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BRAIN Publication-derived

Generation and functional characterization of cerebral organoids from bipolar disorder patient-derived iPSCs

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

👤 Annie Kathuria, Kara Lopez-Lengowski, Magdalena Vater, Donna McPhie, Bruce M. Cohen, Rakesh Karmacharya ⏱ 270 days 📋 14 phases 🧫 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

iPSC reprogramming from fibroblasts 0 to ongoing
  1. 1 Fibroblast collection and patient screening
  2. 2 Fibroblast reprogramming to iPSCs
iPSC culture and maintenance Ongoing prior to organoid differentiation
  1. 3 iPSC culture on Geltrex substrate
Cerebral organoid generation—embryoid body formation Days 1–5
  1. 4 Embryoid body (EB) formation
EB induction and expansion Days 6–7
  1. 5 EB resuspension in induction media
  2. 6 EB embedding in Matrigel via droplet hang method
Cerebral organoid maturation Days 10–180 (6 months)
  1. 7 Media switch to maturation media and orbital shaking
  2. 8 BDNF supplementation
Differentiation of iPSCs to excitatory cortical neurons Days 1–90
  1. 9 iPSC neural induction (Days 1–7)
  2. 10 Early differentiation without SMAD inhibitors (Days 8–29)
  3. 11 Late maturation with Brainphys media (Days 30–90)
Immunohistochemistry sample preparation—OCT method Day of analysis
  1. 12 Organoid fixation and sucrose cryoprotection
  2. 13 OCT embedding and cryosectioning
Immunohistochemistry sample preparation—Paraffin method Day of analysis
  1. 14 Fixation and dehydration series
  2. 15 Paraffin embedding and microtome sectioning
Immunohistochemistry staining Day of analysis
  1. 16 Slide washing and permeabilization
  2. 17 Blocking and primary antibody incubation
  3. 18 Secondary antibody incubation and mounting
  4. 19 High-content imaging
Proximity ligation assay (PLA) for ER–mitochondria interactions Day of analysis
  1. 20 Cortical neuron fixation and permeabilization
  2. 21 Blocking and primary antibody incubation (PLA)
  3. 22 PLA probe incubation
  4. 23 Ligation, amplification, and detection
RNA extraction and transcriptome analysis Day of harvest (6-month organoids)
  1. 24 RNA extraction from organoids
  2. 25 RNA-seq library construction
  3. 26 Illumina NovaSeq sequencing
Bioinformatic analysis: read alignment and gene expression quantification Post-sequencing
  1. 27 Read trimming and alignment
  2. 28 FPKM quantification and normalization
  3. 29 Differential expression testing
Gene ontology, enrichment, and network analyses Post-sequencing analysis
  1. 30 Gene ontology and KEGG pathway analysis
  2. 31 Protein–protein interaction (PPI) network construction
  3. 32 Overlap analysis with disease-associated gene databases
Microelectrode array (MEA) functional recording Days 180–270 (6–9 month organoids)
  1. 33 MEA plate preparation and organoid attachment
  2. 34 Organoid culture on MEA until maturation
  3. 35 Baseline spontaneous activity recording
  4. 36 Electrical stimulation and recording
  5. 37 KCl depolarization and recording
  6. 38 Tetrodotoxin validation recording

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Kathuria et al., 2020. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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