Definitive Hematopoietic Stem Cells Research Articles

An interactive resource of molecular signalling in the developing human haematopoietic stem cell niche.

The emergence of definitive human haematopoietic stem cells (HSCs) from Carnegie Stage (CS) 14 to CS17 in the aorta-gonad-mesonephros (AGM) region is a tightly regulated process. Previously, we conducted spatial transcriptomic analysis of the human AGM region at the end of this period (CS16/CS17) and identified secreted factors involved in HSC development. Here, we extend our analysis to investigate the progression of dorso-ventral polarised signalling around the dorsal aorta over the entire period of HSC emergence. Our results reveal a dramatic increase in ventral signalling complexity from the CS13-CS14 transition, coinciding with the first appearance of definitive HSCs. We further observe stage-specific changes in signalling up to CS17, which may underpin the step-wise maturation of HSCs described in the mouse model. The data-rich resource is also presented in an online interface enabling in silico analysis of molecular interactions between spatially defined domains of the AGM region. This resource will be of particular interest for researchers studying mechanisms underlying human HSC development as well as those developing in vitro methods for the generation of clinically relevant HSCs from pluripotent stem cells.

Capturing Endothelial-to-Hematopoietic Transition Using a Highly Efficient in Vitro Platform

Introduction De novo generation of hematopoietic stem and progenitor cells (HSPCs) for therapeutic use has been a long-standing goal of regenerative medicine. During development, the first definitive hematopoietic stem cells (HSCs) derive from a small number of specialized vascular endothelial cells (EC), named hemogenic endothelial cells (HECs), located in the Aorta-Gonado-Mesonephrose (AGM). The process of HEC conversion to HSPCs is known as endothelial-to-hematopoietic transition (EHT). During EHT, endothelial features are lost while switching on hematopoietic transcriptional programs leading to drastic functional and morphological changes that generate free-floating, round hematopoietic cells from large, flat, anchorage-dependent ECs. Key steps regulating EHT are difficult to elucidate using in vivo models because the hemogenic capacity of HECs occurs within a 2-day developmental window. Only a tiny number of cells undergo EHT in vivo. New model systems that overcome current in vivo obstacles are required so the details of EHT and the mechanisms that lead to the generation of HSPCs can be better understood. We previously reported that fully-committed ECs can be converted to functional HSPCs by ectopic expression of transcription factors Spi1, FOSB, GFI1, and Runx1 (SFGR) and an appropriate vascular niche (Sandler et al. Nature, 2014; Lis et al. Nature, 2017). But conversion using this method required several weeks and was associated with variable and low efficiency. Here, we show that our new in vitro EHT platform (ivEHT), very closely recapitulates the process of EHT in vivo and can be used to uncover heretofore unrecognized steps necessary for EC conversion to HSPCs. Methods All steps in the in vitro conversion of ECs to HSPCs were systematically analyzed and optimized. The refined model utilized new media constituents and polycistronic constructs to ensure ectopic expression of transcription factors Spi1, FOSB, GFI1 and Runx1 (SFGR)in human umbilical vein-ECs (HUVECs) in appropriate ratios and sequence. Results The new ivEHT platform yields efficient EC conversion to hematopoietic like CD45+ cells (≥80%) and high temporal reproducibility of conversion that is largely complete within 96 hrs. The ivEHT sequence of conversion closely follows the timing and sequence of EHT in vivo. Initially, transduced HUVECs are large, flat cells with CDH5-marked intercellular junctions and firm adhesion to basement membrane. Within the first 24hrs of induction, SFGR-HUVECs downregulate ECs markers CD31, KDR, CDH5 and activate the endogenous RUNX1 locus detected by the expression of nuclear GFP (GFPn) under control of the human RUNX1+24 enhancer (detected using a lentiviral reporter vector). Day 2 (D2) is characterized by complete clearance of endothelial-specific Weibel-Palade Bodies (WPBs, marked by Von Willebrand Factor, VWF) and excess mitochondria. This window also initiates morphologic changes in RUNX1-GFPn marked cells with loss of intercellular junctions and attachment to basement membrane and initial expression of hematopoietic markers such as CD45. Days 3-4 are marked by loss of cell volume and cytoplasm, cellular rounding and detachment from matrix, and increased expression of CD45 and other hematopoietic markers like cKIT, MPL, CD43. These stages of conversion are only identified in RUNX1-GFPn marked cells and by the end of D5, ~80% of cells have fully converted to CD45+ cells. Conversion is not accompanied by cell proliferation, but after D5 cell numbers expand rapidly. The ivEHT platform is scalable and highly reproducible making it amenable to higher-throughput testing in 96-well format using flow cytometry readouts. Using the ivEHT platform, we have captured and validated in AGM explants previously unobserved aspects of early EHT that is complete between D2-D3. Discussion This work promises a reproducible, faithful, and scalable platform which generates hematopoietic like cells while successfully recapitulating early steps of the EHT in the AGM. Importantly, this platform is readily accessible and is easily customized for discovery of EHT molecular details currently hidden to science, allowing significant advances in the understanding of how HSPCs emergence in vivo.

