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Therapeutics of the future: Navigating the pitfalls of extracellular vesicles research from an osteoarthritis perspective 未来的疗法:从骨关节炎的角度看细胞外囊泡研究的陷阱。
IF 15.5 1区 医学 Q1 CELL BIOLOGY Pub Date : 2024-06-28 DOI: 10.1002/jev2.12435
Antoine Karoichan, Sarah Boucenna, Maryam Tabrizian

Extracellular vesicles have gained wide momentum as potential therapeutics for osteoarthritis, a highly prevalent chronic disease that still lacks an approved treatment. The membrane-bound vesicles are secreted by all cells carrying different cargos that can serve as both disease biomarkers and disease modifiers. Nonetheless, despite a significant peak in research regarding EVs as OA therapeutics, clinical implementation seems distant. In addition to scalability and standardization challenges, researchers often omit to focus on and consider the proper tropism of the vesicles, the practicality and relevance of their source, their low native therapeutic efficacy, and whether they address the disease as a whole. These considerations are necessary to better understand EVs in a clinical light and have been comprehensively discussed and ultimately summarized in this review into a conceptualized framework termed the nanodiamond concept. Future perspectives are also discussed, and alternatives are presented to address some of the challenges and concerns.

细胞外囊泡作为治疗骨关节炎的潜在疗法已获得广泛关注。膜结合囊泡由所有细胞分泌,携带不同的载体,可作为疾病生物标志物和疾病调节剂。然而,尽管有关将 EVs 作为 OA 治疗药物的研究达到了一个显著的高峰,但临床应用似乎还很遥远。除了可扩展性和标准化方面的挑战外,研究人员往往忽略了对囊泡的适当滋养性、囊泡来源的实用性和相关性、囊泡的低原生疗效以及囊泡是否能从整体上治疗疾病等问题的关注和考虑。要从临床角度更好地理解 EVs,就必须考虑这些因素,本综述对这些因素进行了全面讨论,并最终将其总结为一个概念化框架,称为纳米钻石概念。本综述还讨论了未来的前景,并提出了解决某些挑战和问题的替代方案。
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引用次数: 0
Adult cardiomyocytes-derived EVs for the treatment of cardiac fibrosis 用于治疗心脏纤维化的成人心肌细胞衍生 EVs。
IF 15.5 1区 医学 Q1 CELL BIOLOGY Pub Date : 2024-06-28 DOI: 10.1002/jev2.12461
Marta Prieto-Vila, Yusuke Yoshioka, Naoya Kuriyama, Akihiko Okamura, Yusuke Yamamoto, Asao Muranaka, Takahiro Ochiya

Cardiac fibrosis is a common pathological feature of cardiovascular diseases that arises from the hyperactivation of fibroblasts and excessive extracellular matrix (ECM) deposition, leading to impaired cardiac function and potentially heart failure or arrhythmia. Extracellular vesicles (EVs) released by cardiomyocytes (CMs) regulate various physiological functions essential for myocardial homeostasis, which are disrupted in cardiac disease. Therefore, healthy CM-derived EVs represent a promising cell-free therapy for the treatment of cardiac fibrosis.

To this end, we optimized the culture conditions of human adult CMs to obtain a large yield of EVs without compromising cellular integrity by using a defined combination of small molecules. EVs were isolated by ultracentrifugation, and their characteristics were analysed. Finally, their effect on fibrosis was tested.

Treatment of TGFβ-activated human cardiac fibroblasts with EVs derived from CMs using our culture system resulted in a decrease in fibroblast activation markers and ECM accumulation. The rescued phenotype was associated with specific EV cargo, including multiple myocyte-specific and antifibrotic microRNAs, although their effect individually was not as effective as the EV treatment. Notably, pathway analysis showed that EV treatment reverted the transcription of activated fibroblasts and decreased several signalling pathways, including MAPK, mTOR, JAK/STAT, TGFβ, and PI3K/Akt, all of which are involved in fibrosis development. Intracardiac injection of CM-derived EVs in an animal model of cardiac fibrosis reduced fibrotic area and increased angiogenesis, which correlated with improved cardiac function.

These findings suggest that EVs derived from human adult CMs may offer a targeted and effective treatment for cardiac fibrosis, owing to their antifibrotic properties and the specificity of cargo.

