Project description:Analysis of highly purified long-term hematopoietic stem cells (LT-HSCs) irradiated at 0Gy, 0.02Gy, 0.1Gy and 0.5Gy six months after transplantation. Results provide insight into the molecular mechanisms underlying multiple aspects of LT-HSCs premature ageing after low doses of γ-irradiation (0.02Gy). Four samples were analyzed and correlated with the control group (0Gy).
Project description:Analysis of highly purified long-term hematopoietic stem cells (LT-HSCs) 2 hours after irradiation at 0Gy, 0.02Gy and 2.5Gy. Results provide insight into the molecular mechanisms underlying LT-HSCs immediate response to low doses of γ-irradiation compared to high doses. Three samples were analyzed and correlated with the control group (0Gy).
Project description:The aim of the study was to analyse gene expression differences in LT and ST HSCs in BM of TEL-SYK transplanted versus control C57BL/6mice
Project description:Murine long-term hematopoietic stem cells (HSCs), short-term HSCs and multipotent progenitor cells (MPPs) were isolated from bone marrow and expression profiled on Affy chips. The behavior of maternal-specific imprinting genes, particularly in the H19-Igf2 locus, was focused on, to see if any might be involved in maintaining quiescence of long-term stem cells.
Project description:In blood, the transcription factor C/EBPa is essential for myeloid differentiation and has been implicated in regulating self-renewal of fetal liver hematopoietic stem cells (HSCs). However, its function in adult HSCs is unknown. Here, using an inducible knockout model, we found that C/EBPa deficient adult HSCs underwent a pronounced expansion with enhanced proliferation, characteristics resembling fetal liver HSCs. Consistently, transcription profiling of C/EBPa deficient HSCs revealed a gene expression program similar to fetal liver HSCs. Moreover we observed that age-specific C/EBPa expression correlated with its inhibitory effect on the HSC cell cycle. Mechanistically, we identified N-Myc as a C/EBPa downstream target. C/EBPa upregulation during HSC transition from an active fetal state to a quiescent adult state was accompanied by down-regulation of N-Myc, and loss of C/EBPa resulted in de-repression of NMyc. Our data establish that C/EBPa acts as a molecular switch between fetal and adult states of HSC in part via transcriptional repression of the proto-oncogene N-Myc. HSCs of Pu.1 knock-in (PU.1ki/ki) mice were used for RNA extraction and hybridization on Affymetrix microarrays. We compared these microarray samples with the corresponding wild type.
Project description:FoxM1, a mammalian Forkhead Box M1 protein, is known as a typical proliferation-associated transcription factor that regulates of G1/S and G2/M transition in the proliferating cells. However, the in vivo function of FoxM1 in adult stem cells remains unknown. Here, we found that FoxM1 is highly expressed in hematopoietic stem cells (HSCs) and is essential for maintaining quiescence and self-renewal of HSCs in vivo. FoxM1-deficient mice developed leukopenia, thrombocytopenia and neutropenia with an approximately 6-fold decrease in HSC pool size, which is associated with a failure of G0 cell cycle regulation and increased cell cycling in HSCs. FoxM1 absence did not affect lineage commitment of HSCs and progenitors. However, FoxM1 loss significantly reduced the repopulating capacity and self-renewal of long-term HSC in a cell-autonomous manner. Mechanistically, FoxM1 loss markedly down-regulates the expression of orphan nuclear receptor Nurr1, known to regulate HSC quiescence. We found that FoxM1 directly bound the promoter region of Nurr1 and induced transcriptional activity of Nurr1 promoter in vitro, and forced expression of Nurr1 rescued FoxM1-deletion-induced G0 loss of HSC-enriched population in vitro. Thus, our studies show a previously unrecognized role of FoxM1 as a critical regulator of HSC quiescence and self-renewal by controlling Nurr1-mediated pathways. The Hematopoietic Stem Cells (HSCs) were sorted from FoxM1[fl/fl] and Tie2-Cre FoxM1[fl/fl] mice, then amplified with Ovation Pico WTA System V2 before microarray analysis. There are 3 samples from FoxM1[fl/fl]mice and 3 samples from Tie2-Cre FoxM1[fl/fl] mice.
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.