Pre-Clinical Developmental Hematopoiesis Demonstrates the Heterogeneity of KMT2A-Rearranged Infant Acute Lymphoblastic Leukemic Populations

KMT2A-rearranged (KMT2A-r) infant acute lymphoblastic leukemia (ALL) has a poor prognosis with increased risk of relapse when diagnosed at younger ages. Understanding the origin of these leukemic cells is crucial to comprehend disease development and prognosis differences. The unclear delineation of the identity of early blood populations in humans is a challenge in defining the cell type(s) driving this infant ALL. Recent pre-clinical studies revealed that definitive hematopoietic stem cells (HSCs) have minimal contribution to embryonic hematopoiesis, while independently arising embryonic hematopoietic progenitors (HPCs) that possess more limited self-renewal and/or multilineage differentiation capacities, are both necessary and sufficient to sustain the developing embryo. Here, we aim to leverage pre-clinical knowledge on the markers that distinguish HSCs from HPCs to investigate the potential contribution of HPCs in driving prenatal and infant leukemias. We defined transcriptional signatures from developmental HSC and HPC populations using thirteen single cell RNA sequencing (scRNA-seq) datasets from zebrafish, eight datasets generated by our research group and five from independent published studies. We then employed a neural network approach to define the orthologous cell type signatures most likely correlating with human healthy and blast cells from publicly available data of 18 KMT2A-r infant ALL patients. In accordance with previous literature, HSC and lymphomyeloid progenitor signatures are both highly represented in the leukemic blast populations. Additionally, we found subpopulation differences in gene expression within leukemic blasts cells including lymphoid progenitor, granulocytic, and T cell signatures, possibly suggesting heterogeneity in either their cell type of origin or developmental differentiation potential. Our findings support the ability to use orthologous scRNA-seq to decipher new understanding on the role of HSCs and HPCs in hematological malignances affecting infants and children.

Spatial Transcriptomics Reveals Distinct Hematopoietic Stem Cell Niches in Mouse Fetal Liver

The hematopoietic stem cell (HSC) microenvironment, termed the niche, supports the proliferation, self-renewal, and differentiation abilities of HSCs. The definitive HSCs emerge from the hemogenic endothelium in the aorta-gonad-mesonephros (AGM) region after E10.5, and then migrate to the fetal liver (FL) after E12.5 for expansion. After E18.5, HSCs migrate to the bone marrow where they reside for the postnatal stage and adulthood. Because the fetal liver is thought to be a harbor for the rapid expansion of HSCs, numerous studies have focused on the FL-HSC niche in the search for novel niche factors and niche cells that support HSC expansion. However, to our knowledge thus far, there have been no successes in translating the niche factors to a clinical application for the expansion of HSCs ex vivo. In this study, we use cutting-edge spatial transcriptomics to comprehensively investigate the transcriptomics and interactions between HSCs and the niche cells in the fetal liver. As a result, we have uncovered two distinct niches: the portal-vessel (PV) niche and the sinusoidal niche. To understand the spatial distribution and interactions between FL-HSCs and niche cells, we introduced 2 spatial transcriptomic methods, Slide-seq (10uM resolution), and 10x Genomics Visium (55uM resolution), in our study on E14.5 and E16.5 mouse fetal liver. By integrating with single-cell RNA sequencing, we discovered the spatial transcriptomics of HSCs and potential niche cells, including hepatoblasts, endothelial cells, macrophages, megakaryocytes, and mesenchymal stromal cells (MSCs) in E14.5 and E16.5 mouse fetal liver. Interestingly, we found that MSCs and hepatoblasts were characterized by enriched N-cadherin expression. Both slide-seq and 10x Visium showed that the N-cadherin-expressing MSCs are enriched in the portal vessel area. Importantly, the majority of FL-HSCs are in close proximity to N-cadherin-expressing MSCs and endothelial cells, compared with other potential niche cells, indicating a supportive role of N-cadherin-expressing MSCs and endothelial cells in HSC maintenance. Subsequent CellPhoneDB (CPDB) analysis demonstrated that the N-cadherin-expressing MSCs are major niche-signaling senders with an enriched expression of niche factors and stemness pathway-related ligands. This finding is consistent with our previous finding that N-cadherin-expressing MSCs can maintain reserve HSCs in the adult bone marrow. To investigate the potential role of N-cadherin-expressing cells in supporting FL-HSCs, we generated an N-cadCreER;Cxcl12 and an N-cadCreER;Kitl mouse model to conditionally knockout the well-studied niche factors, Cxcl12 and Kitl (Scf), in N-cadherin-expressing cells. Interestingly, deletion of either Cxcl12 or Kitl through N-cad-CreERT induction resulted in the expansion of myeloid-biased FL-HSCs. Slide-seq further showed that depletion of Cxcl12 through N-cad-CreERT induction led to the repositioning of FL-HSCs from the PV region, which is enriched with MSCs, to the hepatic sinusoidal region, which lacks MSCs. These findings suggested the existence of distinct niches in the fetal liver: the PV niche is composed of N-cadherin + MSCs and peri-PV endothelial cells, and it maintains FL-HSCs with multilineage potential. In contrast, the sinusoidal niche preliminary consists of sinusoidal endothelial cells, hepatocytes, and hepatoblasts, which facilitates the proliferation of FL-HSCs but with a bias toward myeloid lineage. Previous studies suggested that the two forms of Kitl, soluble Kitl (sKitl) and membrane-bound Kitl (mKitl), may have different functions in supporting HSCs. Here, we discovered that mKitl is highly enriched in MSCs, whereas hepatoblasts are the major source of the sKitl. This finding suggests that mKitl may serve as the key niche factor that contributes to the homing and maintenance of FL-HSCs. In summary, by using cutting-edge spatial transcriptomics, we uncovered the existence of distinct niches, the portal-vessel (PV) niche and the sinusoidal niche, in mouse fetal liver and the critical role of N-cadherin + MSCs in maintaining FL-HSCs and a role of hepatoblasts in promoting proliferation. The insight learned from this study enhances our understanding of the complex regulation of HSCs and opens new avenues for potential clinical applications.

