Human Microglia Extracellular Vesicles Derived from Different Microglia Cell Lines: Similarities and Differences

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.

Secreted endogenous macrosomes reduce Aβ burden and ameliorate Alzheimer’s disease

“…6 (C to E), in which remarkable variations in both the 1 H and 15 N dimensions were detected for the residues Phe 19 , Ala 21 , Asp 23 , Leu 34 , Gly 37 , Val 39 , and Val 40 , which have been reported to be involved in Aβ fibrillation (41,42). In comparison, small EVs induced only weak changes in the chemical shifts of Phe 19 , Ala 21 , and Gly 37 (fig. S8, C to E).…”

“…As shown in Fig. 1A, EVs released by microglia were extracted through gradient centrifugation at 11,000g to isolate the macrosomes and at 100,000g to isolate the small EVs (21). We used Aβ40 peptide as the objective pathological target, as its misfolding and aggregation are widely prevalent in the brains of patients with AD (22) and initiate neurotoxic events including neuroinflammation, synaptic and neuronal loss, and tau-associated pathology (23)(24)(25).…”

Nanomaterials as Microglia Modulators in the Treatment of Central Nervous System Disorders

“…Inspired by this idea, researchers started to consider the possibility of exploiting M1polarized microglia as donor cells of exosomes as novel NPs for glioma therapy. [210] Grimaldi et al [211] demonstrated that EVs derived from LPS/ interferon-𝛾 (IFN-𝛾) -treated microglia (referred to as LPS/IFN-𝛾-EVs) injected in the brain of glioma-bearing mice reduced the anti-inflammatory phenotype of TAMs. The reduction of the immunosuppressive phenotypes of TAMs caused a significantly reduced tumor size and tumor-induced neurotoxicity.…”

“…Inspired by this idea, researchers started to consider the possibility of exploiting M1‐polarized microglia as donor cells of exosomes as novel NPs for glioma therapy. [ 210 ]…”

Advances in proteomic phenotyping of microglia in neurodegeneration

“…Proteomic analyses of microglial‐derived exosomes are currently limited by very low protein yield for MS, and lack of definitive verification of their cell of origin. Despite these challenges, several in vitro proteomic studies on microglial‐derived exosomes released in response to inflammatory stimuli have been published [187]. Proteomic analysis of exosomes derived from the N9 microglial cell line identified the composition of microglia‐derived exosomes from cell culture medium [188].…”