Grantee Research Project Results
2022 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 , Romero, J. Carolina
Current 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, 2021 through July 31,2022
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 finished the preparation of a paper that is currently submitted to iScience where we show:
Reverse transfection improves homozygous incorporation of fluorescent tags into hiPSCs.
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 optimized the medium conditions to improve astrocytes and glia differentiation. Figure 1 demonstrates presence of PLP1-sfGFP positive mature oligodendrocytes (green) with their processes wrapped around axons (purple). We also detected significant increase in the number of astrocytes. This was a significant improvement of differentiation conditions in comparison to the previous model.
Combination of the fluorescent fusion tags with imaging, provides more robust information about differences in morphology of cells at different stages of differentiation as well as the effects of drugs/chemicals at the cellular level than quantification of reporter expression.
Objective 3 Part 1: We successfully completed first phase of developing the multielectrode shells for electrophysiology measurement. In addition, we conducted first preliminary experiments using high-density multielectrode system from MaxWell, which allows recording on the level of individual cells (Fig. 2), axon velocity tracking, electrical stimulation in relative higher throughput (24-well format). We are acquiring the system and will conduct the electrical activity recording upon chemical treatment in the MaxWell system in addition to the 3D shell electrodes.
Objective 3 Part 2: This year, we continued to establish and optimize the high-content imaging (HCI) and image analysis pipeline, which we will use to quantify the effects of toxicants on BrainSpheres. We developed customized algorithms to quantify different metrics related to the fluorescent reporter expressed in the organoid. Using the brightfield image from the first imaging pass, a mask around the organoid was generated. As initial measurements, we can then determine the organoid area, as well as the total and average intensities of the expressed fluorescent reporters. In addition, we use a spot-detecting algorithm that identifies and quantifies metrics related to puncti (spots). Based on these metrics, we determine a variety of organoid characteristics, including total and average number of fluorescent cells/organoid, the average area of the spots per organoid, as well as the average intensity and area of the fluorescence of the identified spot. As another approach, we have attempted to measure the fluorescent cell projections. We optimized protocols for cryo-sectioning, optical clearing and light-sheet imaging of the organoids.
Objective 3 Part 3: 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.
Comparison of actual accomplishments with the goals and objectives (outputs/outcomes): Although we are still behind the original schedule with respect to cell line generation due to later hire and COVID and some further delays due to technical issues with CRISPR/Cas9 and lab supply shortage, we made substantial progress and are planning to finish QC and differentiation to BrainSpheres of three from four tags in 2023 Through the troubleshooting process, our lab gained substantial experience in CRISPR/Cas9 gene-editing, which is summarized in a methodological publication and the SOP. We are generating two multicolor iPSC lines instead of one line as proposed originally.
Future Activities:
Finalize cell line generation and QC. Establish HCI pipeline, establish electrophysiology recording upon chemical stimuli and toxicant treatment.
Journal Articles on this Report : 4 Displayed | Download in RIS Format
| Other project views: | All 9 publications | 9 publications in selected types | All 9 journal articles |
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Pistollato F, Bal-Price A, Coecke S, Parvatam S, Pamies D, Czysz K, Hao J, Kee K, Teo AK, Niu S, Wilmes A. Quality criteria for in vitro human pluripotent stem cell-derived models of tissue-based cells. Reproductive Toxicology 2022;112:36-50. |
R839505 (2022) |
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Huang Q, Tang B, Romero JC, Yang Y, Elsayed SK, Pahapale G, Lee TJ, Morales Pantoja IE, Han F, Berlinicke C, Xiang T. Shell microelectrode arrays (MEAs) for brain organoids. Science advances 2022;8(33):eabq5031. |
R839505 (2022) |
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Hogberg HT, Smirnova L. The future of 3D brain cultures in developmental neurotoxicity testing. Frontiers in Toxicology 2022;4:808620. |
R839505 (2022) |
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Smirnova L, Stacey GN. Progress in human stem cell-derived models for developmental and reproductive toxicology studies. Reproductive toxicology 2022:S0890-6238. |
R839505 (2022) |
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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.