Aorta-gonad-mesonephros Research Articles

IκBα controls dormancy in hematopoietic stem cells via retinoic acid during embryonic development

Recent findings suggest that Hematopoietic Stem Cells (HSC) and progenitors arise simultaneously and independently of each other already in the embryonic aorta-gonad mesonephros region, but it is still unknown how their different features are established. Here, we uncover IκBα (Nfkbia, the inhibitor of NF-κB) as a critical regulator of HSC proliferation throughout development. IκBα balances retinoic acid signaling levels together with the epigenetic silencer, PRC2, specifically in HSCs. Loss of IκBα decreases proliferation of HSC and induces a dormancy related gene expression signature instead. Also, IκBα deficient HSCs respond with superior activation to in vitro culture and in serial transplantation. At the molecular level, chromatin regions harboring binding motifs for retinoic acid signaling are hypo-methylated for the PRC2 dependent H3K27me3 mark in IκBα deficient HSCs. Overall, we show that the proliferation index in the developing HSCs is regulated by a IκBα-PRC2 axis, which controls retinoic acid signaling.

A subset of megakaryocytes regulates development of hematopoietic stem cell precursors

Understanding the regulatory mechanisms facilitating hematopoietic stem cell (HSC) specification during embryogenesis is important for the generation of HSCs in vitro. Megakaryocyte emerged from the yolk sac and produce platelets, which are involved in multiple biological processes, such as preventing hemorrhage. However, whether megakaryocytes regulate HSC development in the embryonic aorta-gonad-mesonephros (AGM) region is unclear. Here, we use platelet factor 4 (PF4)-Cre;Rosa-tdTomato+ cells to report presence of megakaryocytes in the HSC developmental niche. Further, we use the PF4-Cre;Rosa-DTA (DTA) depletion model to reveal that megakaryocytes control HSC specification in the mouse embryos. Megakaryocyte deficiency blocks the generation and maturation of pre-HSCs and alters HSC activity at the AGM. Furthermore, megakaryocytes promote endothelial-to-hematopoietic transition in a OP9-DL1 coculture system. Single-cell RNA-sequencing identifies megakaryocytes positive for the cell surface marker CD226 as the subpopulation with highest potential in promoting the hemogenic fate of endothelial cells by secreting TNFSF14. In line, TNFSF14 treatment rescues hematopoietic cell function in megakaryocyte-depleted cocultures. Taken together, megakaryocytes promote production and maturation of pre-HSCs, acting as a critical microenvironmental control factor during embryonic hematopoiesis.

A multistep computational approach reveals a neuro-mesenchymal cell population in the embryonic hematopoietic stem cell niche.

The first hematopoietic stem and progenitor cells (HSPCs) emerge in the Aorta-Gonad-Mesonephros (AGM) region of the mid-gestation mouse embryo. However, the precise nature of their supportive mesenchymal microenvironment remains largely unexplored. Here, we profiled transcriptomes of laser micro-dissected aortic tissues at three developmental stages and individual AGM cells. Computational analyses allowed the identification of several cell subpopulations within the E11.5 AGM mesenchyme, with the presence of a yet unidentified subpopulation characterized by the dual expression of genes implicated in adhesive or neuronal functions. We confirmed the identity of this cell subset as a neuro-mesenchymal population, through morphological and lineage tracing assays. Loss of function in the zebrafish confirmed that Decorin, a characteristic extracellular matrix component of the neuro-mesenchyme, is essential for HSPC development. We further demonstrated that this cell population is not merely derived from the neural crest, and hence, is a bona fide novel subpopulation of the AGM mesenchyme.

Runx1+ vascular smooth muscle cells are essential for hematopoietic stem and progenitor cell development in vivo.

Hematopoietic stem cells (HSCs) produce all essential cellular components of the blood. Stromal cell lines supporting HSCs follow a vascular smooth muscle cell (vSMC) differentiation pathway, suggesting that some hematopoiesis-supporting cells originate from vSMC precursors. These pericyte-like precursors were recently identified in the aorta-gonad-mesonephros (AGM) region; however, their role in the hematopoietic development in vivo remains unknown. Here, we identify a subpopulation of NG2+Runx1+ perivascular cells that display a sclerotome-derived vSMC transcriptomic profile. We show that deleting Runx1 in NG2+ cells impairs the hematopoietic development in vivo and causes transcriptional changes in pericytes/vSMCs, endothelial cells and hematopoietic cells in the murine AGM. Importantly, this deletion leads also to a significant reduction of HSC reconstitution potential in the bone marrow in vivo. This defect is developmental, as NG2+Runx1+ cells were not detected in the adult bone marrow, demonstrating the existence of a specialised pericyte population in the HSC-generating niche, unique to the embryo.

