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BRAIN Publication-derived

Multiple Sclerosis Brain Organoids

Full SOP with Academic or a plan

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

Authors: 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 Duration: 56 days 5 phases Cell source: 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

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

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Attribution

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

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