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BRAIN Publication-derived

Schizophrenia Cerebral Organoids for Developmental Studies

Full SOP with Academic or a plan

Source Notaras et al., 2021 · Center for Neurogenetics, Feil Family Brain and Mind Research Institute, Weill Cornell Medical College, Cornell University · 10.1038/s41398-021-01664-5

Authors: Michael Notaras, Aiman Lodhi, Haoyun Fang, David Greening, Dilek Colak Duration: 40 days 8 phases Cell source: Patient-Derived iPSC (Schizophrenia), Human iPSC (Control)

Abstract

This protocol describes the generation of 3D cerebral organoids from schizophrenia patient-derived and control human iPSCs, followed by comprehensive quantitative proteomics using TMTpro 16-plex isobaric barcoding and liquid chromatography-mass spectrometry. The procedure yields a proteomic map of developing brain tissue that reveals alterations in neuronal development factors and GWAS-implicated proteins in schizophrenia.

Cell source
Patient-Derived iPSC (Schizophrenia), Human iPSC (Control)
Application
Disease modeling and proteomic profiling

Protocol overview

35 steps across 8 phases

Phase 1 · iPSC Culture and Maintenance Ongoing
  1. a Acquire iPSC lines
  2. b Culture iPSC colonies on vitronectin-coated plates
Phase 2 · Embryoid Body Formation and Neural Induction Days 1-10 in vitro (DIV)
  1. a Dissociate iPSC colonies to single cells
  2. b Culture iPSC suspensions in ultra-low attachment plates to form embryoid bodies
  3. c Isolate healthy embryoid bodies and transfer to neural induction culture
  4. d Perform neural induction for up to 7 days
Phase 3 · Organoid Maturation and Expansion Days 8-40 DIV
  1. a Embed organoids in Matrigel droplets
  2. b Culture organoids in terminal organoid media without agitation
  3. c Initiate orbital shaker agitation
  4. d Maintain organoids until harvest at 35-40 DIV
Phase 4 · Sample Preparation for Proteomics Days 35-40 DIV (harvest) + same day
  1. a Harvest organoids and prepare for lysis
  2. b Lyse organoid tissue and extract protein
  3. c Reduce protein disulfide bonds
  4. d Alkylate cysteine residues
  5. e Perform enzymatic digestion with trypsin
Phase 5 · Isobaric Barcoding and Sample Multiplexing Same day as digestion completion
  1. a Prepare peptide suspensions
  2. b Label samples with TMTpro 16-plex reagents
  3. c Quench unreacted TMT reagent
  4. d Pool labeled samples
Phase 6 · Sample Desalting and Purification Same day as multiplexing
  1. a Desalt multiplexed peptide samples using C18 stage-tips
  2. b Dry desalted samples
Phase 7 · Liquid Chromatography-Mass Spectrometry Analysis Days after sample preparation
  1. a Set up EASY-nLC 1200 liquid chromatography system
  2. b Perform peptide separation via gradient LC
  3. c Acquire full-scan mass spectra
  4. d Select and fragment precursor ions
  5. e Acquire tandem mass spectra (MS/MS) of fragment ions
Phase 8 · Data Processing and Bioinformatics Post-analysis
  1. a Pre-process raw mass spectra
  2. b Perform peptide identification using MaxQuant
  3. c Filter identifications and exclude contaminants
  4. d Quantify proteins using TMT reporter ion intensity
  5. e Log2-transform and normalize protein intensities
  6. f Perform principal component analysis (PCA) and quality assessment
  7. g Impute missing values
  8. h Perform statistical testing for differential expression
  9. i Perform functional enrichment analysis

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Attribution

This SOP was written by Organthis based on the published method in Notaras et al., 2021. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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