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BRAIN Publication-derived

Schizophrenia Cortical Organoids

Full SOP with Academic or a plan

Source Ye et al., 2017 · Johns Hopkins University School of Medicine; University of Pennsylvania; Hong Kong University of Science and Technology · 10.1016/j.neuron.2017.10.010

Authors: Fei Ye, Eunchai Kang, Chuan Yu, Xuyu Qian, Fadi Jacob, Cong Yu, Mao Mao, Randy Y. C. Poon, Jieun Kim, Hongjun Song, Guo-li Ming, Mingjie Zhang 7 phases Cell source: Human iPSC, Patient-Derived iPSC (Schizophrenia with DISC1 mutation)

Abstract

This protocol describes methods for generating and characterizing forebrain-specific organoids derived from human iPSCs to study the DISC1/Ndel1 interaction in neural stem cell proliferation and its role in schizophrenia pathogenesis. The organoids enable investigation of how DISC1 mutations affect radial glial cell cycle progression during human brain development.

Cell source
Human iPSC, Patient-Derived iPSC (Schizophrenia with DISC1 mutation)
Application
Disease modeling

Protocol overview

26 steps across 7 phases

Phase 1 · iPSC Colony Preparation and Dissociation Day 0-1
  1. a Passage and Detach iPSC Colonies
  2. b Transfer to Ultra-Low Attachment Plate
Phase 2 · Organoid Induction and Matrigel Embedding Day 5-14
  1. a Medium Exchange with Induction Medium
  2. b Matrigel Embedding
  3. c Mechanical Dissociation from Matrigel
Phase 3 · Organoid Culture and Differentiation Day 14-47
  1. a Culture in Differentiation Medium
  2. b Monitor Organoid Development
Phase 4 · Plasmid Electroporation into Organoids Day 45-47
  1. a Prepare Organoids for Electroporation
  2. b Inject Plasmids into Ventricular Structures
  3. c Apply Electroporation Pulses
  4. d Return Organoids to Bioreactor Culture
Phase 5 · EdU Pulsing and Sample Collection Day 49-50 (post-electroporation days 4-5)
  1. a Pulse with EdU
  2. b Replace Medium and Continue Culture
  3. c Fix Organoids
Phase 6 · Cryosectioning and Immunohistochemistry Post-fixation
  1. a Cryoprotection and Embedding
  2. b Cryosection Organoids
  3. c Permeabilization
  4. d Blocking
  5. e Primary Antibody Incubation
  6. f Washing
  7. g Secondary Antibody Incubation
  8. h EdU Detection
  9. i Nuclear Counterstaining
  10. j Mounting and Imaging
Phase 7 · Image Quantification and Analysis Post-imaging
  1. a Quantify Cell Populations
  2. b Statistical Analysis

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Attribution

This SOP was written by Organthis based on the published method in Ye et al., 2017. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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