Abstract
BackgroundExtracellular vesicles (EVs) play important roles in intercellular communication by delivering RNA, lipid, and proteins to neighboring or distant cells. Identification and classification of EVs secreted from diverse cell types are essential for understanding their signaling properties.MethodsIn this study, EVs from the culture media were isolated by ultracentrifugation and analyzed by electron microscopy (EM) and nanoparticle tracking analyses. Conditioned media (CM) from HEK293 cells culture grown either in serum-free (SF) or 10% fetal bovine serum (FBS) containing media were centrifuged at 100,000×g to separate the SNΔ supernatant and the P100 pellet in which exosomes are enriched. Then, the SNΔ fraction was centrifuged at 200,000×g to yield the P200 pellet fraction containing novel EVs smaller than exosomes. The exosomal markers in the EV subgroups were examined by western blotting and immune-EM, and the functional analyses of EVs were conducted on HEK293 and THP-1 cell culture.ResultsWe identified a new group of EVs in the P200 fraction that was smaller than exosomes in size. Typical exosome markers such as Hsp70, TSG101, and CD63 were found in both P100 exosomes and the P200 vesicles, but CD81 was highly enriched in exosomes but not in the P200 vesicles. Furthermore, chemicals that inhibit the major exosome production pathway did not decrease the level of P200 vesicles. Therefore, these small EVs indeed belong to a distinguished group of EVs. Exosomes and the P200 vesicles were found in CM of human cell lines as well as FBS. Addition of the exosomes and the P200 vesicles to human cell cultures enhanced exosome production and cell proliferation, respectively.ConclusionsOur study identifies a novel population of EVs present in the P200 fraction. This EV population is distinguished from exosomes in size, protein contents, and biogenesis pathway. Furthermore, exosomes promote their own production whereas the P200 vesicles support cell proliferation. In sum, we report a new group of EVs that are distinct physically, biologically and functionally from exosomes.
Highlights
Extracellular vesicles (EVs) play important roles in intercellular communication by delivering RNA, lipid, and proteins to neighboring or distant cells
Vesicles identified in the P200 fraction are smaller than exosomes Numerous previous studies have considered the P100 fraction obtained from ultracentrifugation at 100,000×g as an exosome fraction [19, 27, 28, 30]
We directly visualized the vesicles in the P100 and P200 fractions prepared from HEK293 cell culture media, using transmission electron microscopy (TEM) and environmental scanning electron microscopy (ESEM)
Summary
Extracellular vesicles (EVs) play important roles in intercellular communication by delivering RNA, lipid, and proteins to neighboring or distant cells. Intercellular communication plays a pivotal role in all multicellular organisms, and membrane-bound extracellular vesicles (EVs) are essential components for modulating the cellular functions of neighboring and remote cells by delivering multiple components such as proteins, RNA, and lipids [1,2,3] Such EVs have been identified from almost all mammalian cell types including stem cells [4,5,6], immune cells [7, 8], and the primary cells of the nervous system [9, 10] as well as numerous cancer cell lines [11, 12]. Size ranges of these EVs overlap considerably and their proper classification and nomenclature are still being developed
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