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.

Characterization of Niche Cells and Signals Regulating Stepwise Embryonic Hematopoiesis Using Slide-Seq and Merfish Spatial Transcriptomics

Stagewise HSC development initiates in the aorta-gonad-mesonephros (AGM) region during mouse embryonic day (E)9.5-E12.5 and progresses through stages including hemogenic endothelial cell (HEC) specification, the endothelial to hematopoietic transition (EHT), and maturation. The nascent HSCs (pre-HSCs) form in the AGM region and subsequently migrate to the fetal liver where they undergo further maturation and expansion. Recent advances in single cell technologies have significantly enhanced our understanding of intrinsic cellular programs involved in HSC development. However, our knowledge about niche cells and the signals they emanate in the AGM region, as well as their contribution to HSC development, remain limited. Here we've conducted spatial transcriptomics analysis using Slide-seq on mouse trunk tissues, including both the AGM region and the fetal liver, at E10.5, E11.5 and E12.5. In addition, we performed scRNA-seq on the cells collected from the AGM and trunk at E10.5, E11.5 and E12.5, as well as the fetal liver at E12.5. To integrate the Slide-seq and scRNA-seq data, we developed an algorithm that leverages capabilities from both spatial and in-depth transcriptional sequencing. As a result, we were able to successfully reconstitute the structures of the trunk tissues and obtain gene expression profiles of individual cells at 10 µm resolution. From the analysis, we identified 7 cell clusters surrounding the aorta, with one cluster comprised of Nfgr + cells predominantly occupying the dorsal aorta. The ventral part of aorta displayed a complex and multilayered cellular architecture, featuring pre-HSCs, endothelial cells, mesonephric cells, and macrophages. Notably, we identified a unique mesenchymal stromal cell (MSC) population that co-express Cdh2 (encodes N-cadherin or N-cad) and Pdgfra; thus, we call these cells N-cad + MSCs. These N-cad + MSCs were particularly enriched at the ventral part of the aorta, forming a single cell layer positioned between an inner layer of aortic endothelial cells and an outer layer of mesonephric cells. Next, we utilized several bioinformatics tools (CellphoneDB, CellChat, NicheChat, and StringDB) in effort to identify potential signals that could impact embryonic hematopoiesis. The results revealed intricate interactions between N-cad + MSCs and pre-HSCs, for example, involving JAG1-NOTCH1 and SDF-1 (encoded by Cxcl12)-CXCR4 ligand-receptor pairs. To validate the findings obtained by the Slide-seq data, we selected and designed customized RNA probes for 140 genes of interest, including cell markers and potential signals. We then conducted MERFISH (Multiplexed Error-Robust Fluorescence in situ Hybridization) on 18 E11.5 trunk sections. The MERFISH data enabled us to characterize the spatially resolved niche cells and their derived signals in the AGM region at the single-cell level, which largely corroborated the results obtained from the Slide-seq data. Specifically, the MERFISH data indicates that N-cad + MSCs are in direct contact with aortic endothelial cells and are the primary cellular source of Jag1 and Cxcl12 around the aorta. Given the unique position of N-cad + MSCs and their intricate interactions with pre-HSCs, we formulated the hypothesis that N-cad + MSCs play a crucial role in embryonic hematopoiesis. To functionally validate our hypothesis, we are conducting a series of experiments using Ncad CreER; Cxcl12 flox/floxand Ncad CreER; Jag1 flox/flox mouse models. We anticipate that embryos lacking N-cad + MSC derived Jag1 or Cxcl12 will exhibit a substantial decline in intra-aortic hematopoietic clusters, phenotypic pre-HSCs, and functional HSCs. In summary, our study employed sequencing-based (Slide-seq) and imaging-based (MERFISH) spatial transcriptomics technologies to successfully characterize dynamic, spatially resolved profiles of niche components and associated signaling in the AGM region at the single cell level. This work significantly enhances our understanding of niche signaling and its role in regulating embryonic hematopoiesis and may aid the development of novel methods for inducing the transition from pluripotent stem cells to HSCs.

