Chapter 1
Mesenchymal Stem Cell-Derived Extracellular Vesicle
Isolation and Their Protein Cargo Characterization
Miriam Morente-Lo ´ pez, Juan A. Fafia ´ n-Labora, Mo ´ nica Carrera,
Francisco J. de Toro, Concha Gil, Jesu ´ s Mateos, and Marı ´a C. Arufe
Abstract
In the present protocol, extracellular vesicles (EVs) released from a primary culture of human umbilical cord
mesenchymal stem cells (MSCs) were isolated by ultracentrifugation processes, characterized by transmission electron microscopy (TEM) and measured by nanoparticle tracking analysis (NTA). Protein was
extracted from EVs using RIPA buffer and then was assessed for integrity. The proteomic content of the
total EV protein samples was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS)
after labeling by tandem mass tag (TMT). This combined approach allowed the development of an effective
strategy to study the protein cargo from MSC-derived EVs.
Key words Mesenchymal stem cells, Tandem mass tag, Extracellular vesicles, Transmission electron
microscopy
1 Introduction
During the last decade, extracellular vesicles (EVs) have gained
significant interest due to their role in paracrine responses and
intercellular communication. EVs are derived from the endosomal
system to form multivesicular structures, which fuse with the
plasma membrane and then are released to the extracellular space
[1]. EVs reach recipient cells in the local environment (paracrine
mode) or are transported to distant tissues via the circulation
system (endocrine mode). Numerous investigations have revealed
an important role of EVs in intercellular communication under
both normal and pathological conditions [2–5]. To study the function of EVs, it is essential to characterize their molecular content,
which specifically reflects the phenotype of the cells-of-origin.
Comprehensive proteomic analysis has demonstrated that EVs
derived from mesenchymal stem cells (MSCs) are packaged with
significantly higher fractions of specific protein compared with their
cells-of-origin, indicating a regulation of their contents, which
Mo ´ nica Carrera and Jesu ´ s Mateos (eds.), Shotgun Proteomics: Methods and Protocols, Methods in Molecular Biology, vol. 2259,
https://doi.org/10.1007/978-1-0716-1178-4_1, © Springer Science+Business Media, LLC, part of Springer Nature 2021
3
Mesenchymal Stem Cell-Derived Extracellular Vesicle
Isolation and Their Protein Cargo Characterization
Miriam Morente-Lo ´ pez, Juan A. Fafia ´ n-Labora, Mo ´ nica Carrera,
Francisco J. de Toro, Concha Gil, Jesu ´ s Mateos, and Marı ´a C. Arufe
Abstract
In the present protocol, extracellular vesicles (EVs) released from a primary culture of human umbilical cord
mesenchymal stem cells (MSCs) were isolated by ultracentrifugation processes, characterized by transmission electron microscopy (TEM) and measured by nanoparticle tracking analysis (NTA). Protein was
extracted from EVs using RIPA buffer and then was assessed for integrity. The proteomic content of the
total EV protein samples was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS)
after labeling by tandem mass tag (TMT). This combined approach allowed the development of an effective
strategy to study the protein cargo from MSC-derived EVs.
Key words Mesenchymal stem cells, Tandem mass tag, Extracellular vesicles, Transmission electron
microscopy
1 Introduction
During the last decade, extracellular vesicles (EVs) have gained
significant interest due to their role in paracrine responses and
intercellular communication. EVs are derived from the endosomal
system to form multivesicular structures, which fuse with the
plasma membrane and then are released to the extracellular space
[1]. EVs reach recipient cells in the local environment (paracrine
mode) or are transported to distant tissues via the circulation
system (endocrine mode). Numerous investigations have revealed
an important role of EVs in intercellular communication under
both normal and pathological conditions [2–5]. To study the function of EVs, it is essential to characterize their molecular content,
which specifically reflects the phenotype of the cells-of-origin.
Comprehensive proteomic analysis has demonstrated that EVs
derived from mesenchymal stem cells (MSCs) are packaged with
significantly higher fractions of specific protein compared with their
cells-of-origin, indicating a regulation of their contents, which
Mo ´ nica Carrera and Jesu ´ s Mateos (eds.), Shotgun Proteomics: Methods and Protocols, Methods in Molecular Biology, vol. 2259,
https://doi.org/10.1007/978-1-0716-1178-4_1, © Springer Science+Business Media, LLC, part of Springer Nature 2021
3