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Development of a Functional Assay using Human Brain Organoids to Study Developmental Neurotoxicity
Citation:
Conley, J., S. Boyd, E. Hunter, AND T. Shafer. Development of a Functional Assay using Human Brain Organoids to Study Developmental Neurotoxicity. SOT Conference 2024: A New Approach Method (NAM) to Screen for the Impact of Endogenous Stress on Chemical Toxicity, Salt Lake City, UT, March 10 - 14, 2024. https://doi.org/10.23645/epacomptox.25488151
Impact/Purpose:
Presentation to SOT Conference 2024: A New Approach Method (NAM) to Screen for the Impact of Endogenous Stress on Chemical Toxicity
Description:
Background and Purpose: Human in vitro models of the developing brain are important for studying neurodevelopment and the potential developmental neurotoxicity (DNT) of environmental and pharmacological compounds. Exposure to these compounds during neurodevelopment can cause adverse effects such as morphological alterations and/or functional changes in the developing brain. Over the past 15 years, in-vitro two-dimensional models have been developed as assays to characterize chemicals for potential DNT hazard. Organoids are a three-dimensional (3D) cell culture system that mimic the complex structure and development of the human brain better than their two-dimensional counterparts. Here, the time course for the development of spontaneous electrical activity in the organoids was evaluated as well as the response of that activity to different pharmacological manipulations. Methods: A human induced pluripotent stem cell (iPSC)-derived brain organoid model (PMID: 27883356) that develops mature neurons and glial cells was established and characterized. Over a 10-week differentiation period, immunocytochemistry of the organoids was performed using neural progenitor markers (Nestin, PAX6, SOX2), neuronal markers (β3Tub, Tbr2), astrocyte markers (S100β, GFAP), and an oligodendrocyte marker (Olig1). Neural organoids were plated on a high-density microelectrode array (hdMEA) at different ages to measure the spontaneous electric field potentials produced as networks developed. The organoids were treated acutely with three common pharmacological agents: picrotoxin (PTX), tetrodotoxin (TTX) and nicotine (NIC). PTX (25μM), TTX (1μM), and NIC (300μM) were added to the organoids to target GABAA receptors, voltage-gated sodium channels, and nicotinic acetylcholine receptors respectively. Treatment was followed by consecutive 10-minute recordings for an hour post-exposure. Results: Immunocytochemistry and high-content imaging of the organoids showed a decrease in proliferation and neural progenitor markers (Nestin, PAX6, SOX2) and an increase in neuronal (β3Tub, Tbr2), astrocyte (S100β, GFAP), and oligodendrocyte (Olig1) markers during differentiation (weeks 0-10), indicating that during this time the organoids progressed along a neurodevelopmental ontogeny. Time course analysis of the developing organoids showed complex network formation, characterized by an increase in mean firing rate, spike amplitude, and neuronal outgrowth (percent active electrodes) from week 3 to week 10. Treatment with PTX, TTX, and NIC caused functional changes (burst frequency, synchronicity, and inter-burst interval) in the organoids’ spontaneous network activity. Exposing an immature ( < week 6) organoid to PTX resulted in a decrease in network activity while exposing a mature organoid ( > week 7.5) resulted in an increase in network activity and synchronicity. This recapitulates the developmental change of the GABAA receptors from excitatory to inhibitory as the neurons in the organoid mature. As expected, treatment with TTX resulted in decreased network activity following the acute one-hour exposure. NIC caused an initial decrease in network activity of 5-7 minutes followed by an increase in synchronicity and decrease in inter-burst . . .
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DOI: Development of a Functional Assay using Human Brain Organoids to Study Developmental Neurotoxicity
JCONLEY.24SOTPOSTER.FINAL.PDF (PDF, NA pp, 909.893 KB, about PDF)