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BRAIN Publication-derived

Modeling medulloblastoma in vivo and with human cerebellar organoids

Source Ballabio et al., 2020 · University of Trento · 10.1038/s41467-019-13989-3

👤 Claudio Ballabio, Marica Anderle, Matteo Gianesello, Chiara Lago, Evelina Miele, Marina Cardano, Giuseppe Aiello, Silvano Piazza, Davide Caron, Francesca Gianno, Andrea Ciolfi, Lucia Pedace, Angela Mastronuzzi, Marco Tartaglia, Franco Locatelli, Elisabetta Ferretti, Felice Giangaspero, Luca Tiberi ⏱ 120 days 📋 6 phases 🧫 Human iPSC

Abstract

This protocol describes the generation of human cerebellar organoids and their modification via electroporation with oncogenic gene combinations (Otx2/c-MYC and Gfi1/c-MYC) to model Group 3 medulloblastoma. The organoids recapitulate human Group 3 MB molecular and pathological features, including DNA methylation signatures, and can be used for drug screening and therapeutic validation.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

24 steps across 6 phases

iPSC maintenance and cerebellar organoid differentiation Days 0-35
  1. 1 Culture and maintain human iPSC
  2. 2 Initiate cerebellar organoid differentiation
  3. 3 Culture organoids to day 35 of differentiation
Plasmid preparation and organoid electroporation Day 35
  1. 1 Prepare electroporation plasmids
  2. 2 Prepare electroporation mix for OM or GM conditions
  3. 3 Electroporate cerebellar organoids
  4. 4 Recover organoids post-electroporation
Post-electroporation organoid culture and characterization Days 35-60
  1. 1 Culture modified organoids
  2. 2 Analyze proliferation and differentiation at day 40
  3. 3 Analyze proliferation and differentiation at day 60
  4. 4 Optional: Treat organoids with EZH2 inhibitors
In vivo transplantation into nude mice Days 24–120 post-electroporation
  1. 1 Prepare dissociated organoid cells for injection
  2. 2 Perform intracranial injection into nude mice
  3. 3 Monitor mice and assess tumor formation
  4. 4 Harvest brain tissue and prepare for analysis
Immunofluorescence and immunohistochemistry analysis Post-harvest
  1. 1 Perform immunofluorescence staining on brain sections
  2. 2 Perform immunohistochemistry on paraffin sections
  3. 3 Assess marker expression in tumors
  4. 4 Image and quantify results
DNA methylation profiling and molecular classification Post-harvest
  1. 1 Extract genomic DNA from tumor tissue and organoids
  2. 2 Prepare DNA for methylation profiling
  3. 3 Perform array hybridization and scanning
  4. 4 Analyze methylation data and classify tumors
  5. 5 Interpret classification results

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Ballabio et al., 2020. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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