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BRAIN Publication-derived

Self-Formation of Cortical Layers by Telencephalic Neural

Source Kadoshima et al. · RIKEN Center for Developmental Biology, Kobe, Japan · 10.1073/pnas.1315710110

👤 Kadoshima T, Sakaguchi H, Nakano T, Soen M, Ando S, Eiraku M, Sasai Y ⏱ 112 days 📋 7 phases 🧫 Human ESC

Abstract

Kadoshima et al. demonstrated that human ESC-derived cortical neuroepithelium can self-organize into layered structures recapitulating the ventricular zone, subventricular zone, and cortical plate using the SFEBq method. This RIKEN/Sasai lab protocol generated human-specific outer radial glia cells and represented a parallel foundational advance alongside Lancaster's cerebral organoid work.

Cell source
Human ESC
Application
Cortical Layering Studies

Protocol overview

29 steps across 7 phases

hESC Maintenance on Feeder Layer Ongoing maintenance prior to differentiation
  1. 1 Prepare feeder layer with inactivated MEFs
  2. 2 Culture hESCs on MEF feeder in maintenance medium
  3. 3 Passage hESCs by enzymatic digestion
  4. 4 Re-plate hESCs at optimal split ratio
Initiation of SFEBq Culture and Cortical Specification (Days 0–18) Days 0–18
  1. 1 Dissociate hESCs to single cells
  2. 2 Quick reaggregate hESCs in low-adhesion plates
  3. 3 Prepare cortex differentiation medium
  4. 4 Add morphogens for cortical specification (Days 0–18)
  5. 5 Monitor aggregate morphology
Transfer to Suspension Culture and Early Expansion (Days 18–35) Days 18–35
  1. 1 Transfer aggregates to non-adhesive Petri dish
  2. 2 Switch to N2-supplemented medium
  3. 3 Culture under elevated oxygen (40% O₂/5% CO₂)
  4. 4 Add FBS, heparin, and Matrigel (from Day 35)
  5. 5 Perform medium changes every 2–3 days
Mechanical Dissection and Long-Term Culture (Days 35–56) Days 35–56
  1. 1 Cut aggregates to prevent central cell death
  2. 2 Maintain medium composition
  3. 3 Prepare for transition to high-O₂ dishes (Day 56+)
Maturation and Extended Culture (Days 56–112+) Days 56–112+
  1. 1 Switch to lumox dishes for enhanced oxygen supply
  2. 2 Increase Matrigel concentration (Day 70)
  3. 3 Continue mechanical dissection and medium changes
  4. 4 Maintain final medium composition
Optional Treatments: Rostralization, Ventralization, and ROCK Inhibition Days 15–42 (rostralization); Days 15–21 (ventralization); Days 26–30 (ROCK inhibition)
  1. 1 FGF8b treatment for rostralization (Days 24–42)
  2. 2 Smoothened agonist (SAG) treatment for ventralization (Days 15–21)
  3. 3 Y-27632 treatment for ROCK inhibition (Days 26–30)
Generation of Knock-In hESC Lines (foxg1::Venus and pax6::Venus) Variable; performed prior to experimental use
  1. 1 Design zinc finger nucleases (ZFNs) for gene targeting
  2. 2 Construct targeting vector with homology arms and venus
  3. 3 Dissociate hESCs and prepare for electroporation
  4. 4 Electroporate targeting vector and ZFN-coding mRNAs
  5. 5 Select homologous recombinant clones

Full SOP

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Attribution

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

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