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MUSCLE Publication-derived

Neuromuscular organoids model spinal neuromuscular pathologies in C9orf72 amyotrophic lateral sclerosis

Source Gao et al., 2024 · Westlake University, Hangzhou, Zhejiang, China · 10.1016/j.celrep.2024.113892

👤 Chong Gao, Qinghua Shi, Xue Pan, Jiajia Chen, Yuhong Zhang, Jiali Lang, Shan Wen, Xiaodong Liu, Tian-Lin Cheng, Kai Lei ⏱ 105 days 📋 5 phases 🧫 Patient-Derived iPSC (C9orf72-ALS)

Abstract

This protocol describes the differentiation of C9orf72-ALS patient-derived iPSCs into neuromuscular organoids (NMOs) containing trunk spinal cord motor neurons, interneurons, glial cells, and skeletal muscle. The resulting NMOs model ALS-related spinal neuromuscular pathologies including muscle contraction weakness, neural denervation, loss of Schwann cells, RNA foci, and dipeptide repeat protein aggregation.

Cell source
Patient-Derived iPSC (C9orf72-ALS)
Application
Disease modeling

Protocol overview

29 steps across 5 phases

iPSC Culture and Quality Control Days -5 to 0
  1. 1 iPSC Culture
  2. 2 Genomic DNA Extraction and C9orf72 Verification
Neuromesodermal Progenitor (NMP) Induction Days 0-2
  1. 1 iPSC Dissociation and Plating for NMP Induction
  2. 2 NMP Induction Medium Preparation
  3. 3 Add NMP Induction Medium
  4. 4 NMP Induction Culture
3D Neuromuscular Organoid (NMO) Formation and Maturation Days 2-50
  1. 1 NMPs Dissociation and Seeding into Ultra-Low Attachment Plates
  2. 2 Day 2–3: Initial Medium Supplementation
  3. 3 Day 3: First Medium Change
  4. 4 Days 4–5: Continued Medium Changes
  5. 5 Days 5–10: Basal Medium Refreshment
  6. 6 Day 10: Transfer to Larger Vessel (6 cm dish)
  7. 7 Days 10–30: Growth and Maturation in 6 cm Dish
  8. 8 Day 30: Transfer to 10 cm Dish
  9. 9 Days 30–50: Final Maturation and Quality Control
Organoid Characterization and Functional Assays Days 50–100+
  1. 1 Day 50: Immunofluorescence Characterization
  2. 2 Day 50: Skeletal Muscle Contraction Analysis
  3. 3 Day 100: Advanced Immunofluorescence and NMJ Analysis
  4. 4 Day 100: Fluorescent In Situ Hybridization (FISH) for RNA Foci
  5. 5 Day 100: Autophagy Assessment
  6. 6 Day 100: Dipeptide Repeat Protein (DPR) Detection
  7. 7 Day 50 or Day 150: Microelectrode Array (MEA) Electrophysiology
  8. 8 Day 100: Calcium Imaging
Drug Treatment (GSK2606414) Days 100–105
  1. 1 GSK2606414 Preparation
  2. 2 18-Hour Acute Treatment
  3. 3 5-Day Prolonged Treatment
  4. 4 Post-Treatment Skeletal Muscle Contraction Analysis
  5. 5 Post-Treatment Immunofluorescence
  6. 6 Post-Treatment NMJ Assessment

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Gao et al., 2024. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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