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BRAIN Publication-derived

Human iPSC-Derived Dorsal Forebrain Organoid Generation and Characterization

Source <UNKNOWN>

👤 <UNKNOWN> ⏱ 70 days 📋 15 phases 🧫 Human iPSC

Abstract

This protocol describes the generation of dorsal forebrain organoids from human iPSC lines (BIONi010-C and HMGU1) through a multi-phase differentiation process spanning 70 days. Organoids are characterized via immunohistochemistry, Western blotting, and RNA sequencing to assess cellular composition, laminar organization, and gene expression patterns relevant to cortical development and neuroinflammatory responses.

Cell source
Human iPSC
Application
Disease modeling and developmental study

Protocol overview

111 steps across 15 phases

iPSC Culture and Maintenance Pre-differentiation
  1. 1 Culture iPSC lines in standard conditions
  2. 2 Passage iPSC colonies
  3. 3 Test for mycoplasma contamination
  4. 4 Verify pluripotency status
Initial Organoid Induction (Days 0–6) Days 0–6
  1. 1 Dissociate iPSCs to single cells
  2. 2 Plate cells in low-adhesion 96-well plates
  3. 3 Prepare cortical differentiation medium I (CDMI)
  4. 4 Add cells to CDMI with morphogen inhibitors
  5. 5 Perform media changes every three days (Days 0–6)
  6. 6 Withdraw Y-27632 at Day 6
Early Organoid Maturation (Days 6–18) Days 6–18
  1. 1 Continue CDMI culture with media changes
  2. 2 Transfer organoids to 24-well low-adhesion plates at Day 18
  3. 3 Transition to orbital shaker culture
  4. 4 Switch to cortical differentiation medium II (CDMII)
Intermediate Organoid Development (Days 18–35) Days 18–35
  1. 1 Culture in CDMII on orbital shaker
  2. 2 Switch to cortical differentiation medium III (CDMIII) at Day 35
Late Organoid Maturation (Days 35–70) Days 35–70
  1. 1 Culture in CDMIII on orbital shaker
  2. 2 Switch to cortical differentiation medium IV (CDMIV) at Day 70
  3. 3 Continue CDMIV culture
IL-6 and Hyper-IL-6 Treatment (Days 45–55) Days 45–55
  1. 1 Prepare IL-6 and Hyper-IL-6 stock solutions
  2. 2 Begin IL-6/Hyper-IL-6 treatment at Day 45
  3. 3 Maintain treatment with daily half-volume media changes
  4. 4 Withdraw treatment at Day 55
Immunohistochemistry and Sectioning Variable (typically Days 50–70)
  1. 1 Fix whole organoids in paraformaldehyde
  2. 2 Wash organoids in PBS
  3. 3 Cryoprotection in sucrose solution
  4. 4 Embed organoids in OCT compound
  5. 5 Section organoids on cryostat
  6. 6 Thaw and rehydrate slides
Immunofluorescence Staining Protocol Variable
  1. 1 Permeabilize and block tissue
  2. 2 Perform antigen retrieval (if required)
  3. 3 Apply primary antibodies
  4. 4 Wash sections
  5. 5 Apply secondary antibodies
  6. 6 Wash sections after secondary antibody
  7. 7 Apply nuclear counterstains
  8. 8 Mount and image sections
Western Blotting Variable
  1. 1 Lyse organoid tissue
  2. 2 Incubate lysates on rotating wheel
  3. 3 Sonicate lysates
  4. 4 Clarify lysates by centrifugation
  5. 5 Concentrate protein samples
  6. 6 Quantify total protein
  7. 7 Prepare samples in loading buffer
  8. 8 Resolve proteins on precast gels
  9. 9 Transfer proteins to PVDF membrane
  10. 10 Perform total protein staining
  11. 11 Block membranes
  12. 12 Incubate with primary antibodies
  13. 13 Wash membranes
  14. 14 Incubate with fluorescently labeled secondary antibodies
  15. 15 Wash membranes after secondary antibody
  16. 16 Image blots
RNA Isolation and Sequencing Preparation Variable
  1. 1 Isolate mRNA from organoids
  2. 2 Quality check RNA
  3. 3 Send samples for library preparation
Bulk RNA Sequencing Data Analysis Post-sequencing
  1. 1 Sequence samples on Illumina NovaSeq 6000
  2. 2 Align reads to reference genome
  3. 3 Filter reads
  4. 4 Quantify gene expression
  5. 5 Perform principal component analysis (PCA)
  6. 6 Perform hierarchical clustering
  7. 7 Perform differential gene expression (DGE) analysis
  8. 8 Perform functional analysis
  9. 9 Perform weighted gene co-expression analysis (WGCNA)
  10. 10 Reconstruct regulatory networks
Single-Cell Dissociation and Multiplexing Variable (typically at harvest timepoint)
  1. 1 Cut organoid tissue
  2. 2 Prepare papain dissociation medium
  3. 3 Enzymatic dissociation with papain
  4. 4 First trituration
  5. 5 Continue enzymatic digestion
  6. 6 Second trituration
  7. 7 Remove debris
  8. 8 Centrifuge cell suspension
  9. 9 Resuspend cells in BSA-PBS
  10. 10 Filter through cell strainer
  11. 11 Remove dead cells
  12. 12 Final centrifugation
  13. 13 Multiplex samples
  14. 14 Quantify and pool cells
  15. 15 Load cells onto Chromium chip
  16. 16 Generate scRNA-seq libraries
  17. 17 Pool and sequence libraries
Single-Cell RNA-seq Quality Control and Preprocessing Post-sequencing
  1. 1 Align reads to reference genome
  2. 2 Load data into Seurat
  3. 3 Apply quality control filters
  4. 4 Normalize gene expression
  5. 5 Integrate samples
  6. 6 Perform dimensionality reduction
  7. 7 Perform Louvain clustering
  8. 8 Generate UMAP plots
  9. 9 Remove doublets
  10. 10 Identify cluster markers
  11. 11 Manually assign cell types
  12. 12 Assess brain regional identity
Single-Cell Differential Analysis and Trajectory Inference Post-QC
  1. 1 Analyze cell type proportions
  2. 2 Infer differentiation trajectories
  3. 3 Assess trajectory quality
  4. 4 Identify differentially expressed genes (DGE) by cell type and condition
  5. 5 Perform gene set enrichment analysis (GSEA)
  6. 6 Calculate differential module expression
  7. 7 Test ASD risk gene enrichment
  8. 8 Construct regulatory networks (SCENIC)
Immunohistochemistry Image Analysis Variable
  1. 1 Acquire confocal images
  2. 2 Select regions of interest (ROIs)
  3. 3 Quantify cell populations
  4. 4 Quantify ventricular zone (VZ) markers
  5. 5 Quantify VZ architecture
  6. 6 Quantify cortical plate (CP) lamination
  7. 7 Analyze CTIP2/SATB2 co-expression
  8. 8 Quality control for imaging data

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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