Hematopoietic Stem And Progenitor Cells Fate Research Articles

Reprogramming of 3D genome structure underlying HSPC development in zebrafish

BackgroundDevelopment of hematopoietic stem and progenitor cells (HSPC) is a multi-staged complex process that conserved between zebrafish and mammals. Understanding the mechanism underlying HSPC development is a holy grail of hematopoietic biology, which is helpful for HSPC clinical application. Chromatin conformation plays important roles in transcriptional regulation and cell fate decision; however, its dynamic and role in HSPC development is poorly investigated.MethodsWe performed chromatin structure and multi-omics dissection across different stages of HSPC developmental trajectory in zebrafish for the first time, including Hi-C, RNA-seq, ATAC-seq, H3K4me3 and H3K27ac ChIP-seq.ResultsThe chromatin organization of zebrafish HSPC resemble mammalian cells with similar hierarchical structure. We revealed the multi-scale reorganization of chromatin structure and its influence on transcriptional regulation and transition of cell fate during HSPC development. Nascent HSPC is featured by loose conformation with obscure structure at all layers. Notably, PU.1 was identified as a potential factor mediating formation of promoter-involved loops and regulating gene expression of HSPC.ConclusionsOur results provided a global view of chromatin structure dynamics associated with development of zebrafish HSPC and discovered key transcription factors involved in HSPC chromatin interactions, which will provide new insights into the epigenetic regulatory mechanisms underlying vertebrate HSPC fate decision.

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

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

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

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

Integrins, anchors and signal transducers of hematopoietic stem cells during development and in adulthood.

Hematopoietic stem cells (HSCs), the apex of the hierarchically organized blood cell production system, are generated in the yolk sac, aorta-gonad-mesonephros region and placenta of the developing embryo. To maintain life-long hematopoiesis, HSCs emigrate from their site of origin and seed in distinct microenvironments, called niches, of fetal liver and bone marrow where they receive supportive signals for self-renewal, expansion and production of hematopoietic progenitor cells (HPCs), which in turn orchestrate the production of the hematopoietic effector cells. The interactions of hematopoietic stem and progenitor cells (HSPCs) with niche components are to a large part mediated by the integrin superfamily of adhesion molecules. Here, we summarize the current knowledge regarding the functional properties of integrins and their activators, Talin-1 and Kindlin-3, for HSPC generation, function and fate decisions during development and in adulthood. In addition, we discuss integrin-mediated mechanosensing for HSC-niche interactions, ex vivo protocols aimed at expanding HSCs for therapeutic use, and recent approaches targeting the integrin-mediated adhesion in leukemia-inducing HSCs in their protecting, malignant niches.

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

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

Sequential regulation of hemogenic fate and hematopoietic stem and progenitor cell formation from arterial endothelium by Ezh1/2.

SummaryAcross species, hematopoietic stem and progenitor cells (HSPCs) arise during embryogenesis from a specialized arterial population, termed hemogenic endothelium. Here, we describe a mechanistic role for the epigenetic regulator, Enhancer of zeste homolog-1 (Ezh1), in vertebrate HSPC production via regulation of hemogenic commitment. Loss of ezh1 in zebrafish embryos favored acquisition of hemogenic (gata2b) and HSPC (runx1) fate at the expense of the arterial program (ephrinb2a, dll4). In contrast, ezh1 overexpression blocked hematopoietic progression via maintenance of arterial gene expression. The related Polycomb group subunit, Ezh2, functioned in a non-redundant, sequential manner, whereby inhibition had no impact on arterial identity, but was capable of blocking ezh1-knockdown-associated HSPC expansion. Single-cell RNA sequencing across ezh1 genotypes revealed a dropout of ezh1+/− cells among arterial endothelium associated with positive regulation of gene transcription. Exploitation of Ezh1/2 modulation has potential functional relevance for improving in vitro HSPC differentiation from induced pluripotent stem cell sources.

Manufacturing a Bone Marrow-On-A-Chip Using Maskless Photolithography.

