Identification of osteolineage cell-derived extracellular vesicle cargo implicated in hematopoietic support
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE145178 | GEO | 2020/02/13
REPOSITORIES: GEO
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Project description:Osteolineage cell-derived extracellular vesicles (EVs) play a regulatory role in hematopoiesis and have been shown to promote the ex vivo expansion of human hematopoietic stem and progenitor cells (HSPCs). Here, we demonstrate that EVs from different human osteolineage sources do not have the same HSPC expansion promoting potential. Comparison of stimulatory and non-stimulatory osteolineage EVs by next-generation sequencing and mass spectrometry analyses revealed distinct microRNA and protein signatures identifying EV-derived candidate regulators of ex vivo HSPC expansion. Accordingly, the treatment of umbilical cord blood-derived CD34+ HSPCs with stimulatory EVs altered HSPC transcriptome, including genes with known roles in cell proliferation. An integrative bioinformatics approach, which connects the HSPC gene expression data with the candidate cargo in stimulatory EVs, delineated the potentially targeted biological functions and pathways during hematopoietic cell expansion and development. In conclusion, our study gives novel insights into the complex biological role of EVs in osteolineage cell-HSPC crosstalk and promotes the utility of EVs and their cargo as therapeutic agents in regenerative medicine.
2020-02-28 | PXD017172 | Pride
Project description:Osteolineage cell-derived extracellular vesicles (EVs) play a regulatory role in hematopoiesis and have been shown to promote the ex vivo expansion of human hematopoietic stem and progenitor cells (HSPCs). Here, we demonstrate that EVs from different human osteolineage sources do not have the same HSPC expansion promoting potential. Comparison of stimulatory and non-stimulatory osteolineage EVs by next-generation sequencing and mass spectrometry analyses revealed distinct microRNA (miRNA) and protein signatures identifying EV-derived candidate regulators of ex vivo HSPC expansion. Accordingly, the treatment of umbilical cord blood-derived CD34+ HSPCs with stimulatory EVs altered HSPC transcriptome, including genes with known roles in cell proliferation. An integrative bioinformatics approach, which connects the HSPC gene expression data with the candidate cargo in stimulatory EVs, delineated the potentially targeted biological functions and pathways during hematopoietic cell expansion and development. In conclusion, our study gives novel insights into the complex biological role of EVs in osteolineage cell-HSPC crosstalk and promotes the utility of EVs and their cargo as therapeutic agents in regenerative medicine.
2020-03-01 | GSE143613 | GEO
Project description:Osteolineage cells represent one of the critical bone marrow niche components that support maintenance of hematopoietic stem and progenitor cells (HSPCs). Recent studies demonstrate that extracellular vesicles (EVs) regulate stem cell development via horizontal transfer of bioactive cargo, including microRNAs (miRNAs). Here, we characterize the miRNA profile of EVs secreted by human osteoblasts and study their biological effect of on human umbilical cord blood-derived CD34+ HSPCs by sequencing, gene expression and biochemical analyses. Using next-generation sequencing we show that osteoblast-derived EVs contain highly abundant miRNAs specifically enriched in EVs, including critical regulators of hematopoietic proliferation (e.g., miR-29a). EV treatment of CD34+ HSPCs alters the expression of candidate miRNA targets, such as HBP1, BCL2 and PTEN. Furthermore, EVs enhance proliferation of CD34+ cells and their immature subsets in growth factor-driven ex vivo expansion cultures. Importantly, EV-expanded cells retain their differentiation capacity in vitro and show successful engraftment in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice in vivo. These discoveries reveal a novel osteoblast-derived EV-mediated mechanism for regulation of HSPC proliferation and warrant consideration of EV-miRNAs for the development of expansion strategies to treat hematological disorders.
2016-10-05 | E-MTAB-4560 | biostudies-arrayexpress
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.
