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BRAIN Publication-derived

An Organoid-Based Model of Cortical Development for Miller-Dieker Syndrome Studies

Source Iefremova et al. · <UNKNOWN>

👤 Vira Iefremova, George Manikakis, Olivia Krefft, Ammar Jabali, Kevin Weynans, Ruven Wilkens, Fabio Marsoner, Björn Brändl, Franz-Josef Müller, Philipp Koch, Julia Ladewig ⏱ 33 days 📋 11 phases 🧫 Patient-Derived iPSC (Miller-Dieker Syndrome)

Abstract

This protocol describes the generation of human iPSC-derived cortical organoids to model Miller-Dieker Syndrome (MDS), a neuronal migration disorder caused by mutations in LIS1 and YWHAE genes. The organoids recapitulate early cortical development and exhibit Wnt signaling defects contributing to impaired neuronal organization and migration.

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

Protocol overview

56 steps across 11 phases

iPSC Maintenance and Characterization Ongoing
  1. 1 Culture iPSCs on Geltrex-coated plates
  2. 2 Dissociate iPSCs and add ROCK inhibitor
  3. 3 Verify mycoplasma status
SNP Analysis and Karyotyping Before differentiation
  1. 1 Prepare genomic DNA
  2. 2 Perform whole-genome SNP genotyping
  3. 3 Analyze SNP data
Germ Layer Differentiation for Validation Days 1–28
  1. 1 Dissociate iPSCs and plate in ultra-low-binding plates
  2. 2 Plate EBs onto Geltrex-coated dishes
  3. 3 Culture EBs for differentiation
Generation of LIS1 and 14.3.3ε Rescue Lines Days 1–7+
  1. 1 PCR amplify AAVS1 homology regions
  2. 2 Subclone PCR products into TOPO vector
  3. 3 Clone HR-L into PB-TetON vector
  4. 4 Clone HR-R to generate PB-TetON-AAVS1
  5. 5 PCR amplify LIS1 and YWHAE
  6. 6 Clone LIS1 and YWHAE into PB-TetON-AAVS1
  7. 7 Nucleofect MDS-derived iPSCs
  8. 8 Select for successfully transfected cells
iPSC-Derived Cortical Organoid Generation Days 1–33+
  1. 1 Initiate neural induction
  2. 2 Dissociate and replate cells
  3. 3 Passage rosettes for organoid development
Organoid Characterization: Immunofluorescence Days 10–33
  1. 1 Immunostain for pluripotency markers (TRA-1-60, TRA-1-81, SSEA-4)
  2. 2 Immunostain for general markers (all other antibodies)
  3. 3 Immunostain for cryosections
  4. 4 Image organoids
Organoid Quantification and Analysis Days 10–33
  1. 1 Measure overall organoid size
  2. 2 Quantify loop parameters
  3. 3 Quantify cell death
  4. 4 Quantify Tbr2+ intermediate progenitors
  5. 5 Measure acetylated α-tubulin strand density
  6. 6 Quantify mitotic planes
  7. 7 Quantify apical membrane alignment
RT-PCR and Gene Expression Analysis At specified timepoints
  1. 1 Extract total mRNA
  2. 2 Perform reverse transcription
  3. 3 Perform semiquantitative PCR
  4. 4 Perform quantitative real-time PCR (q-rt-PCR)
Immunoblot Analysis At specified timepoints
  1. 1 Prepare cell lysates
  2. 2 Clarify lysates
  3. 3 Determine protein concentration
  4. 4 Prepare samples for SDS-PAGE
  5. 5 Electrophoresis and blotting
  6. 6 Block and incubate with primary antibody
  7. 7 Incubate with secondary antibody
  8. 8 Visualize blots
Wnt Signaling Modulation and Luciferase Assay Days 20+
  1. 1 Treat organoids with GSK3β inhibitor
  2. 2 Generate Wnt reporter lines
  3. 3 Differentiate reporter lines into cortical rosettes
  4. 4 Seed cortical cells for luciferase assay
  5. 5 Culture cells with test conditions
  6. 6 Perform luciferase assay
  7. 7 Normalize luciferase signals
N-cadherin Functional Studies Days 5–6 of rosette passage
  1. 1 Plate cells for N-cadherin blocking experiments
  2. 2 Change media daily
  3. 3 Quantify neurogenesis in N-cadherin blocked cultures
  4. 4 Plate cells for N-cadherin activation experiments
  5. 5 Change media daily during N-cadherin activation
  6. 6 Quantify neurogenesis in N-cadherin activated cultures

Full SOP

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Attribution

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

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