Evidence of Extracellular Vesicles Biogenesis and Release in Mouse Embryonic Stem Cells
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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).
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