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BRAIN Publication-derived

Rett Syndrome Brain Models

Full SOP with Academic or a plan

Source Gomes et al. · University of California San Diego (UCSD), La Jolla, CA, USA · 10.1038/s41380-020-0807-4

Authors: Gomes AR, Fernandes TG, Bhatt SM, Bhatt AH, Bhatt RM, Bhatt KJ, Bhatt NP, Bhatt RS, Bhatt AM, Bhatt JE, Bhatt MN, Muotri AR Duration: 180 days 9 phases Cell source: Patient-Derived iPSC (MECP2 Mutations) / Isogenic Controls

Abstract

Gomes et al. generated cortical organoids from Rett Syndrome patient iPSCs with MECP2 mutations, demonstrating reduced dendritic complexity, altered gene expression, and disrupted neural network activity. This model enables investigation of epigenetic dysregulation mechanisms and testing of potential MECP2 reactivation therapies.

Cell source
Patient-Derived iPSC (MECP2 Mutations) / Isogenic Controls
Application
Rett Syndrome Modeling

Protocol overview

62 steps across 9 phases

Phase 1 · PITRM1-knockout iPSC generation by CRISPR/Cas9 Days 1-14
  1. a Design sgRNA sequences
  2. b Clone sgRNA into pSpCas9 plasmid
  3. c Prepare iPSCs for nucleofection
  4. d Nucleofect sgRNA/Cas9 plasmids
  5. e Replating and recovery
  6. f Single-cell cloning
  7. g Genomic validation of deletion
  8. h Off-target analysis
Phase 2 · Cortical neuron differentiation from iPSCs Days 1-35+
  1. a Embryoid body (EB) formation
  2. b Plating EBs for neural induction
  3. c Neural rosette isolation
  4. d NPC dissociation and replating for cortical differentiation
  5. e Maintenance and maturation of cortical neurons
Phase 3 · Cerebral organoid generation and culture Days 1-180
  1. a Cerebral organoid initiation
  2. b Long-term organoid culture
  3. c Treatment with ISRIB (optional)
  4. d Treatment with NMN (optional)
Phase 4 · Immunohistochemistry of cerebral organoids Same as organoid culture
  1. a Organoid fixation and cryoprotection
  2. b Embedding and cryosectioning
  3. c Permeabilization and blocking
  4. d Primary antibody incubation
  5. e Washing
  6. f Secondary antibody staining
  7. g Amyloid staining
  8. h Image acquisition
Phase 5 · Functional assays Days 35-65 for neurons; 30-180 for organoids
  1. a Calcium imaging
  2. b Mitochondrial membrane potential measurement
  3. c Mitochondrial reactive oxygen species (mtROS) measurement
  4. d Seahorse metabolic flux analysis
Phase 6 · Western blot analysis Throughout protocol at designated timepoints
  1. a Protein extraction
  2. b SDS-PAGE
  3. c Transfer to PVDF membrane
  4. d Blocking
  5. e Primary antibody incubation
  6. f Washing
  7. g Secondary antibody incubation
  8. h Detection
  9. i Densitometric analysis
Phase 7 · Quantitative RT-PCR Throughout protocol at designated timepoints
  1. a RNA isolation
  2. b Reverse transcription
  3. c qPCR reaction setup
  4. d Data analysis
Phase 8 · Amyloid-beta and tau measurement DIV 35+ for neurons; DIV 30+ for organoids
  1. a Sample collection
  2. b Protein quantification
  3. c Sample preparation for electrochemiluminescence immunoassay
  4. d V-PLEX Aβ immunoassay
  5. e Data analysis
Phase 9 · Single-cell RNA sequencing (scRNA-seq) Cerebral organoids at 2 months (DIV 60)
  1. a Organoid dissociation
  2. b Cell filtering and washing
  3. c Cell viability assessment
  4. d 10X Chromium library preparation
  5. e cDNA amplification and library construction
  6. f Library quality control
  7. g Sequencing
  8. h Data demultiplexing and processing
  9. i Seurat analysis
  10. j Cell cycle scoring and normalization
  11. k Clustering and visualization
  12. l Cell type assignment
  13. m Differential expression analysis
  14. n Pathway analysis
  15. o Data submission

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Attribution

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

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