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BRAIN Publication-derived

Human Sensorimotor Organoid Differentiation from Healthy and ALS-Derived iPSCs

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

👤 João D. Pereira, Daniel M. DuBreuil, Anna-Claire Devlin, Aaron Held, Yechiam Sapir, Eugene Berezovski, James Hawrot, Katherine Dorfman, Vignesh Chander, Brian J. Wainger ⏱ 15 days 📋 4 phases 🧫 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

iPSC Preparation and Sphere Formation Day 0-7
  1. 1 iPSC Enzymatic Dissociation
  2. 2 Sphere Formation in AggreWell 800 Plates
  3. 3 Day 1 Media Supplementation
  4. 4 Daily Media Changes (Days 2-6)
  5. 5 Sphere Counting and Plating Preparation (Day 7)
Sphere Plating and Organoid Culture Initiation Day 7-14
  1. 6 Plate Spheres at Fixed Density
  2. 7 Transition to Adherent Culture Media (Day 9 onwards)
  3. 8 Monitor Cell Migration and Organoid Formation
Organoid Maturation and Cell Type Specification Week 2-15
  1. 9 Early Immunocytochemistry Analysis (Week 2)
  2. 10 Single-Cell RNA-Seq Analysis (Week 2)
  3. 11 Weeks 3-9 Longitudinal Imaging
  4. 12 Week 4 Immunocytochemistry for Neuronal Subtypes
  5. 13 Week 4 FACS Sorting of Neuronal Cells
  6. 14 Week 4-8 Functional Characterization
  7. 15 Week 6 Immunocytochemistry for Supporting Cell Types
  8. 16 Week 7-8 Contraction Assay Setup
  9. 17 Week 8-9 Pharmacological Inhibition of Contractions
  10. 18 Week 9 Electron Microscopy
  11. 19 Week 10 qPCR Gene Expression Analysis
  12. 20 Week 13-15 NMJ Analysis and Neurite Tracking
Optogenetic and Extended Functional Studies (Optional) Week 7-9
  1. 21 Optogenetic Stimulation of Motor Neurons

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Pereira et al., 2021. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

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