心脏纤维化是心血管疾病的常见病理特征,它源于成纤维细胞的过度活化和细胞外基质(ECM)的过度沉积,从而导致心脏功能受损,并可能引发心力衰竭或心律失常。心肌细胞(CM)释放的细胞外囊泡(EVs)调节着心肌稳态所必需的各种生理功能,而这些功能在心脏疾病中会受到破坏。因此,健康 CM 衍生的 EVs 是治疗心脏纤维化的一种前景广阔的无细胞疗法。为此,我们优化了人类成体 CM 的培养条件,通过使用特定的小分子组合,在不损害细胞完整性的情况下获得大量 EVs。我们通过超速离心法分离出了 EVs,并分析了它们的特征。最后,测试了它们对纤维化的影响。利用我们的培养系统,用从 CMs 提取的 EVs 处理 TGFβ 激活的人心脏成纤维细胞,结果发现成纤维细胞活化标记物和 ECM 积累减少。获救的表型与特定的 EV 货物有关,包括多种肌细胞特异性和抗纤维化的 microRNA,尽管它们的单独作用不如 EV 处理有效。值得注意的是,通路分析表明,EV处理可逆转活化成纤维细胞的转录,并减少几种信号通路,包括MAPK、mTOR、JAK/STAT、TGFβ和PI3K/Akt,所有这些通路都参与纤维化的发展。在心脏纤维化动物模型中,心内注射 CM 衍生的 EV 可减少纤维化面积并增加血管生成,这与心脏功能的改善相关。这些研究结果表明,从人类成体CM中提取的EVs由于其抗纤维化特性和货物的特异性,可为心脏纤维化提供有针对性的有效治疗。
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引用次数: 0
Isolation and quantification of L1CAM-positive extracellular vesicles on a chip as a potential biomarker for Parkinson's Disease 在芯片上分离和量化 L1CAM 阳性细胞外囊泡,作为帕金森病的潜在生物标记物。
IF 15.5 1区 医学 Q1 Medicine Pub Date : 2024-06-19 DOI: 10.1002/jev2.12467
Danyu Li, Siyi Zou, Ziyang Huang, Congcong Sun, Guozhen Liu

Extracellular vesicles (EVs) carry disease-specific molecular profiles, demonstrating massive potential in biomarker discovery. In this study, we developed an integrated biochip platform, termed EVID-biochip (EVs identification and detection biochip), which integrates in situ electrochemical protein detection with on-chip antifouling-immunomagnetic beads modified with CD81 antibodies and zwitterion molecules, enabling efficient isolation and detection of neuronal EVs. The capability of the EVID-biochip to isolate common EVs and detect neuronal EVs associated with Parkinson's disease in human serum is successfully demonstrated, using the transmembrane protein L1-cell adhesion molecule (L1CAM) as a target biomarker. The EVID-biochip exhibited high efficiency and specificity for the detection of L1CAM with a sensitivity of 1 pg/mL. Based on the validation of 76 human serum samples, for the first time, this study discovered that the level of L1CAM/neuronal EV particles in serum could serve as a reliable indicator to distinguish Parkinson's disease from control groups with AUC = 0.973. EVID-biochip represents a reliable and rapid liquid biopsy platform for the analysis of complex biofluids offering EVs isolation and detection in a single chip, requiring a small sample volume (300 µL) and an assay time of 1.5 h. This approach has the potential to advance the diagnosis and biomarker discovery of various neurological disorders and other diseases.

细胞外囊泡(EVs)携带疾病特异性分子特征,在生物标记物发现方面具有巨大潜力。在这项研究中,我们开发了一种名为 EVID-生物芯片(EVs identification and detection biochip)的集成生物芯片平台,该平台将原位电化学蛋白检测与经 CD81 抗体和齐聚物分子修饰的芯片抗偶联免疫磁珠整合在一起,实现了对神经元 EVs 的高效分离和检测。以跨膜蛋白 L1-细胞粘附分子(L1CAM)为目标生物标记物,成功证明了 EVID 生物芯片分离普通 EV 和检测人类血清中与帕金森病相关的神经元 EV 的能力。EVID 生物芯片对 L1CAM 的检测具有高效性和特异性,灵敏度为 1 pg/mL。基于对76份人类血清样本的验证,该研究首次发现血清中L1CAM/神经元EV颗粒的水平可作为区分帕金森病和对照组的可靠指标,AUC = 0.973。EVID-生物芯片是一种可靠、快速的液体活检平台,可用于分析复杂的生物流体,在单个芯片中进行EVs分离和检测,只需少量样品(300微升),检测时间为1.5小时。
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引用次数: 0
Stress-induced Rab11a-exosomes induce amphiregulin-mediated cetuximab resistance in colorectal cancer 应激诱导的Rab11a-外泌体在结直肠癌中诱导安非他酮介导的西妥昔单抗抗性。
IF 16 1区 医学 Q1 Medicine Pub Date : 2024-06-18 DOI: 10.1002/jev2.12465
John D. Mason, Ewan Marks, Shih-Jung Fan, Kristie McCormick, Clive Wilson, Adrian L. Harris, Freddie C. Hamdy, Chris Cunningham, Deborah C. I. Goberdhan

Exosomes are secreted vesicles made intracellularly in the endosomal system. We have previously shown that exosomes are not only made in late endosomes, but also in recycling endosomes marked by the monomeric G-protein Rab11a. These vesicles, termed Rab11a-exosomes, are preferentially secreted under nutrient stress from several cancer cell types, including HCT116 colorectal cancer (CRC) cells. HCT116 Rab11a-exosomes have particularly potent signalling activities, some mediated by the epidermal growth factor receptor (EGFR) ligand, amphiregulin (AREG). Mutant activating forms of KRAS, a downstream target of EGFR, are often found in advanced CRC. When absent, monoclonal antibodies, such as cetuximab, which target the EGFR and block the effects of EGFR ligands, such as AREG, can be administered. Patients, however, inevitably develop resistance to cetuximab, either by acquiring KRAS mutations or via non-genetic microenvironmental changes. Here we show that nutrient stress in several CRC cell lines causes the release of AREG-carrying Rab11a-exosomes. We demonstrate that while soluble AREG has no effect, much lower levels of AREG bound to Rab11a-exosomes from cetuximab-resistant KRAS-mutant HCT116 cells, can suppress the effects of cetuximab on KRAS-wild type Caco-2 CRC cells. Using neutralising anti-AREG antibodies and an intracellular EGFR kinase inhibitor, we show that this effect is mediated via AREG activation of EGFR, and not transfer of activated KRAS. Therefore, presentation of AREG on Rab11a-exosomes affects its ability to compete with cetuximab. We propose that this Rab11a-exosome-mediated mechanism contributes to the establishment of resistance in cetuximab-sensitive cells and may explain why in cetuximab-resistant tumours only some cells carry mutant KRAS.

