SBIR-STTR Award

Pre-clinical and pre-field toxicology testing of new drugs and chemicals does not routinely include direct assessment of human developmental neurotoxicity (DNT). Current standards for DNT testing require use of animal models with limited throughput and significant differences from human central nervous system (CNS) development. As such, many chemical products have required post-approval (EPA/FDA) restrictions or cancellations due to human DNT. Such limitations of the current regulatory DNT testing paradigm have prompted increased interest in quantitative high-throughput screening (qHTS) using human pluripotent stem cell (hPSC)-based approaches. Here, we propose to use Rosette Array (RA) technology to develop an hPSC- derived qHTS platform (i.e., qHTS-RAs) for effective and efficient DNT testing. Rosette Array technology standardizes in vitro derivation of human neural rosettes tissues that are mimetic of transverse slices of the human neural tube, the anlage of all CNS tissue. Although neural rosettes are not an exact recapitulation of in vivo primary neurulation, they exhibit the same cell phenotypes, tissue cytoarchitecture, and are derived using morphogenetic signaling pathways endogenous to the in vivo neural tube formation process. Rosette arrays are the first in vitro technology to enable spatial and temporal control of neural rosette emergence in a microarray format. This enables rapid assessment via microscope image analysis. This high-yield, standardized generation of in vitro neural tube analogs enables the repeatability necessary to feasibly incorporate hPSC-based CNS morphogenic readouts into quantitative high-throughput toxicology screening. Thus, we hypothesize that the qHTS-RA platform could increase throughput and accuracy of human DNT risk assessment, allowing consolidation and scale-up of commercial DNT screening. Phase 1 Aims will validate the broad applicability of the qHTS-RA platform, confirming compatibility with human induced pluripotent stem cell (hiPSC) lines and establishing methods for automated image acquisition and batch analysis. Phase 2 Aims to validate assay fitness with a 100 compound DNT reference library screen, in which automated AI image analysis is utilized to further increase accuracy and maximize throughput. If successful, the resulting qHTS-RA platform could replace multiple current DNT assays and increase confidence in toxicological readouts relevant to outcomes unique to human physiology. Thus, the work proposed here could have a transformative effect on DNT research, regulatory efforts to prevent DNT exposure, and future translatability of hPSC-based organoid models for broad study of human development.

Public Health Relevance Statement:
Project Narrative Toxicology screening of commercial chemicals is typically conducted using an assay battery heavily reliant on animal models, which diverge from humans especially in aspects of central nervous system development. Here, we propose to develop and evaluate a quantitative, human pluripotent stem cell-derived, high-throughput screening platform that standardizes neural rosette morphogenesis, modeling early CNS development, for developmental neurotoxicity (DNT) applications. If validated, the novel screening platform would streamline DNT assay batteries and increase the accuracy of chemical safety assessments.

Project Terms:
Affect; Animals; Artificial Intelligence; AI system; Computer Reasoning; Machine Intelligence; Biological Assay; Assay; Bioassay; Biologic Assays; Cell Line; CellLine; Strains Cell Lines; cultured cell line; Cell Nucleus; Nucleus; Cell Survival; Cell Viability; Cells; Cell Body; Pharmaceutical Preparations; Drugs; Medication; Pharmaceutic Preparations; drug/agent; Exhibits; Future; Human; Modern Man; Human Development; In Vitro; Libraries; Manuals; Metabolism; Intermediary Metabolism; Metabolic Processes; Methods; Morphogenesis; morphogenetic process; Organoids; Pesticides; Phenotype; Physiology; Research; Sensitivity and Specificity; Signal Pathway; Standardization; Technology; Testing; Time; Tissues; Body Tissues; Toxicology; Work; Generations; Prosencephalon; Fore-Brain; Forebrain; Agrochemicals; Agricultural Chemicals; Risk Assessment; base; Image Analysis; Image Analyses; image evaluation; image interpretation; Cervical; Phase; Biological; biologic; Chemicals; Evaluation; Neural tube; Fees for Service; Fee-for-Service Plans; analog; Morphology; Spinal; interest; nervous system development; neural control; neural regulation; neuromodulation; neuromodulatory; neuroregulation; Animal Models and Related Studies; model of animal; model organism; Animal Model; neural; relating to nervous system; Neural Development; neurodevelopment; Toxicities; Toxic effect; Chemical Exposure; novel; Modeling; High Throughput Assay; high throughput screening; develop software; developing computer software; software development; Biological Mimetics; Biomimetics; mimetics; Neuraxis; CNS Nervous System; Central Nervous System; nerve stem cell; Neural Stem Cell; neural precursor; neural precursor cell; neural progenitor; neural progenitor cells; neuron progenitors; neuronal progenitor; neuronal progenitor cells; neuronal stem cells; neuroprogenitor; Pharmaceutical Agent; Pharmaceuticals; Pharmacological Substance; Pharmacologic Substance; preventing; prevent; fitness; Data; Detection; Reproducibility; Research Contracts; Resolution; in vitro Model; in vivo; Screening Result; Slice; Tissue Microarray; Tissue Arrays; Tissue Chip; Process; Derivation procedure; Derivation; Development; developmental; Image; imaging; Output; pre-clinical; preclinical; working group; work group; National Toxicology Program; Outcome; scale up; developmental neurotoxicity; novel therapeutics; new drug treatments; new drugs; new therapeutics; new therapy; next generation therapeutics; novel drug treatments; novel drugs; novel therapy; induced pluripotent stem cell; iPS; iPSC; iPSCs; inducible pluripotent stem cell; screening; microscopic imaging; microscope imaging; microscopy imaging; high resolution imaging; human pluripotent stem cell; automated image analysis; analysis pipeline; safety assessment; stem cell based approach

