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BRAIN Publication-derived

Amyotrophic Lateral Sclerosis Spinal Cord Organoids

Full SOP with Academic or a plan

Source Pereira et al., 2021 · Massachusetts General Hospital, Harvard Medical School · 10.1038/s41467-021-24776-4

Authors: João D. Pereira, Daniel M. DuBreuil, Anna-Claire Devlin, Aaron Held, Yechiam Sapir, Eugene Berezovski, James Hawrot, Katherine Dorfman, Vignesh Chander, Brian J. Wainger Duration: 15 days 4 phases Cell source: Human iPSC; Patient-Derived iPSC (ALS)

Abstract

This protocol generates sensorimotor organoid cultures from human iPSCs that contain physiologically functional motor and sensory neurons, skeletal muscle, and supporting cells including astrocytes and microglia, with formation of neuromuscular junctions. The model enables investigation of amyotrophic lateral sclerosis (ALS) pathophysiology across multiple genetic backgrounds and mutations.

Cell source
Human iPSC; Patient-Derived iPSC (ALS)
Application
Disease modeling

Protocol overview

21 steps across 4 phases

Phase 1 · iPSC Preparation and Sphere Formation Day 0-7
  1. a iPSC Enzymatic Dissociation
  2. b Sphere Formation in AggreWell 800 Plates
  3. c Day 1 Media Supplementation
  4. d Daily Media Changes (Days 2-6)
  5. e Sphere Counting and Plating Preparation (Day 7)
Phase 2 · Sphere Plating and Organoid Culture Initiation Day 7-14
  1. a Plate Spheres at Fixed Density
  2. b Transition to Adherent Culture Media (Day 9 onwards)
  3. c Monitor Cell Migration and Organoid Formation
Phase 3 · Organoid Maturation and Cell Type Specification Week 2-15
  1. a Early Immunocytochemistry Analysis (Week 2)
  2. b Single-Cell RNA-Seq Analysis (Week 2)
  3. c Weeks 3-9 Longitudinal Imaging
  4. d Week 4 Immunocytochemistry for Neuronal Subtypes
  5. e Week 4 FACS Sorting of Neuronal Cells
  6. f Week 4-8 Functional Characterization
  7. g Week 6 Immunocytochemistry for Supporting Cell Types
  8. h Week 7-8 Contraction Assay Setup
  9. i Week 8-9 Pharmacological Inhibition of Contractions
  10. j Week 9 Electron Microscopy
  11. k Week 10 qPCR Gene Expression Analysis
  12. l Week 13-15 NMJ Analysis and Neurite Tracking
Phase 4 · Optogenetic and Extended Functional Studies (Optional) Week 7-9
  1. a Optogenetic Stimulation of Motor Neurons

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Attribution

This SOP was written by Organthis based on the published method in Pereira et al., 2021. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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