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Characterization of Immune Cell-derived Extracellular Vesicles and Studying Functional Impact on Cell Environment
Published on: June 2, 2020
Human Microglia Extracellular Vesicles Derived from Different Microglia Cell Lines: Similarities and Differences
Lorenzo Ceccarelli1,2, Laura Marchetti1, Milena Rizzo3
1Department of Pharmacy, University of Pisa, Pisa 56126, Italy.
Abstract:
Microglial cells are a component of the innate immune system in the brain that support cell-to-cell communication via secreted molecules and extracellular vesicles (EVs). EVs can be divided into two major populations: large (LEVs) and small (SEVs) EVs, carrying different mediators, such as proteins, lipids, and miRNAs. The microglia EVs cargo crucially reflects the status of parental cells and can lead to both beneficial and detrimental effects in many physiopathological states. Herein, a workflow for the extraction and characterization of SEVs and LEVs from human C20 and HMC3 microglia cell lines derived, respectively, from adult and embryonic microglia is reported. EVs were gathered from the culture media of the two cell lines by sequential ultracentrifugation steps and their biochemical and biophysical properties were analyzed by Western blot, transmission electron microscopy, and dynamic light scattering. Although the C20- and HMC3-derived EVs shared several common features, C20-derived EVs were slightly lower in number and more polydispersed. Interestingly, C20- but not HMC3-SEVs were able to interfere with the proliferation of U87 glioblastoma cells. This correlated with the different relative levels of eight miRNAs involved in neuroinflammation and tumor progression in the C20- and HMC3-derived EVs, which in turn reflected a different basal activation state of the two cell types. Our data fill a gap in the community of microglia EVs, in which the preparations from human cells have been poorly characterized so far. Furthermore, these results shed light on both the differences and similarities of EVs extracted from different human microglia cell models, underlining the need to better characterize the features and biological effects of EVs for therein useful and correct application.
Insights
Researchers characterized extracellular vesicles (EVs) from human microglia cell lines. Small EVs (SEVs) from C20 cells, but not HMC3 cells, inhibited glioblastoma cell proliferation, linked to miRNA cargo differences.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Microglia, the brain's innate immune cells, communicate via extracellular vesicles (EVs).
- EVs, including small (SEVs) and large (LEVs) populations, carry diverse molecular cargo reflecting parental cell status.
- Microglia-derived EVs influence various physiological and pathological states, but human cell-derived preparations are poorly characterized.
Purpose of the Study:
- To develop and validate a workflow for extracting and characterizing SEVs and LEVs from human C20 and HMC3 microglia cell lines.
- To compare the biochemical and biophysical properties of EVs derived from adult (C20) and embryonic (HMC3) human microglia.
- To investigate the functional differences in microglia-derived EVs, particularly their impact on glioblastoma cell proliferation.
Main Methods:
- Sequential ultracentrifugation was used to isolate EVs from C20 and HMC3 cell culture media.
- EV characterization involved Western blot, transmission electron microscopy, and dynamic light scattering.
- Functional assays assessed the effect of EVs on U87 glioblastoma cell proliferation, with miRNA profiling performed.
Main Results:
- EVs from both cell lines shared common features, but C20-derived EVs were less numerous and more polydispersed.
- C20-derived SEVs, unlike HMC3-derived SEVs, inhibited U87 glioblastoma cell proliferation.
- Differential miRNA cargo in SEVs correlated with distinct basal activation states of C20 and HMC3 cells and functional effects.
Conclusions:
- This study provides a characterized workflow for human microglia-derived EVs, addressing a gap in the field.
- EVs from different human microglia models exhibit distinct properties and biological functions, influenced by miRNA content.
- Accurate characterization of microglia EV features is crucial for their reliable application in research and therapeutics.
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