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BRAIN Publication-derived

Generation and Characterization of C9ORF72-HRE Cerebral Organoids from Patient-Derived iPSCs

Source van der Geest et al., 2024 · University Medical Center Utrecht · 10.1186/s40478-024-01857-1

👤 Astrid T. van der Geest, Channa E. Jakobs, Tijana Ljubikj, Christiaan F. M. Huffels, Marta Cañizares Luna, Renata Vieira de Sá, Youri Adolfs, Marina de Wit, Daan H. Rutten, Marthe Kaal, Maria M. Zwartkruis, Mireia Carcolé, Ewout J. N. Groen, Elly M. Hol, Onur Basak, Adrian M. Isaacs, Henk-Jan Westeneng, Leonard H. van den Berg, Jan H. Veldink, Domino K. Schlegel, R. Jeroen Pasterkamp ⏱ 90 days 📋 13 phases 🧫 Patient-Derived iPSC (C9ORF72-ALS/FTD), Presymptomatic C9-HRE Carriers, Healthy Control iPSC

Abstract

This protocol describes the generation and comprehensive characterization of three-dimensional cerebral organoids derived from induced pluripotent stem cells (iPSCs) obtained from C9ORF72 hexanucleotide repeat expansion (C9-HRE) carriers with amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD), presymptomatic C9-HRE carriers, and healthy controls. The protocol enables the study of molecular pathology, developmental changes, cellular architecture, and synaptic dysfunction in human brain tissue models.

Cell source
Patient-Derived iPSC (C9ORF72-ALS/FTD), Presymptomatic C9-HRE Carriers, Healthy Control iPSC
Application
Disease modeling

Protocol overview

61 steps across 13 phases

iPSC Reprogramming and Culture Variable; cultures maintained long-term
  1. 1 Obtain and culture human dermal fibroblasts
  2. 2 Reprogramm fibroblasts to iPSCs using Sendai virus
  3. 3 Culture iPSCs in StemFlex medium
iPSC Characterization 1-7 days per assay
  1. 1 Perform karyotyping analysis
  2. 2 Perform trilineage differentiation
  3. 3 Perform immunocytochemistry for pluripotency markers
  4. 4 Perform short tandem repeat (STR) analysis
  5. 5 Determine C9-HRE length by repeat-primed PCR (RP-PCR)
  6. 6 Determine C9-HRE length by variable number tandem repeat PCR (VNTR-PCR) for repeats <30
  7. 7 Determine C9-HRE length by nanopore sequencing
Cerebral Organoid Culture 0-90+ days
  1. 1 Dissociate iPSCs and form embryoid bodies (EBs)
  2. 2 Maintain embryoid bodies and initiate differentiation (Days 0-4)
  3. 3 Initiate neural induction (Day 6)
  4. 4 Transfer to matrigel and initiate cerebral differentiation (Day 13)
  5. 5 Continue cerebral differentiation on orbital shaker (Day 17 onwards)
  6. 6 Fix and prepare organoids for analysis (Selected timepoints)
Organoid Size Analysis Days 2, 6, 10, 90
  1. 1 Capture brightfield images
  2. 2 Quantify organoid area
Immunohistochemistry and Immunofluorescence 1-3 days per experiment
  1. 1 Prepare cryosections
  2. 2 Block and stain with primary antibodies
  3. 3 Wash and stain with secondary antibodies
  4. 4 Image sections
Quantitative Real-Time PCR (RT-qPCR) 1 day
  1. 1 Extract RNA from organoids
  2. 2 Synthesize cDNA
  3. 3 Perform RT-qPCR
Western Blot Analysis 1 day
  1. 1 Prepare protein lysates
  2. 2 Prepare and load gel
  3. 3 Transfer proteins to membrane
  4. 4 Block and incubate with primary antibodies
  5. 5 Wash and incubate with secondary antibodies
  6. 6 Image and quantify
Locked Nucleic Acid Fluorescence In Situ Hybridization (LNA-FISH) 2-3 days
  1. 1 Prepare tissue sections
  2. 2 Pre-treat sections
  3. 3 Perform prehybridization and hybridization
  4. 4 Wash and detect probe
  5. 5 Amplify and visualize signal
  6. 6 Mount and image
Single-Cell RNA Sequencing (scRNA-seq) 3-5 days (sample prep and sequencing)
  1. 1 Dissociate organoids to single cells
  2. 2 Freeze cells
  3. 3 Prepare library using 10X Genomics platform
  4. 4 Sequence libraries
  5. 5 Process sequencing data
  6. 6 Perform dimensionality reduction and clustering
  7. 7 Annotate cell types
  8. 8 Analyze cell composition and differential expression
3D Fluorescent Light Sheet Microscopy 2-3 days
  1. 1 Fix and prepare organoids
  2. 2 Perform iDISCO clearing and immunostaining
  3. 3 Tissue clearing
  4. 4 Image with light sheet microscopy
  5. 5 Analyze organoid structure
Synapse Quantification 2 days
  1. 1 Image synaptic puncta
  2. 2 Quantify synaptic puncta
  3. 3 Statistical analysis
Patch-Clamp Electrophysiology 2 days (slice preparation and recording)
  1. 1 Prepare air-liquid interface (ALI) organoid slices
  2. 2 Prepare recording chamber and solutions
  3. 3 Prepare recording electrodes and pipette solution
  4. 4 Perform whole-cell patch-clamp recordings
  5. 5 Record and analyze electrophysiology data
Cell Cycle Phase Analysis 1 day
  1. 1 Perform EdU pulse-labeling
  2. 2 Prepare cells for flow cytometry
  3. 3 Analyze cell cycle by flow cytometry

Full SOP

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Attribution

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

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