation, long-lived HSCs, in particular, must be protected from exogenous and endogenous stress, which cause severe DNA damage. When DNA is damaged, distinct DNA repair mechanisms and cell fate controls occur in adult HSCs compared with committed cells. Growth arrest and DNA damage-inducible 45 alpha (GADD45A) is known to coordinate a variety of cellular stress responses, indicating the molecule is an important stress mediator. So far, the function of GADD45A in hematopoietic stem and progenitor cells is controversial and appears highly dependent on the cell type and stress stimulus. Recent studies have analyzed its role in cell fate decision control of prospectively isolated HSCs and have revealed unexpected functions of GADD45A, as discussed here. The upregulation of GADD45A by DNA damage-causing conditions results in enhanced HSC differentiation, probably to efficiently eliminate aberrant HSCs from the system. These findings, in concert with a few studies on other stem cell systems, have led us to propose DNA damage-induced differentiation as a novel DNA damage response mechanism in stem cells that circumvents the fatal consequences of cumulative DNA damage in the stem cell compartment.

Hematopoietic stem cell-specific GFP-expressing transgenic mice generated by genetic excision of a pan-hematopoietic reporter gene.

Selective labeling of specific cell types by expression of green fluorescent protein (GFP) within the hematopoietic system would have great utility in identifying, localizing, and tracking different cell populations in flow cytometry, microscopy, lineage tracing, and transplantation assays. In this report, we describe the generation and characterization of a new transgenic mouse line with specific GFP labeling of all nucleated hematopoietic cells and platelets. This new "Vav-GFP" mouse line labels the vast majority of hematopoietic cells with GFP during both embryonic development and adulthood, with particularly high expression in hematopoietic stem and progenitor cells (HSPCs). With the exception of transient labeling of fetal endothelial cells, GFP expression is highly selective for hematopoietic cells and persists in donor-derived progeny after transplantation of HSPCs. Finally, we also demonstrate that the loxP-flanked reporter allows for specific GFP labeling of different hematopoietic cell subsets when crossed to various Cre reporter lines. By crossing Vav-GFP mice to Flk2-Cre mice, we obtained robust and highly selective GFP expression in hematopoietic stem cells (HSCs). These data describe a new mouse model capable of directing GFP labeling exclusively of hematopoietic cells or exclusively of HSCs.

Fetal Hematopoietic Stem Cell Transplantation Fails to Fully Regenerate the B-Lymphocyte Compartment.

SummaryB cells are key components of cellular and humoral immunity and, like all lymphocytes, are thought to originate and renew from hematopoietic stem cells (HSCs). However, our recent single-HSC transfer studies demonstrate that adult bone marrow HSCs do not regenerate B-1a, a subset of tissue B cells required for protection against pneumonia, influenza, and other infections. Since B-1a are regenerated by transfers of fetal liver, the question arises as to whether B-1a derive from fetal, but not adult, HSCs. Here we show that, similar to adult HSCs, fetal HSCs selectively fail to regenerate B-1a. We also show that, in humanized mice, human fetal liver regenerates tissue B cells that are phenotypically similar to murine B-1a, raising the question of whether human HSC transplantation, the mainstay of such models, is sufficient to regenerate human B-1a. Thus, our studies overtly challenge the current paradigm that HSCs give rise to all components of the immune system.

SATB1 Expression Marks Lymphoid-Lineage-Biased Hematopoietic Stem Cells in Mouse Bone Marrow

