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BRAIN Publication-derived

Fused dorsal-ventral cerebral organoids model complex interactions between diverse brain regions

Source Bagley et al., 2017 · Institute of Molecular Biotechnology of the Austrian Academy of Science (IMBA) · 10.1038/nmeth.4304

👤 Joshua A Bagley, Daniel Reumann, Shan Bian, Julie Lévi-Strauss, Juergen A Knoblich ⏱ 80 days 📋 9 phases 🧫 Human iPSC

Abstract

This protocol describes a method for generating fused dorsal-ventral cerebral organoids that model complex interactions between different brain regions. By combining drug-patterned ventral forebrain organoids with dorsal forebrain organoids in a co-culture fusion assay, the protocol enables the study of directional GABAergic interneuron migration from ventral to dorsal regions, with applications to understanding neurodevelopmental disorders and testing therapeutic compounds.

Cell source
Human iPSC
Application
Disease modeling; Developmental study; Drug screening

Protocol overview

45 steps across 9 phases

hPSC Culture and Maintenance Ongoing
  1. 1 Culture feeder-dependent hiPSCs
  2. 2 Culture feeder-free hESCs
Reporter Gene Integration via TALEN Days 1–14
  1. 1 Prepare hPSCs for nucleofection
  2. 2 Perform nucleofection with TALEN constructs
  3. 3 Plate nucleofected cells
  4. 4 Select clones with puromycin
  5. 5 Transfer and expand surviving clones
  6. 6 Genotype targeted clones
  7. 7 Archive verified clones
Cerebral Organoid Generation and Patterning Days 5–40
  1. 1 Initiate embryoid body (EB) formation
  2. 2 Apply drug-patterning treatments (Days 5–11, neural induction phase)
  3. 3 Embed EBs in Matrigel
  4. 4 Maintain organoid culture and supplement medium
  5. 5 Assess organoid quality by qPCR (Days 30–40)
Organoid Fusion Co-culture Days 11–80
  1. 1 Prepare EBs for fusion
  2. 2 Embed two EBs in a single Matrigel droplet
  3. 3 Monitor fusion process
  4. 4 Continue culture of fused organoids
Tissue Preparation and Immunofluorescence Days 30–80 (variable based on experimental timepoint)
  1. 1 Fix organoid tissue
  2. 2 Cryoprotect tissue
  3. 3 Embed tissue in OCT and freeze
  4. 4 Cryostat section tissue
  5. 5 Permeabilization and blocking
  6. 6 Primary antibody incubation
  7. 7 Wash after primary antibodies
  8. 8 Secondary antibody incubation
  9. 9 DAPI staining and final wash
  10. 10 Mount coverslips
Slice Culture and Drug Treatment Days 30–80
  1. 1 Prepare organoid fusion slices by vibratome sectioning
  2. 2 Culture slices for time-lapse imaging (short-term, 1–2 days)
  3. 3 Culture slices for drug treatment (long-term, 3 weeks)
  4. 4 Fix slices for analysis
Time-lapse Imaging of Migration Days 60–80
  1. 1 Mount slices for spinning disk confocal microscopy
  2. 2 Acquire z-stack time-lapse movies
  3. 3 Process time-lapse data
  4. 4 Analyze migratory behavior
Cell Counting and Density Quantification Variable (post-sectioning)
  1. 1 Image immunostained cryosections
  2. 2 Stitch and process tile-scan images
  3. 3 Manually count migrated GFP+ cells
  4. 4 Calculate cell density
Marker Expression Quantification Variable (post-immunostaining)
  1. 1 Identify and mark VZ-like regions
  2. 2 Score VZ regions for marker expression
  3. 3 Calculate percentage of VZ regions expressing each marker
  4. 4 Quantify coexpression in migrated GFP+ cells
  5. 5 Calculate percentage of GFP+ cells expressing each marker

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Bagley et al., 2017. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

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