Skip to content
← Back to browse
BRAIN Publication-derived

Generation of Glia-Enriched Brain Organoids with Oligodendrocyte Differentiation and Microglia Integration for MS Disease Modeling

Source Fagiani et al., 2024 · IRCCS San Raffaele Scientific Institute, Milan, Italy · 10.1016/j.xcrm.2024.101680

👤 Francesca Fagiani, Edoardo Pedrini, Stefano Taverna, Elena Brambilla, Valentina Murtaj, Paola Podini, Francesca Ruffini, Erica Butti, Clarissa Braccia, Annapaola Andolfo, Roberta Magliozzi, Lena Smirnova, Tanja Kuhlmann, Angelo Quattrini, Peter A. Calabresi, Daniel S. Reich, Gianvito Martino, Paola Panina-Bordignon, Martina Absinta ⏱ 56 days 📋 5 phases 🧫 Human iPSC-derived Neural Precursor Cells (NPCs) and iPSC-derived Microglia

Abstract

This protocol generates glia-enriched 3D forebrain organoids from hiPSC-derived neural precursor cells using doxycycline-inducible SOX10 expression to accelerate oligodendrocyte differentiation. Mature organoids containing neurons, astrocytes, oligodendrocytes, and integrated hiPSC-derived microglia are obtained within 8 weeks and can be exposed to MS-patient CSF to model chronic active lesion phenotypes for drug screening.

Cell source
Human iPSC-derived Neural Precursor Cells (NPCs) and iPSC-derived Microglia
Application
Disease modeling - Multiple sclerosis inflammatory neurodegeneration phenotyping

Protocol overview

21 steps across 5 phases

NPC Infection and GFP+ Cell Sorting Day -1 to Day 7
  1. 1 Culture and expansion of hiPSC-derived NPCs
  2. 2 Seed NPCs in T25 flasks for viral infection
  3. 3 First viral infection of NPCs
  4. 4 Second viral infection of NPCs
  5. 5 Doxycycline induction and GFP expression
  6. 6 FACS sorting of GFP+ cells
Organoid Formation and Glial Induction (Days 1-5) Day 1 to Day 5
  1. 1 Plate NPCs for organoid formation
  2. 2 Culture NPCs in Reinhardt medium
  3. 3 Switch to glial induction medium (GIM)
  4. 4 Maintain GIM culture with doxycycline
  5. 5 Switch to glial differentiation medium (GDM)
Oligodendrocyte Maturation and Doxycycline Withdrawal (Days 5-14) Day 5 to Day 14
  1. 1 Continue GDM culture with doxycycline
  2. 2 Add BDNF and GDNF at day 14
  3. 3 Discontinue doxycycline at day 14
Final Maturation and Microglia Integration (Days 14-56) Day 14 to Day 56 (8 weeks)
  1. 1 Continue GDM culture without doxycycline
  2. 2 Differentiate hiPSC-derived microglia in parallel
  3. 3 Integrate microglia into 8-week-old organoids
MS-Inflamed CSF Exposure and Inflammatory Challenge Day 56 onward (optional inflammatory challenge)
  1. 1 Prepare MS-inflamed cerebrospinal fluid (CSF)
  2. 2 Expose organoids to MS-inflamed CSF
  3. 3 Harvest organoids for molecular analysis at 24 hours
  4. 4 Harvest organoids for cellular and microscopy analysis at 6 days

Full SOP

🔬

Create a free account to access this protocol

Join OrganMatch to unlock step-by-step procedures, reagent concentrations, QC checklists, and downloadable batch record templates.

Create free account

Already registered? Log in

Attribution

This SOP was authored by Organthis based on the published method in Fagiani 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.

Something wrong with this entry? Report an issue with this protocol

Need a commercial licence?
Use this protocol in your therapeutic or diagnostic pipeline.
Request Commercial Licence