Background: Lifelong hematopoiesis is maintained by cell differentiation in which signaling pathways and transcription factors coordinately induce step-wise maturation of hematopoietic stem cells (HSCs) toward downstream effector cells. In addition, the organization of chromatin structure that creates accessible sites of target genes is also essential so as to ensure temporally and spatially adequate control of internal gene expression. Murine HSCs can be isolated with high efficiency using surface molecules including lineage-related markers, c-Kit, Sca-1, Flt3 and SLAM family proteins. However, even the highly enriched HSC fraction is still heterogeneous regarding differentiation potential, and how the HSC diversity reflects the heterogeneity of intrinsic gene-expression in HSCs is as-yet-unknown. We previously identified Special AT-rich Sequence Binding protein 1 (SATB1), a global chromatin regulator, as a lymphoid-related gene in the HSC differentiation (Satoh and Yokota et al. Immunity 2013). Indeed, SATB1 overexpression strongly enhanced both T and B lymphopoietic potential of murine HSCs whereas SATB1 deficiency caused malfunctions of HSCs in the lymphopoietic activity. Furthermore, another report showed that SATB1-deficient HSCs were less quiescent in transplanted recipients and more prone to differentiate preferentially to myeloid-erythroid lineages (Will et al. Nat Immunol 2013). These results suggested that SATB1 is likely indispensable not only for the lymphopoietic potential but also for the integrity of HSCs. Here, to better understand the mechanism how SATB1 influences homeostatic HSC functions in adult bone marrow (BM), we have developed a new mouse model in which SATB1 expression can be precisely monitored along the HSC differentiation.Methods: The Tomato gene, coding a red fluorescent protein, was knock-in to the coding region of endogenous Satb1 gene. The heterozygous SATB1/Tomato knock-in mice in which one Satb1 allele was replaced with the Tomato were used to sort HSCs in adult BM. The sorted cells were evaluated for the differentiation potential with methylcellulose colony assays and co-cultures with MS5 stromal cells. Further, the long-term reconstitution ability was evaluated by transplantation to lethally irradiated mice. To obtain transcriptome information, total RNA was isolated from SATB1/Tomato- and SATB1/Tomato+ HSCs, and then next-generation sequencing was performed. The data were analyzed with the Ingenuity Pathway Analysis software.Results: We defined Lin- Sca1+ c-KitHi (LSK) CD150+ Flt3- cells as HSCs, especially adopting FLT3- to exclude FLT3+ lymphoid-primed multipotent progenitors from our functional analyses. We found that the LSK CD150+ Flt3- fraction contains substantial number of SATB1/Tomato+ cells. While both SATB1/Tomato- and SATB1/Tomato+ HSCs produced numerous CFU-Mix and CFU-GM/G/M colonies, the latter were less potent to produce BFU-E. In co-culture with MS5 stromal cells that support B and myeloid lineages, the output of B lineage cells from SATB1+ HSCs was more robust than that of SATB1- HSCs. Upon transplantation, enhanced B-lineage engraftment was observed in the SATB1+ HSC-transplanted recipients. Interestingly, while the two types of HSCs showed obvious difference in the differentiation potential toward lymphoid or myeloid lineage, both HSCs reconstituted the LSK CD150+ Flt3- fraction that similarly contained SATB1/Tomato- and SATB1/Tomato+ cells. With the RNA-sequencing data of SATB1- and SATB1+ HSCs, biological pathway analyses revealed that the "Hematological System Development and Function" pathway was remarkably up-regulated in the SATB1+ HSCs. Among subcategories of the "Hematological System Development and Function" pathway, the "quantity of lymphocytes" pathway was increased whereas "quantity of myeloid cells" and "quantity of granulocytes" pathways were decreased.Conclusion: We have developed a new mouse system that can be used to identify and isolate viable lymphoid-biased HSCs in the most primitive hematopoietic cell fraction of adult BM. While the SATB1- and SATB1+ HSCs differ genetically and functionally, both subtypes have displayed a self-renewal activity with mutual interconversion in transplanted recipients. These findings suggest that functional heterogeneity and variability within the HSC population is, at least in part, a manifestation of SATB1 expression. DisclosuresYokota:SHIONOGI & CO., LTD.: Research Funding.

ARAP3 functions in hematopoietic stem cells.

ARAP3 is a GTPase-activating protein (GAP) that inactivates Arf6 and RhoA small GTPases. ARAP3 deficiency in mice causes a sprouting angiogenic defect resulting in embryonic lethality by E11. Mice with an ARAP3 R302,303A mutation (Arap3KI/KI) that prevents activation by phosphoinositide-3-kinase (PI3K) have a similar angiogenic phenotype, although some animals survive to adulthood. Here, we report that hematopoietic stem cells (HSCs) from rare adult Arap3KI/KI bone marrow are compromised in their ability to reconstitute recipient mice and to self-renew. To elucidate the potential cell-autonomous and non-cell-autonomous roles of ARAP3 in hematopoiesis, we conditionally deleted Arap3 in hematopoietic cells and in several cell types within the HSC niche. Excision of Arap3 in hematopoietic cells using Vav1-Cre does not alter the ability of ARAP3-deficient progenitor cells to proliferate and differentiate in vitro or ARAP3-deficient HSCs to provide multi-lineage reconstitution and to undergo self-renewal in vivo. Thus, our data suggest that ARAP3 does not play a cell-autonomous role in HSPCs. Deletion of Arap3 in osteoblasts and mesenchymal stromal cells using Prx1-Cre resulted in no discernable phenotypes in hematopoietic development or HSC homeostasis in adult mice. In contrast, deletion of Arap3 using vascular endothelial cadherin (VEC or Cdh5)-driven Cre resulted in embryonic lethality, however HSCs from surviving adult mice were largely normal. Reverse transplantations into VEC-driven Arap3 conditional knockout mice revealed no discernable difference in HSC frequencies or function in comparison to control mice. Taken together, our investigation suggests that despite a critical role for ARAP3 in embryonic vascular development, its loss in endothelial cells minimally impacts HSCs in adult bone marrow.

Adipocytic Cells Augment the Support of Primitive Hematopoietic Cells In Vitro But Have No Effect in the Bone Marrow Niche Under Homeostatic Conditions

Mesenchymal stem cells (MSCs), as well as osteoblastic cells derived from these MSCs, have been shown to be key components of the hematopoietic stem cell (HSC) niche. In this study, we wished to examine whether other cell types that are known to differentiate from MSCs similarly regulate the stem cell niche, namely cells of the adipocyte lineage. Recent studies have examined the role that adipocytes play in the biology of the HSCs in different bone locations and in transplantation settings; however, none have examined their role under homeostatic conditions. We compared the ability of adipocytic and nonadipocytic cell lines to support primitive hematopoietic cells in vitro. Preadipocytic cell lines demonstrated enhanced support of hematopoietic cells. Similarly, primary bone marrow (BM) cells treated with troglitazone, a drug that enhances adipogenesis, also demonstrated augmented support over control-treated stromal cells. We further examined the effects of increased adipocyte number in vivo under homeostatic conditions using troglitazone treatment and found that these alterations had no effect on HSC frequency. Taken together, we demonstrate that cells of the adipocyte lineage promote the ability of stromal cells to support primitive hematopoietic cells in vitro, yet alterations of adipocyte number and volume in vivo have no effect. These data suggest that adipocytes are not a component of the adult BM HSC niche under homeostatic conditions.