2021-02-10 | PXD022851 | Pride
Project description:Recently, we and others have illustrated that extracellular vesicles (EVs) have the potential to support hematopoietic stem and progenitor cell (HSPC) expansion; however, the mechanism and processes responsible for the intercellular communication by EVs are still unknown. In the current study, we investigate whether primary human bone marrow derived mesenchymal stromal cells (BMSC) EVs isolated from two different origins, fetal (fEV) and adult (aEV) tissue, can increase the relative low number of HSPCs found in umbilical cord blood (UCB) and which EV-derived components are responsible for ex vivo HSPC expansion. Interestingly, aEVs but and to a lesser extent fEVs, showed supportive ex vivo expansion capacity of UCB-HSPCs. Taking advantage of the two BMSC sources with different supportive effects, we analyzed the EV cargo and investigated how gene expression is modulated in HSPCs after incubation with aEVs and fEVs. Proteomics analyses of the protein cargo composition of the supportive aEV versus the less-supportive fEV identified 90% of the Top100 exosome proteins present in the ExoCarta database. Gene Ontology (GO) analyses illustrated that the proteins overrepresented in aEVs were annotated to oxidation-reduction process, mitochondrial ATP synthesis coupled proton transport, or protein folding. In contrast, the proteins overrepresented in fEVs were annotated to extracellular matrix organization positive regulation of cell migration or transforming growth factor beta receptor (TGFBR) signaling pathway. Small RNA sequencing identified different molecular signatures between aEVs and fEVs. Interestingly, the microRNA cluster miR-99b/let-7e/miR-125a, previously identified to increase the number of HSPCs by targeting multiple pro-apoptotic genes, was highly and significantly enriched in aEVs. Although we identified significant differences in the supportive effects of aEVs and fEVs, RNAseq analyses of the 24hrs treated HSPCs indicated that a limited set of genes was differentially regulated when compared to cells that were treated with cytokines only. Together, our study provides novel insights into the complex biological role of EVs and illustrates that aEVs and fEVs differentially support ex vivo expansion capacity of UCB-HSPCs. Together opening new means for the application of EVs in the discovery of therapeutics for more efficient ex vivo HSPC expansion.
2021-02-10 | GSE165323 | GEO
Project description:Recently, we and others have illustrated that extracellular vesicles (EVs) have the potential to support hematopoietic stem and progenitor cell (HSPC) expansion; however, the mechanism and processes responsible for the intercellular communication by EVs are still unknown. In the current study, we investigate whether primary human bone marrow derived mesenchymal stromal cells (BMSC) EVs isolated from two different origins, fetal (fEV) and adult (aEV) tissue, can increase the relative low number of HSPCs found in umbilical cord blood (UCB) and which EV-derived components are responsible for ex vivo HSPC expansion. Interestingly, aEVs and to a lesser extent fEVs, showed supportive ex vivo expansion capacity of UCB-HSPCs. Taking advantage of the two BMSC sources with different supportive effects, we analyzed the EV cargo and investigated how gene expression is modulated in HSPCs after incubation with aEVs and fEVs. Proteomics analyses of the protein cargo composition of the supportive aEV versus the less-supportive fEV identified 90% of the Top100 exosome proteins present in the ExoCarta database. Gene Ontology (GO) analyses illustrated that the proteins overrepresented in aEVs were annotated to oxidation-reduction process, mitochondrial ATP synthesis coupled proton transport, or protein folding. In contrast, the proteins overrepresented in fEVs were annotated to extracellular matrix organization positive regulation of cell migration or transforming growth factor beta receptor (TGFBR) signaling pathway. Small RNA sequencing identified different molecular signatures between aEVs and fEVs. Interestingly, the microRNA cluster miR-99b/let-7e/miR-125a, previously identified to increase the number of HSPCs by targeting multiple pro-apoptotic genes, was highly and significantly enriched in aEVs. Although we identified significant differences in the supportive effects of aEVs and fEVs, RNAseq analyses of the 24hrs treated HSPCs indicated that a limited set of genes was differentially regulated when compared to cells that were treated with cytokines only. Together, our study provides novel insights into the complex biological role of EVs and illustrates that aEVs and fEVs differentially support ex vivo expansion capacity of UCB-HSPCs. Together opening new means for the application of EVs in the discovery of therapeutics for more efficient ex vivo HSPC expansion.
