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BRAIN Publication-derived

Schizophrenia Cerebral Organoids (2018)

Full SOP with Academic or a plan

Source Srikanth et al., 2018 · Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School · 10.1038/s41398-018-0122-x

Authors: Priya Srikanth, Valentina N. Lagomarsino, Christina R. Muratore, Steven C. Ryu, Amy He, Walter M. Taylor, Constance Zhou, Marlise Arellano, Tracy L. Young-Pearse Duration: 40 days 6 phases Cell source: Human iPSC (isogenic with DISC1 disruption in exon 8)

Abstract

This protocol describes the generation of three-dimensional cerebral organoids from isogenic human induced pluripotent stem cells (iPSCs) with and without DISC1 disruption, to model the neurodevelopmental consequences of DISC1 mutation. The protocol characterizes organoid morphology, neural progenitor proliferation, and gene expression changes, revealing that DISC1 disruption causes disorganized rosette structures and impaired proliferation through elevated WNT signaling.

Cell source
Human iPSC (isogenic with DISC1 disruption in exon 8)
Application
Disease modeling - psychiatric disease (schizophrenia risk)

Protocol overview

18 steps across 6 phases

Phase 1 · Embryoid Aggregate Formation and Neural Induction Days 0-10
  1. a iPSC Dissociation and AggreWell Plating
  2. b Transition to Neural Induction Media (Day 6)
  3. c Matrigel Embedding and Transfer to Non-Adhesive Culture (Day 10)
Phase 2 · Organoid Differentiation and Pharmacological Treatment Days 10-19
  1. a Organoid Culture in Differentiation Media
  2. b WNT Pathway Modulation (Days 6-19)
Phase 3 · EdU Labeling and Cell Proliferation Assessment Days 6-19
  1. a EdU Pulse-Chase Labeling
  2. b EdU Detection by Click Chemistry
  3. c Blinded Quantification of EdU Incorporation
Phase 4 · Immunofluorescence Staining and Morphological Analysis Day 19
  1. a Organoid Fixation and Permeabilization
  2. b Primary Antibody Incubation
  3. c Secondary Antibody Incubation and Mounting
  4. d Image Acquisition and Morphological Quantification
Phase 5 · Gene Expression Analysis via Nanostring and qPCR Day 19 (organoids) or Day 40 (monolayer neurons)
  1. a RNA Extraction from Organoids or Monolayer Neurons
  2. b Nanostring Gene Expression Analysis
  3. c Quantitative PCR (qPCR) Validation
Phase 6 · Cell Line Validation and Quality Control Prior to and after completion of study
  1. a Mycoplasma Testing
  2. b STR Profiling for Cell Line Identity
  3. c Karyotype Analysis

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Attribution

This SOP was written by Organthis based on the published method in Srikanth et al., 2018. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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