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BRAIN Publication-derived

Multiple Sclerosis Brain Organoids with Microglia

Full SOP with Academic or a plan

Source Fagiani et al., 2025 · IRCCS San Raffaele Scientific Institute, Milan, Italy · 10.1038/s41467-025-63371-9

Authors: Francesca Fagiani, Edoardo Pedrini, Maria Sofia Martire, Gaia Gastoldi, Colin Vanden Bulcke, Jing-Ping Lin, Dragan Maric, Elena Brambilla, Francesca Ruffini, Carolina Peri, Peter A. Calabresi, Pietro Maggi, Paola Panina-Bordignon, Gianvito Martino, Daniel S. Reich, Martina Absinta Duration: 56 days 9 phases Cell source: Patient-Derived iPSC (Relapsing-Remitting Multiple Sclerosis)

Abstract

This protocol generates hiPSC-derived neural glia-enriched organoids from MS patients to model inflammation-induced cellular senescence in the brain. Over 8 weeks, organoids develop into complex 3D networks containing astrocytes, oligodendrocytes, neurons, and microglia, enabling in vitro investigation of senescence-like processes triggered by MS cerebrospinal fluid and inflammatory cytokines.

Cell source
Patient-Derived iPSC (Relapsing-Remitting Multiple Sclerosis)
Application
Disease modeling

Protocol overview

45 steps across 9 phases

Phase 1 · NPC Expansion and Organoid Initiation Days 0-1
  1. a Prepare NPC Expansion Medium
  2. b Culture hiPSC-NPCs in Matrigel-Coated Flasks
  3. c Enzymatic Detachment and Cell Counting
  4. d Plate Cells for Organoid Formation
Phase 2 · Initial Differentiation and Glial Induction Days 1-5
  1. a Maintain Initial Culture in Reinhardt Medium
  2. b Switch to Glial Induction Medium
  3. c Continue Glial Induction for Days 1-3
Phase 3 · Glial Differentiation and Maturation (Phase 1) Days 5-14
  1. a Switch to Glial Differentiation Medium
  2. b Maintain GDM with Doxycycline from Days 5-14
  3. c Monitor SOX10-eGFP Expression
Phase 4 · Extended Glial Differentiation and GDNF/BDNF Addition Days 14-56 (8 weeks)
  1. a Remove Doxycycline and Add Neurotrophic Factors
  2. b Continue Long-Term Culture to Week 8
  3. c Assess Myelination by Transmission Electron Microscopy
Phase 5 · hiPSC-Derived Microglia Differentiation and Integration Days 0-38 (parallel with organoid generation)
  1. a Initiate Hematopoietic Progenitor Differentiation
  2. b Differentiate Hematopoietic Progenitors to Microglia
  3. c Mature Microglia Using Maturation Kit
  4. d Co-Culture Microglia with Organoids at Day 38
Phase 6 · Senescence Assessment Baseline and CSF/Cytokine Treatments Week 8 onwards (after organoid maturation and microglia integration)
  1. a Assess Baseline Senescence-Associated β-Galactosidase (SA-β-gal) Activity
  2. b Prepare and Aliquot MS Cerebrospinal Fluid
  3. c Prepare Control CSF (Negative Control)
  4. d Prepare Inflammatory Cytokine Cocktail
  5. e Expose Organoids to MS CSF (24 hours)
  6. f Continue CSF Treatment to 6 Days
  7. g Expose Organoids to Inflammatory Cytokine Cocktail (24 hours)
  8. h Treat Organoids with Human Immunoglobulins (Negative Control)
Phase 7 · Pharmacological Modulation of Senescence Day of treatment (6 h CSF pre-treatment + 18 h drug treatment)
  1. a Pre-Treat Organoids with MS CSF (6 hours)
  2. b Prepare Drug Solutions at Two Concentrations
  3. c Co-Treat Organoids with MS CSF and Drugs (18 hours)
  4. d Harvest Organoids for SA-β-gal Staining at 24 Hours Post-CSF
  5. e Assess 6-Day Drug Effect
  6. f Assess Cell Viability After Drug Treatment
  7. g Assess Oligodendrocyte Toxicity Rescue (Optional)
Phase 8 · Single-Cell RNA Sequencing and Transcriptomic Analysis After organoid treatment (processing on harvest day)
  1. a Harvest Organoids for scRNA-seq
  2. b Count Viable Cells and Prepare for scRNA-seq Library Preparation
  3. c Generate scRNA-seq Libraries Using 10× Chromium
  4. d Sequence Libraries
  5. e Process Raw Sequencing Data with CellRanger
  6. f Perform Quality Control and Preprocessing
  7. g Perform Unsupervised Clustering and Cell-Type Identification
  8. h Score Senescence Using SIT (Senescence Index Tool)
  9. i Perform Differential Expression and Pathway Analysis
Phase 9 · Immunofluorescence Validation After organoid treatment (concurrent with scRNA-seq harvest)
  1. a Prepare Organoid Cryosections
  2. b Perform Multiplex Immunofluorescence Staining
  3. c Mount Slides and Image by Confocal Microscopy
  4. d Quantify Cell Populations and Senescence Markers

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Attribution

This SOP was written by Organthis based on the published method in Fagiani et al., 2025. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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