DEVELOPMENT OF CYTOKINE RELEASE ASSAYS FOR HUMAN IPSC-DERIVED MICROGLIA

Microglia are central nervous system resident immune cells essential for normal brain function, but dysregulated microglia contribute to neurodegenerative diseases. Understanding microglia functional states and their role in modulating inflammation during CNS infection is crucial for identifying therapeutic opportunities. Human induced pluripotent stem cell (iPSC)-derived microglia provide a reliable source for studying microglia function in health and disease, particularly NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome-mediated neuroinflammation. iCell Microglia from FUJIFILM Cellular Dynamics were differentiated from a healthy male donor following the Abud et al. 2017 protocol. Microglia were plated at 30K cells/well (96w) or 10K cells/well (384w), cultured 3-days post-thaw. Multiple endpoint assays were developed: interleukin-6 (IL-6) secretion measured via homogeneous time-resolved fluorescence following overnight lipopolysaccharide (LPS) treatment (100 ng/mL), consistently yielding greater than 1000 pg/mL with EC50 of 1-5 ng/mL; IL-1beta release measured using Lumit Immunoassay after sequential LPS (3h, priming) and adenosine triphosphate or Nigericin (30min, activation) treatment to evaluate NLRP3 inflammasome activity; phosphorylated spleen tyrosine kinase (pSyk) assay evaluating triggering receptor expressed on myeloid cells 2-mediated signaling using time-resolved fluorescence resonance energy transfer (TR-FRET), achieving robust signal with pervanadate (100 μM, 30min). These data establish a foundation for studying cytokine release, NLRP3 inflammasome activation, and cell signaling in human iPSC-derived microglia. This baseline enables future studies on neuroinflammation, co-culture with iPSC-derived neurons and astrocytes, and disease modeling with Alzheimer's-relevant TREM2 or apolipoprotein E mutations. These assays provide robust readouts for discovering microglia-modulating compounds with physiological relevance and therapeutic potential in neurodegenerative diseases.