Characterization of induced Pluripotent Stem Cell (iPSC)-derived microglia from different ALS genetic backgrounds

Objectives: The etiopathology of ALS is still incompletely understood, but it’s becoming evident that both motor neuronal cell-autonomous and non-cell-autonomous mechanisms are involved. Microglia are emerging as key players in driving and regulating neuroinflammation. In fact, defects in this mechanism can lead to ineffective response against harmful stressors while aberrant persistence of a pro-inflammatory state can lead to neurotoxicity. To date, in vitro studies mainly focused on induced pluripotent stem cell-derived microglia (iPSC-microglia) obtained from familial ALS (fALS) patients carrying C9ORF72 hexanucleotide expansion repeat (HRE), the most common genetic cause of ALS (C9-ALS), highlighting that C9-ALS microglia present some alterations, even if results are not consistent. Aim of our work is to characterize and compare the main features of iPSC-microglia derived from ALS patients carrying different genetic backgrounds, in order to broaden the investigation to other genetic forms and sporadic cases (sALS) and to understand if the alterations of C9-ALS microglia are exclusive of C9 HRE-related pathology or shared among different ALS types. Materials and methods: iPSC-microglia were obtained through an embryoid bodies-based differentiation protocol and characterized for the expression of specific markers by immunofluorescence. Microglial functionality was evaluated at basal state and upon stimulation with lipopolysaccharide (LPS) through immunofluorescence and ELISA. Results: Microglia were successfully differentiated from iPSCs of fALS patients (carrying mutations in C9ORF72, TARDBP and SOD1), sALS patients and healthy donors. Expression of specific markers (CD11b, Vimentin, TMEM119, TREM2, IBA1) confirmed microglial phenotype. Microglial functionality was assessed by fluorescent latex-beads internalization, formation of lipid droplets and pro-inflammatory cytokines (IL-6, TNFalpha) release. Experiments are still ongoing to quantify the differences in microglial functionality between ALS patients and healthy controls. Conclusions: This in vitro model will allow us to evaluate if the ALS genetic background can influence microglia features, overall contributing to increase the knowledge about microglia-related non-cell-autonomous mechanisms in ALS.