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Single cell RNA-Seq of mouse bone marrow-derived hematopoietic stem cells after 7-days in viro culture

ABSTRACT: Hematopoietic stem cells (HSCs) are cells able to self-renew and differentiate into all blood lineages. We previously have performed serum-free single-cell culture for HSC1 (CD201+CD150+CD48-CD41-CD34-LSK) and competitive repopulation with cultured cells. Single highly purified HSC1 were cultured with stem cell factor (SCF), SCF + G-CSF, or SCF + thrombopoietin (TPO) for 7 days. SCF directly acted on HSC1 and maintained their long-term multilineage reconstitution after serial transplantation. Compared with SCF alone, SCF+G-CSF neither enhanced the number of divisions of HSC1 nor enhanced their levels of reconstitution after serial transplantation. SCF+TPO significantly increased the number of divisions of HSC1. The levels of reconstitution of each lineage in SCF + TPO culture were significantly increased at early months after primary transplantation, however, it gradually decreased after secondary transplantation. Therefore, we performed single-cell RNA-Seq to examine the gene expression of cells cultured with different cytokine combinations.

ORGANISM(S): Mus musculus

PROVIDER: GSE153430 | GEO | 2023/06/28

REPOSITORIES: GEO

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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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Single cell RNA-Seq of mouse bone marrow-derived hematopoietic stem cells after 7-days in viro culture

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