Mesoderm-Derived PDGFRA+ Cells Regulate the Emergence of Hematopoietic Stem Cells in the Dorsal Aorta
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE163757 | GEO | 2022/04/26
REPOSITORIES: GEO
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Project description:During embryonic development, the first hematopoietic stem cells (HSCs) arise from a transient population of endothelial cells lining the ventral surface of the dorsal aorta, via a process of endothelial to hematopoietic transition (EHT) at embryonic day (E) 10.5. This region contains resident PDGFRA+ stromal cells (PSCs), but their identity and role in HSC generation in the AGM are not well understood. Using a library of compound transgenic mice, we identified a population of PDGFRA+/Nestin-GFP (N-GFP)-/PDGFRB-/CD31- cells with PSC activity in the E10.5 and E11.5 mouse AGM. Freshly isolated PSCs were adept at forming blood vessels with CD31+ luminal endothelium enveloped by PDGFRB+ pericytes, when transplanted subcutaneously into mice. Conditional ablation of PDGFRA+ or Nestin+ cells led to either complete or partial loss of PSCs respectively, with severe loss of endothelial and pericyte-like cells, and concomitant loss of blood formation in the AGM. Lineage tracing studies using tamoxifen induction in PDGFRACreERT2/R26eYFP embryos showed that stromal, sub-endothelial, endothelial and long-term repopulating hematopoietic stem cells (LT-HSCs) cells in the E11.5 AGM were progeny of PDGFRA cells. Using transgenic reporter mice, we showed that MesP1 (mesoderm) derived PDGFRA+ cells dominated the sub-endothelial and deeper ventral stroma in the AGM at E10.5 and E11.5 but were replaced by Wnt1 (neural crest) derived cells at E13.5. Re-aggregation of E11.5 Mesp1 derived PSC cells with E13.5 aortic or adult cardiac non-hemogenic endothelial cells resulted in the generation of endothelial cell derived LT-HSCs. RNA-sequencing analysis of non-hemogenic E13.5 endothelial cells showed up-regulation of EHT genes, WNT, BMP and Notch cell signalling pathways when re-aggregated with E11.5 Mesp1 derived PSCs. LT-HSC generation from these re-aggregates was suppressed by dose-dependent inhibition of PDGF-AA/PDGFRA signalling. Taken together, we report that PSC populations in the AGM are temporally dynamic, and that MesP1-derived PSCs regulate hemogenic potential of the endothelium and that this cooperativity is dependent on PDGF-AA/PDGFRA signalling. This submission represents the RNAseq component of the study.
2022-04-26 | GSE114464 | GEO
Project description:The first HSCs are produced in the aorta-gonadmesonephros (AGM) region of the embryo through endothelial to a hematopoietic transition. BMP4 and Hedgehog affect their production/expansion, but it is unknown whether they act to affect the same HSCs. In this study using the BRE GFP reporter mouse strain that identifies BMP/Smad-activated cells, we find that the AGM harbors two types of adult-repopulating HSCs upon explant culture. Embryonic day 11 AGM are dissected and either analyzed directly, or after explant culture in conditions containing BMP/Hedgehog with or without cyclopamine. EC: endothelial enriched (CD31+Kit-); MC: mesenchymal cell enriched (CD31-Kit-); HPSC: hematopoietic progenitor/stem cell enriched; AGM11: E11 fresh AGMs; AGMex: AGM after explant culture; AGMcy: AGM after explant in presence of cyclopamine; CD31p: CD31 positive; CD31n: CD31 negative; KITp: c-Kit positive; KITn: c-Kit negative; BREp: BRE-GFP positive; BREn: BRE-GFP negative
2015-12-22 | E-GEOD-76253 | biostudies-arrayexpress
Project description:The emergence of definitive human haematopoietic stem cells (HSCs) during Carnegie Stages (CS) 14-17 in the aorta-gonad-mesonephros (AGM) region is a complex and tightly regulated process. In a previous study we conducted spatial transcriptomic analysis of the human AGM region at the end of this period (CS16/17) and identified secreted factors involved in HSC development. Here, we extend our analysis to investigate the progression of dorso-ventral polarized signalling around the dorsal aorta over the entire period of HSC emergence. Our results reveal a dramatic increase in signalling complexity from the CS13 to CS14 transition, coinciding with the activation of endothelial-to-haematopoietic transition (EHT) in the ventral domain of the dorsal aorta. We further observe stage-specific changes in signalling complexity up to CS17. Signalling progression described here may underpin step-wise maturation of HSCs described in the mouse model. A data-rich bioinformatics resource presented here along with an interactive online interface enables 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.