外泌体是内泌体系统在细胞内产生的分泌囊泡。我们之前已经证明,外泌体不仅在晚期内体中产生,也在以单体G蛋白Rab11a为标志的回收内体中产生。这些被称为 Rab11a-外泌体的囊泡在营养压力下优先从几种癌细胞类型中分泌出来,包括 HCT116 大肠癌(CRC)细胞。HCT116 Rab11a-外泌体具有特别强的信号活性,其中一些信号活性是由表皮生长因子受体(EGFR)配体安非拉酮(AREG)介导的。KRAS 是表皮生长因子受体(EGFR)的下游靶标,在晚期 CRC 中经常发现 KRAS 的突变激活形式。如果不存在这种情况,就可以使用西妥昔单抗等单克隆抗体来靶向表皮生长因子受体并阻断表皮生长因子受体配体(如 AREG)的作用。然而,患者不可避免地会对西妥昔单抗产生耐药性,这可能是由于获得了 KRAS 突变,也可能是由于非遗传性的微环境变化。在这里,我们展示了几种 CRC 细胞系中的营养应激会导致携带 AREG 的 Rab11a- 外泌体释放。我们证明,虽然可溶性 AREG 没有影响,但与来自西妥昔单抗抗性 KRAS 突变 HCT116 细胞的 Rab11a- 外泌体结合的 AREG 水平低得多,可以抑制西妥昔单抗对 KRAS 野生型 Caco-2 CRC 细胞的影响。通过使用中和性抗 AREG 抗体和细胞内表皮生长因子受体激酶抑制剂,我们发现这种效应是通过 AREG 激活表皮生长因子受体而不是活化的 KRAS 转移来介导的。因此,AREG在Rab11a-外泌体上的呈现会影响其与西妥昔单抗竞争的能力。我们认为,Rab11a-外泌体介导的这一机制有助于西妥昔单抗敏感细胞耐药性的建立,也可以解释为什么西妥昔单抗耐药肿瘤中只有部分细胞携带突变型 KRAS。
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引用次数: 0
Gut-liver axis: Potential mechanisms of action of food-derived extracellular vesicles 肠肝轴:源自食物的细胞外囊泡的潜在作用机制。
IF 16 1区 医学 Q1 Medicine Pub Date : 2024-06-17 DOI: 10.1002/jev2.12466
Sitong Zhang, Qiyue Wang, Daniel En Liang Tan, Vritika Sikka, Cheng Han Ng, Yan Xian, Dan Li, Mark Muthiah, Nicholas W. S. Chew, Gert Storm, Lingjun Tong, Jiong-Wei Wang

Food-derived extracellular vesicles (FEVs) are nanoscale membrane vesicles obtained from dietary materials such as breast milk, plants and probiotics. Distinct from other EVs, FEVs can survive the harsh degrading conditions in the gastrointestinal tract and reach the intestines. This unique feature allows FEVs to be promising prebiotics in health and oral nanomedicine for gut disorders, such as inflammatory bowel disease. Interestingly, therapeutic effects of FEVs have recently also been observed in non-gastrointestinal diseases. However, the mechanisms remain unclear or even mysterious. It is speculated that orally administered FEVs could enter the bloodstream, reach remote organs, and thus exert therapeutic effects therein. However, emerging evidence suggests that the amount of FEVs reaching organs beyond the gastrointestinal tract is marginal and may be insufficient to account for the significant therapeutic effects achieved regarding diseases involving remote organs such as the liver. Thus, we herein propose that FEVs primarily act locally in the intestine by modulating intestinal microenvironments such as barrier integrity and microbiota, thereby eliciting therapeutic impact remotely on the liver in non-gastrointestinal diseases via the gut-liver axis. Likewise, drugs delivered to the gastrointestinal system through FEVs may act via the gut-liver axis. As the liver is the main metabolic hub, the intestinal microenvironment may be implicated in other metabolic diseases. In fact, many patients with non-alcoholic fatty liver disease, obesity, diabetes and cardiovascular disease suffer from a leaky gut and dysbiosis. In this review, we provide an overview of the recent progress in FEVs and discuss their biomedical applications as therapeutic agents and drug delivery systems, highlighting the pivotal role of the gut-liver axis in the mechanisms of action of FEVs for the treatment of gut disorders and metabolic diseases.

