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BRAIN Publication-derived

Modeling alcohol-induced neurotoxicity using human induced pluripotent stem cell-derived three-dimensional cerebral organoids

Source Arzua et al., 2020 · Medical College of Wisconsin · 10.1038/s41398-020-01029-4

👤 Thiago Arzua, Yasheng Yan, Congshan Jiang, Sarah Logan, Reilly L. Allison, Clive Wells, Suresh N. Kumar, Richard Schäfer, Xiaowen Bai ⏱ 61 days 📋 13 phases 🧫 Human iPSC

Abstract

This protocol describes the generation of 2-month-old human iPSC-derived cerebral organoids and their exposure to ethanol to model binge drinking-induced neurotoxicity in fetal brain development. The organoids are characterized for apoptosis, ultrastructural changes, mitochondrial dysfunction, and gene expression alterations to understand the mechanisms of fetal alcohol spectrum disorder.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

66 steps across 13 phases

Human iPSC Culture and Expansion Days -28 to 0 (pre-organoid generation)
  1. 1 Maintain iPSCs on Matrigel-coated dishes
  2. 2 Change culture medium daily
  3. 3 Passage iPSCs at 80% confluency
  4. 4 Test for mycoplasma contamination
3D Cerebral Organoid Generation from iPSCs Days 0–16 (organoid initiation)
  1. 1 Singularize iPSCs and seed in 96-well ultra-low attachment plates
  2. 2 Culture to form embryoid bodies
  3. 3 Transfer embryoid bodies to 24-well plates
  4. 4 Embed in Matrigel droplets
  5. 5 Transfer to spinner platform for long-term culture
Characterization of iPSCs and Cerebral Organoids by Immunostaining Throughout culture (Days 0–56) for milestone characterization
  1. 1 Fix iPSCs with paraformaldehyde
  2. 2 Stain iPSCs with pluripotency markers
  3. 3 Fix and section cerebral organoid tissue
  4. 4 Stain organoid sections with neural markers
  5. 5 Image samples with confocal or slide scanner microscopy
Ethanol Exposure and Assessment of Apoptosis Days 56–58 (2-month organoid treatment period)
  1. 1 Prepare ethanol-containing culture medium
  2. 2 Seal culture dishes with Parafilm to prevent ethanol evaporation
  3. 3 Expose 2-month organoids to ethanol for 6 hours
  4. 4 Verify ethanol concentration in culture medium
  5. 5 Harvest organoids for downstream analyses
Caspase 3 Activity Assay Day 58 (post-ethanol harvest)
  1. 1 Prepare cell lysates from control and ethanol-treated organoids
  2. 2 Quantify total protein concentration
  3. 3 Perform caspase 3 activity assay
Western Blot Analysis of Activated Caspase 3 Day 58 (post-ethanol harvest)
  1. 1 Prepare protein samples for electrophoresis
  2. 2 Separate proteins by gel electrophoresis
  3. 3 Transfer proteins to membrane
  4. 4 Incubate membrane with primary antibody against activated caspase 3
  5. 5 Wash membrane and incubate with secondary antibody
  6. 6 Detect protein bands using ECL reagent and imaging
Immunofluorescence and TUNEL Staining for Cell-Type-Specific Apoptosis Day 58 (post-ethanol harvest)
  1. 1 Fix control and ethanol-treated organoid sections
  2. 2 Perform immunofluorescence staining with neuron and astrocyte markers
  3. 3 Perform TUNEL staining for apoptotic cells
  4. 4 Counterstain nuclei and mount slides
  5. 5 Image slides using whole-slide scanners
Electron Microscopy Analysis of Ultrastructural Changes Day 58 (post-ethanol harvest)
  1. 1 Primary fixation of organoids with glutaraldehyde
  2. 2 Wash organoids in cold buffer
  3. 3 Post-fixation with osmium tetroxide
  4. 4 Wash organoids in distilled water
  5. 5 Dehydrate organoids through graded ethanol series
  6. 6 Embed in epoxy resin and polymerize
  7. 7 Section and stain ultrathin tissue
  8. 8 Image tissue sections with electron microscope
Mitochondrial Bioenergetics Analysis Day 58 (post-ethanol harvest)
  1. 1 Prepare organoid tissue for bioenergetic assay
  2. 2 Prepare assay medium for mitochondrial oxidation
  3. 3 Measure baseline oxygen consumption rate (OCR)
  4. 4 Sequentially inject ATP synthase inhibitor (oligomycin)
  5. 5 Inject uncoupler (FCCP) to measure maximal respiration
  6. 6 Inject electron transport chain blocker (antimycin A)
  7. 7 Calculate OCR parameters
RNA Extraction and Microarray Analysis Day 58 (post-ethanol harvest)
  1. 1 Extract total RNA from organoids
  2. 2 Quantify and assess RNA quality
  3. 3 Submit RNA to microarray service provider
  4. 4 Receive processed microarray data and analyze
Reverse Transcription-Quantitative PCR (RT-qPCR) Validation Day 58–59 (post-ethanol harvest)
  1. 1 Synthesize complementary DNA (cDNA) from isolated RNA
  2. 2 Set up RT-qPCR reactions for 5 representative genes
  3. 3 Run qPCR on iCycler instrument
  4. 4 Collect Ct values and normalize to endogenous control
  5. 5 Compare RT-qPCR validation data with microarray results
Bioinformatics Analysis of Dysregulated Genes and Signaling Pathways Days 59–60 (post-data generation)
  1. 1 Input dysregulated gene data into Ingenuity Pathway Analysis (IPA) software
  2. 2 Perform Diseases and Functions analysis
  3. 3 Identify gene networks and interaction maps
  4. 4 Perform literature validation of dysregulated genes
Statistical Analysis and Data Visualization Days 59–61 (post-data generation)
  1. 1 Compile all experimental data
  2. 2 Test data for normality and outliers
  3. 3 Compare control and ethanol-treated groups using unpaired t-tests
  4. 4 Assess variation within and between groups
  5. 5 Generate publication-quality figures

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Arzua et al., 2020. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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