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BRAIN Publication-derived

Generation and Characterization of Brain Organoids from hiPSCs and ZIKV Infection with PERK Inhibition

Source Lee et al., 2022 · Seoul National University, Konkuk University · 10.1038/s41420-022-00958-x

👤 Seung-Eun Lee, Hanul Choi, Nari Shin, Dasom Kong, Nam Gyo Kim, Hee-Yeong Kim, Min-Ji Kim, Soon Won Choi, Young Bong Kim, Kyung-Sun Kang ⏱ 98 days 📋 12 phases 🧫 Human iPSC (Wild-type), Patient-Derived iPSC (PSEN1 mutation)

Abstract

This protocol describes the generation of 3D cerebral brain organoids from human iPSCs (wild-type and Alzheimer's disease patient-derived), characterization of cortex-like structures, infection with Zika virus (ZIKV), and treatment with PERK inhibitors. The protocol demonstrates that ZIKV infection accelerates AD pathological features including β-amyloid and phosphorylated-Tau accumulation through endoplasmic reticulum stress and PERK-eIF2α pathway activation.

Cell source
Human iPSC (Wild-type), Patient-Derived iPSC (PSEN1 mutation)
Application
Disease modeling, Viral infection study, Alzheimer's disease pathogenesis

Protocol overview

45 steps across 12 phases

hiPSC Maintenance and Preparation Ongoing, passage every 4-6 days
  1. 1 Maintain wild-type hiPSCs in Essential 8 medium
  2. 2 Maintain AD patient-derived hiPSCs in mTESR plus medium
Embryoid Body Formation Days 0–5
  1. 1 Dissociate and aggregate iPSCs
  2. 2 Change medium on day 3
  3. 3 Transfer embryoid bodies to 24-well plates when diameter exceeds 500 µm
Neural Induction and Early Organoid Formation Days 5–11
  1. 1 Culture in neural induction medium
  2. 2 Embed neuroepithelial tissue in Matrigel
Organoid Differentiation and Maturation Days 11–75+
  1. 1 Culture in cerebral organoid differentiation medium (CODM) – stationary phase
  2. 2 Transfer to orbital shaker with modified CODM
  3. 3 Monitor organoid size and morphology
Viral Stock Preparation Prior to infection
  1. 1 Obtain and propagate ZIKV stock
  2. 2 Perform plaque assay for ZIKV titration
  3. 3 Obtain and propagate vaccinia virus (VACV) control stock
ZIKV and VACV Infection of Brain Organoids Day 60 to 14 dpi (Day 75)
  1. 1 Prepare organoids for infection
  2. 2 Infect organoids with ZIKV
  3. 3 Replace virus-containing medium with fresh CODM
  4. 4 Maintain infected organoids in culture with regular medium changes
  5. 5 Collect supernatant at designated time points for plaque assay
PERK Inhibitor Treatment Day 60 to 14 dpi (Day 75)
  1. 1 Prepare GSK2656157 (PERK inhibitor) solution
  2. 2 Infect organoids with ZIKV (0.2 MOI) and treat with PERK inhibitor
  3. 3 Continue inhibitor treatment for 14 days
Viral Confirmation by RT-qPCR Days 64, 67, 74 (4 dpi, 7 dpi, 14 dpi)
  1. 1 Extract total RNA from organoids
  2. 2 Synthesize complementary DNA (cDNA)
  3. 3 Perform qRT-PCR for ZIKV NS5
Protein Analysis by Western Blotting 14 dpi (Day 75)
  1. 1 Prepare organoid lysates
  2. 2 Quantify total protein
  3. 3 Perform SDS-PAGE and transfer
  4. 4 Block and probe with primary antibodies
  5. 5 Incubate with secondary antibodies and detect
Amyloid-Beta ELISA Day 60 (WT/AD characterization) or 14 dpi (post-infection)
  1. 1 Prepare RIPA lysates for Aβ measurement
  2. 2 Quantify total protein by BCA assay
  3. 3 Measure Aβ₄₀ and Aβ₄₂ by ELISA
Immunofluorescence Staining and 3D Imaging Day 60 (characterization) or 14 dpi (post-infection)
  1. 1 Fix organoids in paraformaldehyde
  2. 2 Cryoprotect and embed organoids
  3. 3 Prepare cryosections
  4. 4 Perform antigen retrieval
  5. 5 Block and incubate with primary antibodies
  6. 6 Incubate with secondary antibodies
  7. 7 Stain nuclei and mount
  8. 8 Image and analyze immunofluorescence
  9. 9 Perform 3D clearing and imaging of whole organoids
RNA Extraction and qRT-PCR for ER Stress and UPR Markers Day 38 post-infection (Day 98) or Day 60
  1. 1 Extract total RNA from organoids
  2. 2 Synthesize cDNA
  3. 3 Perform qRT-PCR for ER stress/UPR genes
  4. 4 Expected results for ZIKV-infected organoids

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Lee et al., 2022. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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