(1294-C) Functional Characterization of Healthy and Alzheimer’s Disease-related 3D Neurospheres Formed Using Human iPSC-derived Glutamatergic Neurons, GABAergic neurons, and Astrocytes.
Tuesday, February 6, 2024
12:00 PM – 1:00 PM EST
Location: Exhibit Halls AB
Abstract: Neural 3D organoids from human induced pluripotent stem cells (iPSC) are a rapidly developing technology with great potential for understanding brain development and neuronal diseases. A promising parallel approach is to assemble similar structures as 3D spheroids or “neurospheres” by using defined combinations of fully differentiated human iPSC-derived cells in tri-culture, including glutamatergic neurons, GABAergic neurons, and astrocytes. In this study, we chose to model Alzheimer’s Disease (AD) by incorporating allelic variants of the ApoE gene (2/2, 3/4, and 4/4) to create disease-specific “neurospheres”. 3D spheroids were formed by combining 20-25,000 cells in ultra-low attachment (ULA) plates. We tracked spheroid morphology over time by live cell imaging. Spheroid diameters ranged from 400-500 µm. Microtissues were also analyzed by confocal fluorescence imaging for cell organization and expression of various neural markers, including TUJ1 and GFAP. Functional performance was tested via calcium oscillation assay and was run on a FLIPR instrument capable of fast kinetic recordings. Importantly, the calcium-sensitive dye used contains a background fluorescence masking technology that enables sensitive detection of neurospheres in 384w format. The calcium oscillation patterns were analyzed for metrics like peak count, amplitude, and width. Distinct differences in pattern and decrease in baseline oscillation frequencies were observed between control and AD-specific neurospheres, specifically for the ApoE 4/4 mutation group. Within each group, the calcium kinetics and patterns were highly consistent. For pharmacological characterization, a panel of select compounds was used to show both the appropriate responses to GABA, AMPA, and NMDA, as well as changes to neuroactive compounds. Some drugs previously shown to affect AD phenotypes (memantine and donepezil) decreased calcium peak amplitude and altered other metrics as visualized by ScreenWorks software and analyzed by PeakPro2. Taken together, this biological system of 3D neurospheres assembled from human iPSC-derived cell types paired with high-content imaging and detailed analysis of calcium oscillations demonstrates a promising tool for disease modeling and compound testing.