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BRAIN Publication-derived

Generation of Telencephalic Organoids with Ventral and Dorsal Identities from Human ESCs for Disease Modeling

Source Galimberti et al., 2024 · University of Milan, Department of Biosciences · 10.1038/s41467-024-50877-x

👤 Maura Galimberti, Maria R. Nucera, Vittoria D. Bocchi, Paola Conforti, Elena Vezzoli, Matteo Cereda, Camilla Maffezzini, Raffaele Iennaco, Andrea Scolz, Andrea Falqui, Chiara Cordiglieri, Martina Cremona, Ira Espuny-Camacho, Andrea Faedo, Dan P. Felsenfeld, Thomas F. Vogt, Valeria Ranzani, Chiara Zuccato, Dario Besusso, Elena Cattaneo ⏱ 120 days 📋 5 phases 🧫 Human ESC (RUES2)

Abstract

This protocol describes the generation of three-dimensional telencephalic organoids from isogenic human ESC lines (RUES2) that recapitulate ventral and dorsal telencephalic identities. The protocol enables the study of neurodevelopmental alterations and cell-autonomous and non-cell autonomous mechanisms in Huntington's disease by allowing co-culture of healthy and disease-bearing cells within a single organoid.

Cell source
Human ESC (RUES2)
Application
Disease modeling - Huntington's disease

Protocol overview

15 steps across 5 phases

hESC maintenance and preparation Before Day 0
  1. 1 Culture human ESCs on Geltrex-coated plates
  2. 2 Prepare cells for organoid aggregation
  3. 3 Seed cells in V-bottom 96-well plate
Neural induction Day 2 - Day 6
  1. 1 Replace medium with Neural Induction Medium
  2. 2 Perform partial medium changes
Ventral/dorsal patterning Day 6 - Day 18
  1. 1 Add morphogens for ventral patterning
  2. 2 Continue partial medium changes during patterning
Embedding and maturation Day 18 - Day 120
  1. 1 Transfer organoids to 6 cm² dish on orbital shaker
  2. 2 Embed organoids in Geltrex matrix
  3. 3 Switch to Neuronal Differentiation Medium
  4. 4 Perform partial medium changes during maturation
  5. 5 Continue culture until Day 120
Mosaic organoid generation (optional - for co-culture studies) Before Day 0 - Day 120
  1. 1 Generate stable GFP and TOMATO-expressing cell lines via lentiviral transduction
  2. 2 Co-aggregate CTRL and HD cells at 1:1 ratio
  3. 3 Harvest mosaic organoids at Day 120 and FACS sort

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Galimberti et al., 2024. 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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