Unknown,Transcriptomics,Genomics,Proteomics

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Bulk RNA sequencing of endothelial-derived and non-endothelial-derived BMSCs and OLCs of VE-Cadherin-Cre;LoxP-tdTomato mice

ABSTRACT: We performed lineage tracing experiments using VE-Cadherin-Cre;LoxP-tdTomato mice. In these mice, endothelial cells (ECs) and their progeny are permanently marked by tdTomato fluorescence. We found that a substantial subset of stromal cells is derived from ECs, as indicated by their tdTomato expression. These findings support the notion that endothelial to mesenchymal transition (EndoMT) contributes to hematopoietic bone marrow niche formation in mice. Here we sought to determine the transcriptomic differences between endothelial-derived (tdTomato-positive) and non-endothelial-derived (tdTomato-negative) bone marrow stromal cells (BMSCs) and osteo/chondrolineage progenitor cells (OLCs). Murine niche populations were obtained from collagenased bone fraction of VE-Cadherin-Cre;LoxP-tdTomato mice at 3 weeks (n=2) or 11 weeks (n=2) of age. BMSCs (CD45-TER119-CD31-CD144-SCA-1+ CD51+ cells) and OLCs (CD45-TER119-CD31-CD144-Sca1-CD51+ cells) were FACS-purified and sequenced.

INSTRUMENT(S): Illumina NovaSeq 6000

ORGANISM(S): Mus musculus

SUBMITTER: Paola Pisterzi

PROVIDER: E-MTAB-10020 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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.

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Bulk RNA sequencing of endothelial-derived and non-endothelial-derived BMSCs and OLCs of VE-Cadherin-Cre;LoxP-tdTomato mice

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