2021-02-10 | GSE165921 | GEO
Project description:Hematopoietic stem cells (HSC) rely on a unique regulatory machinery that facilitates life-long blood production and enables reconstitution of the entire hematopoietic system upon transplantation. However, the biological processes governing human HSC self-renewal and engraftment ability are poorly understood and challenging to recapitulate ex vivo to facilitate robust human HSC expansion. We discovered a novel HSC regulatory protein, MYCT1 (MYCT target 1), that is selectively expressed in endothelial cells (EC) and undifferentiated human HSPCs but becomes drastically downregulated during HSC culture. Lentiviral knockdown of MYCT1 in human foetal liver and cord blood HSPCs revealed a critical role for MYCT1 in governing human HSPC expansion and engraftment ability. Single cell RNAseq of human CB HSPCs after MYCT1 knockdown and overexpression revealed that MYCT1 governs HSC functional competence and modulates cellular properties essential for HSC stemness, such as low mitochondrial metabolic activity. Indeed, restoring the compromised MYCT1 expression in cultured human CB HSPCs improved ex vivo expansion of the most undifferentiated human HSPCs and enhanced their engraftment ability. We found that MYCT1 is localized in the endosomal membrane and interacts with vesicle trafficking regulators and signalling machinery essential for HSC and EC function. Loss of MYCT1 led to excessive endocytosis and hyperactive signalling responses to cytokines, whereas restoring MYCT1 expression in cultured CB HSPCs balanced the abnormal endocytosis associated with prolonged culture and fine-tuned signalling responses. Our work identifies MYCT1-moderated endocytosis and environmental sensing as an essential regulatory mechanism required to preserve human HSC stemness, and pinpoints silencing of MYCT1 as a critical contributor to the dysfunction of cultured human HSCs that needs to be addressed to improve human HSC culture strategies.
2024-04-15 | PXD042257 | Pride
Project description:The Notch signaling pathway plays a critical role in regulating the proliferation and differentiation of stem and progenitor cells including hematopoietic stem and progenitor cells (HSPCs). Notch receptors and ligands are expressed in BM stromal and hematopoietic cells. A large body of evidence has demonstrated that activating Notch signaling enhances HSCs self-renewal and facilitates its expansion ex vivo. We report that an endothelium-targeted soluble Notch ligand, the DSL domain of mouse Delta-like 1 fused with a RGD motif (mD1R), efficiently promotes the expansion ex vivo of mouse bone marrow HSPCs in a Notch signaling and endocytosis dependent manner. HSPCs expanded in the presence of mD1R kept long-term HSPC repopulation capacity. We used microarrays to compare the gene expression profiles of HSPCs expanded in the presence of PBS and mD1R. KSL cells were plated on Human umbilical vein endothelial cells (HUVECs) and cultured in a serum-free medium supplemented with a cocktail containing 5 types of mouse cytokines (m5GF) in the presence of PBS or mD1R for 7 days. Then KSL cells were sorted from these cultured hematopoietic cells for RNA extraction and hybridization on Affymetrix microarrays. The experiments were repeated 3 times.
2012-07-03 | E-GEOD-39082 | biostudies-arrayexpress
Project description:The Notch signaling pathway plays a critical role in regulating the proliferation and differentiation of stem and progenitor cells including hematopoietic stem and progenitor cells (HSPCs). Notch receptors and ligands are expressed in BM stromal and hematopoietic cells. A large body of evidence has demonstrated that activating Notch signaling enhances HSCs self-renewal and facilitates its expansion ex vivo. We report that an endothelium-targeted soluble Notch ligand, the DSL domain of mouse Delta-like 1 fused with a RGD motif (mD1R), efficiently promotes the expansion ex vivo of mouse bone marrow HSPCs in a Notch signaling and endocytosis dependent manner. HSPCs expanded in the presence of mD1R kept long-term HSPC repopulation capacity. We used microarrays to compare the gene expression profiles of HSPCs expanded in the presence of PBS and mD1R.
2012-07-04 | GSE39082 | GEO
Project description:A greater understanding of the molecular pathways that underpin the unique human hematopoietic stem and progenitor cell (HSPC) self-renewal program will improve strategies to expand these critical cell types for regenerative therapies. The post-transcriptional mechanisms guiding HSPC fate during ex vivo expansion have not been closely investigated. Using shRNA-mediated knockdown, we show that the RNA-binding protein (RBP) Musashi-2 (MSI2) is required for human HSPC self-renewal. Conversely, when overexpressed, MSI2 induces multiple pro-self-renewal phenotypes, including significant ex vivo expansion of short- and long-term repopulating cells through direct attenuation of aryl hydrocarbon receptor (AHR) signaling. Using a global analysis of MSI2-RNA interactions, we determined that MSI2 post-transcriptionally downregulates canonical AHR pathway components in cord blood HSPCs. Our study provides new mechanistic insight into RBP-controlled RNA networks that underlie the self-renewal process and provides evidence that manipulating such networks can provide a novel means to enhance the regenerative potential of human HSPCs expanded ex vivo.
2016-04-27 | GSE70685 | GEO