STEM CELL-DERIVED EXOSOMES: BIOGENESIS, CHARACTERIZATION, ISOLATION AND PRECONDITIONING
DOI:
https://doi.org/10.11603/mcch.2410-681X.2026.i3.16728Keywords:
exosomes; extracellular vesicles; mesenchymal stem cells; exosome biogenesis; exosome isolation; preconditioning; regenerative medicineAbstract
Introduction. Exosomes are nanoscale extracellular vesicles of endosomal origin (approximately 30–150 nm) secreted by almost all cell types, which mediate intercellular communication by carrying proteins, lipids and nucleic acids. Because stem cell-derived exosomes reproduce much of the paracrine activity of the parent cell without the drawbacks of cell transplantation, they offer a promising option in cell-free regenerative medicine. The aim of the study – to summarise current data on the structure, molecular markers, cellular sources and isolation of stem cell-derived exosomes, and on the preconditioning strategies used to modulate their properties. Research Methods. The peer-reviewed literature was searched in the PubMed, Web of Science and Scopus databases, with priority given to primary experimental studies, widely cited reviews and the international reporting framework for extracellular vesicles. Results and Discussion. The review distinguishes exosomes from microvesicles and apoptotic bodies; describes exosome structure (the lipid bilayer, the tetraspanins CD9, CD63 and CD81, the proteins ALIX and TSG101, and the internal contents); sets out identification by positive and negative markers; outlines the main stem cell types (mesenchymal cells from bone marrow, adipose tissue, umbilical cord and dental tissues; pluripotent, including induced pluripotent, cells; and neural and other progenitors) and how they are obtained; and compares the isolation methods (ultracentrifugation, size-exclusion chromatography, precipitation and filtration) with reported figures. Exosome composition depends strongly on the source cell and culture conditions. Conclusions. Both the stem cell source and the preconditioning regimen (hypoxia, cytokines, threedimensional culture and pharmacological agents) markedly influence exosome properties. Standardised production remain essential if exosomes are to progress from a research material toward a clinically applicable, cell-free therapeutic.
References
1. Kalluri, R., & LeBleu, V. S. (2020). The biology, function, and biomedical applications of exosomes. Science, 367 (6478), eaau6977. https://doi.org/10.1126/science.aau6977 DOI: https://doi.org/10.1126/science.aau6977
2. Doyle, L. M., & Wang, M. Z. (2019). Overview of extracellular vesicles, their origin, composition, purpose, and methods for exosome isolation and analysis. Cells, 8(7), 727. https://doi.org/10.3390/cells8070727 DOI: https://doi.org/10.3390/cells8070727
3. Théry, C., Zitvogel, L., & Amigorena, S. (2002). Exosomes: Composition, biogenesis and function. Nature Reviews Immunology, 2 (8), 569–579. https://doi.org/10.1038/nri855 DOI: https://doi.org/10.1038/nri855
4. Valadi, H., Ekström, K., Bossios, A., Sjöstrand, M., Lee, J.J., & Lötvall, J.O. (2007). Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nature Cell Biology, 9 (6), 654–659. https://doi.org/10.1038/ncb1596 DOI: https://doi.org/10.1038/ncb1596
