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Experimental artefacts can lead to misattribution of bioactivity from soluble mesenchymal stem cell paracrine factors to extracellular vesicles.
Journal of Extracellular Vesicles ( IF 16.0 ) Pub Date : 2020-08-26 , DOI: 10.1080/20013078.2020.1807674
Thomas E Whittaker 1, 2, 3 , Anika Nagelkerke 1, 2, 3 , Valeria Nele 1, 2, 3 , Ulrike Kauscher 1, 2, 3 , Molly M Stevens 1, 2, 3
Affiliation  

It has been demonstrated that some commonly used Extracellular Vesicle (EV) isolation techniques can lead to substantial contamination with non-EV factors. Whilst it has been established that this impacts the identification of biomarkers, the impact on apparent EV bioactivity has not been explored. Extracellular vesicles have been implicated as critical mediators of therapeutic human mesenchymal stem cell (hMSC) paracrine signalling. Isolated hMSC-EVs have been used to treat multiple in vitro and in vivo models of tissue damage. However, the relative contributions of EVs and non-EV factors have not been directly compared. The dependence of hMSC paracrine signalling on EVs was first established by ultrafiltration of hMSC-conditioned medium to deplete EVs, which led to a loss of signalling activity. Here, we show that this method also causes depletion of non-EV factors, and that when this is prevented proangiogenic signalling activity is fully restored in vitro. Subsequently, we used size-exclusion chromatography (SEC) to separate EVs and soluble proteins to directly and quantitatively compare their relative contributions to signalling. Non-EV factors were found to be necessary and sufficient for the stimulation of angiogenesis and wound healing in vitro. EVs in isolation were found to be capable of potentiating signalling only when isolated by a low-purity method, or when used at comparatively high concentrations. These results indicate a potential for contaminating soluble factors to artefactually increase the apparent bioactivity of EV isolates and could have implications for future studies on the biological roles of EVs.



中文翻译:

实验性伪影可能导致生物活性从可溶性间充质干细胞旁分泌因子到细胞外囊泡的错误分配。

已经证明,一些常用的细胞外囊泡(EV)分离技术可能导致非EV因素的大量污染。虽然已经确定这会影响生物标志物的鉴定,但尚未研究对表观EV生物活性的影响。细胞外囊泡被认为是治疗性人间充质干细胞(hMSC)旁分泌信号传导的关键介质。分离的hMSC-EV已用于治疗多种体外体内组织损伤模型。但是,尚未直接比较电动汽车和非电动汽车因素的相对贡献。hMSC旁分泌信号传导对EV的依赖性首先是通过hMSC条件培养基超滤以耗尽EV来建立的,这导致信号传导活性的丧失。在这里,我们表明,该方法还导致非EV因子的耗竭,并且在预防这种情况时,促血管生成信号的活性在体外已完全恢复。随后,我们使用尺寸排阻色谱(SEC)来分离EV和可溶性蛋白,以直接定量地比较它们对信号传导的相对贡献。非EV因子被发现是必要且充分的血管生成的刺激和伤口愈合体外。发现孤立的电动汽车只有在通过低纯度方法分离或以较高浓度使用时才能够增强信号传导。这些结果表明有可能污染可溶性因子以人为地增加EV分离株的表观生物活性,并且可能对未来有关EV生物学作用的研究产生影响。

更新日期:2020-08-27
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