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BRAIN Publication-derived

Huntington's Disease Cortical Organoids

Full SOP with Academic or a plan

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

Authors: 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 Duration: 120 days 5 phases Cell source: 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

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

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Attribution

This SOP was written by Organthis based on the published method in Galimberti et al., 2024. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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