NeuroRepair Department, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland.
Multifactorial Disease and Complex Phenotype Research Area, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy.
Int J Nanomedicine. 2018 Mar 19;13:1653-1664. doi: 10.2147/IJN.S159404. eCollection 2018.
Mesenchymal stem cells have been shown therapeutic in various neurological disorders. Recent studies support the notion that the predominant mechanism by which MSCs act is through the release of extracellular vesicles (EVs). EVs seem to have similar therapeutic activity as their cellular counterparts and may represent an interesting alternative standalone therapy for various diseases. The aim of the study was to optimize the method of EV imaging to better understand therapeutic effects mediated by EVs.
The fluorescent lipophilic stain PKH26 and superparamagnetic iron oxide nanoparticles conjugated with rhodamine (Molday ION Rhodamine B™) were used for the labeling of vesicles in human bone marrow MSCs (hBM-MSCs). The entire cycle from intracellular vesicles to EVs followed by their uptake by hBM-MSCs has been studied. The identity of vesicles has been proven by antibodies against: anti-CD9, -CD63, and -CD81 (tetraspanins). NanoSight particle tracking analysis (NTA), high-resolution flow cytometric analysis, transmission electron microscopy (TEM), ELYRA PS.1 super-resolution microscopy, and magnetic resonance imaging (MRI) were used for the characterization of vesicles.
The PKH26 and Molday ION were exclusively localized in intracellular vesicles positively stained for EV markers: CD9, CD63, and CD81. The isolated EVs represent heterogeneous population of various sizes as confirmed by NTA. The TEM and MRI were capable to show successful labeling of EVs using ION. Co-culture of EVs with hBM-MSCs revealed their uptake by cells in vitro, as visualized by the co-localization of PKH26 or Molday ION with tetraspanins inside hBM-MSCs.
PKH26 and Molday ION seem to be biocompatible with EVs, and the labeling did not interfere with the capability of EVs to re-enter hBM-MSCs during co-culture in vitro. Magnetic properties of IONs provide an additional advantage for the imaging of EV using TEM and MRI.
间充质干细胞在各种神经紊乱疾病中具有治疗作用。最近的研究支持这样一种观点，即间充质干细胞的主要作用机制是通过释放细胞外囊泡 (EVs)。EVs 似乎具有与细胞类似的治疗活性，并可能成为各种疾病的一种有趣的独立治疗选择。本研究的目的是优化 EV 成像方法，以更好地理解 EV 介导的治疗效果。
荧光亲脂性染料 PKH26 和与罗丹明偶联的超顺磁性氧化铁纳米颗粒（Molday ION Rhodamine B™）被用于标记人骨髓间充质干细胞 (hBM-MSCs) 中的囊泡。研究了从细胞内囊泡到 EVs 的整个循环，以及 EVs 被 hBM-MSCs 摄取的过程。通过针对以下标记物的抗体证明了囊泡的身份：抗 CD9、-CD63 和 -CD81（四跨膜蛋白）。纳米粒子跟踪分析 (NTA)、高分辨率流式细胞分析、透射电子显微镜 (TEM)、ELYRA PS.1 超分辨率显微镜和磁共振成像 (MRI) 用于囊泡的表征。
PKH26 和 Molday ION 仅定位于 EV 标志物（CD9、CD63 和 CD81）阳性染色的细胞内囊泡中。分离的 EVs 代表各种大小的异质群体，这通过 NTA 得到证实。TEM 和 MRI 能够显示 ION 成功标记 EVs。EV 与 hBM-MSCs 的共培养显示出 EVs 在体外被细胞摄取，这可以通过 PKH26 或 Molday ION 与 hBM-MSCs 内的四跨膜蛋白共定位来观察到。
PKH26 和 Molday ION 似乎与 EVs 具有生物相容性，并且标记物不会干扰 EVs 在共培养中重新进入 hBM-MSCs 的能力。ION 的磁性特性为 TEM 和 MRI 对 EV 成像提供了额外的优势。
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