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BRAIN Publication-derived

Impaired Neural Stress Resistance and Loss of REST in Bipolar Disorder — Cerebral Organoid Protocol

Source Meyer et al., 2024 · Harvard Medical School, Department of Genetics · 10.1038/s41380-023-02313-7

👤 Katharina Meyer, King-Hwa Ling, Pei-Ling Yeo, Angeliki Spathopoulou, Derek Drake, Jaejoon Choi, Liviu Aron, Mariana Garcia-Corral, Tak Ko, Eunjung Alice Lee, Jenny M. Tam, Roy H. Perlis, George M. Church, Li-Huei Tsai, Bruce A. Yankner ⏱ 160 days 📋 10 phases 🧫 Patient-Derived iPSC (Bipolar Disorder) and Control iPSC

Abstract

This protocol generates cerebral organoids from bipolar disorder (BD) and control iPSC lines to model neural development and identify disease-relevant phenotypes. BD organoids exhibit impaired neural stress resistance, altered early neural development, elevated neural network activity, and reduced REST transcription factor expression and function, which can be rescued by physiological oxygen levels or lithium treatment.

Cell source
Patient-Derived iPSC (Bipolar Disorder) and Control iPSC
Application
Disease modeling

Protocol overview

64 steps across 10 phases

iPSC Reprogramming and Culture Not specified
  1. 1 iPSC Generation from Fibroblasts
  2. 2 Culture iPSCs on Geltrex Matrix
  3. 3 Quality Control of iPSC Lines
Cerebral Organoid Generation and Early Development DIV 0–5
  1. 1 Seed iPSCs to Form Embryoid Bodies
  2. 2 Monitor EB Growth to 600 μm Diameter
  3. 3 Neural Induction Medium
  4. 4 Embed EBs in Matrigel
  5. 5 Grow Embedded EBs Without Shaking
Long-Term Organoid Maintenance and Differentiation DIV 5–90+
  1. 1 Switch to Differentiation Medium with Vitamin A and Begin Shaking
  2. 2 Regular Medium Changes
  3. 3 Monitor Organoid Morphology
Optional Treatment: Oxygen Reduction DIV 0–90
  1. 1 Reduce Oxygen Concentration to 5%
Optional Treatment: Lithium DIV 32–46
  1. 1 Prepare Lithium Carbonate Solution
  2. 2 Apply Lithium Treatment
  3. 3 Assess Phenotype After Lithium Treatment
Sample Collection for Immunofluorescence (DIV 45) DIV 45
  1. 1 Collect Organoid Samples
  2. 2 Fix in Paraformaldehyde
  3. 3 Equilibrate in 15% Sucrose Solution
  4. 4 Transfer to 30% Sucrose Solution
  5. 5 Embed in OCT Compound and Snap-Freeze
Cryosectioning and Immunofluorescence Microscopy After snap-freezing
  1. 1 Section Organoids Using Cryostat
  2. 2 Dry Sections
  3. 3 Rehydrate with PBS
  4. 4 Quench with Glycine
  5. 5 Block and Permeabilize
  6. 6 Wash with PBS
  7. 7 Incubate with Primary Antibody
  8. 8 Wash After Primary Antibody
  9. 9 Incubate with Secondary Antibody
  10. 10 Quench Autofluorescence
  11. 11 Mount Slides
  12. 12 Image Neural Rosettes
  13. 13 Threshold Images Based on Negative Control
  14. 14 Quantify REST Nuclear Intensity
  15. 15 Analyze Architecture with ImageJ
  16. 16 Perform Background Subtraction
  17. 17 Identify Rosette Outline
  18. 18 Measure Rosette Layer Thickness
  19. 19 Perform Watershed Segmentation and Cell Quantification
Calcium Imaging (Optional, DIV 160) DIV 160
  1. 1 Culture PLGA-Containing Organoids
  2. 2 Section PLGA-Containing Organoids
  3. 3 Culture Sections at Air-Liquid Interface
  4. 4 Mature Sections Until DIV 160
  5. 5 Treat with Fluo-4 Calcium Indicator Dye
  6. 6 Perform Live-Cell Spinning Disk Microscopy-Based Calcium Imaging
  7. 7 Apply CNMF-E Signal Processing
  8. 8 Analyze Synchronized and Un-Synchronized Neural Activity
Chromatin Immunoprecipitation (ChIP) DIV 45 or later
  1. 1 Collect Organoids for Chromatin Preparation
  2. 2 Cross-link Chromatin with Paraformaldehyde
  3. 3 Stop Cross-linking with Glycine
  4. 4 Chop and Lyse Organoids
  5. 5 Digest Chromatin with Micrococcal Nuclease
  6. 6 Quantify and Assess Chromatin Quality
  7. 7 Immunoprecipitate with REST Antibody
  8. 8 Recover ChIP DNA
  9. 9 Quantify ChIP DNA by qPCR
  10. 10 Verify PCR Product Purity
RNA Sequencing and Transcriptome Analysis DIV 46 (typical sampling point)
  1. 1 Harvest Organoids for RNA Extraction
  2. 2 Extract Total RNA
  3. 3 Prepare RNA-Sequencing Libraries
  4. 4 Perform RNA Sequencing
  5. 5 Analyze Transcriptome Data
  6. 6 Identify Transcription Factor Binding Sites
  7. 7 Deposit Data in Public Repository

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Meyer et al., 2024. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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