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BRAIN Publication-derived

Hindbrain Organoids for Disease Modelling

Full SOP with Academic or a plan

Source Anderle · CIBIO-University of Trento · 10.1158/0008-5472.can-09-2331

Authors: Marica Anderle Duration: 120 days 8 phases Cell source: Human iPSC

Abstract

This protocol describes the development and genetic modification of human cerebellar organoids derived from induced pluripotent stem cells (iPSCs) to model Group 3 Medulloblastoma. Wild-type organoids are electroporated with PiggyBac transposase and donor plasmids expressing oncogenes (Gfi1/c-Myc or Otx2/c-Myc) to generate medulloblastoma-like organoids that recapitulate molecular and epigenetic features of human tumors. These modified organoids can be used for disease modeling, therapeutic target validation, and drug screening.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

41 steps across 8 phases

Phase 1 · iPSC Maintenance and Expansion Day 0 - Day 20
  1. a iPSC Culture Initialization
  2. b iPSC Dissociation
Phase 2 · Cerebellar Organoid Differentiation Day 1 - Day 35
  1. a Organoid Formation Initiation
  2. b Transfer to Spinning Flask Culture
  3. c Organoid Maturation to Day 35
Phase 3 · Organoid Electroporation with Oncogenes Day 35
  1. a Plasmid Preparation
  2. b Prepare Electroporation Mix - Control (Venus Only)
  3. c Prepare Electroporation Mix - cMyc/Gfi1 Combination (GM)
  4. d Prepare Electroporation Mix - cMyc/Otx2 Combination (OM)
  5. e Prepare Electroporation Mix - SMARCA4 Variants
  6. f Transfer Organoids to Electroporation Cuvettes
  7. g Perform Electroporation
  8. h Post-Electroporation Recovery
Phase 4 · Organoid Expansion and Characterization (Days 36-57) Day 36 - Day 57
  1. a Continue Spinning Flask Culture
  2. b Optional: DZNep Drug Treatment (Days 37-57)
  3. c Optional: Tazemetostat Drug Treatment (Days 36-41)
  4. d Sample Collection for RNA-seq
  5. e RNA-seq Library Preparation
  6. f RNA-seq Sequencing
Phase 5 · Organoid Analysis and Quantification Day 42 - Day 57
  1. a Immunofluorescence Sample Preparation
  2. b Cryosectioning
  3. c Immunofluorescence Staining
  4. d Quantitative Cell Analysis
  5. e Statistical Analysis
Phase 6 · In Vivo Tumor Development and Characterization Day 35 (electroporation) to Day 120+
  1. a Organoid Preparation for Injection
  2. b Stereotactic Injection into Nude Mice
  3. c Post-Operative Care and Monitoring
  4. d Sacrifice and Brain Harvest
  5. e Brain Cryoprotection and Sectioning
  6. f Immunohistochemistry on Tumor Sections
  7. g Tumor Tissue Dissociation for DNA Extraction
Phase 7 · Molecular Analysis: DNA Extraction and Methylation Profiling Days 60-120
  1. a Genomic DNA Extraction from Organoids/Tumors
  2. b DNA Methylation Array Preparation
  3. c DNA Methylation Array Scanning
  4. d Methylation Profile Classification
Phase 8 · RNA-seq Data Analysis and Functional Interpretation Days 90-120
  1. a RNA-seq Read Quality Control
  2. b Read Mapping to Human Genome
  3. c Gene Expression Quantification
  4. d Differential Gene Expression Analysis
  5. e Functional Annotation and Pathway Analysis
  6. f Comparison to Published Medulloblastoma Transcriptomes

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Attribution

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

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