Alzheimer’s Disease modeling using hiPSC neurons and microglia

Abstract

AbstractBackgroundNeuroinflammation is an important topic in neurodegenerative disease research. Scientists have established multiple protocols for differentiating microglia, neurons, and astrocytes from human iPSCs, which enable the study of neuroinflammation in vitro. Here we describe the use of Vala Sciences’ imaging platform, the IC200 Kinetic Image CytometerTM (KIC), to develop screening assays for Alzheimer’s Disease (AD) in cultures with hiPSC‐derived microglia and neurons.MethodWe cultured iCell Glutaneurons with microglia in 384‐well dishes. To detect microglia phagocytosis, we cultured microglia with pHrodo‐Red Zymosan beads or β‐amyloid fragments and then acquired timelapse videos over 5‐24 hours. To measure neuronal activity, we loaded cells with Rhod‐4 calcium dye and imaged at 4fps. We also fixed and immunolabeled cells for neurite and synapse analysis. All assays were scanned using the IC200 KIC. We developed algorithms for our CyteSeer image analysis software to automate neurite tracing, synapse detection, analysis of calcium activity, and microglia phagocytosis and morphology for single cells in a co‐culture system.ResultIn microglia, we demonstrated an increase in uptake of pHrodo‐Red labeled Zymosan beads with increasing concentration of Zymosan particles and in response to IL‐4. We also demonstrated differential engulfment of β‐amyloid 1‐40 and β‐amyloid 1‐42 by microglia. While investigating the effects of microglia on neuronal health, we observed increased neuronal survival, neurite outgrowth, and pre‐synapse count in glutamatergic neurons co‐cultured with microglia when compared to glutamatergic neurons cultured alone. In neuron‐microglia co‐cultures exposed to increasing doses of β‐amyloid 1‐42 fragments, we observed neuronal hyperactivity (increased calcium event frequency), which may reflect disease‐associated stress.ConclusionOur data demonstrate that healthy microglia regulate neuronal heath. Our system can serve as a model for testing inflammatory insults related to AD. To further our research with genetic variants with AD susceptibility, we have created an isogeneic panel of ApoE variants using CRISPR‐Cas9, and plan to characterize them in the culture systems developed above to validate a disease model from these lines.