Fichtel Pascal, von Bonin Malte, Kuhnert Robert, Möbus Kristin, Bornhäuser Martin, Wobus Manja
Department of Medicine I, University Hospital Carl Gustav Carus, Technische Universität, Dresden, Germany.
Center for Regenerative Therapies, Technische Universität, Dresden, Germany.
Front Bioeng Biotechnol. 2022 Jun 1;10:892661. doi: 10.3389/fbioe.2022.892661. eCollection 2022.
Aging of the hematopoietic system is characterized by an expansion of hematopoietic stem and progenitor cells (HSPCs) with reduced capacity for engraftment, self-renewal, and lymphoid differentiation, resulting in myeloid-biased hematopoiesis. This process is mediated by both HSPC intrinsic and extrinsic factors, e.g., the stromal environment. A relevant cellular component of the bone marrow (BM) microenvironment are mesenchymal stromal cells (MSCs) which regulate fate and differentiation of HSPCs. The bi-directional communication with HSPCs is mediated either by direct cell-cell contacts or by extracellular vesicles (EVs) which carry bioactive substances such as small RNA, DNA, lipids and proteins. So far, the impact of MSC-derived EVs on human hematopoietic aging is poorly investigated. BM MSCs were isolated from young (n = 3, median age: 22 years) and aged (n = 3, median age: 70 years) donors and the EVs were isolated after culturing the confluent cell layer in serum-free medium for 48 h. CD34 HSPCs were purified from peripheral blood of healthy donors (n = 3, median age: 65 years) by magnetic sorting. Nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and western blot detection of EV markers CD63, CD81 and Flotillin-1 revealed no significant differences between young and aged MSC-EVs. Interestingly, young MSCs secreted a significantly higher miRNA concentration than aged cells. However, the amount of distinct miRNAs such as miR-29a and miR-34a was significantly higher in aged MSC-EVs. HSPCs incubated with young EVs showed a significant increase in cell number and a higher viability. The expression of the tumor suppressors PTEN, a known target of mir-29a, and CDKN2A was increased in HSPCs incubated with young EVs. The clonogenic assay demonstrated a decreased colony number of CFU-GM after treatment with young EVs and an increased number of BFU-E/CFU-E after incubation with aged MSC-EVs. Xenogenic transplantation experiments showed no significant differences concerning the engraftment of lymphoid or myeloid cell compartments, but the overall human chimerism 8-16 weeks after transplantation was higher after EV treatment. In conclusion, our data suggest that HSPC characteristics such as cell cycle activity and clonogenicity can be modulated by MSC-derived EVs. Further studies have to elucidate the potential therapeutic relevance of our findings.
造血系统衰老的特征是造血干细胞和祖细胞（HSPCs）扩增，但植入、自我更新及淋巴样分化能力降低，导致造血偏向髓系。这一过程由HSPC内在和外在因素介导，例如基质环境。骨髓（BM）微环境的一个相关细胞成分是间充质基质细胞（MSCs），其调节HSPCs的命运和分化。与HSPCs的双向通讯由直接的细胞间接触或携带生物活性物质（如小RNA、DNA、脂质和蛋白质）的细胞外囊泡（EVs）介导。到目前为止，MSC来源的EVs对人类造血衰老的影响研究较少。从年轻供体（n = 3，中位年龄：22岁）和老年供体（n = 3，中位年龄：70岁）分离BM MSCs，在无血清培养基中培养汇合细胞层48小时后分离EVs。通过磁性分选从健康供体（n = 3，中位年龄：65岁）外周血中纯化CD34 HSPCs。纳米颗粒跟踪分析（NTA）、透射电子显微镜（TEM）以及对EV标志物CD63、CD81和Flotillin-1的蛋白质印迹检测显示，年轻和老年MSC-EVs之间无显著差异。有趣的是，年轻MSCs分泌的miRNA浓度明显高于老年细胞。然而，老年MSC-EVs中miR-29a和miR-34a等特定miRNA的量明显更高。与年轻EVs孵育的HSPCs细胞数量显著增加且活力更高。与年轻EVs孵育的HSPCs中，肿瘤抑制因子PTEN（已知的mir-29a靶点）和CDKN2A的表达增加。克隆形成试验表明，用年轻EVs处理后CFU-GM集落数减少，与老年MSC-EVs孵育后BFU-E/CFU-E集落数增加。异种移植实验显示，淋巴样或髓样细胞区室的植入无显著差异，但移植后8 - 16周，EV处理后的总体人嵌合率更高。总之，我们的数据表明，HSPC的特征如细胞周期活性和克隆形成能力可由MSC来源的EVs调节。进一步研究必须阐明我们研究结果的潜在治疗相关性。