The bone marrow (BM) is a complex microenvironment in which hematopoietic stem and progenitor cells (HSPCs) interact with multiple cell types that regulate their quiescence, growth, and differentiation. These cells constitute local niches where HSPCs are confined and subjected to specific set of physical and biochemical cues. Endothelial cells forming the walls of blood capillaries have been shown to establish a vascular niche, whereas osteoblasts lying along the bone matrix organize the endosteal niche with distinct and specific impact on HSPC fate. The observation of the interaction of HSPCs with niche cells, and the investigation of its impact on HSPCs behavior in vivo is hindered by the opacity of the bone matrix. Therefore, various experimental strategies have been devised to reconstitute in vitro the interaction of HSPCs with distinct sets of BM-derived cells. In this chapter, we present a method to manufacture a pseudo BM-on-a-chip with separated compartments mimicking the vascular and the endosteal niches. Such a configuration with connected but distant compartments allowed the investigation of the specific contribution of each niche to the regulation of HSPC behavior. We describe the microfabrication of the chip with a maskless photolithography method that allows the iterative improvement of the geometric design of the chip in order to optimize the adaptation of the multicellular architecture to the specific aim of the study. We also describe the loading and culture of the various cell types in each compartment.

Mineral and Amino Acid Profiling of Different Hematopoietic Populations from the Mouse Bone Marrow

Steady hematopoiesis is essential for lifelong production of all mature blood cells. Hematopoietic stem and progenitor cells (HSPCs) found in the bone marrow ensure hematopoietic homeostasis in an organism. Failure of this complex process, which involves a fine balance of self-renewal and differentiation fates, often result in severe hematological conditions such as leukemia and lymphoma. Several molecular and metabolic programs, internal or in close interaction with the bone marrow niche, have been identified as important regulators of HSPC function. More recently, nutrient sensing pathways have emerged as important modulators of HSC homing, dormancy, and function in the bone marrow. Here we describe a method for reliable measurement of various amino acids and minerals in different rare bone marrow (BM) populations, namely HSPCs. We found that the amino acid profile of the most primitive hematopoietic compartments (KLS) did not differ significantly from the one of their direct progenies (common myeloid progenitor CMP), while granulocyte-monocyte progenitors (GMPs), on the opposite of megakaryocyte-erythroid progenitors (MEPs), have higher content of the majority of amino acids analyzed. Additionally, we identified intermediates of the urea cycle to be differentially expressed in the KLS population and were found to lower mitochondrial membrane potential, an established readout on self-renewal capability. Moreover, we were able to profile for the first time 12 different minerals and detect differences in elemental contents between different HSPC compartments. Importantly, essential dietary trace elements, such as iron and molybdenum, were found to be enriched in granulocyte-monocyte progenitors (GMPs). We envision this amino acid and mineral profiling will allow identification of novel metabolic and nutrient sensing pathways important in HSPC fate regulation.

