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GENERATION OF A PATIENT IPSC-INDUCED 3D SPHEROID MODEL TO STUDY PATHOPHYSIOLOGY OF ALZHEIMER’S DISEASE AND INVESTIGATE NOVEL THERAPEUTICS
Abstract
Aims
Exploration of the pathophysiology of Alzheimer’s disease (AD) has been hampered by lack of systems that accurately recapitulate full profile of disease progression. We have developed a 3-dimentional assembloid model with iPSC induced neurons, astrocyte and microglia derived from AD subjects to investigate the pathophysiology, protein-protein interactions, cellular mechanism and interventional strategies for AD.
Methods
Lines of APP V717I iPSC and isogenic controls were differentiated into neuronal, astrocytic and microglial progenitor cells in 2D culture, and the neuronal cells were seeded with tau oligomers (oTau). The three cell types of progenitor cells were then mixed together to generate 3D Microglia-Astrocyte-Neuronal spheroids (MAstAD).
Results
Analysis of the MAstAD cultures after 1-3 weeks of culture revealed abundant b-amyloid deposition, tau pathology, neurodegeneration, astrogliosis and microglial activation. Immunofluorescence labeling demonstrated abundant Ab deposits (observed by Thiazine red or 4G8 antibody labeling), tau phosphorylation and aggregation, microglial activation (Iba1 labeling and TNF-a- secretion) and neurodegeneration quantified using FluoroJade B, cleaved caspase 3 and LDH measurements. APP V717I mutation and oTau seeding synergistically exacerbated all the phenotypes. Coincident single cell RNA sequencing and mass spectrometry were also performed to explore the cellular and molecular signal pathways in the disease progression.
Conclusions
We have generated a novel 3D assembloid model of AD, which we term MAstAD. The MAstAD system recapitulates many features of AD pathology including microglia/astrocyte gliosis, Ab and tau pathology, and neurodegeneration. This innovative AD model offers an advanced platform to study the cellular and molecular mechanisms of disease progression and develop effective therapeutic strategies for AD.