Scientific Poster

High Content Neuronal Toxicity Assays Using iPS Cell-derived Neurons 

Date | Time: Tuesday, Feb 7 | 1:00 - 3:00 pm

Location:

Presenter: Oksana Sirenko, Molecular Devices

Abstract: The nervous system is a target organ for the toxic effects of chemical compounds, environmental agents and some naturally occurring substances. Neurotoxicity can cause temporary or permanent damage of brain or peripheral nervous system and has been found to be a major cause of neurodegenerative diseases. Therefore, there is a great interest in developing more predictive cell-based models and efficient screening tools for evaluation of qualitative and quantitative risk assessment of the impact of chemical compounds, drug candidates and environmental agents. Human neurons derived from induced pluripotent stem cells (iPSCs), such as iCells^® Neurons, are very attractive for such studies because they exhibit a phenotype and functionality similar to mature neurons. These aspects, coupled with availability of such cells in large quantities, make them useful for the screening of lead compounds and potentially important for reducing animal experimentation and cost of pre-clinical development. We have developed several assays using iCell Neurons and high content imaging for evaluation of impact different compounds on neurogenesis or neural toxicity. In a traditional fixed cell assay, cells were allowed to form neurite networks in 96- or 384-well plates and cultured in the presence of toxic compounds for 48 hours. Cells were then fixed, stained with antibodies against class III beta-tubulin, MAP2, and other neuro-specific markers and fluorescently imaged. High resolution acquisition over a large area is performed with the ImageXpress^® Micro XL system and the images were analyzed with MetaXpress^™ software. Several parameters characterizing neuronal networks including length of neurite outgrowth, branching, number of cells and processes were analyzed. We observed a dose-dependent disintegration of neuronal networks and toxicity due to the kinase inhibitors staurosporin and PD98082, as well as other cytotoxic compounds including Antimycin A, MK571, and Mitomycin C. A second, live cell assay provides additional information for drug discovery. This model assesses positive or negative effects of compounds on the development of neurite networks in real time using time-lapse transmitted light imaging. Initially growth and formation of neuronal networks were characterized over time in an endogenous format. We demonstrated the impact of growth factors, staurosporin and several other kinase inhibitors on cell motility and neurite outgrowth in a time-dependent manner. At desired end-points (30 min, 24h or 48h) live neurons were stained with Calcein AM, allowing for the determination of live cells as well as characterizing development of neuronal networks. Also, we simultaneously utilized Hoechst dye for total cell count, cell cycle, as well as JC-10 for assessing mitochondria integrity allowing for several cytotoxic parameters to be evaluated simultaneously in a multiplexed assay. These methods provide important insights into neurogenesis and neurotoxicity that are not possible with fixed cell assay models.

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