食物衍生细胞外囊泡(FEVs)是从母乳、植物和益生菌等食物材料中提取的纳米级膜囊泡。与其他细胞外小泡不同的是,FEVs 可以在胃肠道恶劣的降解条件下存活下来,并进入肠道。这种独特的特性使 FEVs 成为有前景的健康益生元和治疗肠道疾病(如炎症性肠病)的口服纳米药物。有趣的是,最近在非胃肠道疾病中也观察到了 FEVs 的治疗效果。然而,其机制仍不清楚,甚至是神秘的。据推测,口服的 FEVs 可以进入血液,到达远处的器官,从而发挥治疗作用。然而,新出现的证据表明,到达胃肠道以外器官的 FEV 数量微乎其微,可能不足以解释涉及肝脏等远端器官的疾病所取得的显著治疗效果。因此,我们在此提出,FEVs 主要通过调节肠道微环境(如屏障完整性和微生物群)在肠道局部发挥作用,从而通过肠肝轴对非胃肠道疾病的肝脏产生远程治疗效果。同样,通过快速静脉输液器输送到胃肠道系统的药物也可能通过肠道-肝脏轴发挥作用。由于肝脏是主要的代谢枢纽,肠道微环境可能与其他代谢性疾病有关。事实上,许多非酒精性脂肪肝、肥胖症、糖尿病和心血管疾病患者都患有肠漏和菌群失调。在这篇综述中,我们概述了 FEV 的最新进展,讨论了它们作为治疗剂和给药系统的生物医学应用,强调了肠道-肝脏轴在 FEV 治疗肠道疾病和代谢性疾病的作用机制中的关键作用。
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引用次数: 0
Microglial activation induces nitric oxide signalling and alters protein S-nitrosylation patterns in extracellular vesicles 小胶质细胞活化会诱导一氧化氮信号并改变细胞外囊泡中的蛋白质 S-亚硝基化模式。
IF 16 1区 医学 Q1 Medicine Pub Date : 2024-06-17 DOI: 10.1002/jev2.12455
Natasha Vassileff, Jereme G. Spiers, Sarah E. Bamford, Rohan G. T. Lowe, Keshava K. Datta, Paul J. Pigram, Andrew F. Hill

Neuroinflammation is an underlying feature of neurodegenerative conditions, often appearing early in the aetiology of a disease. Microglial activation, a prominent initiator of neuroinflammation, can be induced through lipopolysaccharide (LPS) treatment resulting in expression of the inducible form of nitric oxide synthase (iNOS), which produces nitric oxide (NO). NO post-translationally modifies cysteine thiols through S-nitrosylation, which can alter function of the target protein. Furthermore, packaging of these NO-modified proteins into extracellular vesicles (EVs) allows for the exertion of NO signalling in distant locations, resulting in further propagation of the neuroinflammatory phenotype. Despite this, the NO-modified proteome of activated microglial EVs has not been investigated. This study aimed to identify the protein post-translational modifications NO signalling induces in neuroinflammation. EVs isolated from LPS-treated microglia underwent mass spectral surface imaging using time of flight-secondary ion mass spectrometry (ToF-SIMS), in addition to iodolabelling and comparative proteomic analysis to identify post-translation S-nitrosylation modifications. ToF-SIMS imaging successfully identified cysteine thiol side chains modified through NO signalling in the LPS treated microglial-derived EV proteins. In addition, the iodolabelling proteomic analysis revealed that the EVs from LPS-treated microglia carried S-nitrosylated proteins indicative of neuroinflammation. These included known NO-modified proteins and those associated with LPS-induced microglial activation that may play an essential role in neuroinflammatory communication. Together, these results show activated microglia can exert broad NO signalling changes through the selective packaging of EVs during neuroinflammation.

神经炎症是神经退行性疾病的一个基本特征,通常出现在疾病病因的早期。小胶质细胞活化是神经炎症的一个重要诱因,可通过脂多糖(LPS)处理诱导小胶质细胞活化,导致一氧化氮合酶(iNOS)的诱导型表达,从而产生一氧化氮(NO)。一氧化氮通过 S-亚硝基化对半胱氨酸硫醇进行翻译后修饰,从而改变目标蛋白质的功能。此外,将这些经 NO 修饰的蛋白质包装到细胞外囊泡 (EVs) 中,可以在远处发出 NO 信号,从而进一步传播神经炎症表型。尽管如此,活化的小胶质细胞EVs的NO修饰蛋白质组尚未得到研究。本研究旨在确定 NO 信号在神经炎症中诱导的蛋白质翻译后修饰。从经 LPS 处理的小胶质细胞中分离出的 EVs 利用飞行时间-二次离子质谱(ToF-SIMS)进行质谱表面成像,此外还进行了碘标记和比较蛋白质组分析,以确定翻译后的 S-亚硝基化修饰。ToF-SIMS 成像成功鉴定了经 LPS 处理的小胶质细胞衍生 EV 蛋白中通过 NO 信号修饰的半胱氨酸硫醇侧链。此外,碘标记蛋白质组分析表明,经 LPS 处理的小胶质细胞 EVs 含有表明神经炎症的 S-亚硝基化蛋白质。这些蛋白包括已知的氮氧化物修饰蛋白和与 LPS 诱导的小胶质细胞活化相关的蛋白,它们可能在神经炎症交流中发挥重要作用。总之,这些结果表明,活化的小胶质细胞在神经炎症过程中可通过选择性包装 EVs 来产生广泛的 NO 信号变化。
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引用次数: 0
Single-extracellular vesicle (EV) analyses validate the use of L1 Cell Adhesion Molecule (L1CAM) as a reliable biomarker of neuron-derived EVs 单细胞外囊泡 (EV) 分析验证了 L1 细胞粘附分子 (L1CAM) 是神经元衍生 EV 的可靠生物标记。
IF 16 1区 医学 Q1 Medicine Pub Date : 2024-06-13 DOI: 10.1002/jev2.12459
Carlos J Nogueras-Ortiz, Erden Eren, Pamela Yao, Elizabeth Calzada, Christopher Dunn, Olga Volpert, Francheska Delgado-Peraza, Maja Mustapic, Alexey Lyashkov, F Javier Rubio, Michael Vreones, Lesley Cheng, Yang You, Andrew F Hill, Tsuneya Ikezu, Erez Eitan, Edward J Goetzl, Dimitrios Kapogiannis