2023-10-31 | GSE233130 | GEO
Project description:The emergence of definitive human haematopoietic stem cells (HSCs) during Carnegie Stages (CS) 14-17 in the aorta-gonad-mesonephros (AGM) region is a complex and tightly regulated process. In a previous study we conducted spatial transcriptomic analysis of the human AGM region at the end of this period (CS16/17) and identified secreted factors involved in HSC development. Here, we extend our analysis to investigate the progression of dorso-ventral polarized signalling around the dorsal aorta over the entire period of HSC emergence. Our results reveal a dramatic increase in signalling complexity from the CS13 to CS14 transition, coinciding with the activation of endothelial-to-haematopoietic transition (EHT) in the ventral domain of the dorsal aorta. We further observe stage-specific changes in signalling complexity up to CS17. Signalling progression described here may underpin step-wise maturation of HSCs described in the mouse model. A data-rich bioinformatics resource presented here along with an interactive online interface enables 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.
2023-10-31 | GSE233126 | GEO
Project description:The first HSCs are produced in the aorta-gonadmesonephros (AGM) region of the embryo through endothelial to a hematopoietic transition. BMP4 and Hedgehog affect their production/expansion, but it is unknown whether they act to affect the same HSCs. In this study using the BRE GFP reporter mouse strain that identifies BMP/Smad-activated cells, we find that the AGM harbors two types of adult-repopulating HSCs upon explant culture.
2015-12-22 | GSE76253 | GEO
Project description:Hematopoietic stem cells (HSC) are generated from specialized endothelial cells of the embryonic aorta. Previously, inflammatory factors have been implicated in regulating mouse HSC development, but it is unclear what cells in the embryonic aorta-gonad- mesonephros (AGM) microenvironment produce these factors. In the adult, macrophages play both pro- and anti-inflammatory roles. We sought to examine whether macrophages or other hematopoietic cells found in the embryo prior to HSC generation are involved in the AGM HSC-generative microenvironment. Our CyTOF results indicate two abundant myeloid cell types - mannose-receptor positive AGM- associated macrophages (AGM-aM) and mannose-receptor negative macrophages/progenitors. We show that the appearance of macrophages in the AGM is dependent on CX3CR1. AGM-aM express a pro-inflammatory signature, localize to the embryonic aorta and dynamically interact with nascent and emerging intra-aortic hematopoietic cells (IAHC). Importantly, upon macrophage depletion, no adult- repopulating HSCs are detected, thus implicating unique pro-inflammatory AGM- associated macrophages in regulating the embryonic development of HSCs.
2019-06-01 | GSE130579 | GEO
Project description:Hematopoietic stem cells (HSCs) are generated via a natural transdifferentiation process known as endothelial-to-hematopoietic cell transition (EHT). Due to small numbers of embryonal arterial cells undergoing EHT and the paucity of markers to enrich for hemogenic endothelial cells, the genetic program driving HSC emergence is largely unknown. Here, we use a highly sensitive RNAseq method to examine the whole transcriptome of small numbers of enriched aortic HSCs (CD31+cKit+Ly6aGFP+), hemogenic endothelial cells (CD31+cKit-Ly6aGFP+) and endothelial cells (CD31+cKit-Ly6aGFP-). Comparison of mRNA profiles of endothelial cells, hemogenic endothelial cells, and hematopoietic stem cells generated by deep-sequencing of sorted populations from pool of embryos, in triplicate.
2014-12-14 | E-GEOD-63316 | biostudies-arrayexpress
Project description:Hematopoietic stem and progenitor cells (HSPCs) are required to establish and maintain the adult blood system in vertebrates. During development, HPSCs are generated from hemogenic endothelial cells that undergo an endothelial-to-hematopoietic transition (EHT). Growth factors and epigenetic changes can promote EHT, but these mechanisms do not explain its tight spatiotemporal regulation during development. Here, we show that microRNA (miR) miR-223-mediated regulation of N-glycan biosynthesis intrinsically restrains EHT, representing a new pathway that prevents excessive HSPC production. We find that miR-223 is uniquely expressed in hemogenic endothelial cells undergoing EHT and in nascent HSPCs. Loss of miR-223 promotes the expansion of these cells in the zebrafish and mouse aorta-gonad-mesonephros (AGM), where EHT occurs. miR-223 targets alg2 (alpha 1,3/ 1,6 mannosyltransferase) in the AGM endothelium to restrict hemogenic and HSPC specification. This enzyme is involved in the attachment of the N-glycans, a common co-translational modification9-12 that influences several pathophysiological processes, but has not yet been implicated in EHT. Using an N-glycosensor, we demonstrate that abundant protein N-glycan attachment occurs in vascular cells during EHT, and this process is required for HSPC production. High-throughput glycome analysis upon loss of miR-223 revealed that terminal alpha 1,3/6 mannose modifications are increased at the expense of alpha 2,3/6 sialic acid sugars. Importantly, pharmacological manipulation targeting these N-glycan types in wild-type embryos phenocopies the loss of miR-223 and enhances EHT as well as HSPC production. Thus, the N-glycome plays a previously unappreciated role as an intrinsic regulator of EHT, with specific mannose and sialic acid modifications serving as key endothelial determinants to restrict the hematopoietic fate.
