Differential Stimulation of Pluripotent Stem Cell-Derived Human Microglia Leads to Exosomal Proteomic Changes Affecting Neurons

Abstract

Microglial exosomes are an emerging communication pathway, implicated in fulfilling homeostatic microglial functions and transmitting neurodegenerative signals. Gene variants of triggering receptor expressed on myeloid cells-2 (TREM2) are associated with an increased risk of developing dementia. We investigated the influence of the TREM2 Alzheimer’s disease risk variant, R47Hhet, on the microglial exosomal proteome consisting of 3019 proteins secreted from human iPS-derived microglia (iPS-Mg). Exosomal protein content changed according to how the iPS-Mg were stimulated. Thus lipopolysaccharide (LPS) induced microglial exosomes to contain more inflammatory signals, whilst stimulation with the TREM2 ligand phosphatidylserine (PS+) increased metabolic signals within the microglial exosomes. We tested the effect of these exosomes on neurons and found that the exosomal protein changes were functionally relevant and influenced downstream functions in both neurons and microglia. Exosomes from R47Hhet iPS-Mg contained disease-associated microglial (DAM) signature proteins and were less able to promote the outgrowth of neuronal processes and increase mitochondrial metabolism in neurons compared with exosomes from the common TREM2 variant iPS-Mg. Taken together, these data highlight the importance of microglial exosomes in fulfilling microglial functions. Additionally, variations in the exosomal proteome influenced by the R47Hhet TREM2 variant may underlie the increased risk of Alzheimer’s disease associated with this variant.

Glucocorticoid receptor activation during human microglial differentiation leads to genomic instability and senescence

“…Using our previously described protocol, iPS-microglia (iPS-Mg) were generated 19,20,21,22 (Garcia-Reitboeck et al 2018; Xiang et al 2018; Piers et al 2020, Cosker et al 2021). All iPSCs were cultured and passaged routinely in Essential 8 medium (Gibco), iPS-myeloid progenitor cells (iPS-MPro) were harvested from the flask of embryoid bodies and further differentiated with IL-34, MCSF and TGF-β1 for 14 days, plus a final maturation with CX3CL1 and CD200 (all growth factors were from Peprotech, London UK) for 3 more days as previously described 19, 23 (Piers et al 2020; Mallach et al 2021).…”

“…All modules demonstrated strong evidence for high quality Supplementary figure 3 A. Quantification of PI staining to determine the % of dead cells in NT, Dex, RU/Dex pretreated iPS-Mg. Using our previously described protocol, iPS-microglia (iPS-Mg) were generated 19,20,21,22 and passaged routinely in Essential 8 medium (Gibco), iPS-myeloid progenitor cells (iPS-MPro) were harvested from the flask of embryoid bodies and further differentiated with IL-34, MCSF and TGF-β1 for 14 days, plus a final maturation with CX3CL1 and CD200 (all growth factors were from Peprotech, London UK) for 3 more days as previously described 19,23 (Piers et al 2020;Mallach et al 2021).…”

The multiple faces of extracellular vesicles released by microglia: Where are we 10 years after?

“…Other human cell lines (i.e., C20) display an altered response to LPS ( Garcia-Mesa et al, 2017 ; Pozzo et al, 2019 ), while establishing primary microglia from post-mortem/cryo-preserved human brain tissue is still challenging. This limitation may be overcome by the analysis of EVs produced by microglia-like cells, differentiated from induced pluripotent stem cells (iPSC), as recently described ( Mallach et al, 2021a , b ; You et al, 2022 ). Several new protocols to generate human microglia in vitro from iPSCs have been reported, which try to mimic the cues that naturally drive microglia differentiation in vivo .…”

“…Analysis of the molecular composition of microglial EVs in different conditions may help understanding their specific functions. Proteomic analysis of microglial EVs has been performed on different samples: EVs from N9 ( Potolicchio et al, 2005 ) and BV2 ( Yang et al, 2018 ) cell lines, primary rat microglia ( Hooper et al, 2012 ; Drago et al, 2017 ; Mukherjee et al, 2020 ), MACS sorted rat microglia ( Murgoci et al, 2020 ), primary leech microglia ( Arab et al, 2019 ; Lemaire et al, 2019 ), microglia-like cells from human iPSCs ( Mallach et al, 2021a , b ; You et al, 2022 ), CD11b + EVs from human brain tissue ( Cohn et al, 2021 ). Their lipid ( Osteikoetxea et al, 2015 ; Cohn et al, 2021 ; Kumar et al, 2021 ) and small non-coding RNA (miRNA) composition ( Cunha et al, 2016 ; Prada et al, 2018 ; Lemaire et al, 2019 ; Tian et al, 2019 ; Cohn et al, 2021 ; Li et al, 2022 ) have also been investigated.…”

“…Inflammation profoundly influences microglial EV composition ( Kumar et al, 2017 ; Yang et al, 2018 ; Murgoci et al, 2020 ; Mallach et al, 2021a , b ). In this regard LPS, a major component of the outer membrane of Gram-negative bacteria, can act in synergy with ATP, a typical danger signal released by damaged cells.…”