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BRAIN Publication-derived

Down Syndrome Cerebral Organoids for Disease Modelling

Full SOP with Academic or a plan

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

Authors: Jan T. Czerminski, Oliver D. King, Jeanne B. Lawrence Duration: 90 days 8 phases Cell source: 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

Phase 1 · iPSC Culture and Maintenance Ongoing, passaged every 3-4 days
  1. a Culture iPSCs on vitronectin-coated plates
  2. b Passage iPSCs every 3-4 days
  3. c Verify chromosome 21 status by FISH
Phase 2 · Cerebral Organoid Differentiation (Qian Protocol - Forebrain) Days 0-90
  1. a Prepare embryoid bodies (EBs)
  2. b Transfer aggregates to Matrigel embedding (Day 7)
  3. c Initiate orbital shaker culture (Day 14)
  4. d Maintain organoid culture through day 90
Phase 3 · Organoid Harvesting and Sample Preparation Day 90
  1. a Collect media for amyloid-beta analysis
  2. b Harvest organoids for RNA extraction
  3. c Homogenize organoids
Phase 4 · RNA Extraction and Library Preparation Day 90 (or from frozen samples)
  1. a Extract RNA from Trizol homogenate
  2. b Treat with DNase I
  3. c Purify RNA with RNeasy MinElute columns
  4. d Assess RNA quality
  5. e Prepare strand-specific cDNA libraries
Phase 5 · High-Throughput Sequencing After library preparation
  1. a Sequence on Illumina platform
Phase 6 · RNA Sequencing Data Analysis After sequencing
  1. a Align reads to reference genome
  2. b Count reads to genes
  3. c Normalize and identify differentially expressed genes
  4. d Perform cell type deconvolution
  5. e Generate publication-quality graphs
Phase 7 · Amyloid-Beta ELISA Assay After media collection (day 90)
  1. a Prepare media samples for ELISA
  2. b Perform ultrasensitive amyloid-beta ELISA
  3. c Read absorbance
  4. d Generate standard curve and calculate concentrations
  5. e Normalize to RNA content
Phase 8 · Reverse Transcription and qPCR Validation After RNA extraction
  1. a Perform reverse transcription
  2. b Set up qPCR reactions
  3. c Perform qPCR on thermal cycler
  4. d Analyze qPCR data

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Attribution

This SOP was written by Organthis based on the published method in Czerminski et al., 2022. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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