Modeling CNS neuronal‐glial interactions relevant to Alzheimer’s Disease and ADRD with microglia and neurons derived from human induced pluripotent stem cells

AbstractBackgroundCNS neurons and microglia participate in neuroimmune pathways linked to Alzheimer’s Disease (AD) and AD Related Dementias (ADRD). However, CNS neurons and microglia cannot be isolated from living people, and primary microglia harvested post‐mortem rapidly de‐differentiate in culture. hiPSC‐neurons and hiPSC‐MG represent a practical alternative for in vitro research, as they are human‐derived, upscalable, and can be made from people with AD/ADRD‐sensitive or ‐resilient genotypes. We are developing high‐throughput assays utilizing hiPSC‐neurons/‐MG, to explore interactions between these cell types and pathways relevant to AD, and to test compounds for beneficial effects on neuronal health.MethodhiPSC‐neurons (excitatory) were purchased from Fujifilm CDI or differentiated in‐house (with kits from Elixirgen). hiPSC‐MG were differentiated in‐house from hiPSCs via a protocol in which yolk sac‐like structures are produced as an intermediary step. Mono‐ or cocultures were prepared in 96‐ or 384‐well dishes. Results were obtained via automated digital microscopy, with cellular structures and biomarkers analyzed via high content analysis (HCA) and cell activity (calcium or voltage transients) analyzed via Kinetic Image Cytometry; in certain experiments, LDH release, a general measure of cytotoxicity, was also assayed.ResultCoculture of hiPSC‐MG with hiPSC‐neurons led to a 2‐fold increase in neurites. hiPSC‐MG also elicited calcium transients in the hiPSC‐neurons via close contact. The APOE‐e4 gene variant confers the greatest risk for late‐onset AD, and proteolysis of ApoE4 may produce neurotoxic fragments. In our hands, a fragment of ApoE4 (ApoECFp17, aa 1‐151) induced LDH release in both hiPSC‐neurons and hiPSC‐MG, indicating cytotoxicity, whereas the corresponding fragment of ApoE3 had no effect; additionally, the toxic effect on hiPSC‐MG was partially blocked by PH002 (an ApoE4 “structure corrector”) and by RAP (a protein that blocks ApoE4 binding to cell‐surface receptors).ConclusionOur data demonstrate that hiPSC‐MG alter neurite expression and calcium transient activity in cocultured hiPSC‐neurons, and that the aa 1‐151 fragment of ApoE4 is toxic to human neurons and microglia, which could potentially play a role in AD etiology. Future research will incorporate hiPSC‐astrocytes into the cultures to further model CNS neuronal‐glial interactions and elucidate pathways relevant to AD/ADRD.