Isolation of neuron-derived extracellular vesicles (NDEVs) with L1 Cell Adhesion Molecule (L1CAM)-specific antibodies has been widely used to identify blood biomarkers of CNS disorders. However, full methodological validation requires demonstration of L1CAM in individual NDEVs and lower levels or absence of L1CAM in individual EVs from other cells. Here, we used multiple single-EV techniques to establish the neuronal origin and determine the abundance of L1CAM-positive EVs in human blood. L1CAM epitopes of the ectodomain are shown to be co-expressed on single-EVs with the neuronal proteins β-III-tubulin, GAP43, and VAMP2, the levels of which increase in parallel with the enrichment of L1CAM-positive EVs. Levels of L1CAM-positive EVs carrying the neuronal proteins VAMP2 and β-III-tubulin range from 30% to 63%, in contrast to 0.8%–3.9% of L1CAM-negative EVs. Plasma fluid-phase L1CAM does not bind to single-EVs. Our findings support the use of L1CAM as a target for isolating plasma NDEVs and leveraging their cargo to identify biomarkers reflecting neuronal function.

用 L1 细胞粘附分子(L1CAM)特异性抗体分离神经元衍生的细胞外囊泡(NDEVs)已被广泛用于鉴定中枢神经系统疾病的血液生物标记物。然而,方法论的全面验证需要在单个 NDEV 中证明 L1CAM,以及在来自其他细胞的单个 EV 中证明 L1CAM 水平较低或不存在。在这里,我们使用多种单个 EV 技术确定了神经元的来源,并确定了人体血液中 L1CAM 阳性 EV 的丰度。结果表明,L1CAM 外结构域的表位与神经元蛋白 β-III-tubulin、GAP43 和 VAMP2 共同表达在单个 EV 上,它们的水平随着 L1CAM 阳性 EV 的富集而增加。携带神经元蛋白 VAMP2 和 β-III-tubulin 的 L1CAM 阳性 EVs 含量为 30% 至 63%,而 L1CAM 阴性 EVs 含量为 0.8% 至 3.9%。血浆液相 L1CAM 不与单个 EV 结合。我们的研究结果支持将 L1CAM 作为分离血浆 NDEVs 的目标,并利用它们的货物来鉴定反映神经元功能的生物标记物。
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引用次数: 0
Inter-laboratory multiplex bead-based surface protein profiling of MSC-derived EV preparations identifies MSC-EV surface marker signatures 对间叶干细胞衍生的EV制剂进行实验室间基于多聚酶珠的表面蛋白分析,确定间叶干细胞-EV表面标记特征。
IF 16 1区 医学 Q1 Medicine Pub Date : 2024-06-13 DOI: 10.1002/jev2.12463
Vivian V. T. Nguyen, Joshua A. Welsh, Tobias Tertel, Andre Choo, Simonides I. van de Wakker, Kyra A. Y. Defourny, Bernd Giebel, Pieter Vader, Jayanthi Padmanabhan, Sai Kiang Lim, Esther N. M. Nolte-'t Hoen, Marianne C. Verhaar, R. Beklem Bostancioglu, Antje M. Zickler, Jia Mei Hong, Jennifer C. Jones, Samir EL Andaloussi, Bas W. M. van Balkom, André Görgens

