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BRAIN Publication-derived

Schizophrenia Brain Organoids with Single-Cell Profiling

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Source Sawada et al., 2024 · Lieber Institute for Brain Development · 10.1176/appi.ajp.20220723

Authors: Tomoyo Sawada, André R. Barbosa, Bruno Araujo, Alejandra E. McCord, Laura D'Ignazio, Kynon J.M. Benjamin, Bonna Sheehan, Michael Zabolocki, Arthur Feltrin, Ria Arora, Anna C. Brandtjen, Joel E. Kleinman, Thomas M. Hyde, Cedric Bardy, Daniel R. Weinberger, Apuã C.M. Paquola, Jennifer A. Erwin Duration: 241 days 8 phases Cell source: Patient-Derived iPSC (Schizophrenia) and Control iPSC

Abstract

This protocol describes the differentiation of iPSCs derived from schizophrenia patients and healthy controls into ventral forebrain organoids (VFOs) to model striatal neuronal development. VFOs recapitulate aspects of developing striatum and reveal accelerated maturation of inhibitory neurons in schizophrenia organoids, providing a human developmental platform to study neurodevelopmental mechanisms of schizophrenia.

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

Protocol overview

41 steps across 8 phases

Phase 1 · iPSC Generation and Maintenance Passage 5-7 fibroblasts to feeder-free iPSC culture
  1. a Reprogramming dura-derived fibroblasts to iPSCs
  2. b iPSC colony selection and initial expansion
  3. c Transfer to feeder-free culture
  4. d Pluripotency confirmation and characterization
Phase 2 · VFO Formation and Early Differentiation (Days 0-12) Days 0-12
  1. a iPSC dissociation and plating for organoid formation
  2. b Medium transition to DFN2
  3. c Addition of ventralization factors
  4. d Medium transition to DFN2B27 and addition of striatal patterning factors
  5. e Matrigel embedding and static culture
Phase 3 · VFO Maturation and Continued Differentiation (Days 16-60) Days 16-60
  1. a Switch to NBB27 medium and agitation
  2. b Addition of neurotrophic factors
  3. c Feeding schedule
  4. d Medium changes after day 60
Phase 4 · Real-Time qPCR Analysis Days 12-150
  1. a Organoid sample preparation
  2. b RNA extraction
  3. c cDNA synthesis
  4. d Real-time qPCR
Phase 5 · Immunostaining and Imaging Days 30-160
  1. a Organoid fixation
  2. b Sucrose cryoprotection
  3. c OCT embedding and freezing
  4. d Tissue section preparation and permeabilization
  5. e Primary antibody incubation
  6. f Secondary antibody incubation
  7. g Image acquisition and analysis
Phase 6 · Western Blotting Days 60-160
  1. a Organoid lysis
  2. b Protein quantification
  3. c SDS-PAGE gel electrophoresis
  4. d Protein transfer and detection
Phase 7 · Multi-Electrode Array (MEA) Recording Days 14-241
  1. a MEA plate preparation and organoid plating
  2. b Medium feeding
  3. c Spontaneous activity recording
  4. d Spike detection and analysis
  5. e Pharmacological manipulation
  6. f Drug treatment experiments
  7. g High-density MEA recording
Phase 8 · Single-Cell RNA Sequencing (scRNA-seq) Sample Preparation Days 70 and 150
  1. a Organoid selection and dissociation
  2. b Cell hashing preparation
  3. c Hashtag antibody staining
  4. d Sample pooling and filtering
  5. e Cell concentration adjustment and loading
  6. f Library preparation

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Attribution

This SOP was written by Organthis based on the published method in Sawada et al., 2024. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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