Int J Med Sci 2021; 18(4):1058-1066. doi:10.7150/ijms.52768

Research Paper

Subpopulations of exosomes purified via different exosomal markers carry different microRNA contents

Shao-Chun Wu, MD1,*, Pao-Jen Kuo, MD2,*, Cheng-Shyuan Rau, MD3, Yi-Chan Wu, MSc2, Chia-Jung Wu, BA2, Tsu-Hsiang Lu, BA2, Chia-Wei Lin, MSc2, Chia-Wen Tsai, MSc2, Ching-Hua Hsieh, MD, PhD, FACS2,✉

1. Department of Anesthesiology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Taiwan
2. Department of Plastic Surgery, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Taiwan
3. Department of Neurosurgery, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Taiwan
* Indicates equal contribution to first authorship

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Citation:
Wu SC, Kuo PJ, Rau CS, Wu YC, Wu CJ, Lu TH, Lin CW, Tsai CW, Hsieh CH. Subpopulations of exosomes purified via different exosomal markers carry different microRNA contents. Int J Med Sci 2021; 18(4):1058-1066. doi:10.7150/ijms.52768. Available from https://www.medsci.org/v18p1058.htm

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Abstract

The heterogeneity of exosome populations presents a great challenge to their study. The current study was designed to investigate the potential heterogeneity miRNA contents in circulating exosomes purified via different exosomal markers. In this study, exosomes from the serum of C57BL/6 mice after cecum ligation and perforation (CLP) or sham operation were isolated by precipitation using ExoQuick-TC and affinity purified with anti-Rab5b, anti-CD9, anti-CD31, and anti-CD44 antibodies using the Exo-Flow Exosome Capture kit to collect exosome subpopulations. RNA extracted from the exosomes isolated by ExoQuick-TC were profiled by next-generation sequencing (NGS). Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) was also employed to determine the expression profiles of four representative exosomal miRNAs (mmu-miR-486-5p, mmu-miR-10a-5p, mmu-miR-143-3p, and mmu-miR-25-3p) selected from the NGS analysis. The results revealed that the expression patterns of these miRNAs in exosomes isolated by ExoQuick-TC as determined by RT-qPCR and NGS were similar, showing upregulation of mmu-miR-10a-5p and mmu-miR-143-3p but downregulation of mmu-miR-25-3p and mmu-miR-486-5p following CLP when compared to the levels in exosomes from sham control mice. However, their expression levels in the antibody-captured exosome subpopulations varied. The miRNAs in the exosomes captured by anti-Rab5b or anti-CD9 antibodies were more similar to those isolated by ExoQuick-TC than to those captured by anti-CD44 antibodies. However, there were no significant differences in these four miRNAs in CD31-captured exosomes. This study demonstrated that purification with different exosomal markers allows the collection of different exosome subpopulations with various miRNA contents. The results of this study demonstrate the heterogeneity of circulating exosomes and suggest the importance of stratifying exosome subpopulations when using circulating exosomes as biomarkers or investigating exosome function. In addition, this study also emphasized the necessity of using a consistent exosome marker across different samples as detecting biomarkers.

Keywords: Biomarkers, Exosome, MicroRNA, Next-generation sequencing, Subpopulation, Surface markers