2032 - DYNAMICS QUANTIFICATION OF TRANSCRIPTION FACTOR SWITCHES IN HSPC LINEAGE CHOICE

Transcription factor (TF) networks regulate the lineage choice of hematopoietic stem and progenitor cells (HSPCs). Previous studies suggested a functional antagonism between the core hematopoietic TFs PU.1 and GATA1, and their autoregulation as the basis for a cell-intrinsic stochastic switch regulating PU.1 expression in lineage choice of HSPCs. Using long-term quantitative time-lapse imaging of HSCs from the PU.1eYFP/GATA1mCherry reporter mouse line, we previously showed that a random PU.1 and GATA1 switch does not initiate myeloid lineage choice. To further analyze the regulation of PU.1, we generated a novel mouse line with an inducible PU.1-ERT2 allele co-expressed with the PU.1eYFP/GATA1mCherry reporter alleles. Using single-cell reprogramming assays, microfluidics facilitated reversible PU.1 induction by TNF and single-cell quantification in response to transgenic PU.1-ERT2 activation, we quantified PU.1 expression in thousands of single HSPCs to measure the short- and long- term autoregulation dynamics. Surprisingly, we do not observe direct autoregulation of PU.1 at many different stages of hematopoietic differentiation. We find PU.1 autoregulation not to be involved in initiating differentiation, but to be an indirect consequence of it. Finally, to dissect the molecular triad of PU.1, GATA1 and GATA2 in regulating HSC lineage choice, we have generated a novel reporter mouse line for GATA2 protein and continuously quantify its expression dynamics, simultaneous with that of GATA1 in living HSPCs along the megakaryocyte-erythrocyte and granulocyte-monocyte differentiation trajectories. These data disprove several previous assumptions about the regulation of HSPC fate control, identify novel regulators, and demonstrate vacancy for other cell-extrinsic and -intrinsic players in regulating hematopoietic TFs specifically and lineage choice of HSCs in general.

CLONAL ETIOLOGY OF EMERGENCY DENDRITIC CELL DEVELOPMENT

Dendritic cells (DCs) are critical mediators of both innate and adaptive immune responses. Increasing DC numbers by administration of Flt3 ligand (FL) provides therapeutic benefit for infection and cancer immunotherapy. However, how FL signals regulate emergency DC development at the clonal level is largely unknown. To interrogate the early cellular and molecular events within hematopoietic stem and progenitor cells (HSPCs) after FL stimulation, we develop a single cell multi-omics profiling approach. This allows simultaneous measurement of surface marker phenotype, division history and the transcriptome of single HSPCs treated with PBS or FL after transfer into non-irradiated recipients. We observe hyperproliferation of most HSPCs after FL treatment. In particular, we identify a unique group of hyper-proliferative cells that co-expresses gene signatures of both ‘multipotent progenitors’ and ‘DC progenitors’, indicative of the earliest molecular and cellular events during emergency DC development. To understand how FL affects HSPC fate towards other lineages at the clonal level, we tag individual HSPCs with unique DNA barcodes and compare their development, with or without FL stimulation in vivo. We observe no evidence of lineage diversion of HSPCs away from alternative fates and instead determine that pre-existing multi-/oligo-potent retain their potency and selectively expand a DC-producing sub-branch, leading to preferential production of DCs. Collectively, our results suggest that lineage programs within single HSPCs are established and stable, and that selective clonal expansion, not fate ‘plasticity’ of HSPCs via extrinsic regulation, explains FL-mediated emergency DC development. These findings provide new insight into the control and regulation of fate during hematopoiesis.

PF456 BETA‐CATENIN/TCF SIGNALING PATHWAY PROMOTES GRANULOCYTIC DIFFERENTIATION OF HEMATOPOIETIC PROGENITORS BY FACILITATING IL‐1 RECEPTOR SIGNALING