2021-09-09 | GSE135246 | GEO
Project description:Hematopoietic stem cells (HSCs) are at the basis of the hematopoietic hierarchy. Their ability to self-renew and differentiate is strictly controlled by molecular signals produced by their surrounding micorenvironments composed of stromal cells. HSCs first emerge in the AGM (Aorta Gonads Mesonephros) region, amplify in the fetal liver (FL) and are maintained in the adult bone marrow (BM). To further characterize the molecular program of the HSC niches, we have compared the global transcriptome of HSC-supportive and non-supportive stromal clones established from the AGM, FL and BM. Hematopoietic stem cells (HSCs) are at the basis of the hematopoietic hierarchy. Their ability to self-renew and differentiate is strictly controlled by molecular signals produced by their surrounding micorenvironments composed of stromal cells. HSCs first emerge in the AGM (Aorta Gonads Mesonephros) region, amplify in the fetal liver (FL) and are maintained in the adult bone marrow (BM). To further characterize the molecular program of the HSC niches, we have compared the global transcriptome of HSC-supportive line from Fetal Calvaria (OP9) and non-supportive stromal clones from fetal liver (BFC). Hematopoietic stem cells (HSCs) are at the basis of the hematopoietic hierarchy. Their ability to self-renew and differentiate is strictly controlled by molecular signals produced by their surrounding micorenvironments composed of stromal cells. HSCs first emerge in the AGM (Aorta Gonads Mesonephros) region, amplify in the fetal liver (FL) and are maintained in the adult bone marrow (BM). To further characterize the molecular program of the HSC niches, we have compared the global transcriptome of HSC-supportive and non-supportive stromal clones established from fetal liver. We took advantage of stromal clones established from the AGM, FL and BM and tested for their ability to support or not HSCs ex vivo. RNA were extracted from confluent stromal cultures or sorted cells and used for hybridization of Affymetrix (mouse gene 1.0 ST) microarrays.
2014-03-01 | E-GEOD-44181 | biostudies-arrayexpress
Project description:Haematopoietic stem cells (HSCs) are derived early from embryonic precursor cells, such as haemogenic endothelial cells and pre-HSCs. However, the identity of precursor cells remains elusive due to their rareness, transience, and inability to be isolated efficiently. Here we employed potent surface markers to capture the nascent pre-HSCs at 30% purity, as rigorously validated by single-cell-initiated serial transplantation assay. Then we applied single-cell RNA-Seq technique to analyse five populations closely related to HSC formation: endothelial cells, CD45- and CD45+ pre-HSCs in E11 aorta-gonad-mesonephros (AGM) region, and mature HSCs in E12 and E14 foetal liver. In comparison, the pre-HSCs showed unique features in transcriptional machinery, apoptosis, metabolism state, signalling pathway, transcription factor network, and lncRNA expression pattern. Among signalling pathways enriched in pre-HSCs, the mTOR activation was uncovered indispensable for the emergence of HSCs but not haematopoietic progenitors from endothelial cells in vivo. By comparing with proximal populations without HSC potential, the core molecular signature of pre-HSCs was identified. Collectively, our work paves the way for dissection of complex molecular mechanisms regulating the step-wise generation of HSCs in vivo, informing future efforts to engineer HSCs for clinical application. RNA-Seq of 181 single-cell samples from 8 FACS sorted cell types: 1. endothelial cells (samples E11.0_EC_xxxx. CD31+ VE-cadherin+CD41-CD43-CD45-Ter119-); 2. T1 pre-HSCs (samples E11.0_T1_xxxx. CD31+CD45-CD41low c-Kit+CD201high); 3. T1 CD201- cells (samples E11.0_T1CD201neg_xxxx, CD31+CD45-CD41low c-Kit+CD201low/-) ; 4. T2 pre-HSCs (samples E11.0_T2_35xx. CD31+CD45+c-Kit+CD201high), 5. T2 CD41low (samples E11.0_T2_21xx, E11.0_T2_24xx and E11.0_T2_27xx. CD31+CD45+CD41low); 6. E12 HSCs (samples E12.5_FL_xxxx. Lin-Sca-1+Mac-1lowCD201+); 7. E14 HSCs (samples E14.5_FL_xxxx. CD45+CD150+CD48-CD201+); 8. Adult HSCs (samples Adult_HSC_xxxx. CD45+CD150+CD48-CD201+). ECs, T1 pre-HSCs, T1 CD201- cells, T2 pre-HSCs, T2 CD41low cells were sorted from E11 AGM region. Mature HSCs were sorted from E12 or E14 fetal liver and adult bonemarrow.
2016-05-19 | E-GEOD-67120 | biostudies-arrayexpress