Haematopoietic stem and progenitor cell heterogeneity is inherited from the embryonic endothelium

Definitive haematopoietic stem and progenitor cells (HSPCs) generate erythroid, lymphoid and myeloid lineages. HSPCs are produced in the embryo via transdifferentiation of haemogenic endothelial cells in the aorta–gonad–mesonephros (AGM). HSPCs in the AGM are heterogeneous in differentiation and proliferative output, but how these intrinsic differences are acquired remains unanswered. Here we discovered that loss of microRNA (miR)-128 in zebrafish leads to an expansion of HSPCs in the AGM with different cell cycle states and a skew towards erythroid and lymphoid progenitors. Manipulating miR-128 in differentiating haemogenic endothelial cells, before their transition to HSPCs, recapitulated the lineage skewing in both zebrafish and human pluripotent stem cells. miR-128 promotes Wnt and Notch signalling in the AGM via post-transcriptional repression of the Wnt inhibitor csnk1a1 and the Notch ligand jag1b. De-repression of cskn1a1 resulted in replicative and erythroid-biased HSPCs, whereas de-repression of jag1b resulted in G2/M and lymphoid-biased HSPCs with long-term consequence on the respective blood lineages. We propose that HSPC heterogeneity arises in the AGM endothelium and is programmed in part by Wnt and Notch signalling.

Heme-deficient primitive red blood cells induce HSPC ferroptosis by altering iron homeostasis during zebrafish embryogenesis.

The crosstalk between hematopoietic lineages is important for developmental hematopoiesis. However, the role of primitive red blood cells (RBCs) in the formation of definitive hematopoietic stem and progenitor cells (HSPCs) is largely unknown. Primitive RBC deficiencies in mammals always lead to early embryonic lethality, but zebrafish lines with RBC deficiencies can survive to larval stage. By taking advantage of a zebrafish model, we find that the survival of nascent HSPCs is impaired in alas2- or alad-deficient embryos with aberrant heme biosynthesis in RBCs. Heme-deficient primitive RBCs induce ferroptosis of HSPCs through the disruption of iron homeostasis. Mechanistically, heme-deficient primitive RBCs cause blood iron-overload via Slc40a1, and an HSPC iron sensor, Tfr1b, mediates excessive iron absorption. Thus, iron-induced oxidative stress stimulates the lipid peroxidation, which directly leads to HSPC ferroptosis. Anti-ferroptotic treatments efficiently reverse HSPC defects in alas2 or alad mutants. HSPC transplantation assay reveals that the attenuated erythroid reconstitution efficiency may result from the ferroptosis of erythrocyte-biased HSPCs. Together, these results illustrate that heme-deficient primitive RBCs are detrimental to HSPC production and may provide potential implications for iron dysregulation-induced hematological malignancies.