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.

De novo hematopoiesis from the fetal lung

AbstractHemogenic endothelial cells (HECs) are specialized cells that undergo endothelial-to-hematopoietic transition (EHT) to give rise to the earliest precursors of hematopoietic progenitors that will eventually sustain hematopoiesis throughout the lifetime of an organism. Although HECs are thought to be primarily limited to the aorta-gonad-mesonephros (AGM) during early development, EHT has been described in various other hematopoietic organs and embryonic vessels. Though not defined as a hematopoietic organ, the lung houses many resident hematopoietic cells, aids in platelet biogenesis, and is a reservoir for hematopoietic stem and progenitor cells (HSPCs). However, lung HECs have never been described. Here, we demonstrate that the fetal lung is a potential source of HECs that have the functional capacity to undergo EHT to produce de novo HSPCs and their resultant progeny. Explant cultures of murine and human fetal lungs display adherent endothelial cells transitioning into floating hematopoietic cells, accompanied by the gradual loss of an endothelial signature. Flow cytometric and functional assessment of fetal-lung explants showed the production of multipotent HSPCs that expressed the EHT and pre-HSPC markers EPCR, CD41, CD43, and CD44. scRNA-seq and small molecule modulation demonstrated that fetal lung HECs rely on canonical signaling pathways to undergo EHT, including TGFβ/BMP, Notch, and YAP. Collectively, these data support the possibility that post-AGM development, functional HECs are present in the fetal lung, establishing this location as a potential extramedullary site of de novo hematopoiesis.

Cross-species transcriptomics reveals bifurcation point during the arterial-to-hemogenic transition

Hemogenic endothelium (HE) with hematopoietic stem cell (HSC)-forming potential emerge from specialized arterial endothelial cells (AECs) undergoing the endothelial-to-hematopoietic transition (EHT) in the aorta-gonad-mesonephros (AGM) region. Characterization of this AECs subpopulation and whether this phenomenon is conserved across species remains unclear. Here we introduce HomologySeeker, a cross-species method that leverages refined mouse information to explore under-studied human EHT. Utilizing single-cell transcriptomic ensembles of EHT, HomologySeeker reveals a parallel developmental relationship between these two species, with minimal pre-HSC signals observed in human cells. The pre-HE stage contains a conserved bifurcation point between the two species, where cells progress towards HE or late AECs. By harnessing human spatial transcriptomics, we identify ligand modules that contribute to the bifurcation choice and validate CXCL12 in promoting hemogenic choice using a human in vitro differentiation system. Our findings advance human arterial-to-hemogenic transition understanding and offer valuable insights for manipulating HSC generation using in vitro models.

A Sox17 downstream gene Rasip1 is involved in the hematopoietic activity of intra-aortic hematopoietic clusters in the midgestation mouse embryo