Background:The canonical Wnt signaling pathway is an essential regulator of differentiation and cell fate decisions in many types of somatic stem cells. However, in hematopoietic stem and progenitor cells (HSPCs), the role of the canonical Wnt signaling remains unclear. A critical event in Wnt‐target gene activation is the interaction of ß‐catenin with TCF/LEF transcription factors which enables transcription of regulated regions. The majority of previous studies relied on ß‐catenin manipulation. Intriguingly, ß‐catenin has been described to have also important cytoplasmic functions and therefore, it is not clear whether the previously observed phenotypes are really caused by the alteration of expression of Wnt‐target genes.Aims:Our aim was to investigate how specific loss of ß‐catenin interaction with TCF/LEF factors will affect the function of HSPCs in vivo and to decipher molecular mechanism that is responsible for the functional changes.Methods:To investigate the function of canonical Wnt signaling pathway in HSPCs, we disrupted ß‐catenin interaction with TCF/LEF factors by expressing a dominant negative form of human TCF4 (dnTCF4) specifically in hematopoietic tissues. dnTCF4 forms a heterodimers with endogenous TCF factors and as it lacks ß‐catenin binding domain, it prevents the transactivation of Wnt‐target genes. The hematopoietic tissues of transgenic mice were analyzed using flow cytometry, while the function of HSPCs was assessed by bone marrow transplantation assays in vivo and colony formation assay in vitro. NF‐κB activation was assessed by intracellular staining of IκBa and analyzed by flow cytometry in various cell populations. RNA sequencing was performed in sorted short‐term HSC, defined as lin−, c‐kit+, Sca1+, CD48+, CD150−, using Illumina platform.Results:dnTCF4 transgenic mice presented decreased levels of granulocytes in peripheral blood accompanied by accumulation of HSPCs in bone marrow. Consistently, dnTCF4 bone marrow cells displayed granulocytic differentiation block, augmented colony‐forming abilities, and retained immature phenotype longer than wild type control in colony formation assays. The accumulation of HSPCs was also reflected by enhanced engraftment rate of dnTCF4‐expressing bone marrow after transplantation. Transcriptomic analysis revealed that dnTCF4‐expressing HSPCs have reduced expression of genes involved in granulocyte differentiation and function, including Csf3r, Il18r, and Il1r1r. Interestingly, IL‐1ß treatment fail to activate NF‐κB signaling pathway, a well described inducer of myeloid differentiation, in dnTCF4‐expressing HSPCs.Summary/Conclusion:Altogether, our data suggest that the canonical Wnt signaling promotes HSPCs differentiation towards granulocytes by upregulating IL‐1 receptor levels that allow proper NF‐κB activation. Since our murine model does present intact cytoplasmic ß‐catenin activity, our data supports that ß‐catenin controls HSPCs fate and differentiation via transactivation of Wnt‐target genes.

Regnase-1-mediated post-transcriptional regulation is essential for hematopoietic stem and progenitor cell homeostasis

The balance between self-renewal and differentiation of hematopoietic stem and progenitor cells (HSPCs) maintains hematopoietic homeostasis, failure of which can lead to hematopoietic disorder. HSPC fate is controlled by signals from the bone marrow niche resulting in alteration of the stem cell transcription network. Regnase-1, a member of the CCCH zinc finger protein family possessing RNAse activity, mediates post-transcriptional regulatory activity through degradation of target mRNAs. The precise function of Regnase-1 has been explored in inflammation-related cytokine expression but its function in hematopoiesis has not been elucidated. Here, we show that Regnase-1 regulates self-renewal of HSPCs through modulating the stability of Gata2 and Tal1 mRNA. In addition, we found that dysfunction of Regnase-1 leads to the rapid onset of abnormal hematopoiesis. Thus, our data reveal that Regnase-1-mediated post-transcriptional regulation is required for HSPC maintenance and suggest that it represents a leukemia tumor suppressor.

Nocturnal Melatonin Renews Bone and Blood Forming Stem Cells Reservoir By Metabolic Reprograming

