A high content imaging screening assay to identify novel neuroinflammation modulators using human patient iPSC derived microglia

Neuroinflammation occurs in various chronic CNS disorders. Although novel targets that influence neuroinflammatory processes have been identified, translation to human based treatments has been slow. In Drug Discovery (DD) various tools including primary cell lines are often used to screen for new chemical matter. However, screening strategies utilising human induced pluripotent stem cells (iPSCs) can humanise early stages of DD improving translation compared to simple/reduced in vitro cell models. We used human iPSC-derived microglia (iMGL) from patients harbouring a loss of function mutation in triggering receptor expressed on myeloid cells 2 (TREM2) an Alzheimer’s disease biomarker and developed/validated a high-content imaging-based in vitro phagocytosis assay to phenotypically screen for & identify novel neuroinflammation modulators. The iMGLs were phenotypically characterised by immunofluorescence, showing expression of human microglial markers Transmembrane protein (TMEM) 119, P2Y purinoceptor 12 (P2RY12), and distribution and expression of TREM2. The confocal imaging-based phagocytosis assay is a fixed timepoint, endpoint assay that allows the % of phagocytosing cells to be accurately quantified. We successfully miniaturised the assay to a 384-well format and used a pHrodo-labelled brain protein as a patho-physiologically relevant bait. Providing deeper insight into cellular mechanisms, up to five different parameters are measured in parallel: (i) total nuclei count, (ii) phagocytosis of pHrodo Amyloid-β bait, (iii) TREM2 expression/distribution, (iv) LAMP-1 co-localisation, and (v) cell morphology. We demonstrated that iMGL LoF TREM2 cells had a marked reduction in the level of basal phagocytosis compared to healthy iMGL cells. We screened 960 compounds from our in-house library using the high-content imaging assay to identify putative phagocytosis activators/enhancers. Compounds were tested at 10uM (N=3), pre-incubated with iMGL LoF TREM cells for 60 min, after which phagocytosis was induced by adding the bait. After 2 hours, cells were fixed with 4% (v:v) paraformaldehyde and read on a Molecular Devices ImageXpress confocal instrument. Data analysis included both compound-induced cytotoxicity and % phagocytosis. We identified compounds that increased basal levels of phagocytosis in iMGL LoF TREM2 cells which are being further characterised to better understand pharmacology and define mechanism of action. Our goal has been to incorporate more predictive humanised screening assays into our DD programmes, deliver more successful projects, and identify neuroinflammation modulators to treat CNS diseases.