BackgroundDuring mouse embryonic development, definitive hematopoiesis is first detected around embryonic day (E) 10.5 in the aorta-gonad-mesonephros (AGM) region. Hematopoietic stem cells (HSCs) arise in the dorsal aorta’s intra-aortic hematopoietic cell clusters (IAHCs). We have previously reported that a transcription factor Sox17 is expressed in IAHCs, and that, among them, CD45lowc-Kithigh cells have high hematopoietic activity. Furthermore, forced expression of Sox17 in this population of cells can maintain the formation of hematopoietic cell clusters. However, how Sox17 does so, particularly downstream signaling involved, remains poorly understood. The purpose of this study is to search for new Sox17 targets which contribute to cluster formation with hematopoietic activity.MethodsRNA-sequencing (RNA-seq) analysis was done to identify genes that are upregulated in Sox17-expressing IAHCs as compared with Sox17-negative ones. Among the top 7 highly expressed genes, Rasip1 which had been reported to be a vascular-specific regulator was focused on in this study, and firstly, the whole-mount immunostaining was done. We conducted luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay to examine whether Sox17 regulates Rasip1 gene expression via binding to its enhancer element. We also analyzed the cluster formation and the multilineage colony-forming ability of Rasip1-transduced cells and Rasip1-knockdown Sox17-transduced cells.ResultsThe increase of the Rasip1 expression level was observed in Sox17-positive CD45lowc-Kithigh cells as compared with the Sox17-nonexpressing control. Also, the expression level of the Rasip1 gene was increased by the Sox17-nuclear translocation. Rasip1 was expressed on the membrane of IAHCs, overlapping with the endothelial cell marker, CD31, and hematopoietic stem/progenitor marker (HSPC), c-Kit. Rasip1 expression was observed in most part of c-Kit+Sox17+ cells in IAHCs. Luciferase reporter assay and ChIP assay indicated that one of the five putative Sox17-binding sites in the Rasip1 enhancer region was important for Rasip1 expression via Sox17 binding. Rasip1 knockdown in Sox17-transduced cells decreased the cluster formation and diminished the colony-forming ability, while overexpression of Rasip1 in CD45lowc-Kithigh cells led to a significant but transient increase in hematopoietic activity.ConclusionsRasip1 knockdown in Sox17-transduced CD45lowc-Kithigh cells displayed a significant decrease in the multilineage colony-forming ability and the cluster size. Rasip1 overexpression in Sox17-untransduced CD45lowc-Kithigh cells led to a significant but transient increase in the multilineage colony-forming ability, suggesting the presence of a cooperating factor for sustained hematopoietic activity.

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.

Cell competition for neuron-derived trophic factor controls the turnover and lifespan of microglia.

Microglia are brain-resident macrophages capable of long-term maintenance through self-renewal. Yet the mechanism governing the turnover and lifespan of microglia remains unknown. In zebrafish, microglia arise from two sources, rostral blood island (RBI) and aorta-gonad-mesonephros (AGM). The RBI-derived microglia are born early but have a short lifespan and diminish in adulthood, while the AGM-derived microglia emerge later and are capable of long-term maintenance in adulthood. Here, we show that the attenuation of RBI microglia is due to their less competitiveness for neuron-derived interleukin-34 (Il34) caused by age-dependent decline of colony-stimulating factor-1 receptor a (csf1ra). Alterations of Il34/Csf1ra levels and removal of AGM microglia revamp the proportion and lifespan of RBI microglia. The csf1ra/CSF1R expression in zebrafish AGM-derived microglia and murine adult microglia also undergo age-dependent decline, leading to the elimination of aged microglia. Our study reveals cell competition as a general mechanism controlling the turnover and lifespan of microglia.

Characterization of Dynamic Niche Signaling Modules during Embryonic Hematopoietic Development Using Spatial Transcriptomics