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.

Human fetal T cells: Insights into developmental specialization and mechanisms of lineage transition

The switch from primitive to definitive hematopoiesis occurs early in development through the emergence of a wave of definitive hematopoietic stem cells from intraembryonic sites, supplanting the original primitive population of extraembryonically derived stem cells. When it became clear that unique features of the fetal immune system could not be reproduced by adult stem cells, it was hypothesized that a lineage of definitive fetal hematopoietic stem cells predominates antenatally, ultimately giving way to an emerging wave of adult stem cells and resulting in a "layered" fetal immune system consisting of overlapping lineages. It is now clear, however, that the transition from human fetal-to-adult T cell identity and function does not occur due to a binary switch between distinct fetal and adult lineages. Rather, recent evidence from single cell analysis suggests that during the latter half of fetal development a gradual, progressive transition occurs at the level of hematopoietic stem-progenitor cells (HSPCs) which is reflected in their T cell progeny. At a transcriptional level, clusters of genes are up- and down-regulated with sequenced timing, suggesting that the transition is under the control of master regulatory factors, including epigenetic modifiers. The net effect is still one of "molecular layering," that is, the continuous layering of iterative generations of HSPCs and T cells that arise through progressive changes in gene expression. This review will focus on recent discoveries that elucidate mechanisms of fetal T cell function and the transition from fetal to adult identity. The epigenetic landscape of fetal T cells facilitates their ability to fulfill the dominant fetal mandate of generating tolerance against self, maternal, and environmental antigens by supporting their predisposition to differentiate into CD25+ FoxP3+ regulatory T cells (TRegs ). We will explore how the coordinated development of two complementary populations of fetal T cells-conventional T cells dominated by TRegs and tissue-associated memory effector cells with innate-like inflammatory potential-is crucial not only for maintaining intrauterine immune quiescence but also for facilitating an immune response that is appropriately tuned for the bombardment of antigen stimulation that happens at birth.

The eighth International Forum on Stem Cells: virtual meeting, October 20-21, 2022.

The eighth International Forum on Stem Cells: virtual meeting, October 20-21, 2022.

Using Spatial Transcriptomics to Reveal Fetal Liver Hematopoietic Stem Cell-Niche Interactions

The hematopoietic stem cell (HSC) microenvironment, termed the niche, supports the proliferation, self-renewal, and differentiation abilities of HSCs. The definitive HSCs emerge from the hemogenic endothelium in the aorta-gonad-mesonephros (AGM) region after E10.5, and then migrate to the fetal liver after E12.5 for expansion. After E18.5, HSCs migrate to the bone marrow where they reside for the postnatal stage and adulthood. Because the fetal liver is thought to be a harbor for the rapid expansion of HSCs, numerous studies have focused on the fetal liver HSC niche in the search for novel niche factors and niche cells that support HSC expansion. However, to our knowledge, there are no successes in translating the niche factors to a clinical application for the expansion of HSCs ex vivo. In this study, we use cutting-edge spatial transcriptomics to comprehensively investigate the transcriptomics and interactions between HSCs and the niche cells in the fetal liver, and to identify the niche cells and factors for HSC expansion. To understand the spatial distribution and interactions between HSCs and niche cells in fetal liver, we introduced 2 spatial transcriptomic methods, Slide-seq (Stickels et al. Nature Biotechnology, 2021), and 10x Genomics Visium Spatial Gene Expression, in our study on E14.5 and E16.5 mouse fetal liver. By integrating with a 10x Genomics' Chromium™ Single Cell 3' Solution, we discovered the spatial transcriptomics of HSCs and potential niche cells, including hepatoblasts, endothelium cells, macrophages, megakaryocytes, and hepatic stellate cells/perivascular mesenchymal cells (PMCs) in E14.5 and E16.5 mouse fetal liver. Interestingly, we found that PMCs and hepatoblasts were characterized by enriched N-cadherin expression. Both slide-seq and 10x Visium showed that the N-cadherin-expressing PMCs are enriched in the portal vessel area. Importantly, the majority of fetal liver HSCs are in close proximity to N-cadherin-expressing PMCs and endothelial cells, indicating a supportive role of N-cadherin-expressing PMCs and endothelial cells in HSC maintenance. Subsequent CellPhoneDB (CPDB) analysis demonstrated that the N-cadherin-expressing PMCs are major niche-signaling senders with an enriched expression of niche factors, such as CXCL12 and KITL, and stemness pathway-related ligands, such as IGFs, TGFβs, JAG2, and DLK1, indicating N-cadherin-expressing PMCs could be the major niche cells in supporting HSCs in the fetal liver. This finding is consistent with our previous finding that N-cadherin-expressing bone and marrow stromal progenitor cells can maintain reserve HSCs in the adult bone marrow. In addition, endothelium cells have enriched expression of several niche factors, including BMPs, KITL, IGFs and TGFβs. To investigate the potential role of N-cadherin-expressing cells in supporting fetal liver HSCs, we generated an N-cadCreER;Cxcl12 and an N-cadCreER;Scf mouse model to conditionally knockout the well-studied niche factors, CXCL12 and SCF, in N-cadherin-expressing cells. Interestingly, conditional knockout of either Cxcl12 or Scf in N-cadherin-expressing cells resulted in an increase in the number of HSCs. Moreover, flowcytometry analysis and scRNA-seq on conditional knockout of Cxcxl12 in N-cadherin-expressing cells showed a myeloid-biased differentiation. Subsequential slide-seq showed that fetal liver HSCs were located close to N-cadherin-expressing PMCs and endothelial cells. Intriguingly, fetal liver HSCs migrated toward and resided in close proximity to megakaryocytes after conditional knockout of Cxcxl12 in N-cadherin-expressing cells. In summary, we postulate that N-cadherin-expressing cells, especially PMCs, maintain fetal liver HSCs. The knockout of Cxcl12 in N-cadherin-expressing cells leads to the migration of HSCs towards megakaryocytes, which may induce HSC expansion and biased differentiation. In summary, by using cutting-edge spatial transcriptomics, we revealed a comprehensive spatial transcriptomics of HSCs and niche cells in E14.5 and E16.5 mouse fetal liver. The N-cadherin-expressing cells in the fetal liver is a major niche in maintaining HSCs, while other potential niches may be responsible for the expansion of HSCs. In the future, we will use multiple approaches to verify the distribution changes of HSCs in N-cadCreER;Cxcl12 mouse, and to reveal the niches in support of the expansion of HSCs.

