Altered Induced Pluripotent Stem Cell (iPSC)-derived Microglia in Amyotrophic Lateral Sclerosis

Microglia, the principal resident immune cells of the central nervous system, are constantly engaged in detecting changes in their environment, maintaining homeostasis and protecting against endogenous or environmental injurious agents [1]. Microglia has emerged as an interesting target in neurodegenerative diseases and deserve to be studied in more detail. Indeed, the mechanisms by which aberrant microglia activation causes neurodegeneration remains unclear. Microglia seems to be implicated in the initiation and in the progression of Amyotrophic Lateral Sclerosis (ALS), switching from a neuroprotective activated state to a neurotoxic one [2]. The aim of our study is to investigate whether ALS patient-derived microglia are defective compared to healthy donors cells and to study their possible neurotoxicity in triggering neuronal death in iPSC-derived spinal cord organoids and eventually the neuroprotective role of healthy microglia in counteracting neurodegeneration. We differentiated microglia from Induced Pluripotent Stem Cells (iPSCs) generated from healthy donor and patients affected by ALS through an embryoid-bodies based protocol [3]. After differentiation, microglia cells were characterized for the expression of specific markers such as CD11b, Vimentin, TREM2, TMEM119, and IBA1, while their functionality was evaluated by assessing fluorescent latex-beads internalization confirming their phagocytic activity. We also evaluated the presence of lipid droplets, organelles increasingly being accepted as markers of inflammation, by using the fluorescent dye Bodipy specific for neutral lipids. In addition, we measured by ELISA the amount of two pro-inflammatory cytokines (IL-6 and TNF-alpha) in the supernatant of microglia cells before and after priming with lipopolysaccharide (LPS, 1ug/ml) or with TNF-alpha (50ng/ml) plus IFN-gamma (20ng/ml). We obtained spinal cord organoids from iPSCs [4] in 21 days expressing neuronal specific markers (SMI-312, βIII-tubulin, Choline Acetyltransferase and HB9). Our preliminary data indicated no differences in the expression of microglia and neuronal specific markers between healthy donor and patient-derived cells. On the contrary, both unstimulated and LPS primed microglia from ALS patients displayed a significant higher amount of lipid droplets compared to healthy donors. Similarly, the level of TNFalpha in the supernatant of both unstimulated and LPS primed microglia from ALS patients was higher than in healthy donors. In addition, we performed initial co-culture experiments demonstrating the ability of microglia to integrate into spinal cord organoids. Given the important modulatory role in neural death in neurodegenerative diseases, we will establish co-cultures of microglia and iPSC-derived organoids combining microglia obtained from patients with organoids derived from controls and vice-versa in order to analyze engraftment of microglia on organoids and to study the interactions between microglial and neural cells.