5. Mulcahy, L. A., Pink, R. C., & Carter, D. R. F. (2014). Routes and mechanisms of extracellular vesicle uptake. Journal of Extracellular Vesicles, 3, 24641. https://doi.org/10.3402/jev.v3.24641 DOI: https://doi.org/10.3402/jev.v3.24641
6. Panda, B., Sharma, Y., Gupta, S., & Mohanty, S. (2021). Mesenchymal stem cell-derived exosomes as an emerging paradigm for regenerative therapy and nanomedicine: A comprehensive review. Life, 11 (8), 784. https://doi.org/10.3390/life11080784 DOI: https://doi.org/10.3390/life11080784
7. Tang, Y., Zhou, Y., & Li, H.J. (2021). Advances in mesenchymal stem cell exosomes: A review. Stem Cell Research & Therapy, 12 (1), 71. https://doi.org/10.1186/s13287-021-02138-7 DOI: https://doi.org/10.1186/s13287-021-02138-7
8. Lotfy, A., AboQuella, N.M., & Wang, H. (2023). Mesenchymal stromal/stem cell (MSC)-derived exosomes in clinical trials. Stem Cell Research & Therapy, 14 (1), 66. https://doi.org/10.1186/s13287-023-03287-7 DOI: https://doi.org/10.1186/s13287-023-03287-7
9. Tan, F., Li, X., Wang, Z., Li, J., Shahzad, K., & Zheng, J. (2024). Clinical applications of stem cell-derived exosomes. Signal Transduction and Targeted Therapy, 9 (1), 17. https://doi.org/10.1038/s41392-023-01704-0 DOI: https://doi.org/10.1038/s41392-023-01704-0
10. van Niel, G., D’Angelo, G., & Raposo, G. (2018). Shedding light on the cell biology of extracellular vesicles. Nature Reviews Molecular Cell Biology, 19 (4), 213–228. https://doi.org/10.1038/nrm.2017.125 DOI: https://doi.org/10.1038/nrm.2017.125
11. Welsh, J. A., Goberdhan, D. C. I., O’Driscoll, L., Buzas, E. I., Blenkiron, C., & Théry, C. (2024). Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches. Journal of Extracellular Vesicles, 13 (2), e12404. https://doi.org/10.1002/jev2.12404 DOI: https://doi.org/10.1002/jev2.12451
12. Skotland, T., Hessvik, N. P., Sandvig, K., & Llorente, A. (2019). Exosomal lipid composition and the role of ether lipids and phosphoinositides in exosome biology. Journal of Lipid Research, 60 (1), 9–18. https://doi.org/10.1194/jlr.R084343 DOI: https://doi.org/10.1194/jlr.R084343
13. Donoso-Quezada, J., Ayala-Mar, S., & González-Valdez, J. (2021). The role of lipids in exosome biology and intercellular communication: Function, analytics and applications. Traffic, 22 (7), 204–220. https://doi.org/10.1111/tra.12803 DOI: https://doi.org/10.1111/tra.12803
14. Andreu, Z., & Yáñez-Mó, M. (2014). Tetraspanins in extracellular vesicle formation and function. Frontiers in Immunology, 5, 442. https://doi.org/10.3389/fimmu.2014.00442 DOI: https://doi.org/10.3389/fimmu.2014.00442
15. Keerthikumar, S., & Mathivanan, S. (2016). ExoCarta: A web-based compendium of exosomal cargo. Journal of Molecular Biology, 428 (4), 688–692. https://doi.org/10.1016/j.jmb.2015.09.019 DOI: https://doi.org/10.1016/j.jmb.2015.09.019
16. Kalra, H., Simpson, R. J., Ji, H., Aikawa, E., Altevogt, P., & Mathivanan, S. (2012). Vesiclepedia: A compendium for extracellular vesicles with continuous community annotation. PLoS Biology, 10 (12), e1001450. https://doi.org/10.1371/journal.pbio.1001450 DOI: https://doi.org/10.1371/journal.pbio.1001450
17. Chigozie, V. U. (2026). Exosomal bioactive molecules: Molecular regulation, functional heterogeneity, and translational implications in disease diagnostics and therapeutics. Clinical and Experimental Medicine, 26 (1), 179. https://doi.org/10.1007/s10238-026-02092-9 DOI: https://doi.org/10.1007/s10238-026-02092-9
