Ghebes Corina A, Morhayim Jess, Kleijer Marion, Koroglu Merve, Erkeland Stefan J, Hoogenboezem Remco, Bindels Eric, van Alphen Floris P J, van den Biggelaar Maartje, Nolte Martijn A, van der Eerden Bram C J, Braakman Eric, Voermans Carlijn, van de Peppel Jeroen
Department of Hematopoiesis, Sanquin Research, Amsterdam, Netherlands.
Department of Hematology, Erasmus MC, University Medical Center, Rotterdam, Netherlands.
Front Bioeng Biotechnol. 2021 Feb 25;9:640419. doi: 10.3389/fbioe.2021.640419. eCollection 2021.
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 HSPC expansion. Interestingly, aEVs and to a lesser extent fEVs, showed supportive 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 vs. 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 24 h 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 expansion capacity of UCB-HSPCs. Together opening new means for the application of EVs in the discovery of therapeutics for more efficient HSPC expansion.
最近，我们和其他研究表明，细胞外囊泡（EVs）有促进造血干细胞和祖细胞（HSPC）扩增的潜力；然而，EVs介导细胞间通讯的机制和过程仍不清楚。在本研究中，我们探究了从两种不同来源（胎儿组织（fEV）和成体组织（aEV））分离得到的原代人骨髓间充质基质细胞（BMSC）来源的EVs，是否能增加脐带血（UCB）中相对较少的HSPC数量，以及哪些EV衍生成分负责HSPC的扩增。有趣的是，aEVs以及程度稍轻的fEVs，显示出对UCB-HSPCs的支持性扩增能力。利用这两种具有不同支持作用的BMSC来源，我们分析了EV的货物成分，并研究了HSPCs在与aEVs和fEVs孵育后基因表达是如何被调节的。对具有支持作用的aEV与支持作用较弱的fEV的蛋白质货物成分进行蛋白质组学分析，鉴定出了ExoCarta数据库中存在的前100种外泌体蛋白中的90%。基因本体论（GO）分析表明，aEVs中高表达的蛋白质被注释为氧化还原过程、线粒体ATP合成偶联质子转运或蛋白质折叠。相比之下，fEVs中高表达的蛋白质被注释为细胞外基质组织、细胞迁移的正调控或转化生长因子β受体（TGFBR）信号通路。小RNA测序确定了aEVs和fEVs之间不同的分子特征。有趣的是，先前已确定通过靶向多个促凋亡基因来增加HSPC数量的微小RNA簇miR-99b/let-7e/miR-125a，在aEVs中高度且显著富集。虽然我们确定了aEVs和fEVs在支持作用上存在显著差异，但对处理24小时的HSPCs进行RNA测序分析表明，与仅用细胞因子处理的细胞相比，只有有限的一组基因受到差异调节。总之，我们的研究为EVs的复杂生物学作用提供了新的见解，并表明aEVs和fEVs对UCB-HSPCs的扩增能力有不同的支持作用。这共同为EVs在发现更有效的HSPC扩增治疗方法中的应用开辟了新途径。
