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BRAIN Publication-derived

Miller-Dieker Syndrome Brain Organoids

Full SOP with Academic or a plan

Source Bershteyn et al., 2017 · Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco · 10.1016/j.stem.2016.12.007

Authors: Marina Bershteyn, Tomasz J. Nowakowski, Alex A. Pollen, Elizabeth Di Lullo, Aishwarya Nene, Anthony Wynshaw-Boris, Arnold R. Kriegstein Duration: 70 days 9 phases Cell source: Patient-Derived iPSC (Miller-Dieker Syndrome)

Abstract

This protocol generates 3D cerebral organoids from control and Miller-Dieker syndrome (MDS) patient-derived iPSCs to model lissencephaly and characterize cellular defects in neural progenitors, including increased apoptosis in neuroepithelial stem cells, defective neuronal migration, and prolonged mitosis in outer radial glia. The organoids recapitulate early human corticogenesis and enable investigation of disease pathogenesis at the cellular level.

Cell source
Patient-Derived iPSC (Miller-Dieker Syndrome)
Application
Disease modeling

Protocol overview

48 steps across 9 phases

Phase 1 · iPSC Dissociation and Aggregate Formation Day 0
  1. a Dissociate iPSCs to single cells
  2. b Prepare cortical differentiation medium with inhibitors
  3. c Form aggregates in lipidure-coated 96-well plates
Phase 2 · Early Neural Differentiation in Suspension Culture Days 0-18
  1. a Maintain aggregates in cortical differentiation medium
  2. b Monitor organoid size and morphology
  3. c Transfer aggregates to ultra-low adhesion plates at day 18
Phase 3 · Extended Organoid Culture with Oxygen Control Days 18-35
  1. a Incubate organoids in controlled oxygen atmosphere
  2. b Perform regular media changes
  3. c Monitor organoid development by immunostaining
Phase 4 · Extended Culture with Growth Factors (Days 35-70) Days 35-70
  1. a Supplement medium with FBS, Matrigel, and heparin from day 35
  2. b Continue oxygen-controlled culture
  3. c Perform media changes every 2-3 days
  4. d Harvest organoids at day 70 (10 weeks) for analysis
Phase 5 · Slice Culture and Live Imaging (Weeks 5 and 10) Weeks 5 and 10
  1. a Embed organoids in low-melting agarose
  2. b Generate vibratome sections
  3. c Incubate slices with adenovirus overnight
  4. d Mount slices on glass-bottom dishes
  5. e Perform live imaging
Phase 6 · Immunostaining Protocol Week 5 and 10 (post-harvest)
  1. a Fix organoids with 4% PFA
  2. b Wash and cryoprotect in sucrose
  3. c Embed in OCT compound and freeze
  4. d Section on cryostat
  5. e Perform heat/citrate antigen retrieval
  6. f Permeabilize and block
  7. g Incubate with primary antibodies
  8. h Wash after primary antibodies
  9. i Incubate with secondary antibodies
  10. j Final washes and mounting
Phase 7 · Neuronal Migration Assay (In Vitro on Matrigel) Days 0-3 of migration assay
  1. a Resuspend intact organoids in Matrigel
  2. b Solidify Matrigel
  3. c Overlay with culture medium
  4. d Monitor process outgrowth
  5. e Perform live imaging from Day 2
  6. f Perform endpoint immunostaining on Day 3
  7. g Analyze cell tracks using Imaris software
Phase 8 · Neuronal Migration Assay (Co-culture with Cortical Tissue Explants) Days 0-4 of migration assay
  1. a Infect week 5 organoids with AAV1-CAG-tdTomato
  2. b Prepare human cortical tissue slices
  3. c Infect cortical slices with CMV-GFP adenovirus
  4. d Co-culture organoid and tissue slices on Day 1
  5. e Embed co-culture in Matrigel
  6. f Culture for 4 days
  7. g Fix and analyze migration endpoint
Phase 9 · Single-Cell RNA Sequencing Weeks 5, 10, and 15
  1. a Dissociate organoids to single cells
  2. b Capture single cells using fluidigm C1
  3. c Prepare single-cell cDNA libraries
  4. d Perform high-throughput sequencing
  5. e Align reads and quantify gene expression
  6. f Identify radial glia-like cells and oRG signature

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Attribution

This SOP was written by Organthis based on the published method in Bershteyn et al., 2017. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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