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BRAIN Publication-derived

Bipolar Disorder Cerebral Organoids for Disease Modelling

Full SOP with Academic or a plan

Source Kathuria et al., 2020 · Massachusetts General Hospital, Harvard Medical School · 10.1186/s13073-020-00733-6

Authors: Annie Kathuria, Kara Lopez-Lengowski, Magdalena Vater, Donna McPhie, Bruce M. Cohen, Rakesh Karmacharya Duration: 270 days 14 phases Cell source: Patient-Derived iPSC (Bipolar Disorder)

Abstract

This protocol describes the generation of cerebral organoids patterned after the dorsal forebrain from patient-derived and control iPSCs, followed by molecular and functional characterization to identify disease-specific transcriptomic differences and synaptic deficits in bipolar disorder using RNA-seq, immunohistochemistry, proximity ligation assays, and microelectrode array recordings.

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

Protocol overview

38 steps across 14 phases

Phase 1 · iPSC reprogramming from fibroblasts 0 to ongoing
  1. a Fibroblast collection and patient screening
  2. b Fibroblast reprogramming to iPSCs
Phase 2 · iPSC culture and maintenance Ongoing prior to organoid differentiation
  1. a iPSC culture on Geltrex substrate
Phase 3 · Cerebral organoid generation—embryoid body formation Days 1-5
  1. a Embryoid body (EB) formation
Phase 4 · EB induction and expansion Days 6-7
  1. a EB resuspension in induction media
  2. b EB embedding in Matrigel via droplet hang method
Phase 5 · Cerebral organoid maturation Days 10-180 (6 months)
  1. a Media switch to maturation media and orbital shaking
  2. b BDNF supplementation
Phase 6 · Differentiation of iPSCs to excitatory cortical neurons Days 1-90
  1. a iPSC neural induction (Days 1-7)
  2. b Early differentiation without SMAD inhibitors (Days 8-29)
  3. c Late maturation with Brainphys media (Days 30-90)
Phase 7 · Immunohistochemistry sample preparation—OCT method Day of analysis
  1. a Organoid fixation and sucrose cryoprotection
  2. b OCT embedding and cryosectioning
Phase 8 · Immunohistochemistry sample preparation—Paraffin method Day of analysis
  1. a Fixation and dehydration series
  2. b Paraffin embedding and microtome sectioning
Phase 9 · Immunohistochemistry staining Day of analysis
  1. a Slide washing and permeabilization
  2. b Blocking and primary antibody incubation
  3. c Secondary antibody incubation and mounting
  4. d High-content imaging
Phase 10 · Proximity ligation assay (PLA) for ER–mitochondria interactions Day of analysis
  1. a Cortical neuron fixation and permeabilization
  2. b Blocking and primary antibody incubation (PLA)
  3. c PLA probe incubation
  4. d Ligation, amplification, and detection
Phase 11 · RNA extraction and transcriptome analysis Day of harvest (6-month organoids)
  1. a RNA extraction from organoids
  2. b RNA-seq library construction
  3. c Illumina NovaSeq sequencing
Phase 12 · Bioinformatic analysis: read alignment and gene expression quantification Post-sequencing
  1. a Read trimming and alignment
  2. b FPKM quantification and normalization
  3. c Differential expression testing
Phase 13 · Gene ontology, enrichment, and network analyses Post-sequencing analysis
  1. a Gene ontology and KEGG pathway analysis
  2. b Protein–protein interaction (PPI) network construction
  3. c Overlap analysis with disease-associated gene databases
Phase 14 · Microelectrode array (MEA) functional recording Days 180-270 (6-9 month organoids)
  1. a MEA plate preparation and organoid attachment
  2. b Organoid culture on MEA until maturation
  3. c Baseline spontaneous activity recording
  4. d Electrical stimulation and recording
  5. e KCl depolarization and recording
  6. f Tetrodotoxin validation recording

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Attribution

This SOP was written by Organthis based on the published method in Kathuria et al., 2020. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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