Mesenchymal stromal cells (MSCs) are promising regenerative therapeutics that primarily exert their effects through secreted extracellular vesicles (EVs). These EVs – being small and non-living – are easier to handle and possess advantages over cellular products. Consequently, the therapeutic potential of MSC-EVs is increasingly investigated. However, due to variations in MSC-EV manufacturing strategies, MSC-EV products should be considered as highly diverse. Moreover, the diverse array of EV characterisation technologies used for MSC-EV characterisation further complicates reliable interlaboratory comparisons of published data. Consequently, this study aimed to establish a common method that can easily be used by various MSC-EV researchers to characterise MSC-EV preparations to facilitate interlaboratory comparisons. To this end, we conducted a comprehensive inter-laboratory assessment using a novel multiplex bead-based EV flow cytometry assay panel. This assessment involved 11 different MSC-EV products from five laboratories with varying MSC sources, culture conditions, and EV preparation methods. Through this assay panel covering a range of mostly MSC-related markers, we identified a set of cell surface markers consistently positive (CD44, CD73 and CD105) or negative (CD11b, CD45 and CD197) on EVs of all explored MSC-EV preparations. Hierarchical clustering analysis revealed distinct surface marker profiles associated with specific preparation processes and laboratory conditions. We propose CD73, CD105 and CD44 as robust positive markers for minimally identifying MSC-derived EVs and CD11b, CD14, CD19, CD45 and CD79 as reliable negative markers. Additionally, we highlight the influence of culture medium components, particularly human platelet lysate, on EV surface marker profiles, underscoring the influence of culture conditions on resulting EV products. This standardisable approach for MSC-EV surface marker profiling offers a tool for routine characterisation of manufactured EV products in pre-clinical and clinical research, enhances the quality control of MSC-EV preparations, and hopefully paves the way for higher consistency and reproducibility in the emerging therapeutic MSC-EV field.

间充质基质细胞(MSCs)是一种很有前景的再生疗法,主要通过分泌的细胞外囊泡(EVs)发挥其作用。与细胞产品相比,这些EVs体积小、无生命,更易于处理,而且具有优势。因此,间充质干细胞EVs的治疗潜力正受到越来越多的研究。然而,由于间充质干细胞-EV 制造策略的不同,间充质干细胞-EV 产品应被视为高度多样化。此外,用于间充质干细胞-EV表征的EV表征技术多种多样,这使得可靠的实验室间已发表数据比较变得更加复杂。因此,本研究旨在建立一种通用方法,方便不同间充质干细胞-EV 研究人员对间充质干细胞-EV 制剂进行表征,以促进实验室间的比较。为此,我们使用一种新型的基于多重珠蛋白的EV流式细胞术检测板进行了一次全面的实验室间评估。这项评估涉及五个实验室的 11 种不同间充质干细胞-EV 产品,它们的间充质干细胞来源、培养条件和 EV 制备方法各不相同。通过该检测面板(主要涵盖一系列间充质干细胞相关标志物),我们确定了一组细胞表面标志物,这些标志物在所有探索的间充质干细胞-EV制备的EV上始终呈阳性(CD44、CD73和CD105)或阴性(CD11b、CD45和CD197)。层次聚类分析揭示了与特定制备过程和实验室条件相关的不同表面标记特征。我们建议将 CD73、CD105 和 CD44 作为可靠的阳性标记物,用于最小化鉴定间充质干细胞衍生的 EV;将 CD11b、CD14、CD19、CD45 和 CD79 作为可靠的阴性标记物。此外,我们还强调了培养基成分(尤其是人血小板裂解液)对EV表面标志物特征的影响,突出了培养条件对EV产物的影响。这种可标准化的间充质干细胞-EV 表面标记谱分析方法为临床前和临床研究中制备的 EV 产品的常规表征提供了工具,加强了间充质干细胞-EV 制剂的质量控制,并有望为提高新兴治疗性间充质干细胞-EV 领域的一致性和可重复性铺平道路。
{"title":"Inter-laboratory multiplex bead-based surface protein profiling of MSC-derived EV preparations identifies MSC-EV surface marker signatures","authors":"Vivian V. T. Nguyen,&nbsp;Joshua A. Welsh,&nbsp;Tobias Tertel,&nbsp;Andre Choo,&nbsp;Simonides I. van de Wakker,&nbsp;Kyra A. Y. Defourny,&nbsp;Bernd Giebel,&nbsp;Pieter Vader,&nbsp;Jayanthi Padmanabhan,&nbsp;Sai Kiang Lim,&nbsp;Esther N. M. Nolte-'t Hoen,&nbsp;Marianne C. Verhaar,&nbsp;R. Beklem Bostancioglu,&nbsp;Antje M. Zickler,&nbsp;Jia Mei Hong,&nbsp;Jennifer C. Jones,&nbsp;Samir EL Andaloussi,&nbsp;Bas W. M. van Balkom,&nbsp;André Görgens","doi":"10.1002/jev2.12463","DOIUrl":"10.1002/jev2.12463","url":null,"abstract":"<p>Mesenchymal stromal cells (MSCs) are promising regenerative therapeutics that primarily exert their effects through secreted extracellular vesicles (EVs). These EVs – being small and non-living – are easier to handle and possess advantages over cellular products. Consequently, the therapeutic potential of MSC-EVs is increasingly investigated. However, due to variations in MSC-EV manufacturing strategies, MSC-EV products should be considered as highly diverse. Moreover, the diverse array of EV characterisation technologies used for MSC-EV characterisation further complicates reliable interlaboratory comparisons of published data. Consequently, this study aimed to establish a common method that can easily be used by various MSC-EV researchers to characterise MSC-EV preparations to facilitate interlaboratory comparisons. To this end, we conducted a comprehensive inter-laboratory assessment using a novel multiplex bead-based EV flow cytometry assay panel. This assessment involved 11 different MSC-EV products from five laboratories with varying MSC sources, culture conditions, and EV preparation methods. Through this assay panel covering a range of mostly MSC-related markers, we identified a set of cell surface markers consistently positive (CD44, CD73 and CD105) or negative (CD11b, CD45 and CD197) on EVs of all explored MSC-EV preparations. Hierarchical clustering analysis revealed distinct surface marker profiles associated with specific preparation processes and laboratory conditions. We propose CD73, CD105 and CD44 as robust positive markers for minimally identifying MSC-derived EVs and CD11b, CD14, CD19, CD45 and CD79 as reliable negative markers. Additionally, we highlight the influence of culture medium components, particularly human platelet lysate, on EV surface marker profiles, underscoring the influence of culture conditions on resulting EV products. This standardisable approach for MSC-EV surface marker profiling offers a tool for routine characterisation of manufactured EV products in pre-clinical and clinical research, enhances the quality control of MSC-EV preparations, and hopefully paves the way for higher consistency and reproducibility in the emerging therapeutic MSC-EV field.</p>","PeriodicalId":15811,"journal":{"name":"Journal of Extracellular Vesicles","volume":null,"pages":null},"PeriodicalIF":16.0,"publicationDate":"2024-06-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jev2.12463","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141310829","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
The Na+/Ca2+ exchanger NCX3 mediates Ca2+ entry into matrix vesicles to facilitate initial steps of mineralization in osteoblasts Na+/Ca2+交换子NCX3介导Ca2+进入基质囊泡,促进成骨细胞矿化的初始步骤。
IF 16 1区 医学 Q1 Medicine Pub Date : 2024-06-11 DOI: 10.1002/jev2.12450
Irshad A. Sheikh, Monica T. Midura-Kiela, André Herchuelz, Sophie Sokolow, Pawel R. Kiela, Fayez K. Ghishan