18. Zhang, Y., Liu, Y., Liu, H., & Tang, W.H. (2019). Exosomes: Biogenesis, biologic function and clinical potential. Cell & Bioscience, 9, 19. https://doi.org/10.1186/s13578-019-0282-2 DOI: https://doi.org/10.1186/s13578-019-0282-2
19. Mathieu, M., Névo, N., Jouve, M., Valenzuela, J. I., Maurin, M., & Théry, C. (2021). Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9. Nature Communications, 12, 4389. https://doi.org/10.1038/s41467-021-24384-2 DOI: https://doi.org/10.1038/s41467-021-24384-2
20. Fan, Y., Pionneau, C., Cocozza, F., Boëlle, P. Y., Chardonnet, S., & Rubinstein, E. (2023). Differential proteomics argues against a general role for CD9, CD81 or CD63 in the sorting of proteins into extracellular vesicles. Journal of Extracellular Vesicles, 12 (8), e12352. https://doi.org/10.1002/jev2.12352 DOI: https://doi.org/10.1002/jev2.12352
21. Dominici, M., Le Blanc, K., Mueller, I., Slaper-Cortenbach, I., Marini, F., & Horwitz, E. (2006). Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy, 8 (4), 315–317. https://doi.org/10.1080/14653240600855905 DOI: https://doi.org/10.1080/14653240600855905
22. Takahashi, K., & Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 126 (4), 663–676. https://doi.org/10.1016/j.cell.2006.07.024 DOI: https://doi.org/10.1016/j.cell.2006.07.024
23. Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., & Yamanaka, S. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 131 (5), 861–872. https://doi.org/10.1016/j.cell.2007.11.019 DOI: https://doi.org/10.1016/j.cell.2007.11.019
24. Khan, M., Nickoloff, E., Abramova, T., Johnson, J., Verma, S.K., & Kishore, R. (2015). Embryonic stem cell-derived exosomes promote endogenous repair mechanisms and enhance cardiac function following myocardial infarction. Circulation Research, 117 (1), 52–64. https://doi.org/10.1161/CIRCRESAHA.117.305990 DOI: https://doi.org/10.1161/CIRCRESAHA.117.305990
25. Yuan, P., Ding, L., Chen, H., Wang, Y., Li, C., & Zhao, S. (2021). Neural stem cell-derived exosomes regulate neural stem cell differentiation through miR-9-Hes1 axis. Frontiers in Cell and Developmental Biology, 9, 601600. https://doi.org/10.3389/fcell.2021.601600 DOI: https://doi.org/10.3389/fcell.2021.601600
26. Stevanato, L., Thanabalasundaram, L., Vysokov, N., & Sinden, J.D. (2016). Investigation of content, stoichiometry and transfer of miRNA from human neural stem cell line derived exosomes. PLoS One, 11 (1), e0146353. https://doi.org/10.1371/journal.pone.0146353 DOI: https://doi.org/10.1371/journal.pone.0146353
27. Ke, X., Yang, D., Liang, J., Wang, X., Wu, S., et al. (2017). Human endothelial progenitor cell-derived exosomes increase proliferation and angiogenesis in cardiac fibroblasts. DNA and Cell Biology, 36 (11), 1018–1028. https://doi.org/10.1089/dna.2017.3836 DOI: https://doi.org/10.1089/dna.2017.3836
28. Ibrahim, A. G. E., Cheng, K., & Marbán, E. (2014). Exosomes as critical agents of cardiac regeneration triggered by cell therapy. Stem Cell Reports, 2 (5), 606–619. https://doi.org/10.1016/j.stemcr.2014.04.006 DOI: https://doi.org/10.1016/j.stemcr.2014.04.006
