Department of Precision Medicine, Translational Medicine Research Center, Naval Medical University, Shanghai, People's Republic of China.
Department of Stem Cell and Regeneration Medicine, Translational Medicine Research Center, Naval Medical University, Xiangyin Road 800, Shanghai, People's Republic of China.
Stem Cell Res Ther. 2023 Sep 25;14(1):272. doi: 10.1186/s13287-023-03491-5.
Extracellular vesicles (EVs) from human umbilical cord mesenchymal stem cells (hUMSCs) are widely considered to be the best mediators for cell-free therapy. An understanding of their composition, especially RNA, is particularly important for the safe and precise application of EVs. Up to date, the knowledge of their RNA components is limited to NGS sequencing and cannot provide a comprehensive transcriptomic landscape, especially the long and full-length transcripts. Our study first focused on the transcriptomic profile of hUMSC-EVs based on nanopore sequencing.
In this study, different EV subtypes (exosomes and microvesicles) derived from hUMSCs were isolated and identified by density gradient centrifugation. Subsequently, the realistic long transcriptomic profile in different subtypes of hUMSC-EVs was systematically compared by nanopore sequencing and bioinformatic analysis.
Abundant transcript variants were identified in EVs by nanopore sequencing, 69.34% of which transcripts were fragmented. A series of full-length and long transcripts was also observed and showed a significantly higher proportion of intact or near-complete transcripts in exosomes than that in microvesicles derived from hUMSCs. Although the composition of RNA biotypes transported by different EV subtypes was similar, the distribution of transcripts and genes revealed the inter-heterogeneity and intra-stability between exosomes and microvesicles. Further, 85 different expressed transcripts (56 genes) and 7 fusion genes were identified. Pathway enrichment analysis showed that upregulated-expressed genes in microvesicles were mainly enriched in multiple neurodegenerative diseases, while upregulated-expressed genes in exosomes were mainly enriched in neutrophil extracellular trap formation, suggesting different functional tendencies of EV subtypes.
This study provides a novel understanding of different types of hUMSC-EVs, which not only suggests different transcriptome sorting mechanisms between exosomes and microvesicles, but also shows that different EV subtypes from the same source have different physiological functions, suggesting distinct clinical application prospects.
人脐带间充质干细胞（hUMSC）的细胞外囊泡（EVs）被广泛认为是无细胞治疗的最佳介导物。了解其组成，尤其是 RNA，对于 EVs 的安全和精确应用尤为重要。迄今为止，对其 RNA 成分的认识仅限于 NGS 测序，无法提供全面的转录组图谱，特别是长的全长转录本。我们的研究首先基于纳米孔测序聚焦于 hUMSC-EVs 的转录组图谱。
在这项研究中，通过密度梯度离心分离和鉴定了源自 hUMSC 的不同 EV 亚型（外泌体和微泡）。随后，通过纳米孔测序和生物信息学分析系统地比较了不同亚型 hUMSC-EVs 中的真实长转录组图谱。
通过纳米孔测序在 EVs 中鉴定出丰富的转录变体，其中 69.34%的转录本是片段化的。还观察到一系列全长和长转录本，并且源自 hUMSC 的外泌体中的完整或近乎完整转录本的比例明显高于微泡。尽管不同 EV 亚型转运的 RNA 生物型组成相似，但转录本和基因的分布揭示了外泌体和微泡之间的异质性和稳定性。此外，鉴定出 85 个不同表达的转录本（56 个基因）和 7 个融合基因。通路富集分析表明，微泡中上调表达的基因主要富集在多种神经退行性疾病中，而外泌体中上调表达的基因主要富集在中性粒细胞胞外诱捕网形成中，这表明 EV 亚型具有不同的功能倾向。
这项研究提供了对不同类型 hUMSC-EVs 的新认识，不仅表明了外泌体和微泡之间不同的转录组分选机制，还表明了来自同一来源的不同 EV 亚型具有不同的生理功能，提示了不同的临床应用前景。