Bone marrow (BM) residing hematopoietic stem and progenitor cells (HSPC) replenish the blood with mature cells with a finite life span on a daily basis while maintaining the reservoir of undifferentiated stem cells. We recently showed that light/darkness onset induce two different BM HSPC peaks. Morning-induced norepinephrine and TNF secretion metabolically facilitate HSPC differentiation and egress to replenish the circulation with new mature leukocytes. Night augmented BM melatonin renews BM CD150+ hematopoietic stem cell (HSC) reservoir and their long-term repopulation potential (Golan et al, Cell Stem Cell, In Press). How melatonin primes BM HSPC to change their phenotype and function to re-acquire an undifferentiated and primitive state, is poorly understood. The hormone melatonin is an important mediator of bone formation and mineralization, and ultimately regulates the balance of bone remodeling (Cardinali DP et al, J. Pineal Res., 2003). The cross talk between HSPC and their BM stromal microenvironment is tightly regulated and determines HSPC fate. Therefore, we examined whether melatonin plays a role in regulation of murine BM mesenchymal stem and progenitor cells (MSPC, CD45-/Sca-1+/PDGFRα+), known to support HSPC maintenance in their BM niches. Mice treated with melatonin for 5h during the morning had increased levels of BM MSPC endowed with higher colony-forming unit fibroblast (CFU-F) potential in vitro. Interestingly, the metabolic state of these progenitor cells was altered by melatonin demonstrating reduced glucose uptake ability and lower mitochondria content. To test if differences in stromal cells content exist between day and night, we examined BM MSPC and found increased levels at 11PM, the time of melatonin BM peak, with higher Sca-1high surface expression levels, as compared to daylight 11AM. These changes were associated with augmented CFU-F levels by MSPC harvested at 11PM and accompanied by reduced glucose uptake levels and mitochondria content. Our preliminary results suggest that melatonin at night increases BM MSPC levels and reduces their metabolic activity to maintain them in a primitive and undifferentiated state. Moreover, we found that melatonin-elevated HSPC at 11PM also share lower glucose uptake ability with reduced mitochondria content and lower mitochondrial membrane potential (evaluated by TMRE). We hypothesize that melatonin reprograms the metabolic state of both HSPC and their stromal MSPC microenvironment to renew and maintain a primitive state of both populations at night. One of the factors inhibited by melatonin is the bioactive lipid Sphingosine 1-Phosphate (S1P), which in turn inhibits melatonin production. We found that mice with low S1P levels (S1Plow) due to lack of the SPHK1 enzyme have high BM melatonin levels also during the day in contrast to wild type (WT) mice. S1Plow mice had higher levels of primitive stromal progenitor cells including CFU-F and lower levels of differentiating osteoblast precursors compared to WT mice. In addition, these mice had less BM Reactive Oxygen Species (ROS)high committed hematopoietic progenitor cells, but more primitive ROSlow EPCR+ HSC endowed with higher long-term repopulation capacity in both primary and serially transplanted recipients. Next, we examined how light/dark cues affect the homing of transplanted BM HSPC into the BM of irradiated hosts 18h after transplantation. We found that donor HSPC harvested at 11PM have elevated homing ability compared to 11AM harvested cells. Importantly, MSPC also better homed to the BM of irradiated recipients when we transplanted donor BM cells obtained at 11PM compared with 11AM. As a result, accelerated BM repopulation kinetics was documented one week post transplantation in mice transplanted with BM cells harvested at 11 PM.Taken together, our results reveal that in vivo melatonin renews and maintains the BM reservoir and function of primitive MSPC and HSPC by metabolically reprogramming these cells during the night on a daily basis. Since the primed HSPC and MSPC at night showed improved function and BM homing potential, these features might be mimicked by human BM cells in order to harness them for improved clinical transplantation protocols. DisclosuresNo relevant conflicts of interest to declare.

Prostaglandin E2 Stimulates CREB-Mediated Modification of Histone Variant Nucleosomes at Enhancers to Promote Hematopoietic Stem Cell Fate