29. González-Cubero, E., González-Fernández, M. L., Gutiérrez-Velasco, L., Navarro-Ramírez, E., & Villar-Suárez, V. (2021). Isolation and characterization of exosomes from adipose tissue-derived mesenchymal stem cells. Journal of Anatomy, 238 (5), 1203–1217. https://doi.org/10.1111/joa.13365 DOI: https://doi.org/10.1111/joa.13365
30. Tracy, S. A., Ahmed, A., Tigges, J. C., Ericsson, M., Pal, A. K., & Fauza, D. O. (2019). A comparison of clinically relevant sources of mesenchymal stem cell-derived exosomes: Bone marrow and amniotic fluid. Journal of Pediatric Surgery, 54 (1), 86–90. https://doi.org/10.1016/j.jpedsurg.2018.10.020 DOI: https://doi.org/10.1016/j.jpedsurg.2018.10.020
31. Mai, Z., Chen, H., Ye, Y., Hu, Z., Sun, W., Cui, L., & Zhao, X. (2021). Translational and clinical applications of dental stem cell-derived exosomes. Frontiers in Genetics, 12, 750990. https://doi.org/10.3389/fgene.2021.750990 DOI: https://doi.org/10.3389/fgene.2021.750990
32. Wang, A. Y. L. (2021). Human induced pluripotent stem cell-derived exosomes as a new therapeutic strategy for various diseases. International Journal of Molecular Sciences, 22 (4), 1769. https://doi.org/10.3390/ijms22041769 DOI: https://doi.org/10.3390/ijms22041769
33. Jeske, R., Bejoy, J., Marzano, M., & Li, Y. (2020). Human pluripotent stem cell-derived extracellular vesicles: Characteristics and applications. Tissue Engineering Part B: Reviews, 26 (2), 129–144. https://doi.org/10.1089/ten.TEB.2019.0252 DOI: https://doi.org/10.1089/ten.teb.2019.0252
34. Wang, S., Hou, Y., Li, X., Song, Z., Sun, B., Li, X., & Zhang, H. (2020). Comparison of exosomes derived from induced pluripotent stem cells and mesenchymal stem cells as therapeutic nanoparticles for treatment of corneal epithelial defects. Aging, 12 (19), 19546–19562. https://doi.org/10.18632/aging.103904 DOI: https://doi.org/10.18632/aging.103904
35. Kurian, T.K., Banik, S., Gopal, D., Chakrabarti, S., & Mazumder, N. (2021). Elucidating methods for isolation and quantification of exosomes: A review. Molecular Biotechnology, 63 (4), 249–266. https://doi.org/10.1007/s12033-021-00300-3 DOI: https://doi.org/10.1007/s12033-021-00300-3
36. Baranyai, T., Herczeg, K., Onódi, Z., Voszka, I., Módos, K., et al. (2015). Isolation of exosomes from blood plasma: Qualitative and quantitative comparison of ultracentrifugation and size exclusion chromatography methods. PLoS One, 10 (12), e0145686. https://doi.org/10.1371/journal.pone.0145686 DOI: https://doi.org/10.1371/journal.pone.0145686
37. An, M., Wu, J., Zhu, J., & Lubman, D. M. (2018). Comparison of an optimized ultracentrifugation method versus size-exclusion chromatography for isolation of exosomes from human serum. Journal of Proteome Research, 17 (10), 3599–3605. https://doi.org/10.1021/acs.jproteome.8b00479 DOI: https://doi.org/10.1021/acs.jproteome.8b00479
38. Gardiner, C., Ferreira, Y. J., Dragovic, R. A., Redman, C. W. G., & Sargent, I. L. (2013). Extracellular vesicle sizing and enumeration by nanoparticle tracking analysis. Journal of Extracellular Vesicles, 2, 19671. https://doi.org/10.3402/jev.v2i0.19671 DOI: https://doi.org/10.3402/jev.v2i0.19671
39. Wang, Z. G., He, Z. Y., Liang, S., Yang, Q., Cheng, P., & Chen, A. M. (2020). Comprehensive proteomic analysis of exosomes derived from human bone marrow, adipose tissue, and umbilical cord mesenchymal stem cells. Stem Cell Research & Therapy, 11 (1), 511. https://doi.org/10.1186/s13287-020-02032-8 DOI: https://doi.org/10.1186/s13287-020-02032-8
