Quantitative Proteomic Analysis of Mouse Fetal and Adult Hematopoietic Progenitor Cells
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion
ORGANISM(S): Mus Musculus (ncbitaxon:10090)
SUBMITTER: 
Jenny Hansson
PROVIDER: MSV000085672 | MassIVE | Thu Jul 02 11:44:00 BST 2020
SECONDARY ACCESSION(S): PXD020154
REPOSITORIES: MassIVE
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Project description:To facilitate comparative genomic analyses of human fetal and adult cells undergoing erythropoiesis, we employed a serum-free two-phase liquid culture system to expand and differentiate primary human CD34+ hematopoietic stem/progenitor cells (HSPCs) ex vivo. In this experimental context, highly enriched populations of stage-matched, differentiating, primary proerythroblasts (ProEs) were generated. We selected four time points (day 0, CD34+ HSPCs; day 3, 5, and 7, differentiating ProEs) that represented similar stages differentiation for gene expression profiling using microarrays. Primary maturing fetal or adult erythroblasts were generated ex vivo from CD34+ hematopoietic stem/progenitor cells (HSPCs) using a serum-free two-phase liquid culture system. Total RNA from primary fetal and adult HSPCs (day 0) and differentiating proerythroblasts (ProEs; day 3, 5, and 7) were extracted and used to hybridize to Affymetrix expression arrays using the HG-U133 Plus 2.0 platform.
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Project description:Innate immune signaling protects against pathogens, controls hematopoietic development and functions in oncogenesis, yet the relationship between these mechanisms is incompletely defined. Downregulating the GATA2 transcription factor in fetal hematopoietic progenitor cells upregulates genes encoding innate immune regulators, increases Interferon-g (IFNg) signaling and disrupts differentiation. Here, we demonstrate that deletion of an enhancer that confers GATA2 expression in fetal progenitor cells elevated Toll-Like Receptor (TLR) TLR1/2 and TLR2/6 expression and signaling. Genetic rescue by expressing GATA2 downregulated the elevated TLR signaling. IFNg amplified TLR1/2 and TLR2/6 signaling in GATA2-deficient progenitor cells, synergistically activating cytokine/chemokine genes and elevating cytokine/chemokine production in their myeloid cell progeny. Genome-wide analysis of how IFNg and TLR signaling remodels the progenitor cell transcriptome in GATA2-deficient cells revealed exaggerated responses at innate immune genes harboring motifs for signal-dependent transcription factors. Thus, GATA2 establishes a transcriptome that constrains innate immune signaling, and insufficient GATA2 renders fetal progenitor cells hypersensitive to innate immune signaling.
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Project description:Hematopoietic stem cell transplantation (HSCT) is a common practice in the treatment of (malignant-) hematopoietic diseases. Umbilical Cord Blood (UCB) has become an important hematopoietic stem and progenitor cell (HSPC) source for allogeneic HSCT. However, the relatively low number of HSPCs that are present in a single UCB unit is associated with a delayed engraftment and a higher risk for graft failure. To overcome these limitations, the discovery of new therapeutics and the development of efficient ex vivo HSPC expansion conditions will help towards the successful treatment of malignant hematopoietic disease. Bone marrow derived mesenchymal stromal cells (BMSCs) are known to support the characteristic properties of HSPCs in the bone marrow microenvironment. Recently, we and others have illustrated that extracellular vesicles (EVs) have the potential to support HSPC expansion, however, the mechanism and processes responsible for the cell-cell communication of EVs are still unknown. In the current study, we investigate whether primary human BMSC EVs isolated from two different origins, fetal (fEV) and adult (aEV) tissue respectively, can increase the relative low number of HSPC found in UCB and which EV-derived components are responsible for ex vivo HSPC expansion. Interestingly, aEVs but not fEVs showed supportive ex vivo expansion capacity of UCB-HSPC. Taking advantage of the two BMSC sources with different supportive effects, we performed small RNA sequencing and proteomics analyses on the EV cargo and investigated how gene expression is modulated in HSPC after incubation with aEVs and fEVs. Proteomics analyses of the protein cargo composition of the supportive aEV versus the non-supportive fEV identified 90% of the Top100 exosome proteins present in the ExoCarta database. We identified well-defined EV markers such as ANXA1, ANXA2, ANXA5 and GAPDH as the most abundant proteins detected in both aEV and fEV. Gene Ontology (GO) analyses of the enriched proteins in aEVs and fEVs illustrated that of the proteins overrepresented in aEVs were annotated to oxidation-reduction process (eg. HADHA, HADHB), mitochondrial ATP synthesis coupled proton transport (eg. ATP5A1, ATP5B and ATP5O) or protein folding (eg. FKBP11, FKBP10, FKBP2, MESDC2). In contrast, the proteins overrepresented in fEVs were annotated to extracellular matrix organization (eg. FN1, COL12A1, COL1A1, COL6A1, CCDC80) or positive regulation of cell migration (eg. ITGA4, ITGA6, GDF15, SEMA3, APDSD6, MMP14, ICAM1, FGFR1, PDGFRA, ADAMTS1). Interestingly, we found various proteins that were overrepresented in the non-supportive fEV cargo, that are involved in transforming growth factor beta receptor (TGFBR) signaling pathway (TGFBR1, TGFBR2, TGFB1, TGFB2, LTBP1, LTBP2, BMPR1A, GDF5, GDF15, PARP1, RPS27A and COL3A1). Together this illustrates the large differences in the protein content of EV populations derived from two different origins. Small RNA sequencing identified different molecular signatures between the supportive aEVs and the non-supportive fEVs. miRNA, yRNA and piRNA were abundantly found in aEV, while fEVs contained a significant enrichment of snoRNAs. Interestingly, the microRNA cluster miR-99b/let-7e/miR-125a, previously identified to increase the number of HSPC by targeting multiple proapoptotic genes, was highly and significantly enriched in aEV in comparison to fEV. Although we identified a significant difference in EV cargo and supportive effects of aEVs and fEVs, RNAseq analyses of 24hrs treated HSPCs with aEVs or fEVs indicated that only a limited set of genes was differentially regulated when compared to cells that were only treated with cytokines. Interestingly, we identified 5 genes, (eg MTF1, PER1, HOMER1, HSPA6, ENSG00000260534) that were significantly upregulated upon aEV compared to fEV treatment of HSPC. Moreover, HSPC treated with fEVs, resulted in increased expression of genes (SKIL, SMAD7, ENG, LDLRAD4) that are involved in the negative regulation of the TGFbeta signalling pathway. Together with the TGFbeta pathway proteins that were specifically identified in the fEVs, our results suggest that HSPCs may activate a negative feedback loop upon fEV treatment that consolidate HSPC expansion. In conclusion, our study gives novel insights into the complex biological role of EVs in the bone marrow microenvironment. Systematic analyses of supportive and non-supportive human BMSC derived extracellular vesicles indicated known molecules, eg. microRNA cluster miR-99b/let-7e/miR-125a, and the presence of TGFbeta pathway components that are important regulators in the cell-cell communication via EVs and open new means for the application of EVs in the discovery of therapeutics for more efficient ex vivo HSPC expansion.
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