Assay Development for Measuring Chemical Effects on the Epigenome in Stem Cells

7. Project Summary The recent advances in pluripotent stem cell (PSC) technology enable researchers to establish human tissue derived cellular platforms for toxicity testing that are capable of providing information on mechanism of action, while facilitating the incorporation of broad genetic diversity and clinically validated disease conditions. We and other investigators have shown that it is possible run neurotoxicity screens with primary cells derived from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) and obtain consistent and reliable results that offer advantages over screens run in cell lines or in rodent cells. The limiting factors in translating this technology into a commercial product have been an inability to establish large-scale reproducible protocols for generation and differentiation of PSCs and access to large collections of genetically diverse well-phenotyped cell lines. In this proposal, we aim to develop a fully automated platform for production of PSC-derived cells in a 96-well format utilizing a genetically diverse bank of cell lines. The large-scale approach will improve the statistical precision in the readouts and provide for parallel production not previously obtainable, which will translate into more predictive models. The product will be available to customers as a service for in-house screening of compounds or we can provide 96-well plates of iPSC-derived NSCs and DA neurons - vials of cells with instructions for plating and initial characterization will also be available. Plates will be generated from either a single cell line for initial screening of large numbers of compounds or independent cell lines from 96 healthy tissue donors representing the genetic diversity of the US population for hit validation. Analysis will include high-content image analysis of cell survival as well as transcriptional profiling. Our goal is to significantly reduce the costs of generating stem cell screening platforms and provide information that is more effective at predicting how human cells will respond to exposure to various toxicants during differentiation and at mature cell states. Our product will support the goals of the funding mechanisms, while also establishing a baseline of variation of transcription for neurallineages that will be critical when determining the significance of toxic effects of chemical compounds or developmental differences when analyzing diseased lines. PUBLIC HEALTH RELEVANCE PUBLIC HEALTH RELEVANCE: In this application, we aim to develop a fully automated platform for production of PSC-derived neuronal cells in a 96-well format from one (initial screen) and 96 (validation) individuals representing the genetic diversity of the US population. The product will be available to customers as a service for in-house screening of compounds or we can provide 96-well plates of iPSC-derived NSCs, and DA neurons - vials of cells with instructions for plating and initial characterization will also be available. Analysis will include high-content imaging analysis of cell survival and transcriptional analysis.