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BRAIN Publication-derived

Schizophrenia Brain Organoids (2023)

Full SOP with Academic or a plan

Source Kathuria et al., 2023 · Massachusetts General Hospital, Center for Genomic Medicine · 10.1007/s44192-023-00031-8

Authors: Annie Kathuria, Kara Lopez-Lengowski, Donna McPhie, Bruce M. Cohen, Rakesh Karmacharya 10 phases Cell source: Patient-Derived iPSC (schizophrenia); Patient-Derived iPSC (bipolar disorder)

Abstract

This protocol generates iPSC-derived cerebral organoids and cortical neurons from patients with schizophrenia and bipolar disorder to identify disease-specific differences in gene expression, mitochondrial respiration, and mitochondria–endoplasmic reticulum interactions. The work compares transcriptomic profiles and functional parameters between the two psychiatric disorders using RNA-seq, Seahorse metabolic assays, and proximity ligation assays.

Cell source
Patient-Derived iPSC (schizophrenia); Patient-Derived iPSC (bipolar disorder)
Application
Disease modeling; Comparative transcriptomic analysis

Protocol overview

32 steps across 10 phases

Phase 1 · iPSC Culture Maintenance and Preparation Ongoing until confluency
  1. a Maintain iPSC cultures in NutriStem hPSC XF Medium
Phase 2 · Cortical Neuron Differentiation – Initial Differentiation Phase Days 0-7
  1. a Plate iPSCs on Geltrex-coated plates
  2. b Prepare N2/B27 differentiation medium for first 7 days
  3. c Culture cells in N2/B27 media with morphogens for 7 days
Phase 3 · Cortical Neuron Differentiation – Intermediate Phase Days 8-29
  1. a Daily medium changes in N2/B27 media without morphogens
  2. b Monitor morphological differentiation
Phase 4 · Cortical Neuron Differentiation – Maturation Phase Day 30 onwards
  1. a Transition to Brainphys Neuronal Medium
  2. b Perform medium changes twice weekly
Phase 5 · Cerebral Organoid Generation and Culture 0-9 months
  1. a Initiate cerebral organoid differentiation
  2. b Culture organoids for 6-9 months
Phase 6 · RNA Extraction and Library Preparation Upon organoid harvest
  1. a Extract RNA from cerebral organoids
  2. b Construct RNA-seq libraries
Phase 7 · RNA Sequencing and Data Processing Library prep completion to data analysis
  1. a Perform paired-end RNA sequencing
  2. b Trim reads and align to reference genome
  3. c Quantify gene expression
  4. d Perform differential expression testing
Phase 8 · Gene Ontology and Gene Set Enrichment Analysis Data analysis phase
  1. a Perform KEGG and GO analysis
  2. b Perform gene set enrichment analysis (GSEA)
  3. c Map DEGs to mitochondrial pathways
Phase 9 · Seahorse Mitochondrial Respiration Assay Organoid age: 9 months
  1. a Prepare organoid sections and microplate coating
  2. b Plate organoid sections on coated microplates
  3. c Perform Seahorse XF Cell Mito Stress Test
  4. d Collect and analyze Seahorse data
Phase 10 · Proximity Ligation Assay (PLA) for Mitochondria–ER Interactions Neurons at 90 days differentiation
  1. a Prepare and fix cortical neurons
  2. b Permeabilize cells
  3. c Apply blocking solution
  4. d Incubate with primary antibodies
  5. e Wash and add PLA probes
  6. f Perform ligation reaction
  7. g Perform amplification reaction
  8. h Final wash and mounting
  9. i Image and quantify PLA signal

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Attribution

This SOP was written by Organthis based on the published method in Kathuria et al., 2023. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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