Directed differentiation of pluripotent stem cells (PSCs) into functional hematopoietic stem cells (HSCs) remains elusive. A better understanding of HSC development, both intrinsically and extrinsically, will provide insights to guide PSC-HSC differentiation. During mouse embryogenesis, a subpopulation of endothelial cells in the aorta-gonad-mesonephros (AGM) region are specified at embryonic day (E) 9.5 to hemogenic endothelial cells (HECs). HECs then undergo an endothelial to hematopoietic transition (EHT) between E9.5 and E10.5. Among these hematopoietic cells, only a few are nascent HSCs (pre-HSCs) that migrate to the fetal liver for maturation and expansion. Recent advances in single cell RNA sequencing (scRNA-seq) substantially improved our understanding of intrinsic cellular programs of human and mouse HSC development; however, much less is known about the hematopoietic niche cells and niche-emanated signals in the AGM region and fetal liver. To characterize the niche components and related signaling of HSC development, we conducted a spatial transcriptomics analysis using Slide-seq (with 10 µm resolution) on mouse E10.5, E11.5 and E12.5 trunk tissues covering both the AGM region and fetal liver. We also performed scRNA-seq on seven samples collected from trunk tissues, including E10.5, E11.5 and E12.5 AGM regions; E10.5, E11.5 and E12.5 trunks; and E12.5 fetal liver. We developed an algorithm to integrate the Slide-seq and scRNA-seq data that takes advantage of their spatial and in-depth transcriptional sequencing capacity, respectively. Using E11.5 Slide-seq data as an example, we successfully identified most, if not all, of the tissue structures at a near single cell level in trunk tissue (see Figure). Furthermore, we could determine the expression of thousands of genes on individual beads, providing an invaluable tool for investigating spatial-characterized niche cells and signals. Compared to the dorsal aorta, which was dominated by one cell cluster expressing Nfgr, a complex and multilayered cellular architecture was revealed in the ventral part of aorta where EHT occurs, including pre-HSCs, endothelial cells, neural crest derived cells, mesonephric cells, and macrophages. We found a rare population of mesenchymal stromal cells (MSCs) that co-express Cdh2 (encodes N-cadherin or N-cad) and Pdgfra. Of note, these N-cad+ MSCs exclusively localize to the ventral part of the aorta as a single cell layer and are sandwiched between an inner layer of aortic endothelial cells and an outer layer of mesonephric cells. Secondly, we identified multiple genes enriched in the ventral aorta compared to the dorsal aorta, including Dlk1, Wnt4, Igf1, and Ptn. Gene Ontology analysis and KEGG signaling pathway analysis were also performed to investigate the biological processes and signaling pathways enriched in individual cell clusters. Potential signaling modules representing ligand-receptor interactions between niche cells and HECs/pre-HSCs were analyzed by CellphoneDB; the identified spatial location of niche cells relative to pre-HSCs allows us to exclude predicted interactions in which niche cells were located too far from pre-HSCs to be relevant. As a result, we generated a list of known and potentially novel signals critical for EHT, including DLK1, JAG1 and TGFβ emanating from N-cad+ MSCs, endothelial cell-derived JAG2 and DLL1/4, mesonephric cell-secreted WNT2b, GREM1 (a BMP antagonist) and R-spondin1, gonad cell-derived SPP1, and macrophage-produced TNFα. A thorough analysis on both AGM and fetal liver at different developmental stages is ongoing. In summary, we dissected dynamic and near single cell-resolved spatial characteristics of the niche components and signals for HSC development in the AGM region and fetal liver. These findings will deepen our knowledge of embryonic hematopoiesis and guide the in vitro induction of human PSCs to functional HSCs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

De Novo Hematopoiesis from the Fetal LUNG

Hemogenic endothelial cells (HECs) are specialized cells that undergo endothelial to hematopoietic transition (EHT) to give rise to the earliest precursors of hematopoietic progenitors that will eventually sustain hematopoiesis throughout the lifetime of an organism. Although HECs are thought to be primarily limited to the aorta gonad mesonephros (AGM) during early development, EHT has been described in various other hematopoietic organs and embryonic vessels. Though not traditionally seen as a hematopoietic organ, the lung houses many resident hematopoietic cells, aids in platelet biogenesis and is a reservoir for hematopoietic stem and progenitor cells (HSPCs). However, lung HECs have never been described. Here we demonstrate that the fetal lung is a novel source of HECs that have the functional capacity to undergo EHT to produce de novo HSPCs and their resultant progeny. Explant cultures of murine and human fetal lungs display adherent endothelial cells transitioning into floating hematopoietic cells accompanied by the gradual loss of an endothelial signature. Flow cytometric and functional assessment of fetal lung explants showed the production of multipotent HSPCs that expressed the EHT and pre-HSPC markers EPCR, CD41, CD43 and CD44. scRNA-Seq and small molecule modulation demonstrated that fetal lung HECs rely on canonical signaling pathways to undergo EHT, including TGFβ/BMP, Notch and YAP. Immunofluorescent stains of fixed frozen fetal lung samples showed discrete regions of clustered hematopoietic cells expressing VE-cadherin, CD41 and CD45 adjacent to fetal lung vasculature. Notably, these clusters were oriented away from the vascular lumen, suggesting they may be fated to remain within the lung interstitium. Collectively, these data strongly support the concept that post-AGM development, functional HECs are present in the fetal lung establishing this location as a potential extramedullary site of de-novo hematopoiesis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Meis1 Establishes the Pre-Hemogenic Endothelial State Prior to Runx1 Expression