DIAGNOSIS AND MANAGEMENT OF CTLA-4 VARIANT IMMUNE DYSREGULATION

<h3>Introduction</h3> Cytotoxic T-lymphocyte antigen 4 (CTLA-4) is a negative regulator of the immune system and when deficient, can result in immune dysregulation. Here we present a patient with progressive immune dysregulation and confirmed CTLA-4 haploinsufficiency. <h3>Case Description</h3> A 25-year-old male diagnosed with Evans syndrome and CNS vasculitis in childhood was stabilized on mycophenolate mofetil (MMF). At age 14, he developed pneumonia and had a lung biopsy consistent with lymphocytic interstitial pneumonia. Immune evaluation confirmed common variable immunodeficiency with dysregulated T and B cell subsets, and he was started on immunoglobulin replacement therapy. Genetic evaluation revealed 2 CTLA-4 variants of uncertain significance and functional assays confirmed CTLA-4 haploinsufficiency. Subsequently, he developed splenomegaly, chronic lung disease, and EBV viremia. A repeat lung biopsy was consistent with polymorphic EBV-positive lymphoproliferative disorder, prompting rituximab therapy. CTLA-4 Ig could not be utilized because of the potential of worsening chronic EBV. He was prescribed sirolimus. Acyclovir and trimethoprim-sulfamethoxazole were prescribed prophylactically for T cell lymphopenia. <h3>Discussion</h3> This patient with EBV and CTLA-4-driven lung disease is susceptible to worsening lung and lymphoproliferative diseases and recurrent infections. Management options include rituximab for EBV, sirolimus for immune dysregulation, and when possible, CTLA-4 Ig once EBV is controlled. The manifestations of CTLA-4 haploinsufficiency are unique and patients require close clinical follow up. Additional biomarkers of dysregulation may include monitoring of dysregulated T and B cell subsets. Management aims include targeted therapy to control immune dysregulation and potentially definitive hematopoietic stem cell transplantation.

Lifelong multilineage contribution by embryonic-born blood progenitors.

Haematopoietic stem cells (HSCs) arise in the embryo from the arterial endothelium through a process known as the endothelial-to-haematopoietic transition (EHT)1-4. This process generates hundreds of blood progenitors, of which a fraction go on to become definitive HSCs. It is generally thought that most adult blood is derived from those HSCs, but to what extent other progenitors contribute to adult haematopoiesis is not known. Here we use in situ barcoding and classical fate mapping to assess the developmental and clonal origins of adult blood in mice. Our analysis uncovers an early wave of progenitor specification-independent of traditional HSCs-that begins soon after EHT. These embryonic multipotent progenitors (eMPPs) predominantly drive haematopoiesis in the young adult, have a decreasing yet lifelong contribution over time and are the predominant source of lymphoid output. Putative eMPPs are specified within intra-arterial haematopoietic clusters and represent one fate of the earliest haematopoietic progenitors. Altogether, our results reveal functional heterogeneity during the definitive wave that leads to distinct sources of adult blood.

