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BRAIN Publication-derived

PTCH1-mutant human cerebellar organoids: CRISPR-edited iPSC differentiation and characterization

Source van Essen et al., 2024 · Nuffield Department of Clinical Neurosciences, University of Oxford · 10.1242/dmm.050323

👤 Max J. van Essen, Elizabeth J. Apsley, Joey Riepsaame, Ruijie Xu, Paul A. Northcott, Sally A. Cowley, John Jacob, Esther B. E. Becker ⏱ 90 days 📋 11 phases 🧫 Human iPSC

Abstract

This protocol describes the generation and differentiation of PTCH1-mutant human induced pluripotent stem cells (iPSCs) into cerebellar organoids using CRISPR/Cas9 gene editing. The method combines iPSC culture, targeted genetic editing, directed cerebellar differentiation, and comprehensive molecular characterization to model early pathophysiological events in medulloblastoma tumorigenesis without animal models.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

63 steps across 11 phases

iPSC culture and maintenance Ongoing
  1. 1 Culture iPSCs on Matrigel-coated plates
  2. 2 Apply Rho kinase inhibitor after passaging
CRISPR-mediated PTCH1 gene editing Days 1–14
  1. 1 Design and prepare guide RNAs
  2. 2 Prepare crRNA/tracrRNA hybrids
  3. 3 Complex ribonuclease proteins with Cas9
  4. 4 Electroporate iPSCs with ribonucleoprotein complexes
  5. 5 Culture electroporated cell pool
  6. 6 Extract genomic DNA and PCR genotyping
  7. 7 Generate monoclonal iPSC lines by single-cell cloning
  8. 8 Confirm clones by Sanger sequencing
CRISPR clone quality control Days 14–21
  1. 1 Perform SNP genotyping array analysis
  2. 2 Check for off-target CRISPR effects by sequencing
  3. 3 Test for mycoplasma contamination
  4. 4 Measure pluripotency by flow cytometry
Trilineage differentiation validation Days 22–29
  1. 1 Differentiate iPSCs into three germ layers
  2. 2 Change medium to lineage-specific medium
  3. 3 Fix cells for immunostaining
EdU proliferation assay on iPSCs Days 22–29
  1. 1 Prepare cells for EdU labeling
  2. 2 Perform flow cytometry with EdU and Hoechst
Cerebellar organoid differentiation Days 30–90
  1. 1 Prepare embryoid bodies
  2. 2 Initiate cerebellar lineage induction
  3. 3 Perform weekly medium changes (days 2–14)
  4. 4 Transfer organoids to larger wells
  5. 5 Switch to differentiation medium
  6. 6 Culture on transwell membranes from day 35 onward
  7. 7 Harvest organoids at designated timepoints
SHH pathway manipulation and drug treatment (optional) Days 2–35
  1. 1 Prepare SMO inhibitor cyclopamine treatment
  2. 2 Prepare SMO agonist SAG treatment
  3. 3 Test drug withdrawal effects
  4. 4 Harvest treated organoids
RNA extraction and RT-qPCR analysis Days 21–90
  1. 1 Extract RNA from organoid samples
  2. 2 Perform reverse transcription
  3. 3 Determine primer efficiency and optimal cDNA input
  4. 4 Perform RT-qPCR
  5. 5 Analyze RT-qPCR data
Immunostaining and imaging Days 21–90
  1. 1 Fix organoids in paraformaldehyde
  2. 2 Embed in Optimal Cutting Temperature compound
  3. 3 Cryosection organoids
  4. 4 Permeabilize cryosections
  5. 5 Block non-specific binding
  6. 6 Incubate with primary antibodies
  7. 7 Incubate with secondary antibodies
  8. 8 Visualize nuclei and mount slides
  9. 9 Image stained sections
Flow cytometry analysis Days 35–50
  1. 1 Dissociate organoids to single cells
  2. 2 Wash cells in FACS buffer
  3. 3 Fix cells in paraformaldehyde
  4. 4 Permeabilize cells for intracellular staining
  5. 5 Stain with primary antibody
  6. 6 Wash and stain with nuclear dye
  7. 7 Acquire data by flow cytometry
  8. 8 Analyze mean fluorescence intensity
RNA sequencing and transcriptome analysis Days 35–90 (sequencing) + data analysis
  1. 1 Extract RNA for RNA-seq
  2. 2 Assess RNA quality
  3. 3 Measure RNA concentration
  4. 4 Prepare RNA-seq libraries
  5. 5 Perform RNA-seq
  6. 6 Quantify gene expression using Salmon
  7. 7 Import transcript quantifications into R
  8. 8 Perform differential expression analysis
  9. 9 Normalize expression data and perform PCA
  10. 10 Perform gene ontology and pathway analysis
  11. 11 Compare organoid transcriptomes with published datasets

Full SOP

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Attribution

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

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