Hematopoietic Stem Cells (HSCs) are the most primitive cells of the hematopoietic system, defined by their ability to sustain lifelong hematopoiesis and differentiate to all mature hematopoietic lineages. HSC transplant (HSCT) is the first-line treatment for many patients diagnosed with a serious hematological disease, however HSCs are an exceedingly rare cell population that are challenging to expand ex vivo. Generating functional HSCs ex vivo from reprogrammed somatic cells or pluripotent stem cells (PSC) is a promising alternative. However, a gap remains in the knowledge of the developmental pathways regulating HSC ontogenesis, making it challenging to reproducibly engineer bona fide HSCs. Hematopoietic stem and progenitor cells (HSPCs) originate from an endothelial-to-hematopoietic transition (EHT) during embryogenesis. EHT is a step-wise process initiating when a specialized subset of endothelial cells (EC), termed hemogenic endothelial cells (HEC) undergo dyshesion from the endothelial bed and form intra-aortic hematopoietic clusters within the major arteries of the embryo. The processes driving the specification of these HEC rather than a vascular fate is not well understood, despite representing a critical need to replicate EHT in vitro. To define factors in the endothelium that prime EC towards a hemogenic phenotype, we performed Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) on CD31+ FACS-isolated cells from the aorta-gonad-mesonephros (AGM) of E10.5 wildtype mouse embryos (Figure 1). CITE-seq can simultaneously label cells based on surface protein markers and profile single-cell transcriptomes, therefore bypassing the need for isolation of rare cells by FACS. Unsupervised clustering based on single cell transcriptomes showed distinct populations of EC (composed of venous and arterial subpopulations), cells undergoing EHT that expressed both endothelial and hematopoietic genes, and mature hematopoietic cells. In addition to EC and EHT clusters, the CITE-seq data revealed a small population of pre-hemogenic endothelial cells (pre-HE) that was distinct from HE cells, which clustered with "EHT" cells (Figure 1). Pre-HE cells expressed high levels endothelial markers and Cd44, a marker recently associated with hemogenic potential, but lacked expression of the prototypic HE markers Runx1 and Gfi1(Figure 1). To look for drivers of hematopoietic cell fate we identified 12 transcription factors (TFs) that were upregulated between the arterial endothelial cells (aEC) and pre-HE clusters identified by CITE-Seq, and that persisted through EHT. Within this list, Meis1 ranked as being the most significantly enriched, with its binding partner Pbx1 ranked fourth, therefore we hypothesized that Meis1 may be required to drive early hemogenic specification. We generated embryos with endothelial-specific deletion of Meis1 to investigate the functional requirement for Meis1 in EC. Endothelial-specific deletion of Meis1 impaired the formation of functional Runx1-expressing HE which significantly impeded the emergence of pre-HSPC via EHT. In summary, we used a combination of CITE-seq and bulk RNA-seq to identify Meis1 as a candidate early functional marker of hemogenic specification. We show that Meis1 primes arterial endothelial cells towards the hemogenic trajectory, and that it is required in the mouse embryo for the formation of early pre-hemogenic cells prior to the emergence of functional HE and pre-HSPCs. Our findings implicate Meis1 in a critical fate-determining step for establishing EHT potential in endothelial cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

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.

Mimicry of embryonic circulation enhances the hoxa hemogenic niche and human blood development

Precursors of the adult hematopoietic system arise from the aorta-gonad-mesonephros (AGM) region shortly after the embryonic circulation is established. Here, we develop a microfluidic culture system to mimic the primitive embryonic circulation and address the hypothesis that circulatory flow and shear stress enhance embryonic blood development. Embryonic (HOXA+) hematopoiesis was derived from human pluripotent stem cells and induced from mesoderm by small-molecule manipulation of TGF-β and WNT signaling (SB/CHIR). Microfluidic and orbital culture promoted the formation of proliferative CD34+RUNX1C-GFP+SOX17-mCHERRY+ precursor cells that were released into the artificial circulation from SOX17+ arterial-like structures. Single-cell transcriptomic analysis delineated extra-embryonic (yolk sac) and HOXA+ embryonic blood differentiation pathways. SB/CHIR and circulatory flow enhance hematopoiesis by the formation of proliferative HOXA+RUNX1C+CD34+ precursor cells that differentiate into monocyte/macrophage, granulocyte, erythrocyte, and megakaryocyte progenitors.

