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BRAIN Publication-derived

Modeling Down Syndrome neurodevelopment with isogenic cerebral organoids

Source Czerminski et al., 2022 · University of Massachusetts Medical School · 10.1101/2022.05.25.493459;

👤 Jan T. Czerminski, Oliver D. King, Jeanne B. Lawrence ⏱ 90 days 📋 8 phases 🧫 Patient-Derived iPSC (Down Syndrome, Trisomy 21)

Abstract

This protocol generates isogenic trisomic and disomic cerebral organoids from Down syndrome patient-derived iPSCs to model early fetal brain development. The organoids are characterized through transcriptomics and media analysis to identify neurodevelopmental and neurodegenerative phenotypes associated with trisomy 21, including increased amyloid-beta secretion and subtle transcriptional changes in key developmental genes.

Cell source
Patient-Derived iPSC (Down Syndrome, Trisomy 21)
Application
Disease modeling

Protocol overview

30 steps across 8 phases

iPSC Culture and Maintenance Ongoing, passaged every 3-4 days
  1. 1 Culture iPSCs on vitronectin-coated plates
  2. 2 Passage iPSCs every 3-4 days
  3. 3 Verify chromosome 21 status by FISH
Cerebral Organoid Differentiation (Qian Protocol - Forebrain) Days 0-90
  1. 1 Prepare embryoid bodies (EBs)
  2. 2 Transfer aggregates to Matrigel embedding (Day 7)
  3. 3 Initiate orbital shaker culture (Day 14)
  4. 4 Maintain organoid culture through day 90
Organoid Harvesting and Sample Preparation Day 90
  1. 1 Collect media for amyloid-beta analysis
  2. 2 Harvest organoids for RNA extraction
  3. 3 Homogenize organoids
RNA Extraction and Library Preparation Day 90 (or from frozen samples)
  1. 1 Extract RNA from Trizol homogenate
  2. 2 Treat with DNase I
  3. 3 Purify RNA with RNeasy MinElute columns
  4. 4 Assess RNA quality
  5. 5 Prepare strand-specific cDNA libraries
High-Throughput Sequencing After library preparation
  1. 1 Sequence on Illumina platform
RNA Sequencing Data Analysis After sequencing
  1. 1 Align reads to reference genome
  2. 2 Count reads to genes
  3. 3 Normalize and identify differentially expressed genes
  4. 4 Perform cell type deconvolution
  5. 5 Generate publication-quality graphs
Amyloid-Beta ELISA Assay After media collection (day 90)
  1. 1 Prepare media samples for ELISA
  2. 2 Perform ultrasensitive amyloid-beta ELISA
  3. 3 Read absorbance
  4. 4 Generate standard curve and calculate concentrations
  5. 5 Normalize to RNA content
Reverse Transcription and qPCR Validation After RNA extraction
  1. 1 Perform reverse transcription
  2. 2 Set up qPCR reactions
  3. 3 Perform qPCR on thermal cycler
  4. 4 Analyze qPCR data

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Czerminski et al., 2022. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

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