Robust temporal map of human in vitro myelopoiesis using single-cell genomics

Myeloid cells are central to homeostasis and immunity. Characterising in vitro myelopoiesis protocols is imperative for their use in research, immunotherapies, and understanding human myelopoiesis. Here, we generate a >470K cells molecular map of human induced pluripotent stem cells (iPSC) differentiation into macrophages. Integration with in vivo single-cell atlases shows in vitro differentiation recapitulates features of yolk sac hematopoiesis, before definitive hematopoietic stem cells (HSC) emerge. The diversity of myeloid cells generated, including mast cells and monocytes, suggests that HSC-independent hematopoiesis can produce multiple myeloid lineages. We uncover poorly described myeloid progenitors and conservation between in vivo and in vitro regulatory programs. Additionally, we develop a protocol to produce iPSC-derived dendritic cells (DC) resembling cDC2. Using CRISPR/Cas9 knock-outs, we validate the effects of key transcription factors in macrophage and DC ontogeny. This roadmap of myeloid differentiation is an important resource for investigating human fetal hematopoiesis and new therapeutic opportunities.

Mapping human haematopoietic stem cells from haemogenic endothelium to birth.

The ontogeny of human haematopoietic stem cells (HSCs) is poorly defined owing to the inability to identify HSCs as they emerge and mature at different haematopoietic sites1. Here we created a single-cell transcriptome map of human haematopoietic tissues from the first trimester to birth and found that the HSC signature RUNX1+HOXA9+MLLT3+MECOM+HLF+SPINK2+ distinguishes HSCs from progenitors throughout gestation. In addition to the aorta-gonad-mesonephros region, nascent HSCs populated the placenta and yolk sac before colonizing the liver at 6 weeks. A comparison of HSCs at different maturation stages revealed the establishment of HSC transcription factor machinery after the emergence of HSCs, whereas their surface phenotype evolved throughout development. The HSC transition to the liver marked a molecular shift evidenced by suppression of surface antigens reflecting nascent HSC identity, and acquisition of theHSC maturity markers CD133 (encoded byPROM1) and HLA-DR. HSC origin was tracked to ALDH1A1+KCNK17+ haemogenic endothelial cells, which arose from an IL33+ALDH1A1+ arterial endothelial subset termed pre-haemogenic endothelial cells. Using spatial transcriptomics and immunofluorescence, we visualized this process in ventrally located intra-aortic haematopoietic clusters. The in vivo map of human HSC ontogeny validated the generation of aorta-gonad-mesonephros-like definitive haematopoietic stem and progenitor cells from human pluripotent stem cells, and serves as a guide to improve their maturation to functional HSCs.

Developmental maturation of the hematopoietic system controlled by a Lin28b-let-7-Cbx2 axis.

SUMMARYHematopoiesis changes over life to meet the demands of maturation and aging. Here, we find that the definitive hematopoietic stem and progenitor cell (HSPC) compartment is remodeled from gestation into adulthood, a process regulated by the heterochronic Lin28b/let-7 axis. Native fetal and neonatal HSPCs distribute with a pro-lymphoid/erythroid bias with a shift toward myeloid output in adulthood. By mining transcriptomic data comparing juvenile and adult HSPCs and reconstructing coordinately activated gene regulatory networks, we uncover the Polycomb repressor complex 1 (PRC1) component Cbx2 as an effector of Lin28b/let-7’s control of hematopoietic maturation. We find that juvenile Cbx2−/− hematopoietic tissues show impairment of B-lymphopoiesis, a precocious adult-like myeloid bias, and that Cbx2/PRC1 regulates developmental timing of expression of key hematopoietic transcription factors. These findings define a mechanism of regulation of HSPC output via chromatin modification as a function of age with potential impact on age-biased pediatric and adult blood disorders.

