Microglia derived from human induced pluripotent stem cells are regulated by osteopontin, an endogenous extracellular matrix protein maintaining immune homeostasis

IntroductionMicroglia are brain-resident immune cells responsible for maintaining homeostasis, coordinating responses to injury and disease, and mediating regeneration. Upon activation, they undergo dynamic changes in morphology, gene expression, and function, reflecting the nature and context of the stimuli encountered. Although pharmacological modulation of microglia holds great promise for treating various neurological disorders, its development is hampered by a major translational roadblock: Human microglial cell lines commonly used in preclinical studies, as well as primary rodent microglia, substantially limit the translatability of results. Here, we aimed to generate microglia from human induced pluripotent stem cells (hiPSCs) and to demonstrate their physiological responsiveness to the brain-endogenous, context-relevant ligand osteopontin (OPN).Materials and methodsMicroglia generated from two healthy hiPSC lines were stimulated with OPN, lipopolysaccharide (LPS), or their combination for 24 h and subsequently analyzed. Microglial identity and the expression of the phagocytic cell marker cluster of differentiation 68 (CD68) were determined by immunocytochemistry. Cell viability was assessed by propidium iodide (PI)/Hoechst staining, morphological activation was evaluated using Sholl analysis, and inflammatory gene expression changes were assessed by RT-qPCR.ResultshiPSC-derived microglia acquired a native central nervous system (CNS)-specific immunophenotype, expressing the microglia-specific markers ionized calcium-binding adapter molecule 1 (IBA1), transmembrane protein 119 (TMEM119), PU.1, and Spalt-like transcription factor 1 (SALL1), while remaining negative for Myb and membrane-spanning 4-domains, subfamily A, member 7 (MS4A7) at the protein level. Exposure to LPS led hiPSC-derived microglia to adopt a rounded, process-retracted shape and to increase CD68 protein intensity, a surrogate marker of lysosomal and phagocytic activity, while downregulating the anti-inflammatory marker cluster of differentiation 206 (CD206) at the transcriptional level. OPN induced a distinct microglial functional state characterized by intermediate morphology, increased CD68 intensity, and reduced homeostatic gene expression, without eliciting robust inflammatory gene expression. Intriguingly, OPN prevented LPS-induced microglial cell death, and when hiPSC-derived microglia exposed to LPS were additionally treated with OPN, the morphological effects of LPS were reversed.ConclusionOPN induced a distinct early response profile in hiPSC-derived microglia, characterized by intermediate morphological remodeling, increased CD68 intensity, and reduced homeostatic gene expression, without overt pro-inflammatory gene expression. These findings support the role of OPN as a physiological priming signal in microglia and highlight hiPSC-derived microglia as a model for studying regulators of microglial modulation.