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BRAIN Publication-derived

Neural stem cell homeostasis in cortical organoids carrying Angiogenin mutations: Disease modeling and characterization

Source Ferguson et al., 2024 · Department of Life Sciences, University of Bath · 10.1002/path.6244

👤 Ross Ferguson, Michael A van Es, Leonard H van den Berg, Vasanta Subramanian ⏱ 50 days 📋 12 phases 🧫 Patient-Derived iPSC (ALS-FTD)

Abstract

This protocol generates patient-derived iPSC lines from skin fibroblasts of an ALS-FTD family with Angiogenin and/or TARDBP mutations, then differentiates them into cortical organoids and cortical neurons to model neurodevelopmental and neuronal stress defects associated with frontotemporal lobar degeneration. The protocol characterizes abnormal neural stem cell dynamics, rosette disorganization, altered neuronal subtype specification, and impaired stress responses.

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

Protocol overview

74 steps across 12 phases

Fibroblast culture and genomic characterization Day 0 (baseline)
  1. 1 Culture human fibroblasts
  2. 2 Isolate genomic DNA and sequence ANG and TARDBP genes
iPSC reprogramming Day 0–Day 25
  1. 1 Reprogramming fibroblasts to iPSCs
  2. 2 Pick and mechanically disassociate iPSC clones
  3. 3 Passage iPSC colonies on feeder layers
  4. 4 Characterize iPSC clones for pluripotency
  5. 5 Test iPSC clones for multi-lineage differentiation
Embryoid body formation and characterization Day 0–Day 14
  1. 1 Initiate embryoid body (EB) differentiation
  2. 2 Culture embryoid bodies and change media
  3. 3 Harvest embryoid bodies
Cortical organoid differentiation Day 0–Day 50
  1. 1 Initiate cortical differentiation
  2. 2 Generate aggregates in low-attachment plates
  3. 3 Continue organoid maturation until day 25
  4. 4 Switch to mature neural differentiation media (day 25 onwards)
  5. 5 Maintain organoids until day 50
Directed cortical neuron differentiation Day 0–Day 20+
  1. 1 Initiate neural differentiation (days 0–8)
  2. 2 Passage onto Matrigel-coated plates (day 8)
  3. 3 Continue NPC expansion until day 13
  4. 4 Switch to differentiation media (day 13 onwards)
  5. 5 Maintain mature cortical neurons until day 20+
RNA isolation and RT-PCR As needed for characterization
  1. 1 Lyse cells in TRIzol
  2. 2 Treat RNA with DNase
  3. 3 Reverse transcription
  4. 4 Perform RT-PCR
  5. 5 Analyze PCR products
Immunofluorescence on monolayer cultures As needed for marker characterization
  1. 1 Fix cells on coverslips
  2. 2 Dehydrate and store fixed cells
  3. 3 Rehydrate and block
  4. 4 Incubate with primary antibody
  5. 5 Wash and incubate with secondary antibody
  6. 6 Final washes and mount
  7. 7 Image cells
Immunofluorescence and immunohistochemistry on organoid cryosections Day 20–Day 50 (organoid time points)
  1. 1 Prepare organoids for cryosectioning
  2. 2 Embed in OCT and freeze
  3. 3 Cut cryosections
  4. 4 Warm slides and remove OCT
  5. 5 Perform antigen retrieval
  6. 6 Block and incubate with primary antibody
  7. 7 Wash and incubate with secondary antibody
  8. 8 Final washes and mount with DAPI
  9. 9 Image stained organoid sections
Haematoxylin and eosin (H&E) staining of organoid sections As needed for morphological assessment
  1. 1 Prepare sections for H&E staining
  2. 2 Stain with haematoxylin
  3. 3 Blue sections in ammonia water
  4. 4 Dehydrate through ethanol series
  5. 5 Stain with eosin
  6. 6 Complete dehydration
  7. 7 Mount and cure
  8. 8 Image and analyze
O-propargyl-puromycin (OPP) incorporation assay for protein synthesis At differentiation time points (day 6, 12, 20)
  1. 1 Prepare cells for OPP incorporation
  2. 2 Pulse cells with O-propargyl-puromycin (OPP)
  3. 3 Fix cells
  4. 4 Perform additional marker immunostaining (optional)
  5. 5 Perform Click-iT reaction for OPP detection
  6. 6 Wash and mount
  7. 7 Image and quantify OPP incorporation
  8. 8 Quantify mean OPP intensity per nucleus
Neuronal stress assays and viability assessment Day 20–Day 50 (mature neuron stage)
  1. 1 Treat cortical organoids with stress agent
  2. 2 Assess neuronal viability by MTT assay
  3. 3 Fix organoids for stress granule imaging
  4. 4 Stain for stress granule markers
  5. 5 Quantify stress granule frequency
  6. 6 Stain for apoptotic markers (optional)
Western blotting for protein expression and processing As needed for mechanistic studies
  1. 1 Prepare cell lysates under stress or control conditions
  2. 2 Lyse cells
  3. 3 Quantify protein concentration
  4. 4 Load and run gel electrophoresis
  5. 5 Transfer proteins to PVDF membrane
  6. 6 Block membrane
  7. 7 Incubate with primary antibody
  8. 8 Wash membrane
  9. 9 Incubate with secondary antibody
  10. 10 Final wash and detection
  11. 11 Quantify band intensity

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Ferguson et al., 2024. 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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