Stimulation of hematopoietic stem and progenitor cells (HSPCs) with the inflammatory mediator Prostaglandin E2 (PGE2) enhances self-renewal and stem cell engraftment following transplantation. Currently, the long-acting derivative of PGE2, 16,16-dimethyl-PGE2 (dmPGE2) is in its fourth clinical trial to improve HSC engraftment and reduce graft versus host disease. To understand the effect of dmPGE2, we assessed genome-wide chromatin reorganization and gene expression changes in human CD34+ HSPCs after 2 hours of dmPGE2 treatment, the time period of treatment in the clinical trials. Enhancers are known to regulate gene expression changes in specific environmental contexts such as stress or inflammation, however the regulatory principles by which subsets of enhancers become activated are poorly understood. Here, we mapped active enhancers by ChIP-seq for H2K27ac and found that dmPGE2 activates a discrete set of enhancers in HSPCs. To investigate enhancer chromatin remodeling, we performed micrococcal nuclease digestion followed by high-throughput sequencing (MNase-seq) to map the occupancy and position of nucleosomes. We found that, contrary to the predominant assumption that open chromatin structures are essentially nucleosome-free, MNase-accessible nucleosomes are retained at inducible enhancers following dmPGE2 stimulation. Through ATAC-seq analysis we mapped changes in open chromatin and found that induced enhancers gain chromatin accessibility following stimulation while maintaining their nucleosome configuration. Surprisingly, this indicates that nucleosomes present at the center of dmPGE2-responsive enhancers play an important function in enhancer accessibility and activity. We then correlated enhancers with gene expression changes by performing RNA-seq and found that genes associated with dmPGE2-induced enhancers display higher gene expression changes after stimulation compared to genes associated with non-responsive enhancers. Transcripts upregulated after dmPGE2 treatment include previously identified regulators of self-renewal and migration such as NR4A2, EGR1 and CXCR4. Moreover, inflammatory chemokines including CXCL2 and CXCL8 as well as members of the activating protein 1 (AP-1) transcription factor gene family such as FOS, FOSL2 and JUNB are increasingly expressed upon stimulation. The gene expression profile included a signature implying CREB as the main transcription factor responsible for the acute dmPGE2 response. Western blot revealed dmPGE2-mediated activation of the signaling transcription factor CREB through phosphorylation in HSPCs. Using ChIP-seq, we found increased genomic binding of phospho-CREB (pCREB) after dmPGE2 treatment in the enhancers. Surprisingly, the binding of pCREB coincided directly with variant histone H2A.Z containing labile nucleosomes in enhancers. We validated the interaction between pCREB and H2A.Z on chromatin in dmPGE2-responsive U937 cells through chromatin fractionation followed by complex immunoprecipitation. This suggests that labile nucleosomes provide sufficient DNA access to allow for binding of pCREB at enhancers. Taken together, our study proposes a novel model for stimulus-mediated activation of enhancers by the inflammatory mediator dmPGE2. dmPGE2 induces the phosphorylation of CREB and subsequently leads to a specific interaction of pCREB with previously deposited H2A.Z-rich nucleosomes at inducible enhancers who regulate genes that promote HSPC fate. This new mechanism of variant histone deposition followed by the interaction with a signaling transcription factor at enhancers supports a rapid inducible response from the environment. DisclosuresNo relevant conflicts of interest to declare.

Proteinase 3 Limits the Number of Hematopoietic Stem and Progenitor Cells in Murine Bone Marrow

SummaryHematopoietic stem and progenitor cells (HSPCs) undergo self-renewal and differentiation to guarantee a constant supply of short-lived blood cells. Both intrinsic and extrinsic factors determine HSPC fate, but the underlying mechanisms remain elusive. Here, we report that Proteinase 3 (PR3), a serine protease mainly confined to granulocytes, is also expressed in HSPCs. PR3 deficiency intrinsically suppressed cleavage and activation of caspase-3, leading to expansion of the bone marrow (BM) HSPC population due to decreased apoptosis. PR3-deficient HSPCs outcompete the long-term reconstitution potential of wild-type counterparts. Collectively, our results establish PR3 as a physiological regulator of HSPC numbers. PR3 inhibition is a potential therapeutic target to accelerate and increase the efficiency of BM reconstitution during transplantation.

“Sea”-ing Is Believing: In Vivo Imaging of Hematopoietic Stem Cells and Cancer Using Zebrafish

Communication between cells and their environment regulates their behaviors. Like a naturalist who watches animals to understand them, visualizing cells within their native ecosystems provides clues into how they work. In this review, we discuss recent findings using in vivo imaging in zebrafish to understand the behaviors of hematopoietic stem and progenitor cells (HSPCs) and cancer. Recent studies visualizing the birth and migration of HSPCs throughout development and adulthood have revealed the contributions of signals from somites, immune cells, and stromal cells in modulating HSPC fate decisions. New studies in zebrafish cancer have also revealed the earliest initiation of tumorigenesis, the complex interplay of heterogenous tumor cells with each other and their environment, and how these interactions influence tumor migration and metastasis. These insights aid our understanding of cell-cell communications that are critical for making advances in regenerative medicine and cancer therapeutics.