Pre-configuring chromatin architecture with histone modifications guides hematopoietic stem cell formation in mouse embryos

The gene activity underlying cell differentiation is regulated by a diverse set of transcription factors (TFs), histone modifications, chromatin structures and more. Although definitive hematopoietic stem cells (HSCs) are known to emerge via endothelial-to-hematopoietic transition (EHT), how the multi-layered epigenome is sequentially unfolded in a small portion of endothelial cells (ECs) transitioning into the hematopoietic fate remains elusive. With optimized low-input itChIP-seq and Hi-C assays, we performed multi-omics dissection of the HSC ontogeny trajectory across early arterial ECs (eAECs), hemogenic endothelial cells (HECs), pre-HSCs and long-term HSCs (LT-HSCs) in mouse embryos. Interestingly, HSC regulatory regions are already pre-configurated with active histone modifications as early as eAECs, preceding chromatin looping dynamics within topologically associating domains. Chromatin looping structures between enhancers and promoters only become gradually strengthened over time. Notably, RUNX1, a master TF for hematopoiesis, enriched at half of these loops is observed early from eAECs through pre-HSCs but its enrichment further increases in HSCs. RUNX1 and co-TFs together constitute a central, progressively intensified enhancer-promoter interactions. Thus, our study provides a framework to decipher how temporal epigenomic configurations fulfill cell lineage specification during development.

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.

Lin28b-Let-7-PRC1 Axis Guides Developmental Maturation of the Hematopoietic System

Polycomb group (PcG) proteins are a well-studied group of chromatin modifiers belonging to one of two distinct multi-protein complexes: Polycomb repressive complex 1 (PRC1) and PRC2. With definitive hematopoiesis, PRCs contribute to many aspects of fetal and adult blood formation. However, it is largely unknown how many of the age-specific effects of PRCs in hematopoiesis are regulated. Here, we show that the definitive hematopoietic stem and progenitor cell (HSPC) compartment is remodeled from the fetus to the neonate and into young adulthood coordinated with changes in mature blood cell output. This process is in part dependent on the PRC1 component Cbx2, which is regulated by the heterochronic Lin28b/let-7 axis.First, we quantified various population of definitive hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) using midgestation fetal liver (FL, embryonic day 14.5 (E14.5)), newborn bone marrow (BM, postnatal day 0-1), or young adult (postnatal age 6 to 8 weeks) BM. The lymphoid biased multipotent progenitor 4 (MPP4, ~0.9-fold) declined as the mice matured and aged. We also found erythroid-biased MPP2 diminished (~0.7-fold) while myeloid-biased MPP3 increased (~1.7-fold) with maturation. Using isolated long-term (LT) HSCs from these three stages, we found that E14.5 FL (~8.0-fold) and neonatal LT-HSC (~4.0-fold) showed more rapid B-cell reconstitution compared to young adult LT-HSCs upon transplantation. We found that many of these effects were regulated by Lin28b/let-7.Next, we aimed to determine the downstream mediators of Lin28/let-7's effect on HSPCs maturation. By interrogating gene regulatory subnetworks differentially active across mouse HSPC maturation and mining these subnetworks for predicted let-7 target transcripts, we found Cbx2 enriched in E14.5 FL (P=0.003) and adult HSPCs ectopically expressing LIN28B relative to wild-type adult HSPCs. In cell-based assays, we confirmed that let-7 microRNAs directly regulated CBX2 protein levels. Thus, the Lin28b/let-7 axis governs CBX2 protein levels, leading us to hypothesize that this axis exerts its wide-ranging effects on hematopoietic maturation by regulating PRC1 by controlling Cbx2 levels.As CBX2's developmental stage-specific functions have not been investigated, we generated Cbx2-/-embryos and investigated definitive FL hematopoiesis. We observed skewing of myeloerythorid progenitors to an adult-like myeloid-predominant distribution in Cbx2-/- embryos (P=0.0002), and B-cells in Cbx2-/- neonatal spleens were diminished (P=0.04). We further examined this effect using transplanted Cbx2-/- MPP4 from E14.5 FL which resulted in a decreased donor derived B-lymphoid output compared to wild-type littermates (~0.7-fold).To understand the functional role of Cbx2/PRC1 in juvenile hematopoiesis, we next investigated the role of Cbx2 in maintaining histone H2A monoubiquitinylation (H2AK119Ub) - the histone modification placed by PRC1 - in FL HSPCs. In Cbx2-/- FL HSPCs, the global distribution of H2AK119Ub localization did not change, but several specific H2AK119Ub peaks were altered. We observed differential H2AK119Ub abundance associated with a candidate enhancer within the Erg gene, suggestive of control of Erg expression by Lin28b/let-7/Cbx2. We confirmed that this enhancer activated transcription from a minimal promoter (~8-fold). Erg expression was increased in perinatal spleens of Cbx2-/- mice compared to Cbx2+/+ littermates (~4-fold). Moreover, we found that Cbx2 could repress ERG expression as well as other master HSPC transcription factors.Overall, our findings show that the Lin28b/let-7-axis controls developmental stage-specific hematopoietic output through PRC1-mediated chromatin remodeling. These findings demonstrate a key mechanism by which HSPCs alter their properties during developmental maturation with relevance to age-skewed blood disorders. DisclosuresNo relevant conflicts of interest to declare.