Pre-clinical and pre-field toxicology testing of new drugs and chemicals does not routinely include direct assessment of human developmental neurotoxicity (DNT). Current standards for DNT testing require use of animal models with limited throughput and significant differences from human central nervous system (CNS) development. As such, many chemical products have required post-approval (EPA/FDA) restrictions or cancellations due to human DNT. Such limitations of the current regulatory DNT testing paradigm have prompted increased interest in quantitative high-throughput screening (qHTS) using human pluripotent stem cell (hPSC)-based approaches. Here, we propose to use Rosette Array (RA) technology to develop an hPSC- derived qHTS platform (i.e., qHTS-RAs) for effective and efficient DNT testing. Rosette Array technology standardizes in vitro derivation of human neural rosettes tissues that are mimetic of transverse slices of the human neural tube, the anlage of all CNS tissue. Although neural rosettes are not an exact recapitulation of in vivo primary neurulation, they exhibit the same cell phenotypes, tissue cytoarchitecture, and are derived using morphogenetic signaling pathways endogenous to the in vivo neural tube formation process. Rosette arrays are the first in vitro technology to enable spatial and temporal control of neural rosette emergence in a microarray format. This enables rapid assessment via microscope image analysis. This high-yield, standardized generation of in vitro neural tube analogs enables the repeatability necessary to feasibly incorporate hPSC-based CNS morphogenic readouts into quantitative high-throughput toxicology screening. Thus, we hypothesize that the qHTS-RA platform could increase throughput and accuracy of human DNT risk assessment, allowing consolidation and scale-up of commercial DNT screening. Phase 1 Aims will validate the broad applicability of the qHTS-RA platform, confirming compatibility with human induced pluripotent stem cell (hiPSC) lines and establishing methods for automated image acquisition and batch analysis. Phase 2 Aims to validate assay fitness with a 100 compound DNT reference library screen, in which automated AI image analysis is utilized to further increase accuracy and maximize throughput. If successful, the resulting qHTS-RA platform could replace multiple current DNT assays and increase confidence in toxicological readouts relevant to outcomes unique to human physiology. Thus, the work proposed here could have a transformative effect on DNT research, regulatory efforts to prevent DNT exposure, and future translatability of hPSC-based organoid models for broad study of human development.

Public Health Relevance Statement:
Project Narrative Toxicology screening of commercial chemicals is typically conducted using an assay battery heavily reliant on animal models, which diverge from humans especially in aspects of central nervous system development. Here, we propose to develop and evaluate a quantitative, human pluripotent stem cell-derived, high-throughput screening platform that standardizes neural rosette morphogenesis, modeling early CNS development, for developmental neurotoxicity (DNT) applications. If validated, the novel screening platform would streamline DNT assay batteries and increase the accuracy of chemical safety assessments.

Project Terms:
Affect; Animals; Artificial Intelligence; AI system; Computer Reasoning; Machine Intelligence; Biological Assay; Assay; Bioassay; Biologic Assays; Cell Line; CellLine; Strains Cell Lines; cultured cell line; Cell Nucleus; Nucleus; Cell Survival; Cell Viability; Cells; Cell Body; Pharmaceutical Preparations; Drugs; Medication; Pharmaceutic Preparations; drug/agent; Exhibits; Future; Human; Modern Man; Human Development; In Vitro; Libraries; Manuals; Metabolism; Intermediary Metabolism; Metabolic Processes; Methods; Morphogenesis; morphogenetic process; Organoids; Pesticides; Phenotype; Physiology; Research; Sensitivity and Specificity; Signal Pathway; Standardization; Technology; Testing; Time; Tissues; Body Tissues; Toxicology; Work; Generations; Prosencephalon; Fore-Brain; Forebrain; Agrochemicals; Agricultural Chemicals; Risk Assessment; base; Image Analysis; Image Analyses; image evaluation; image interpretation; Cervical; Phase; Biological; biologic; Chemicals; Evaluation; Neural tube; Fees for Service; Fee-for-Service Plans; analog; Morphology; Spinal; interest; nervous system development; neural control; neural regulation; neuromodulation; neuromodulatory; neuroregulation; Animal Models and Related Studies; model of animal; model organism; Animal Model; neural; relating to nervous system; Neural Development; neurodevelopment; Toxicities; Toxic effect; Chemical Exposure; novel; Modeling; High Throughput Assay; high throughput screening; develop software; developing computer software; software development; Biological Mimetics; Biomimetics; mimetics; Neuraxis; CNS Nervous System; Central Nervous System; nerve stem cell; Neural Stem Cell; neural precursor; neural precursor cell; neural progenitor; neural progenitor cells; neuron progenitors; neuronal progenitor; neuronal progenitor cells; neuronal stem cells; neuroprogenitor; Pharmaceutical Agent; Pharmaceuticals; Pharmacological Substance; Pharmacologic Substance; preventing; prevent; fitness; Data; Detection; Reproducibility; Research Contracts; Resolution; in vitro Model; in vivo; Screening Result; Slice; Tissue Microarray; Tissue Arrays; Tissue Chip; Process; Derivation procedure; Derivation; Development; developmental; Image; imaging; Output; pre-clinical; preclinical; working group; work group; National Toxicology Program; Outcome; scale up; developmental neurotoxicity; novel therapeutics; new drug treatments; new drugs; new therapeutics; new therapy; next generation therapeutics; novel drug treatments; novel drugs; novel therapy; induced pluripotent stem cell; iPS; iPSC; iPSCs; inducible pluripotent stem cell; screening; microscopic imaging; microscope imaging; microscopy imaging; high resolution imaging; human pluripotent stem cell; automated image analysis; analysis pipeline; safety assessment; stem cell based approach