Grantee Research Project Results
2023 Progress Report: 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 Period Covered by this Report: August 1, 2022 through July 31,2023
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
Objective:
1.Design an iPS cell line with fluorescently tagged fusion glial fibrillary acidic protein (GFAP), proteolipid protein 1 (PLP), synaptophysin (SYP), and b-III-tubulin using CRISPR/CAS9 gene-editing technology.
2.Generate 3D multi-fluorescent BrainSpheres from the iPSC line created in objective 1 and characterize them in terms of neural differentiation and fusion-protein fidelity.
3.Expose the multi-fluorescent BrainSpheres to five chemicals for the 6-in-1 BrainSphere assay optimization; establish the 3D electrophysiological recording and high content imaging (HCI) workflow.
4.Establish test strategy workflow for the toxicity screening using the 6-in-1 BrainSphere assay and test further 30 compounds. Develop SOPs.
Progress Summary:
Objective 1-2: This year we published two papers (Romero et al, Front in Cell Neuroscie 2023 and Morales Pantoja et al., Adv, Biology 2023, where we show:
- Brain organoids containing PLP1-sfGFP expressing Oligodendrocytes (OLs) are a suitable model to trace oligodendrogenesis and monitor chemical effects on OLs at different stages of differentiation. We currently have following lines: N8-PLP:GFP, CS2-PLP:GFP, N8-PLP:GFP/SYP:BFP and N8-SYP:GFP. All of the lines underwent all the QCs and are ready to use for chemical testing. In spite of the seveal attempts and extensive troubleshooting, we were not able to perform the transfection of additional two reporters yet. We will use help from a core facility, which has extensive experience in CRISPR/Cas9 cloning to continue troubleshooting the approach.
- We optimized the medium conditions to improve astrocytes and glia differentiation (Figure 1).
Figure 1. Quantification of glial and neuronal markers in NIBSC8 iPSC line differentiated to bMPS in standard differentiation medium (CTR) and glia enrichment medium (GEM) for 10 weeks. Data represents mean intensity ± SEM of O4- (A, green), GFAP- (B, red), and NF-H- (C, purple) positive cell from 5-7 bMPS per condition and time point obtained from 8-11 stacks from z-stacks of one-micron intervals. Mann-Whitney test was used for significance testing. Representative images from at least 3 experiments are shown. The scale bar represents 50 µm
Objective 3 Part 1: We further optimized the 3D Shell electrodes. We addressed two additional elements in our current cycle: long-term monitoring and high-resolution surface probing. In addition, we established high-density MEA arrays and characterized differentiation and pharmacology of brain organoid electrical activity. Currently we are testing heavy metal and domoic acid as reference compounds. Importantly, we transferred our model to EPA (Drs. T. Shafer and S. Hunter laboratories) through the active CRADA. Both laboratories purchased the same MEA system that will allow us to screen the compounds in two laboratories and show the inter and intralaboratory variability.
Objective 3 Part 2: We continue to expose BrainSpheres to model toxicants to derive preliminary data and effective concentrations to be used in the HCI high-throughput screening pipeline. Compounds tested: chlorpyrifos, cuprizone, selected flame retardants (TBBPA, TPHP, IPP, BDE-47), heavy metals and their mixture (Pb, As, Cd, Cr), PAHs, acetaminophen, LPC, paroxetine.
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Figure 2. SYP:GFP brain organoids exposed to 100 nM arsenic, 32 nM Cd, 100 nM chromium or 320 nM lead for four weeks from 4 to 8 weeks of
differentiation. Green puncta are synaprophysin:GFP positive synapses.
Comparison of actual accomplishments with the goals and objectives (outputs/outcomes): We are still behind the original schedule with respect to cell line generation due to later hire of Dr. Romero, COVID lockdown, failure in designing the CRISPR/Cas9 constructs with efficient gRNA from the first attempt. We made, however, substantial progress in electrophysiology and pre-testing of the existing two reporters. Since we are working on all aims in parallel, we are still making substantial progress regardless that the four-color cell line is not ready yet. We are conducting imaging optimizations and toxicant exposures to meet the original goals.
Future Activities:
Finalize cell line generation and QC. Establish HCI pipeline, establish electrophysiology recording upon toxicant treatment.
Journal Articles on this Report : 3 Displayed | Download in RIS Format
Other project views: | All 9 publications | 9 publications in selected types | All 9 journal articles |
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Romero JC, Berlinicke C, Chow S, Duan Y, Wang Y, Chamling X, Smirnova L. Oligodendrogenesis and myelination tracing in a CRISPR/Cas9-engineered brain microphysiological system. Frontiers in Cellular Neuroscience 2023;16:1094291. |
R839505 (2023) |
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Morales Pantoja IE, Ding L, Leite PE, Marques SA, Romero JC, Alam El Din DM, Zack DJ, Chamling X, Smirnova L. A Novel Approach to Increase Glial Cell Populations in Brain Microphysiological Systems. Advanced Biology 2023:2300198. |
R839505 (2023) |
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Smirnova L, Caffo BS, Gracias DH, Huang Q, Morales Pantoja IE, Tang B, Zack DJ, Berlinicke CA, Boyd JL, Harris TD, Johnson EC. Organoid intelligence (OI):the new frontier in biocomputing and intelligence-in-a-dish. Frontiers in Science 2023:0. |
R839505 (2023) |
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Progress and Final Reports:
Original AbstractThe 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.