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BRAIN Publication-derived

Spatially-Restricted Inflammation-Induced Senescent-Like Glia in Multiple Sclerosis: hiPSC-Derived Neural Glia-Enriched Organoid Protocol

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

👤 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 ⏱ 56 days 📋 9 phases 🧫 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

NPC Expansion and Organoid Initiation Days 0-1
  1. 1 Prepare NPC Expansion Medium
  2. 2 Culture hiPSC-NPCs in Matrigel-Coated Flasks
  3. 3 Enzymatic Detachment and Cell Counting
  4. 4 Plate Cells for Organoid Formation
Initial Differentiation and Glial Induction Days 1-5
  1. 5 Maintain Initial Culture in Reinhardt Medium
  2. 6 Switch to Glial Induction Medium
  3. 7 Continue Glial Induction for Days 1-3
Glial Differentiation and Maturation (Phase 1) Days 5-14
  1. 8 Switch to Glial Differentiation Medium
  2. 9 Maintain GDM with Doxycycline from Days 5-14
  3. 10 Monitor SOX10-eGFP Expression
Extended Glial Differentiation and GDNF/BDNF Addition Days 14-56 (8 weeks)
  1. 11 Remove Doxycycline and Add Neurotrophic Factors
  2. 12 Continue Long-Term Culture to Week 8
  3. 13 Assess Myelination by Transmission Electron Microscopy
hiPSC-Derived Microglia Differentiation and Integration Days 0-38 (parallel with organoid generation)
  1. 14 Initiate Hematopoietic Progenitor Differentiation
  2. 15 Differentiate Hematopoietic Progenitors to Microglia
  3. 16 Mature Microglia Using Maturation Kit
  4. 17 Co-Culture Microglia with Organoids at Day 38
Senescence Assessment Baseline and CSF/Cytokine Treatments Week 8 onwards (after organoid maturation and microglia integration)
  1. 18 Assess Baseline Senescence-Associated β-Galactosidase (SA-β-gal) Activity
  2. 19 Prepare and Aliquot MS Cerebrospinal Fluid
  3. 20 Prepare Control CSF (Negative Control)
  4. 21 Prepare Inflammatory Cytokine Cocktail
  5. 22 Expose Organoids to MS CSF (24 hours)
  6. 23 Continue CSF Treatment to 6 Days
  7. 24 Expose Organoids to Inflammatory Cytokine Cocktail (24 hours)
  8. 25 Treat Organoids with Human Immunoglobulins (Negative Control)
Pharmacological Modulation of Senescence Day of treatment (6 h CSF pre-treatment + 18 h drug treatment)
  1. 26 Pre-Treat Organoids with MS CSF (6 hours)
  2. 27 Prepare Drug Solutions at Two Concentrations
  3. 28 Co-Treat Organoids with MS CSF and Drugs (18 hours)
  4. 29 Harvest Organoids for SA-β-gal Staining at 24 Hours Post-CSF
  5. 30 Assess 6-Day Drug Effect
  6. 31 Assess Cell Viability After Drug Treatment
  7. 32 Assess Oligodendrocyte Toxicity Rescue (Optional)
Single-Cell RNA Sequencing and Transcriptomic Analysis After organoid treatment (processing on harvest day)
  1. 33 Harvest Organoids for scRNA-seq
  2. 34 Count Viable Cells and Prepare for scRNA-seq Library Preparation
  3. 35 Generate scRNA-seq Libraries Using 10× Chromium
  4. 36 Sequence Libraries
  5. 37 Process Raw Sequencing Data with CellRanger
  6. 38 Perform Quality Control and Preprocessing
  7. 39 Perform Unsupervised Clustering and Cell-Type Identification
  8. 40 Score Senescence Using SIT (Senescence Index Tool)
  9. 41 Perform Differential Expression and Pathway Analysis
Immunofluorescence Validation After organoid treatment (concurrent with scRNA-seq harvest)
  1. 42 Prepare Organoid Cryosections
  2. 43 Perform Multiplex Immunofluorescence Staining
  3. 44 Mount Slides and Image by Confocal Microscopy
  4. 45 Quantify Cell Populations and Senescence Markers

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Fagiani et al., 2025. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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