Gauging relevance and reproducibility of human iPSC-derived 3D neuralspheroids for high throughput neuroscience drug discovery and toxicology testing

The human cerebral cortex is a highly sophisticated three-dimensional structure which comprises multiple different neural cell types intricately interconnected to form a functional organ. Recent work has focused on the development of in vitro models capable of recapitulating brain composition and function. We have developed a three-dimensional human-induced Pluripotent Stem Cell (hiPSC)-derived model amenable to high-throughput drug discovery and toxicology studies. This platform is comprised by neurospheroids measuring approximately 600 µm, populated by cortical neurons and astrocytes. Transcriptional analysis confirms a neuronal expression pattern, immunocytochemistry analysis demonstrates the complex interconnection of cortical neurons and astrocytes and the expression of maturity markers and glutamate transporters typically present in human cortical tissue, and functional analysis demonstrates appropriate neuronal behavior. The functional assays highlight the utility of the platform where it can be used as a phenotypic readout for drug efficacy and toxicity. Specifically, kinetic calcium flux analysis, assayed using a Fluorometric Imaging Plate Reader (FLIPR), provides a measurement of the underlying neuronal activity and highlights the high-throughput capabilities of this platform for the detection and quantification of calcium oscillations both at unstimulated baseline and when exposed to known reference compounds, pathway modulator, and/or toxicants. Variability studies investigating spheroid size, calcium flux and immunocytochemistry, demonstrate the robustness of the platform for high-throughput screening. We also demonstrate that this platform is a highly stable model with a broad assay window of use once differentiated. Taken together, the model described herein provides a highly reliable and stable in vitro platform for high-throughput drug discovery and toxicology investigations.

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