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BRAIN Publication-derived

Generation of PTEN-Mutant Forebrain Organoids from hiPSCs for ASD Disease Modeling

Source Cleveland Clinic Β· 10.21203/rs.3.rs-2787151/v1

πŸ‘€ <UNKNOWN>, <UNKNOWN> ⏱ 72 days πŸ“‹ 8 phases 🧫 Patient-Derived iPSC (PTEN germline mutations: PHTS-ASD and PHTS-cancer)

Abstract

This protocol generates forebrain organoids from gene-edited hiPSCs carrying PTEN mutations (G132D associated with ASD or M134R associated with cancer) to model neurodevelopmental pathology. The protocol recapitulates early forebrain formation, neuroepithelial organization, and cortical layering, enabling investigation of how distinct PTEN missense mutations disrupt neurodevelopment at different developmental stages.

Cell source
Patient-Derived iPSC (PTEN germline mutations: PHTS-ASD and PHTS-cancer)
Application
Disease modeling - PTEN-associated autism spectrum disorder and Hamartoma Tumor Syndrome

Protocol overview

68 steps across 8 phases

hiPSC Maintenance and Preparation Before Day 0
  1. 1 Maintain PTEN-mutant hiPSC lines
Embryoid Body Formation (Days 0–1) Day 0–1
  1. 1 Dissociate hiPSC monolayer with Collagenase IV
  2. 2 Collect and wash clusters
  3. 3 Resuspend clusters in first FB induction medium
  4. 4 Culture embryoid bodies for 24 hours
  5. 5 Change forebrain induction medium on day 1
Forebrain Induction and Maturation (Days 3–7) Day 3–7
  1. 1 Perform medium changes on days 3–4
  2. 2 Transition to second FB induction medium (days 5–6)
  3. 3 Embed organoids in Geltrex matrix (day 7)
  4. 4 Solidify gel matrix
  5. 5 Add second FB medium and culture to day 14
  6. 6 Apply AKT inhibitor (optional, days 0–7)
Bioreactor Culture and Maturation (Days 14–72+) Day 14–72+
  1. 1 Remove organoids from Geltrex matrix
  2. 2 Transfer to SpinΞ©β„’ bioreactor wells
  3. 3 Establish bioreactor spin conditions
  4. 4 Culture in third FB medium (days 14–35)
  5. 5 Add Matrigel coating (starting day 35)
  6. 6 Continue culture to day 72 and beyond
Immunofluorescence Staining (All Time Points) Days 7, 14, 28, 72+
  1. 1 Fix organoids with paraformaldehyde
  2. 2 Wash fixed organoids
  3. 3 Incubate in sucrose overnight
  4. 4 Embed organoids in cryomold
  5. 5 Cryosection organoids
  6. 6 Permeabilize sections
  7. 7 Block nonspecific binding
  8. 8 Incubate with primary antibodies
  9. 9 Wash after primary antibody
  10. 10 Incubate with secondary antibodies
  11. 11 Final washing and mounting
  12. 12 Acquire confocal microscopy images
  13. 13 Alternative: whole-mount staining for early-stage organoids
Western Blot Analysis Days 0–7 (or as specified)
  1. 1 Harvest and collect organoids
  2. 2 Wash organoids with PBS
  3. 3 Lyse organoids with M-PER buffer
  4. 4 Clarify lysates by centrifugation
  5. 5 Quantify total protein by BCA assay
  6. 6 Prepare samples for gel electrophoresis
  7. 7 Load samples and run polyacrylamide gel
  8. 8 Transfer proteins to membrane
  9. 9 Block membrane with BSA
  10. 10 Incubate with primary antibodies
  11. 11 Wash membrane after primary antibody
  12. 12 Incubate with secondary antibodies
  13. 13 Final washing
  14. 14 Detect immunoreactive bands
  15. 15 Quantify band intensity
Single-Cell RNA Sequencing (scRNAseq) Day 7 (or specified time point)
  1. 1 Harvest organoids and dissociate to single cells
  2. 2 Enzymatic dissociation with TrypLE Select
  3. 3 Stop enzymatic dissociation
  4. 4 Transfer and centrifuge cell suspension
  5. 5 Resuspend cells in PBS
  6. 6 Filter cells through cell strainer
  7. 7 Assess cell viability and count
  8. 8 Prepare 10X Chromium Single Cell chip
  9. 9 Generate droplets and perform reverse transcription
  10. 10 Recover cDNA from emulsion
  11. 11 Clean cDNA with Silane DynaBeads
  12. 12 Amplify cDNA
  13. 13 Cleanup amplified cDNA
  14. 14 Quantify cDNA
  15. 15 Construct 3' libraries
scRNAseq Data Analysis Post-sequencing
  1. 1 Sequence libraries to target depth
  2. 2 Process FASTQ files with cellranger
  3. 3 Quality control filtering
  4. 4 Normalization and clustering
  5. 5 Trajectory analysis with Monocle3
  6. 6 Differential expression analysis
  7. 7 Ingenuity Pathway Analysis

Full SOP

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Attribution

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

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