40. Chen, Y., Qi, W., Wang, Z., & Niu, F. (2025). Exosome source matters: A comprehensive review from the perspective of diverse cellular origins. Pharmaceutics, 17 (2), 147. https://doi.org/10.3390/pharmaceutics17020147 DOI: https://doi.org/10.3390/pharmaceutics17020147
41. Long, R., & Wang, S. (2024). Exosomes from preconditioned mesenchymal stem cells: Tissue repair and regeneration. Regenerative Therapy, 25, 355–366. https://doi.org/10.1016/j.reth.2024.01.009 DOI: https://doi.org/10.1016/j.reth.2024.01.009
42. Zhuo, H., Chen, Y., & Zhao, G. (2024). Advances in application of hypoxia-preconditioned mesenchymal stem cell-derived exosomes. Frontiers in Cell and Developmental Biology, 12, 1446050. https://doi.org/10.3389/fcell.2024.1446050 DOI: https://doi.org/10.3389/fcell.2024.1446050
43. Luo, Z., Wu, F., Xue, E., Huang, L., Yan, P., et al. (2019). Hypoxia preconditioning promotes bone marrow mesenchymal stem cells survival by inducing HIF-1α in injured neuronal cells derived exosomes culture system. Cell Death & Disease, 10 (2), 134. https://doi.org/10.1038/s41419-019-1410-y DOI: https://doi.org/10.1038/s41419-019-1410-y
44. Cheng, A., Choi, D., Lora, M., Shum-Tim, D., Rak, J., & Colmegna, I. (2020). Human multipotent mesenchymal stromal cells cytokine priming promotes RAB27B-regulated secretion of small extracellular vesicles with immunomodulatory cargo. Stem Cell Research & Therapy, 11 (1), 539. https://doi.org/10.1186/s13287-020-02050-6 DOI: https://doi.org/10.1186/s13287-020-02050-6
45. Kang, M., Huang, C. C., Gajendrareddy, P., Lu, Y., Shirazi, S., Ravindran, S., & Cooper, L. F. (2022). Extracellular vesicles from TNFα preconditioned MSCs: Effects on immunomodulation and bone regeneration. Frontiers in Immunology, 13, 878194. https://doi.org/10.3389/fimmu.2022.878194 DOI: https://doi.org/10.3389/fimmu.2022.878194
46. Cavallero, S., Dekali, S., Guitard, N., Théry, H., Hélissey, C., & François, S. (2023). Effects of preconditioning with TNFα and IFNγ in angiogenic potential of mesenchymal stromal cell-derived extracellular vesicles. Frontiers in Cell and Developmental Biology, 11, 1291016. https://doi.org/10.3389/fcell.2023.1291016 DOI: https://doi.org/10.3389/fcell.2023.1291016
47. Lee, S.Y., & Lee, J.W. (2022). 3D spheroid cultures of stem cells and exosome applications for cartilage repair. Life, 12 (7), 939. https://doi.org/10.3390/life12070939 DOI: https://doi.org/10.3390/life12070939
48. Amini, H., Rezabakhsh, A., Heidarzadeh, M., Hassanpour, M., Hashemzadeh, S., & Reiter, R.J. (2021). An examination of the putative role of melatonin in exosome biogenesis. Frontiers in Cell and Developmental Biology, 9, 686551. https://doi.org/10.3389/fcell.2021.686551 DOI: https://doi.org/10.3389/fcell.2021.686551
49. Oses, C., Olivares, B., Ezquer, M., Acosta, C., Bosch, P., Donoso, M., Léniz, P., & Ezquer, F. (2017). Preconditioning of adipose tissue-derived mesenchymal stem cells with deferoxamine increases the production of pro-angiogenic, neuroprotective and anti-inflammatory factors. PLoS One, 12 (5), e0178011. https://doi.org/10.1371/journal.pone.0178011 DOI: https://doi.org/10.1371/journal.pone.0178011
