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BRAIN Publication-derived

Primary Progressive Multiple Sclerosis Cerebral Organoids

Full SOP with Academic or a plan

Source Daviaud et al., 2022 · Tisch Multiple Sclerosis Research Center of New York · 10.1101/2022.06.24.497517

Authors: Nicolas Daviaud, Eric Chen, Tara Edwards, Saud A Sadiq Duration: 42 days 6 phases Cell source: Patient-Derived iPSC (Primary Progressive Multiple Sclerosis, Secondary Progressive Multiple Sclerosis, Relapsing-Remitting Multiple Sclerosis) and Healthy Control

Abstract

This protocol describes the derivation and characterization of cerebral organoids from induced pluripotent stem cells (iPSCs) generated from peripheral blood mononuclear cells of multiple sclerosis patients and healthy controls. The organoids model dorsal forebrain development and enable investigation of genetic and cellular dysregulation underlying different MS subtypes, particularly the disruption of stem cell proliferation and oligodendrocyte differentiation observed in primary progressive MS.

Cell source
Patient-Derived iPSC (Primary Progressive Multiple Sclerosis, Secondary Progressive Multiple Sclerosis, Relapsing-Remitting Multiple Sclerosis) and Healthy Control
Application
Disease modeling

Protocol overview

35 steps across 6 phases

Phase 1 · Patient Selection and iPSC Reprogramming from CD34+ Cells Days 1-21
  1. a Collect and Isolate CD34+ Hematopoietic Stem and Progenitor Cells
  2. b Expand CD34+ Cells In Vitro
  3. c Reprogram CD34+ Cells to iPSCs by Electroporation
  4. d Culture Electroporated Cells on Matrigel
  5. e Isolate and Expand iPSC Colonies
  6. f Quality Control: Test for Mycoplasma, Pluripotency, and Karyotype
Phase 2 · iPSC Maintenance and Neural Precursor Cell Differentiation Days 1-35 (NPC generation timeline)
  1. a Maintain iPSCs in Pluripotent State
  2. b Dissociate iPSCs into Single Cells
  3. c Generate Embryoid Bodies (EBs)
  4. d Initiate Neural Induction
  5. e Plate EBs on Matrigel and Continue Neural Induction
  6. f Switch to NPC Medium
  7. g Expand and Passage NPCs
Phase 3 · Cerebral Organoid Generation and Culture Days 1-42
  1. a Dissociate iPSCs and Generate Initial Embryoid Bodies
  2. b Culture EBs in mTeSR Plus Without Thiazovivin
  3. c Initiate Neural Induction
  4. d Embed EBs in Matrigel and Switch to Organoid Differentiation Medium
  5. e Switch to Orbital Shaker Culture with Vitamin A Supplementation
  6. f Culture Organoids to Day 42
Phase 4 · Organoid Fixation, Cryoprotection, and Sectioning Day 42
  1. a Wash Organoids in D-PBS
  2. b Fix Organoids in Paraformaldehyde
  3. c Wash Fixed Organoids
  4. d Cryoprotect in Sucrose
  5. e Embed in OCT Compound and Flash Freeze
  6. f Section Organoids on Cryostat
Phase 5 · Immunofluorescence Staining and Microscopy Day 42 (or later post-sectioning)
  1. a Thaw Slides and Create Hydrophobic Barrier
  2. b Permeabilize and Block Non-Specific Binding
  3. c Incubate with Primary Antibodies
  4. d Wash Slides
  5. e Incubate with Secondary Antibodies and Counterstain
  6. f Mount and Preserve
  7. g Image on Fluorescent Microscope
Phase 6 · Neural Differentiation of NPCs (Optional Parallel Analysis) Days 1-10
  1. a Plate NPCs on Matrigel
  2. b Initiate Mitogen Withdrawal Differentiation
  3. c Fix and Analyze Differentiated Cultures

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Attribution

This SOP was written by Organthis based on the published method in Daviaud et al., 2022. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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