SBIR-STTR Award

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 neural lineages that will be critical when determining the significance of toxic effects of chemical compounds or developmental differences when analyzing diseased lines.

Public Health Relevance Statement:


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.

NIH Spending Category:
Bioengineering; Genetics; Neurosciences; Regenerative Medicine; Stem Cell Research; Transplantation

Project Terms:
1-Methyl-4-phenylpyridinium; Biological Assay; Cardiac Myocytes; Cell Count; Cell Line; Cell Survival; cell type; Cells; Chemicals; Collaborations; Collection; cost; Data; Databases; Development; Disease; dopaminergic neuron; Exposure to; Foundations; Funding Mechanisms; Future; Generations; Genetic Transcription; Goals; Housing; Human; human embryonic stem cell; human tissue; Image; Image Analysis; improved; Individual; induced pluripotent stem cell; Instruction; magnetic beads; Marketing; Measurement; Measures; Messenger RNA; Modeling; nerve stem cell; Neurons; neurotoxic; neurotoxicity; New York; novel; Phase; Phenotype; Pluripotent Stem Cells; Population; predictive modeling; Procedures; Product Packaging; Production; Protocols documentation; public health relevance; relating to nervous system; Research Personnel; Robotics; Rodent; Rotenone; Running; scale up; screening; Services; Shipping; Ships; Sorting - Cell Movement; stem cell technology; Stem cells; Technology; Time; Tissue Donors; Toxic effect; toxicant; Toxicity Tests; transcriptomics; Translating; Validation; Variant; Variation (Genetics); Vial device

Despite the fact that animal-based neurotoxicology models have relatively low sensitivity, and are burdened by high workload, cost and animal ethics, they have been the mainstay of evaluating neurotoxicology. However, toxicology today is looking for alternatives as it faces enormous challenges to the use of animals. On one hand there is enormous societal pressure to reduce the use of animals, and on the other hand the demand for testing is increasing. More than 30,000 chemicals are estimated in use without adequate toxicological information in the USA and Europe, and the task of testing thousands of chemicals systematically with classical animal assays likely exceeds our present capabilities. The recent advance in pluripotent stem cell (PSC)-based technology and the ability to generate truly large numbers of allelically diverse cells and use uniform methods of differentiation into al the major types of cells in the nervous system offer a potential new tool for improved understanding of chemically induced toxicity. This is especially useful for developmental neurotoxicity, because neural cells differentiate early during development and this process is relatively easily recapitulated in vitro via rosette formation and isolation of neural stem cells (NSC), which can subsequently be differentiated into neurons and glia. We have in a Phase I grant provided proof-of-concept for iPSC-based neurotoxicity assays. However several issues need to be addressed before such assays can be used routinely to test neurotoxicity. For example, there is a lack of neural reporters in referenced lines of both genders, which will be invaluable for further assay development and refinement. There is also a lack of datasets that can serve as baseline for toxicity assays, and a lack of reference response to a reference set of compounds that can be used to calibrate the response of future lines and compare with the rodent data. The objective of this Phase II application is to develop a neurotoxicity tool kit that addresses these issues and to commercialize it.

Public Health Relevance Statement:


Public Health Relevance:
One emerging field in toxicology is the use of iPSC for neurotoxicology and developmental neurotoxicity. If the proposed aims are achieved, this project will provide the community a complete neurotoxicity tool kit with a database of baseline for toxicity assays and response to reference compounds. This will help provide answers to a key question about the feasibility of using iPSC-derived cells for neurotoxicity and to replace the in vivo animal tests.

Project Terms:
Acute; Address; Animal Testing; Animals; assay development; base; Biological Assay; Brain; Cell Count; Cell Line; cell type; Cells; Chemically Induced Toxicity; Chemicals; Chronic; Communities; Complex; cost; Data; Data Set; Databases; Development; developmental neurotoxicity; Ethics; Europe; Exposure to; Face; Female; Future; Gender; Generations; Genes; Grant; Human; improved; In Vitro; in vivo; induced pluripotent stem cell; male; Methods; Modeling; Morphology; Nature; nerve stem cell; Nervous system structure; Neuroglia; Neurons; neurotoxic; neurotoxicity; neurotoxicology; Organ; Pathway interactions; Pharmaceutical Preparations; Phase; Pluripotent Stem Cells; pressure; Procedures; Process; public health relevance; relating to nervous system; Reporter; response; Rodent; Run-On Assays; Specificity; Technology; Testing; Time; tool; Toxic effect; Toxic Environmental Substances; Toxicology; Workload