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BRAIN Publication-derived

Brain Ribosomopathy Cerebral Organoids

Full SOP with Academic or a plan

Source Zhang et al., 2024 · University of Southern California · 10.1126/sciadv.adk1034

Authors: Wei Zhang, Minjie Zhang, Li Ma, Supawadee Jariyasakulroj, Qing Chang, Ziying Lin, Zhipeng Lu, Jian-Fu Chen Duration: 15 days 16 phases Cell source: Human iPSC

Abstract

This protocol generates human iPSC-derived cerebral organoids and mouse models carrying SNORD118 mutations to recapitulate brain ribosomopathy defects. The resulting models exhibit protein synthesis loss, proteotoxic stress, neural progenitor cell dysfunction, and brain growth retardation, enabling mechanistic studies and small-molecule drug screening for therapeutic intervention.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

70 steps across 16 phases

Phase 1 · Human iPSC Culture and Maintenance Ongoing
  1. a iPSC Culture Setup
  2. b iPSC Passaging
Phase 2 · Genome Editing of SNORD118 5-7 days
  1. a Plasmid and ssODN Preparation
  2. b ROCK Inhibitor Pre-treatment
  3. c Cell Dissociation
  4. d Electroporation
  5. e Antibiotic Selection
  6. f Colony Isolation and Genotyping
Phase 3 · Human Neural Progenitor Cell (NPC) Generation 3-4 weeks
  1. a Embryoid Body (EB) Formation
  2. b Neural Rosette Formation
  3. c Neural Rosette Manual Dissection
  4. d NPC Maintenance and Expansion
Phase 4 · Cerebral Organoid Generation 8 weeks
  1. a Embryoid Body (EB) Formation from iPSCs
  2. b EB Culture Days 4-7
  3. c Neural Induction (Days 7-12)
  4. d Geltrex Embedding (Days 12-16)
  5. e Spinner Flask Culture (Day 16 onwards)
  6. f Organoid Maturation and Analysis (Weeks 4-8)
Phase 5 · Organoid Treatment with 2BAct Variable based on treatment timepoint
  1. a 2BAct Preparation and Addition
  2. b Continuous Treatment Through Differentiation
  3. c Analysis of Treated Organoids
Phase 6 · Immunohistochemistry (IHC) and Immunofluorescence Staining 1-2 days
  1. a Organoid Fixation
  2. b Cryoprotection and Embedding
  3. c Sectioning
  4. d Immunostaining
  5. e Image Acquisition and Quantification
Phase 7 · In Vitro NPC Analysis: BrdU and EdU Incorporation Assays 1-2 days
  1. a BrdU Labeling of Organoids
  2. b EdU Assay
  3. c Quantification
Phase 8 · O-Propargyl-Puromycin (OPP) Incorporation Assay for Protein Synthesis Measurement 1 day
  1. a OPP Treatment
  2. b Fixation and Permeabilization
  3. c Click Chemistry Detection
  4. d DAPI Counterstaining and Imaging
  5. e Quantification
Phase 9 · 5-Ethynyl Uridine (5-EU) Incorporation Assay for rRNA Synthesis Measurement 1 day
  1. a 5-EU Labeling
  2. b Fixation and Click Chemistry
  3. c Nucleolar Marker Immunostaining
  4. d Image Acquisition and Quantification
Phase 10 · Real-time Quantitative PCR (qPCR) for U8 and rRNA Expression 1 day
  1. a RNA Extraction
  2. b Reverse Transcription
  3. c qRT-PCR Setup
  4. d Data Analysis
Phase 11 · Mouse SNORD118 Conditional Knockout Model Generation Variable (months to years for breeding)
  1. a SNORD118 Flox Mouse Line Generation
  2. b Breeding to EMX1-Cre Line
  3. c Breeding Strategy for Experiments
  4. d Animal Care and Compliance
Phase 12 · Mouse Brain Analysis at Embryonic Day 14.5 (E14.5) 1 day (experimental day; performed on timed-pregnant dams)
  1. a BrdU Labeling of Embryonic Brains
  2. b Cryoprotection and Embedding
  3. c Sectioning and Nissl Staining
  4. d Immunohistochemistry for Neural Markers
  5. e Image Acquisition and Quantification
Phase 13 · In Vivo 2BAct Treatment in Pregnant Mice 5 days (E11.5 to E15.5)
  1. a 2BAct Stock Preparation
  2. b Daily Injections E11.5 to E15.5
  3. c Embryo Dissection and Analysis at E16.5
  4. d Tissue Processing and Analysis
Phase 14 · Cell Cycle Synchronization and Analysis 1-2 days
  1. a Nocodazole Synchronization
  2. b Nocodazole Washout and Cell Cycle Release
  3. c Propidium Iodide (PI) Staining and Flow Cytometry
  4. d Immunofluorescence for Cell Cycle Markers
Phase 15 · Immunoprecipitation for MDM2 Protein Interaction Analysis 1 day
  1. a Cell Lysis
  2. b Protein Concentration Determination
  3. c MDM2 Immunoprecipitation
  4. d Washing and Elution
  5. e Western Blotting
  6. f Quantification
Phase 16 · Small-Molecule Chemical Screening of NPCs 3-5 days
  1. a Compound Library Preparation
  2. b NPC Treatment and EdU Incorporation Assay
  3. c EdU Labeling
  4. d EdU Detection and Quantification
  5. e Hit Identification and Dose-Response Analysis

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Attribution

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

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