Induced Pluripotent Stem Cell (iPSC)-derived Microglia for ALS modeling

Amyotrophic Lateral Sclerosis (ALS) is a progressive and fatal disease, primarily characterized by the degeneration of upper/lower motoneurons. ALS has a still incompletely understood etiopathology, but it is thought to be caused by the combination of neuronal cell-autonomous and non-cell autonomous mechanisms involving microglia. Microglia, the innate immune cells of the central nervous system, regulate neuroinflammation and their uncontrolled activation, consisting in the aberrant persistence of a pro-inflammatory state, can promote neurotoxicity in ALS. Aim of our work is to investigate the possible effects of ALS patient-derived microglia in promoting neurotoxicity and the potential neuroprotective role of healthy donor-derived microglia by co-culturing microglial cells obtained from induced pluripotent stem cells (iPSC-microglia) with iPSC-derived motoneurons (iPSC-MNs) and iPSC-derived spinal cord organoids (iPSC-spORGs). Our work first focused on the preliminary optimization of the differentiation protocols to obtain all the iPSC-derived models to be further used in co-culture conditions. In particular, iPSC-microglia were obtained from healthy donor iPSCs after 55 days of differentiation. Microglia cells were characterized for the expression of specific markers (CD11b, Vimentin, TREM2, TMEM119, IBA1) by immunofluorescence, while their functionality was evaluated by assessing fluorescent latex-beads internalization which confirmed their phagocytic activity. iPSC-MNs were differentiated from embryoid bodies for 34 days and iPSC-spORGs were obtained in 21 days. Both iPSC-MNs and iPSC-spORGs tested positive for the expression of neuronal and motoneuronal markers (SMI-312, Choline Acetyltransferase and HB9) by immunofluorescence and RT-PCR. Our data indicate that we can efficiently obtain all the iPSC-derived in vitro models to be now used in co-culture systems also from ALS patients’ cells. This will allow to study the possible neurotoxicity of human ALS iPSC-microglia in triggering neuronal death in iPSC-MNs/spORGs and the neuroprotective role of healthy microglia in counteracting neurodegeneration, further contributing to elucidate the interplay of cell-autonomous and non-cell autonomous mechanisms in ALS pathogenesis.