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BRAIN Publication-derived

Self-Organized Formation of Polarized Cortical Tissues from ESCs and Its Active Manipulation by Extrinsic Signals

Source Eiraku et al. · <UNKNOWN>

👤 Mototsugu Eiraku, Kiichi Watanabe, Mami Matsuo-Takasaki, Masako Kawada, Shigenobu Yonemura, Michiru Matsumura, Takafumi Wataya, Ayaka Nishiyama, Keiko Muguruma, Yoshiki Sasai ⏱ 46 days 📋 8 phases 🧫 Mouse ESC (EB3, EB5, Sox1-GFP 46C) and Human ESC

Abstract

This protocol describes the generation of self-organized, polarized cortical tissues from mouse and human ES cells using the SFEBq (serum-free floating culture of embryoid body-like aggregate with quick reaggregation) method. The protocol enables the reproducible formation of neuroepithelial rosettes with cortical neuronal differentiation, and allows manipulation of regional identity through extrinsic patterning signals.

Cell source
Mouse ESC (EB3, EB5, Sox1-GFP 46C) and Human ESC
Application
Developmental study and cortical tissue modeling

Protocol overview

36 steps across 8 phases

Generation of Bf1::Venus ES Cell Line Preparation phase (prior to differentiation)
  1. 1 Prepare targeting construct using rapid two-recombination (EG) method
  2. 2 Electroporate linearized vector into EB3 ES cells
  3. 3 Select homologous recombinant ES cells
  4. 4 Confirm targeted clones and select working clone
Mouse ES Cell Culture and SFEBq Differentiation Days 0–18
  1. 1 Prepare ES cell aggregates
  2. 2 Add recombinant patterning factors from day 0
  3. 3 Apply Wnt-3A treatment during days 6–10
  4. 4 Change medium every 2–3 days until day 18
  5. 5 Transfer aggregates to non-adhesive dish on day 18
Human ES Cell Culture and SFEBq Differentiation Days 0–46
  1. 1 Maintain hES cells and prepare for differentiation
  2. 2 Pretreat hES cells with Y27632 (optional)
  3. 3 Dissociate and reaggregate hES cells
  4. 4 Add patterning factors from day 0 to day 24
  5. 5 Use DMEM/F12/KSR as differentiation medium for hES cells
  6. 6 Transfer aggregates on day 18
  7. 7 Replate aggregates on day 25
Neuronal Dissociation Culture Days 12–42
  1. 1 Dissociate cortical tissue on day 12
  2. 2 Plate dissociated cells on day 12
  3. 3 Change medium to differentiation medium on day 14
  4. 4 Perform half-volume medium changes every 5 days
Cell Dissociation and FACS Preparation Days 7, 9, 12
  1. 1 Dissociate aggregates to single cells
  2. 2 Analyze cells by FACS on day 7, 9, or 12
Transplantation into Neonatal Mouse Cortex Day 11 (aggregate harvest) → P2 (transplant day) → 4 weeks (analysis)
  1. 1 Prepare P2 mouse pups
  2. 2 Dissociate donor Bf1::Venus cell aggregates
  3. 3 Inject cell suspension into host cortex
  4. 4 Revive and recovery
  5. 5 Analyze graft integration at 4 weeks
  6. 6 Alternative: Transplant neuronal masses
Calcium Imaging of Neuronal Activity Days 18–24
  1. 1 Set up filter culture on day 18
  2. 2 Change half-volume of medium every 3 days
  3. 3 Change medium to ACSF on day 21 or 24
  4. 4 Load calcium indicator dye
  5. 5 Perform calcium imaging
  6. 6 Apply pharmacological agents
Electron Microscopy Throughout differentiation (variable)
  1. 1 Fix tissue for electron microscopy
  2. 2 Process for transmission electron microscopy

Full SOP

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Attribution

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

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