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.

Using Spatial Transcriptomics to Reveal Fetal Liver Hematopoietic Stem Cell-Niche Interactions

The hematopoietic stem cell (HSC) microenvironment, termed the niche, supports the proliferation, self-renewal, and differentiation abilities of HSCs. The definitive HSCs emerge from the hemogenic endothelium in the aorta-gonad-mesonephros (AGM) region after E11.5, and then migrate to the fetal liver after E12.5 for expansion. After E17.5, HSCs migrate to the bone marrow and reside in the bone marrow for the postnatal stage and adulthood. Because the fetal liver is thought to be a harbor for the rapid expansion of HSCs, numerous studies have focused on the fetal liver HSC niche in the search for novel niche factors and niche cells that support HSC expansion. However, to our knowledge, there are no successes in translating the niche factors to a clinical application for the expansion of HSCs ex vivo. In this study, we are using cutting-edge spatial transcriptomics to comprehensively study the transcriptomics and interactions between HSCs and the niche cells in the fetal liver, and in search of the niche cells and factors for HSC expansion.To understand the spatial distribution and interactions between HSCs and niche cells in the fetal liver, we introduced 2 spatial transcriptomic methods, slide-seq, and 10x Visium, in our study on E14.5 mouse fetal liver. By integrating with a parallel single-cell sequencing analysis, we revealed the spatial transcriptomics of HSCs and potential niche cells, including hepatoblasts, endothelium cells, macrophages, megakaryocytes, and hepatic stellate cells/perivascular mesenchymal cells (PMCs) in E14.5 mouse fetal liver. Interestingly, we found that the PMCs were characterized by enriched N-cadherin expression. Both slide-seq and 10x Visium showed that the N-cadherin-expressing PMCs are enriched in the portal vessel area. Importantly, the majority of fetal liver HSCs are in close proximity to N-cadherin-expressing PMCs, indicating a supportive role of N-cadherin-expressing PMCs in HSC maintenance. Subsequent CellPhoneDB (CPDB) analysis demonstrated that the N-cadherin-expressing PMCs are major niche-signaling senders with an enriched expression of niche factors, such as CXCL12 and KITL, and stemness pathway-related ligands, such as IGF1, IGF2, TGFβ2, TGFβ3, JAG2, and DLK1, indicating N-cadherin-expressing PMCs could be the major niche cells in supporting HSCs in the fetal liver. This finding was consistent with our previous finding that N-cadherin-expressing bone and marrow stromal progenitor cells can maintain reserve HSCs in the adult bone marrow. Moreover, CPDB analysis indicated that other potential niche cells, such as endothelium cells, macrophages, and megakaryocytes, may support HSCs in different signal transduction pathways. For example, endothelium cells have an enriched expression of KITL, IGF2, DLL1, TGFβ1, and TGFβ2; macrophages have enriched expression of KITL, IFNγ, and TGFβ1; megakaryocytes have enriched expression of PF4, JAG2 and TGFβ1. Intriguingly, our previous studies showed that megakaryocytes could promote HSC expansion under stress conditions in the bone marrow. To investigate the potential role of N-cadherin-expressing cells in supporting fetal liver HSCs, we generated an N-cad CreER;Cxcl12 and an N-cad CreER;Scf mouse model to conditionally knockout the well-studied niche factors, CXCL12 and SCF, in N-cadherin-expressing cells. Conditional knockout of either Cxcl12 or Scf in N-cadherin-expressing cells resulted in an increase in the number of HSCs. Moreover, conditional knockout of Cxcxl12 in N-cadherin-expressing cells also resulted in a myeloid-biased differentiation. We postulate that the knockout of Cxcl12 or Scf in N-cadherin-expressing cells leads to the migration of HSCs towards other potential niche cells, such as macrophages and megakaryocytes, which may induce HSC expansion and biased differentiation.In summary, by using cutting-edge spatial transcriptomics, we revealed a comprehensive spatial transcriptomics of HSCs and niche cells in E14.5 mouse fetal liver. The N-cadherin-expressing cells in the fetal liver is a major niche in maintaining HSCs, while other potential niches may be responsible for the expansion of HSCs. In the future, we will use multiple approaches, such as spatial transcriptomics and fluorescence in situ hybridization (FISH), to verify the distribution changes of HSCs in N-cad CreER;Cxcl12 mouse, and to reveal the niches in support of the expansion of HSCs. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.