Matrix vesicles (MVs) provide the initial site for amorphous hydroxyapatite (HA) formation within mineralizing osteoblasts. Although Na+/Ca2+ exchanger isoform-3 (NCX3, SLC8A3) was presumed to function as major Ca2+ transporter responsible for Ca2+ extrusion out of osteoblast into the calcifying bone matrix, its presence and functional role in MVs have not been investigated. In this study, we investigated the involvement of NCX3 in MV-mediated mineralization process and its impact on bone formation. Using differentiated MC3T3-E1 cells, we demonstrated that NCX3 knockout in these cells resulted in a significant reduction of Ca2+ deposition due to reduced Ca2+ entry within the MVs, leading to impaired mineralization. Consequently, the capacity of MVs to promote extracellular HA formation was diminished. Moreover, primary osteoblast isolated from NCX3 deficient mice (NCX3−/−) exhibits reduced mineralization efficacy without any effect on osteoclast activity. To validate this in vitro finding, μCT analysis revealed a substantial decrease in trabecular bone mineral density in both genders of NCX3−/− mice, thus supporting the critical role of NCX3 in facilitating Ca2+ uptake into the MVs to initiate osteoblast-mediated mineralization. NCX3 expression was also found to be the target of downregulation by inflammatory mediators in vitro and in vivo. This newfound understanding of NCX3's functional role in MVs opens new avenues for therapeutic interventions aimed at enhancing bone mineralization and treating mineralization-related disorders.

基质囊泡 (MV) 是矿化成骨细胞内形成无定形羟基磷灰石 (HA) 的初始场所。尽管Na+/Ca2+交换异构体-3(NCX3,SLC8A3)被认为是主要的Ca2+转运体,负责将Ca2+从成骨细胞挤出到钙化骨基质中,但其在MV中的存在和功能作用尚未得到研究。在本研究中,我们研究了 NCX3 在中胚层介导的矿化过程中的参与及其对骨形成的影响。通过使用分化的 MC3T3-E1 细胞,我们证实在这些细胞中敲除 NCX3 会导致 Ca2+ 沉积的显著减少,这是由于 Ca2+ 进入中胚层的减少导致矿化受损。因此,MV促进细胞外HA形成的能力减弱。此外,从 NCX3 缺乏的小鼠(NCX3-/-)中分离出的原发性成骨细胞显示出矿化效率降低,而对破骨细胞的活性没有任何影响。为了验证这一体外研究结果,μCT 分析表明,NCX3-/- 小鼠的骨小梁矿物质密度大幅下降,从而证明了 NCX3 在促进 Ca2+ 摄取到中微粒以启动成骨细胞介导的矿化过程中的关键作用。研究还发现,在体外和体内,NCX3 的表达也是炎症介质下调的目标。对 NCX3 在骨小梁中功能作用的这一新发现,为旨在增强骨矿化和治疗矿化相关疾病的治疗干预开辟了新途径。
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引用次数: 0
Quantification of urinary podocyte-derived migrasomes for the diagnosis of kidney disease 用于诊断肾病的尿液荚膜衍生移行体定量分析
IF 16 1区 医学 Q1 Medicine Pub Date : 2024-06-09 DOI: 10.1002/jev2.12460
Rong Yang, Heng Zhang, Si Chen, Kaibin Lou, Meng Zhou, Mingchao Zhang, Rui Lu, Chunxia Zheng, Limin Li, Qihan Chen, Zhihong Liu, Ke Zen, Yanggang Yuan, Hongwei Liang

