Evidence of Extracellular Vesicles Biogenesis and Release in Mouse Embryonic Stem Cells
Extracellular vesicles (EVs) released by mouse embryonic stem cells (mESCs) are considered a source of bioactive molecules that modulate their microenvironment by acting on intercellular communication. Either intracellular endosomal machinery or their derived EVs have been considered a relevant system of signal circuits processing. Herein, we show that these features are found in mESCs. Ultrastructural analysis revealed structures and organelles of the endosomal system such as coated pits and endocytosis-related vesicles, prominent rough endoplasmic reticulum and Golgi apparatus, and multivesicular bodies (MVBs) containing either few or many intraluminal vesicles (ILVs) that could be released as exosomes to extracellular milieu. Besides, budding vesicles shed from the plasma membrane to the extracellular space is suggestive of microvesicle biogenesis in mESCs. mESCs and mouse blastocyst express specific markers of the Endosomal Sorting Complex Required for Transport (ESCRT) system. Ultrastructural analysis and Nanoparticle Tracking Analysis (NTA) of isolated EVs revealed a heterogeneous population of exosomes and microvesicles released by mESCs. These vesicles contain Wnt10b and the Notch ligand Delta-like 4 (DLL4) and also the co-chaperone stress inducible protein 1 (STI1) and its partner Hsp90. Wnt10b and Dll4 colocalize with EVs biogenesis markers in mESCs. Overall, the present study supports the function of the mESCs endocytic network and their EVs as players in stem cell biology.
KeywordsMouse embryonic stem cells Extracellular vesicles Vesicles biogenesis Endosomal trafficking Transmission electron microscopy
We are very grateful to Dr. Vilma R. Martins, Research Superintendent of International Research Center at A. C. Camargo Cancer Center and her group, specially, Marcos Vinicios Salles Dias and Fernanda Giudice for technical support in NanoSight equipment. We also are thankful to Camila Lopes Ramos from Sirio-Libanes Hospital Teaching and Research Institute for technical support in RNA assays. We also thank Mario Costa Cruz (CEFAP-USP, Confocal Microscopy technician); Gaspar Ferreira de Lima, Edson Rocha de Oliveira, Victor E. Arana-Chavez, Márcia Tanakai, André Aguillera and Rita S. (CEME) for technical assistance in electron microscopy.
Compliance with Ethical Standards
Conflict of interest
The authors declare that they have no conflicts of interest.
This study was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP, Processes numbers: 2011/13906-2, 2013/22078-1 and 2014/17385-5).
- 16.Kowal, J., et al. (2016) Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes. Proceedings of the National Academy of Sciences of the United States of America 113(8):E968-77.Google Scholar
- 20.Li, L., et al. (2010). A unique interplay between Rap1 and E-cadherin in the endocytic pathway regulates self-renewal of human embryonic stem cells. Stem cells (Dayton, Ohio), 28(2), 247–257.Google Scholar
- 21.Théry, C., Amigorena, S., Raposo, G., & Clayton, A. (2006). Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Current Protocols in Cell Biology Chap, 3, Unit 3.22.Google Scholar
- 27.Wollert, T., Wunder, C., Lippincott-schwartz, J., Hurley, J. H. (2009). Membrane scission by the ESCRT-III complex. Nature, 458(7235), 172–177.Google Scholar
- 38.Ratajczak, J., et al. (2006). Embryonic stem cell-derived microvesicles reprogram hematopoietic progenitors: evidence for horizontal transfer of mRNA and protein delivery. Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U. K, 20(5), 847–856.CrossRefGoogle Scholar
- 46.Raz, R., Lee, C. K., Cannizzaro, L. A., d’Eustachio, P., & Levy, D. E. (1999) Essential role of STAT3 for embryonic stem cell pluripotency. Proceedings of the National Academy of Sciences of the United States of America 96(6):2846–51.Google Scholar
- 55.Falguières, T., Luyet, P. P., Bissig, C., Scott, C. C., Velluz, M. C., & Gruenberg, J. (2008). In vitro budding of intralumenal vesicles into late endosomes is regulated by Alix and Tsg101. Molecular Biology of the Cell, 19(11), 4942–4955. https://doi.org/10.1091/mbc.E08-03-0239.
- 58.Lässle, M., Blatch, G. L., Kundra, V., Takatori, T., & Zetter, B. R. (1997). Stress-inducible, murine protein mSTI1: Characterization of binding domains for heat shock proteins and in vitro phosphorylation by different kinases. The Journal of Biological Chemistry, 272(3), 1876–1884.PubMedCrossRefGoogle Scholar
- 64.Prinsloo, E., Setati, M. M., Longshaw, V. M., & Blatch, G. L. (2009) Chaperoning stem cells: A role for heat shock proteins in the modulation of stem cell self-renewal and differentiation?. BioEssays 31(4):370–377.Google Scholar
- 66.Yuan, A., et al. (2009) Transfer of microRNAs by embryonic stem cell microvesicles. PLoS One 4(3). https://doi.org/10.1371/journal.pone.0004722.
- 78.Gross, J. C., Chaudhary, V., Bartscherer, K., & Boutros, M. (2012). Active Wnt proteins are secreted on exosomes. Science Reporter, 14(10), 1036–1045.Google Scholar