Grantee Research Project Results
Multiplexed human BrainSphere Developmental Neurotoxicity Test for Six Key Events of Neural Development
EPA Grant Number: R839505Title: Multiplexed human BrainSphere Developmental Neurotoxicity Test for Six Key Events of Neural Development
Investigators: Lena, Smirnova , Thomas, Hartung , David, Gracias , Cynthia, Berlinicke
Institution: The Johns Hopkins University
EPA Project Officer: Callan, Richard
Project Period: August 1, 2019 through July 31, 2022 (Extended to July 31, 2024)
Project Amount: $849,277
RFA: Advancing Actionable Alternatives to Vertebrate Animal Testing for Chemical Safety Assessment (2018) RFA Text | Recipients Lists
Research Category: Chemical Safety for Sustainability
Description:
Exposure to environmental chemicals during early life is suspected to contribute to the increasing incidence of neurodevelopmental disorders, especially autism. Currently, 1 in 59 children in US are diagnosed with autism spectrum disorders (ASD), and this cannot be explained only by the genetics, suggesting that environmental exposures contribute. Demanding animal tests for Developmental Neurotoxicity (DNT) have been devised, but because of complex underlying mechanisms, limitations of current approaches are enormous. The high costs and technical difficulties of these tests are prohibitive for routine DNT chemicals screening. Alternatives are needed to change this.
Objective:
The main goal of this project is to develop a testing strategy for DNT, which can be included in a DNT IATA (Integrated Approaches to Testing and Assessment). This strategy will be based on a human 3D iPSC-derived brain model with knocked-in fluorescent tags for neural markers (6-in-1 BrainSphere assay), where six key events of neurodevelopment and there perturbation will be assessed in one assay.
Approach:
Our 3D BrainSphere model has most cell types of the Central Nervous System, myelinated axons and is electrophysiologically active. We have shown the potential of this model for DNT and neurodegeneration applications. We will further modify this model by using CRISPR/Cas9 gene-editing technology to generate an iPSC cell line that contains GFAP, b-III-tubulin, myelin basic protein (MBP) and synaptophysin fluorescently-tagged fusion proteins. Differentiation of this line into BrainSpheres will allow us to visualize and track neurons (b-III-tubulin, Synaptophysin), astrocytes (GFAP) and oligodendrocytes (MBP) within the same spheroid, based on the cells that express these fusion proteins and avoid time-consuming immunohistochemistry. With this system we will establish quantitative image-based assays for neuronal differentiation, myelination, neurite outgrowth, synaptogenesis, glia migration and gliosis, which are key events perturbed by developmental neurotoxicants. Electrophysiological recording will be performed in 3D in a recently developed self-rolling electrode platform.
Expected Results:
As an outcome of this project, we will define a testing strategy for DNT, which covers some key processes of neural development and can be integrated into DNT IATA. The advantage of this strategy over other existing in vitro approaches will be a cost- and time effective in vitro human relevant iPSC-derived 3D brain-model-based assay, where at least six endpoints will be multiplexed and measured from the same sample. This robust and standardized in vitro assay, relevant for the assessment of human toxicity, will reduce the costs and accelerate the prioritization and testing of environmental chemicals, hazard identification and characterization within a risk assessment context.
Publications and Presentations:
Publications have been submitted on this project: View all 9 publications for this projectJournal Articles:
Journal Articles have been submitted on this project: View all 9 journal articles for this projectSupplemental Keywords:
developmental neurotoxicants, environmental chemicals, 3D organotypic cultures, new approach methodologiesProgress and Final Reports:
The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.