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BRAIN Publication-derived

Impact of alcohol exposure on neural development and network formation in human cortical organoids

Source Adams et al., 2023 · University of California San Diego, Department of Pediatrics/Rady Children's Hospital · 10.1038/s41380-022-01862-7

👤 Jason W. Adams, Priscilla D. Negraes, Justin Truong, Timothy Tran, Ryan A. Szeto, Bruno S. Guerra, Roberto H. Herai, Carmen Teodorof-Diedrich, Stephen A. Spector, Miguel Del Campo, Kenneth L. Jones, Alysson R. Muotri, Cleber A. Trujillo ⏱ 84 days 📋 12 phases 🧫 Human iPSC (WT83C6, CVB, 4C1) and primary human fetal neurons (10-11 PCW)

Abstract

This protocol examines the effects of ethanol (EtOH) exposure on human cortical organoid development, neural differentiation, and network formation. hiPSC-derived cortical organoids are exposed to 100 mM EtOH (stabilizing to ~20 mM) for seven days, followed by characterization of proliferation, apoptosis, epigenetic alterations, transcriptomic changes, and electrophysiological activity to model prenatal alcohol exposure and fetal alcohol spectrum disorders.

Cell source
Human iPSC (WT83C6, CVB, 4C1) and primary human fetal neurons (10-11 PCW)
Application
Disease modeling - Fetal Alcohol Spectrum Disorders (FASD) pathophysiology

Protocol overview

69 steps across 12 phases

Cell source preparation and expansion Days 1-21 (prior to EtOH exposure)
  1. 1 Expand hiPSC lines on Matrigel-coated dishes
  2. 2 Prepare primary fetal tissue cultures
  3. 3 Differentiate hiPSC-derived cortical organoids
  4. 4 Differentiate hiPSC-derived astrocytes
Ethanol (EtOH) exposure Days 22-28 (7-day exposure period)
  1. 1 Prepare EtOH-saturated atmosphere
  2. 2 Supplement culture media with 100 mM EtOH
  3. 3 Maintain EtOH exposure for seven days
Post-exposure maintenance (recovery phase) Days 29-84 (variable duration depending on assay endpoint)
  1. 1 Maintain EtOH-exposed cells in normal media (no EtOH)
  2. 2 Feed cultures routinely during recovery period
Cellular phenotyping - Cell cycle analysis Days 36-56 post-EtOH exposure initiation
  1. 1 Manually dissociate and count cells
  2. 2 Fix cells with 70% ethanol
  3. 3 Resuspend in DAPI staining solution
  4. 4 Load cells and quantify by flow cytometry
Cellular phenotyping - Apoptosis and cell death assessment Days 36-56 post-EtOH exposure initiation
  1. 1 Manually dissociate cells
  2. 2 Resuspend in Annexin V binding buffer
  3. 3 Add Annexin V conjugate and Hoechst
  4. 4 Wash and add propidium iodide
  5. 5 Load cells and run Annexin V assay
Epigenetic profiling - ATAC-Seq (chromatin accessibility) Days 50-56 (cortical organoids at two months post-EtOH onset)
  1. 1 Cryopreserve organoid samples
  2. 2 Transfer samples to Active Motif for ATAC-Seq
  3. 3 Perform ATAC-Seq at Active Motif
  4. 4 Call peaks using MACS2
  5. 5 Perform quality control on peaks
  6. 6 Perform motif and functional enrichment analysis
Epigenetic profiling - Histone mass spectrometry Days 36-56 (multiple timepoints)
  1. 1 Harvest and lyse cells for histone extraction
  2. 2 Acid extract histones from cell pellets
  3. 3 Wash and air-dry histone pellets
  4. 4 Resuspend histones and perform propionylation
  5. 5 Digest histones with trypsin
  6. 6 Resuspend histone peptides for MS analysis
  7. 7 Perform LC-MS/MS analysis
  8. 8 Repeat MS analysis three times per sample
  9. 9 Analyze MS data using Skyline
  10. 10 Calculate relative abundance of histone modifications
Synaptic morphology - Synaptic puncta quantification Days 50-56 (cortical organoids at two months post-EtOH onset)
  1. 1 Prepare organoid tissue sections
  2. 2 Perform triple immunofluorescence staining
  3. 3 Incubate secondary antibodies
  4. 4 Mount coverslips and visualize
  5. 5 Quantify synaptic puncta density
Spatial proteomics - Nanostring GeoMx Digital Spatial Profiling (DSP) Days 50-56 (cortical organoids at two months post-EtOH onset)
  1. 1 Prepare organoid tissue sections
  2. 2 Stain tissue with imaging reagents and cell markers
  3. 3 Select protein panels for analysis
  4. 4 Scan slides on GeoMx DSP instrument
  5. 5 Select regions of interest (ROI/AOI)
  6. 6 Perform UV-driven photocleavage and oligonucleotide collection
  7. 7 Quantify oligonucleotides on nCounter
  8. 8 Analyze data using GeoMx DSP software
Electrophysiology - Multi-electrode array (MEA) recording Days 29-49 (cortical organoids at 1 month, then maturation for 2-3 weeks on MEA plates)
  1. 1 Prepare MEA plates with adhesion molecules
  2. 2 Plate cortical organoids on MEA plates
  3. 3 Maintain organoids on MEA plates
  4. 4 Begin MEA recordings 14 days post-plating
  5. 5 Record spontaneous neural activity
  6. 6 Perform spike and burst detection
  7. 7 Analyze network synchrony
  8. 8 Continue weekly MEA recordings
Drug rescue screening Days 64-84 (EtOH-exposed organoids at 2.5 months age, treated for 3 weeks)
  1. 1 Prepare drug compounds
  2. 2 Treat EtOH-exposed organoids with drug compounds
  3. 3 Treat control organoids with vehicle
  4. 4 Harvest organoids immediately after drug treatment
Western blot analysis of synaptic and astrocytic proteins Days 36-56 and post-drug treatment (various timepoints)
  1. 1 Prepare protein lysates from organoids
  2. 2 Quantify total protein concentration
  3. 3 Prepare protein samples for gel loading
  4. 4 Load and run SDS-PAGE gels
  5. 5 Transfer proteins to PVDF membrane
  6. 6 Block membrane with blocking buffer
  7. 7 Incubate with primary antibodies
  8. 8 Wash and incubate with secondary antibodies
  9. 9 Detect protein bands using chemiluminescence
  10. 10 Quantify band intensities

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Adams et al., 2023. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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