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BRAIN Publication-derived

Generation of Cerebral Organoids from iPSCs and Injection with ALS-Derived Spinal Cord Extracts for TDP-43 Pathology Propagation Studies

Source Tamaki et al., 2023 · Montreal Neurological Institute-Hospital, McGill University · 10.1371/journal.pgen.1010606

👤 Yoshitaka Tamaki, Jay P. Ross, Paria Alipour, Charles-Étienne Castonguay, Boting Li, Helene Catoire, Daniel Rochefort, Makoto Urushitani, Ryosuke Takahashi, Joshua A. Sonnen, Stefano Stifani, Patrick A. Dion, Guy A. Rouleau ⏱ 116 days 📋 9 phases 🧫 Human iPSC (from peripheral blood mononuclear cells; ALS patient and healthy control derived)

Abstract

This protocol describes the generation of cerebral organoids from human induced pluripotent stem cells and their use as a three-dimensional human CNS tissue model to study the cell-to-cell propagation of pathogenic TDP-43 derived from postmortem amyotrophic lateral sclerosis spinal cord. The protocol demonstrates that ALS patient-derived pathogenic TDP-43 induces time-dependent formation of cytoplasmic phosphorylated TDP-43 aggregates, astrogliosis, cellular apoptosis, and DNA double-strand breaks in ALS-derived cerebral organoids.

Cell source
Human iPSC (from peripheral blood mononuclear cells; ALS patient and healthy control derived)
Application
Disease modeling (ALS TDP-43 propagation and pathology in human CNS tissue)

Protocol overview

58 steps across 9 phases

iPSC Culture and Maintenance Continuous until 70–80% confluency
  1. 1 Reprogramming of PBMCs into iPSCs
  2. 2 Culture of iPSCs on Matrigel-coated plates
  3. 3 Passage of iPSC colonies
Cerebral Organoid Differentiation (Days 0–10) DIV 0 to DIV 10
  1. 1 Formation of embryoid bodies (EBs)
  2. 2 Transfer to induction medium
  3. 3 Embedding in Matrigel and expansion phase
  4. 4 Transition to maturation phase
  5. 5 Media replacement during maturation
Preparation of Sarkosyl-Insoluble Protein Fractions from Spinal Cord Single working day (for tissue processing)
  1. 1 Sonication and initial extraction with HS-TX buffer
  2. 2 Centrifugation for HS-TX soluble fraction
  3. 3 Myelin removal by sucrose flotation
  4. 4 DNA and RNA removal with benzonase
  5. 5 Centrifugation after benzonase treatment
  6. 6 Sarkosyl extraction of insoluble fractions
  7. 7 Centrifugation to separate sarkosyl-soluble and -insoluble fractions
  8. 8 Final washing and resuspension of sarkosyl-insoluble fraction
  9. 9 Protein concentration measurement
Injection of Sarkosyl-Insoluble Fractions into Cerebral Organoids DIV 60 (organoid age at injection)
  1. 1 Preparation of cerebral organoids for injection
  2. 2 Injection of sarkosyl-insoluble fraction
  3. 3 Return to culture conditions
Protein Extraction from Cerebral Organoids (Post-Injection: 2, 4, and 8 Weeks) DIV 74, DIV 88, and DIV 116 (2, 4, and 8 weeks post-injection respectively)
  1. 1 Collection and washing of organoids
  2. 2 Sonication and lysis in RIPA buffer
  3. 3 Centrifugation for RIPA-soluble fraction
  4. 4 Protein concentration measurement of RIPA-soluble fraction
  5. 5 Preparation of RIPA-insoluble fraction
Western Blot Analysis Single working day (following protein extraction)
  1. 1 Sample preparation and denaturation
  2. 2 Electrophoresis on polyacrylamide gel
  3. 3 Protein transfer to PVDF membrane
  4. 4 Membrane blocking
  5. 5 Primary antibody incubation
  6. 6 Washing with PBS-T
  7. 7 Secondary antibody incubation
  8. 8 Final washing and substrate application
  9. 9 Image acquisition
Cryogenic Sectioning and Immunofluorescence Analysis 2–3 working days (sectioning followed by immunofluorescence)
  1. 1 Fixation of cerebral organoids
  2. 2 Cryoprotection with sucrose
  3. 3 Embedding in gelatin-sucrose solution
  4. 4 Freezing in liquid nitrogen
  5. 5 Cryosectioning
  6. 6 Storage of frozen sections
  7. 7 Gelatin removal from slides
  8. 8 Tissue blocking with BSA
  9. 9 Primary antibody incubation
  10. 10 Washing with PBS-T
  11. 11 Secondary antibody incubation
  12. 12 Final washing and mounting
  13. 13 Image acquisition by confocal microscopy
TUNEL Assay for Apoptosis Detection Single working day (following cryosectioning)
  1. 1 Gelatin removal from slides
  2. 2 Proteinase K treatment
  3. 3 Fixation with paraformaldehyde
  4. 4 Washing after fixation
  5. 5 DNA labeling with TdT enzyme and BrdUTP
  6. 6 Primary antibody incubation (anti-BrdU)
  7. 7 Final washing and mounting
  8. 8 Image acquisition
Quality Control and Statistical Analysis Following completion of all experimental phases
  1. 1 Image quantification and analysis
  2. 2 Western blot densitometry
  3. 3 Statistical analysis

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Tamaki et al., 2023. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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