Migrasomes represent a recently uncovered category of extracellular microvesicles, spanning a diameter range of 500 to 3000 nm. They are emitted by migrating cells and harbour a diverse array of RNAs and proteins. Migrasomes can be readily identified in bodily fluids like serum and urine, rendering them a valuable non-invasive source for disease diagnosis through liquid biopsy. In this investigation, we introduce a streamlined and effective approach for the capture and quantitative assessment of migrasomes, employing wheat germ agglutinin (WGA)-coated magnetic beads and flow cytometry (referred to as WBFC). Subsequently, we examined the levels of migrasomes in the urine of kidney disease (KD) patients with podocyte injury and healthy volunteers using WBFC. The outcomes unveiled a substantial increase in urinary podocyte-derived migrasome concentrations among individuals with KD with podocyte injury compared to the healthy counterparts. Notably, the urinary podocyte-derived migrasomes were found to express an abundant quantity of phospholipase A2 receptor (PLA2R) proteins. The presence of PLA2R proteins in these migrasomes holds promise for serving as a natural antigen for the quantification of autoantibodies against PLA2R in the serum of patients afflicted by membranous nephropathy. Consequently, our study not only pioneers a novel technique for the isolation and quantification of migrasomes but also underscores the potential of urinary migrasomes as a promising biomarker for the early diagnosis of KD with podocyte injury.

迁移体是最近发现的一类细胞外微囊泡,直径范围在 500 纳米到 3000 纳米之间。它们由迁移的细胞释放出来,携带着多种多样的 RNA 和蛋白质。迁移体很容易在血清和尿液等体液中被识别出来,因此是通过液体活检进行疾病诊断的重要非侵入性来源。在这项研究中,我们采用小麦胚芽凝集素(WGA)包被磁珠和流式细胞术(简称 WBFC),介绍了一种捕获和定量评估迁移体的简便有效的方法。随后,我们利用流式细胞术检测了荚膜细胞损伤的肾病(KD)患者和健康志愿者尿液中的移行体水平。结果显示,与健康志愿者相比,荚膜细胞损伤的肾病患者尿液中荚膜细胞衍生的migrasome浓度大幅增加。值得注意的是,研究发现尿液中的荚膜细胞衍生移行体表达大量磷脂酶 A2 受体(PLA2R)蛋白。这些移行小体中存在的 PLA2R 蛋白有望作为一种天然抗原,用于定量检测膜性肾病患者血清中针对 PLA2R 的自身抗体。因此,我们的研究不仅开创了分离和量化移行小体的新技术,还强调了尿液中的移行小体作为早期诊断荚膜细胞损伤的KD生物标志物的潜力。
{"title":"Quantification of urinary podocyte-derived migrasomes for the diagnosis of kidney disease","authors":"Rong Yang,&nbsp;Heng Zhang,&nbsp;Si Chen,&nbsp;Kaibin Lou,&nbsp;Meng Zhou,&nbsp;Mingchao Zhang,&nbsp;Rui Lu,&nbsp;Chunxia Zheng,&nbsp;Limin Li,&nbsp;Qihan Chen,&nbsp;Zhihong Liu,&nbsp;Ke Zen,&nbsp;Yanggang Yuan,&nbsp;Hongwei Liang","doi":"10.1002/jev2.12460","DOIUrl":"10.1002/jev2.12460","url":null,"abstract":"<p>Migrasomes represent a recently uncovered category of extracellular microvesicles, spanning a diameter range of 500 to 3000 nm. They are emitted by migrating cells and harbour a diverse array of RNAs and proteins. Migrasomes can be readily identified in bodily fluids like serum and urine, rendering them a valuable non-invasive source for disease diagnosis through liquid biopsy. In this investigation, we introduce a streamlined and effective approach for the capture and quantitative assessment of migrasomes, employing wheat germ agglutinin (WGA)-coated magnetic beads and flow cytometry (referred to as WBFC). Subsequently, we examined the levels of migrasomes in the urine of kidney disease (KD) patients with podocyte injury and healthy volunteers using WBFC. The outcomes unveiled a substantial increase in urinary podocyte-derived migrasome concentrations among individuals with KD with podocyte injury compared to the healthy counterparts. Notably, the urinary podocyte-derived migrasomes were found to express an abundant quantity of phospholipase A2 receptor (PLA2R) proteins. The presence of PLA2R proteins in these migrasomes holds promise for serving as a natural antigen for the quantification of autoantibodies against PLA2R in the serum of patients afflicted by membranous nephropathy. Consequently, our study not only pioneers a novel technique for the isolation and quantification of migrasomes but also underscores the potential of urinary migrasomes as a promising biomarker for the early diagnosis of KD with podocyte injury.</p>","PeriodicalId":15811,"journal":{"name":"Journal of Extracellular Vesicles","volume":null,"pages":null},"PeriodicalIF":16.0,"publicationDate":"2024-06-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jev2.12460","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141296204","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
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Journal of Extracellular Vesicles
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