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BRAIN Publication-derived

Periventricular Heterotopia Cerebral Organoids

Full SOP with Academic or a plan

Source Klaus et al., 2019 · Max Planck Institute of Psychiatry, Munich, Germany · 10.1038/s41591-019-0371-0

Authors: Johannes Klaus, Sabina Kanton, Christina Kyrousi, Ane Cristina Ayo-Martin, Rossella Di Giaimo, Stephan Riesenberg, Adam C. O'Neill, J. Gray Camp, Chiara Tocco, Malgorzata Santel, Ejona Rusha, Micha Drukker, Mariana Schroeder, Magdalena Götz, Stephen P. Robertson, Barbara Treutlein, Silvia Cappello Duration: 70 days 13 phases Cell source: Patient-Derived iPSC (Periventricular Heterotopia with DCHS1 or FAT4 mutations); Human iPSC (isogenic knockout controls)

Abstract

This protocol describes the generation of three-dimensional cerebral organoids from induced pluripotent stem cells (iPSCs) derived from individuals with periventricular heterotopia (PH) caused by mutations in DCHS1 or FAT4 genes, as well as isogenic knockout control lines. The organoids recapitulate the cortical heterotopia phenotype and reveal defects in neural progenitor cell morphology and neuronal migration dynamics characteristic of PH disease.

Cell source
Patient-Derived iPSC (Periventricular Heterotopia with DCHS1 or FAT4 mutations); Human iPSC (isogenic knockout controls)
Application
Disease modeling - Periventricular Heterotopia

Protocol overview

70 steps across 13 phases

Phase 1 · Fibroblast Collection and iPSC Reprogramming Day 0-16
  1. a Fibroblast Collection and Feeder Cell Setup
  2. b Patient Fibroblast Dissociation and Seeding
  3. c Medium Change and B18R Supplementation
  4. d Modified mRNA (mmRNA) Transfection
  5. e Colony Observation and Early Selection
  6. f Medium Switch to STEMPRO hESC SFM
  7. g iPSC Colony Harvesting and Passaging
  8. h Adaptation to Feeder-Free Culture
Phase 2 · CRISPR-Cas9 Knockout iPSC Line Generation Variable (typically 1-2 weeks)
  1. a doxycycline Induction of Cas9
  2. b gRNA Lipofection for DCHS1 Editing
  3. c gRNA Electroporation for FAT4 Editing
  4. d Edited Cell Selection and Cloning
  5. e Genotyping of Single-Cell Colonies
Phase 3 · Neural Progenitor Cell (NPC) Differentiation from iPSCs Day 0-14
  1. a Embryoid Body Formation
  2. b Embryoid Body Plating and Neural Induction
  3. c Neural Rosette Identification and Manual Isolation
  4. d NPC Expansion and Passaging
Phase 4 · Cerebral Organoid Generation and Culture Day 0-70
  1. a Organoid Initiation from iPSCs
  2. b Organoid Maintenance and Medium Exchanges
  3. c Organoid Analysis Timepoints
Phase 5 · miRNA-Mediated Gene Knockdown in Organoids Variable (7-14 days post-electroporation)
  1. a miRNA Vector Construction and Validation
  2. b Organoid Electroporation Setup
  3. c Electroporation Pulse Delivery
  4. d Post-Electroporation Recovery
Phase 6 · Three-Dimensional Time-Lapse Imaging of Neuronal Migration Typically day 42-46 of organoid culture
  1. a Organoid Slice Preparation
  2. b Slice Culture on Cell Culture Inserts
  3. c Live Imaging Setup and Acquisition
  4. d Neuronal Tracking and Analysis
Phase 7 · Two-Dimensional Neuronal Migration Assay Day 0-10 of neuronal differentiation
  1. a NPC Differentiation in Monolayer
  2. b Live Imaging of Neuronal Migration
  3. c Cell Tracking and Parameter Measurement
  4. d Neuronal Identity Confirmation
Phase 8 · Single-Cell RNA Sequencing (scRNA-seq) - Smart-seq2 Protocol (Organoids) Variable (typically organoids at day 50-60)
  1. a Organoid Microdissection
  2. b Tissue Dissociation and Cell Filtering
  3. c Cell Washing and Counting
  4. d FACS Sorting of Single Cells
  5. e Smart-seq2 cDNA Synthesis
  6. f Library Construction with Nextera XT
  7. g Sequencing on Illumina HiSeq2500
Phase 9 · Single-Cell RNA Sequencing (scRNA-seq) - 10X Genomics Protocol (2D Neuronal Cultures) Day 14 of neuronal differentiation
  1. a Neuronal Culture Dissociation
  2. b Cell Washing and Resuspension
  3. c 10X Chromium Library Preparation Setup
  4. d cDNA Cleanup, Amplification, and Library Preparation
  5. e Library Pooling and Sequencing
Phase 10 · Immunohistochemistry and Imaging Variable (typically at organoid analysis timepoints)
  1. a Organoid Sectioning
  2. b Antigen Retrieval and Blocking
  3. c Primary Antibody Incubation
  4. d Washing and Secondary Antibody Incubation
  5. e Nuclear Staining and Mounting
  6. f Microscopy and Image Acquisition
  7. g Image Quantification
Phase 11 · FACS-Based Quantification of Cell Populations Day 55-60 of organoid culture
  1. a Organoid Collection and Enzymatic Dissociation
  2. b Cell Washing and Filtering
  3. c Cell Fixation in Ethanol
  4. d Antibody Staining
  5. e Secondary Antibody Incubation
  6. f FACS Data Acquisition
Phase 12 · Western Blot Analysis Variable (typically day 40-42 of organoid culture)
  1. a Protein Extraction from NPCs
  2. b Protein Quantification
  3. c SDS-PAGE Separation
  4. d Protein Transfer to Nitrocellulose Membrane
  5. e Blocking and Primary Antibody Incubation
  6. f Washing and Secondary Antibody Incubation
  7. g Chemiluminescent Detection
  8. h Quantification
Phase 13 · In Situ Hybridization Variable (typically at organoid analysis timepoints)
  1. a Probe Generation
  2. b Organoid Sectioning and Slide Preparation
  3. c mRNA Detection by In Situ Hybridization
  4. d Washing and Antibody Detection
  5. e Mounting and Microscopy

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Attribution

This SOP was written by Organthis based on the published method in Klaus et al., 2019. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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