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A limitation of using exosomes to their fullest potential is their limited secretion from cells, a major bottleneck to efficient exosome production and application. This is especially true for mesenchymal stem cells (MSCs), which can self-renew but have a limited expansion capacity, undergoing senescence after only a few passages, with exosomes derived from senescent stem cells showing impaired regenerative capacity compared to young cells. Here, we examined the effects of small molecule modulators capable of enhancing exosome secretion from MSCs. The treatment of MSCs with a combination of N-methyldopamine and norepinephrine robustly increased exosome production by three-fold without altering the ability of the MSC exosomes to induce angiogenesis, polarize macrophages to an anti-inflammatory phenotype, or downregulate collagen expression. These small molecule modulators provide a promising means to increase exosome production by MSCs.
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This content will become publicly available on November 19, 2026
A nucleic acid labeling chemistry reveals surface DNA on exosomes
Abstract Chemical labeling of nucleic acids is essential to pinpoint the structure, localization, and function of RNA and DNA. Yet, reversible sequence-independent chemistries that can label native RNA and DNA remain poorly developed. Here we describe Reversible Uridine Nitrilium-mediated Addition (RUNA), a reversible covalent chemistry that selectively modifies uridine and thymidine residuesviaN3 deprotonation and reaction with a nitrilium ion intermediate generated from an aldehyde and an isonitrile. The reaction forms a stable N3 adduct that can be quantitatively reversed by hydrolysis. By using reagents that are either membrane permeable or impermeable, we demonstrate the localization and function of DNA on exosomes. Although exosomes harbor nucleic acids, whether the latter are encapsulated in the exosome lumen or are surface-adhered is unknown. RUNA revealed that exosomes display DNA on their outer surface. The abundance of such surface DNA increases upon DNA-damage accumulation in cancer cells that are treated with a PARP inhibitor. This surface DNA drives exosome uptake by M2-polarized macrophages through scavenger receptors and triggers a shift toward an M1-like pro-inflammatory state. The selective labeling of surface DNA revealed an unexpected mechanism by which exosomes engage innate immune cells. RUNA is a versatile tool to analyze the nucleic acid content and functionality of extracellular vesicles in health and disease. Significance StatementPinpointing the localization of RNA and DNA in cells and organelles is central to deriving insights into their biological functions in health and disease. We describe a new method, RUNA, for labeling nucleic acids that is sequence-independent and reversible. By varying RUNA reagents, we can distinguish between nucleic acids that are located either inside or outside of membrane compartments. Using RUNA, we showed that DNA is associated with the outer surface of exosomes that are secreted by cancer cells. Further, the amount of surface DNA increases when the cancer cells are treated with an anti-cancer drug. This surface DNA promotes the uptake of exosomes by innate immune cells known as macrophages and modulates their inflammatory response.
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- PAR ID:
- 10700270
- Publisher / Repository:
- bioRxiv
- Date Published:
- Format(s):
- Medium: X
- Institution:
- bioRxiv
- Sponsoring Org:
- National Science Foundation
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