Mesoderm-derived PDGFRA+ cells regulate the emergence of hematopoietic stem cells in the dorsal aorta

Mouse haematopoietic stem cells (HSCs) first emerge at embryonic day 10.5 (E10.5), on the ventral surface of the dorsal aorta, by endothelial-to-haematopoietic transition. We investigated whether mesenchymal stem cells, which provide an essential niche for long-term HSCs (LT-HSCs) in the bone marrow, reside in the aorta–gonad–mesonephros and contribute to the development of the dorsal aorta and endothelial-to-haematopoietic transition. Here we show that mesoderm-derived PDGFRA+ stromal cells (Mesp1der PSCs) contribute to the haemogenic endothelium of the dorsal aorta and populate the E10.5–E11.5 aorta–gonad–mesonephros but by E13.5 were replaced by neural-crest-derived PSCs (Wnt1der PSCs). Co-aggregating non-haemogenic endothelial cells with Mesp1der PSCs but not Wnt1der PSCs resulted in activation of a haematopoietic transcriptional programme in endothelial cells and generation of LT-HSCs. Dose-dependent inhibition of PDGFRA or BMP, WNT and NOTCH signalling interrupted this reprogramming event. Together, aorta–gonad–mesonephros Mesp1der PSCs could potentially be harnessed to manufacture LT-HSCs from endothelium.

Mast Cell Repopulating Ability Is Lost During the Transition From Pre-HSC to FL HSC.

Recent advances in developmental immunology have revealed a hematopoietic stem cell (HSC)-independent origin for various innate immune lineages, including mast cells (MCs). It is now established that adult bone marrow (BM) long-term HSCs do not regenerate MCs but, instead, the physiological production of MCs starts before the emergence of HSCs in the aorta-gonad-mesonephros (AGM) region and is mostly completed before birth. However, while the AGM region represents a major site of MC generation during ontogeny, whether the first emerging HSCs in the AGM or fetal liver (FL) possess the potential to regenerate MCs is unknown. Here, we combined three fate-mapping mouse models with detailed HSC transplantation assays to determine the potential of AGM and FL HSCs to produce MCs. We show that HSCs from E11.5 AGM and E12.5 FL efficiently repopulated MCs in recipients. In stark contrast, HSCs from ≥E14.5 FL failed to reconstitute MCs. An Endothelial (EC) fate-mapping study confirmed the EC origin of the majority of MCs. Additionally, our HSC-labeling showed that HSCs do not produce MCs in a physiological setting. Hence, although most MCs are generated and maintained via an HSC-independent pathway, the earliest HSCs to emerge in the AGM and seed the early FL can produce MCs, but only during a minimal time window. Our results challenge the stem cell theory in hematology and EC-derived mast cells may contribute to the pathogenesis of postnatal mast cell disorders.

P57Kip2 regulates embryonic blood stem cells by controlling sympathoadrenal progenitor expansion

AbstractHematopoietic stem cells (HSCs) are of major clinical importance, and finding methods for their in vitro generation is a prime research focus. We show here that the cell cycle inhibitor p57Kip2/Cdkn1c limits the number of emerging HSCs by restricting the size of the sympathetic nervous system (SNS) and the amount of HSC-supportive catecholamines secreted by these cells. This regulation occurs at the SNS progenitor level and is in contrast to the cell-intrinsic function of p57Kip2 in maintaining adult HSCs, highlighting profound differences in cell cycle requirements of adult HSCs compared with their embryonic counterparts. Furthermore, this effect is specific to the aorta-gonad-mesonephros (AGM) region and shows that the AGM is the main contributor to early fetal liver colonization, as early fetal liver HSC numbers are equally affected. Using a range of antagonists in vivo, we show a requirement for intact β2-adrenergic signaling for SNS-dependent HSC expansion. To gain further molecular insights, we have generated a single-cell RNA-sequencing data set of all Ngfr+ sympathoadrenal cells around the dorsal aorta to dissect their differentiation pathway. Importantly, this not only defined the relevant p57Kip2-expressing SNS progenitor stage but also revealed that some neural crest cells, upon arrival at the aorta, are able to take an alternative differentiation pathway, giving rise to a subset of ventrally restricted mesenchymal cells that express important HSC-supportive factors. Neural crest cells thus appear to contribute to the AGM HSC niche via 2 different mechanisms: SNS-mediated catecholamine secretion and HSC-supportive mesenchymal cell production.