Combined influence of biophysical and biochemical cues on maintenance and proliferation of hematopoietic stem cells.

Homeostasis of hematopoietic stem and progenitor cells (HSPC) is controlled by a combination of biochemical and biophysical environmental cues in the bone marrow (BM) niche, where a tight balance of quiescence and proliferation of HSPC is maintained. Specifically, alongside soluble factors and extracellular matrix (ECM) proteins, spatial confinement and ECM stiffness have been recognized to be critical for regulation of HSPC fate. Here we employ a modular, glycosaminoglycan (GAG)-based biohybrid hydrogel system to balance proliferation of human HSPC and maintenance of quiescent hematopoietic stem cells (HSC) through simultaneous regulation of exogenous biochemical and biophysical cues. Our results demonstrate that HSPC respond to increased spatial confinement with lowered proliferation and cell cycling, which results in higher frequency of quiescent LTC-IC (long-term culture initiating cells), while GAG-rich 3D environments further support maintenance of the cells.

Wnt9a Is Required for the Aortic Amplification of Nascent Hematopoietic Stem Cells

SummaryAll mature blood cell types in the adult animal arise from hematopoietic stem and progenitor cells (HSPCs). However, the developmental cues regulating HSPC ontogeny are incompletely understood. In particular, the details surrounding a requirement for Wnt/β-catenin signaling in the development of mature HSPCs are controversial and difficult to consolidate. Using zebrafish, we demonstrate that Wnt signaling is required to direct an amplification of HSPCs in the aorta. Wnt9a is specifically required for this process and cannot be replaced by Wnt9b or Wnt3a. This proliferative event occurs independently of initial HSPC fate specification, and the Wnt9a input is required prior to aorta formation. HSPC arterial amplification occurs prior to seeding of secondary hematopoietic tissues and proceeds, in part, through the cell cycle regulator myca (c-myc). Our results support a general paradigm, in which early signaling events, including Wnt, direct later HSPC developmental processes.

Cell Adhesion Molecules in Normal and Malignant Hematopoiesis: from Bench to Bedside

Hematopoietic stem and progenitor cells (HSPC) express at their surface an array of cell adhesion receptors that enables them to interact with extracellular matrix components and transmembrane cell adhesion molecules expressed within stem cell niches in the bone marrow. Herein, we review the different cell adhesive interactions taking place within the bone marrow between HSPC and their niches (via integrins, CD44, selectin ligands, and Eph receptors) and how they support HSPC anchoring in the bone marrow and regulate HSPC fate during hematopoiesis. We review how malignant hematopoietic cells utilize adhesive interactions to resist chemotherapy treatments. Finally, we review how these adhesive interactions can be therapeutically targeted to increase HSPC mobilization for transplantation and to chemosensitize malignant hematopoietic cells to chemotherapy.

Space constraints govern fate of hematopoietic stem and progenitor cells in vitro

Deciphering exogenous cues that determine stem cell fate decisions is a persisting challenge of cell biology and bioengineering. In an effort to unravel the role of spatial constraints in the cell-instructive characteristics of bone marrow microenvironments, murine hematopoietic stem and progenitor cells (HSPC) were exposed to fibronectin-coated microcavities in vitro. Microcavity sizes were chosen to allow for the inclusion of either individual or multiple cells. Repopulation experiments using lethally irradiated mice showed that the maintenance of functional HSPC in culture critically depends on cavity dimensions. Short-term repopulating hematopoietic stem cells (ST-HSC) were found to be best supported within single-cell sized compartments while long-term repopulating HSC (LT-HSC) were maintained within both cavity sizes. In sum, the reported data reveal spatial restriction to be a simple but powerful means for directing HSPC fate ex vivo.