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BRAIN Publication-derived

Cortical Organoids for Disease Modelling (2008)

Full SOP with Academic or a plan

Source Eiraku et al., 2008 · RIKEN Center for Developmental Biology, Kobe, Japan · 10.1016/j.stem.2008.09.002

Authors: Mototsugu Eiraku, Kiichi Watanabe, Mami Matsuo-Takasaki, Masako Kawada, Shigenobu Yonemura, Michiru Matsumura, Takafumi Wataya, Ayaka Nishiyama, Keiko Muguruma, Yoshiki Sasai Duration: 46 days 7 phases Cell source: Mouse ESC (EB5), Human ESC

Abstract

This protocol describes the SFEBq (serum-free culture of embryoid body-like aggregates, quick) method for efficient generation of polarized cortical neuroepithelia and layer-specific cortical neurons from mouse and human embryonic stem cells. The method recapitulates spatial and temporal aspects of early corticogenesis and enables regional and temporal control of cortical tissue identity through manipulation of extrinsic signaling factors (Fgf, Wnt, BMP).

Cell source
Mouse ESC (EB5), Human ESC
Application
Developmental study, Disease modeling, Neural tissue engineering

Protocol overview

16 steps across 7 phases

Phase 1 · ESC Dissociation and Quick Reaggregation Day 0
  1. a Dissociate ESCs to single cells
  2. b Prepare SFEBq aggregates in 96-well low cell-adhesion plates
Phase 2 · Neural Differentiation in Suspension (Days 1-7) Days 1-7
  1. a Culture aggregates in differentiation medium
  2. b Monitor aggregate formation
Phase 3 · Transfer to Adhesion Culture and Continued Differentiation (Days 7-10) Days 7-10
  1. a Transfer aggregates to bacterial-grade dish with N2 medium
  2. b Culture in N2 medium for 3 days
Phase 4 · Neuroepithelial Rosette Formation and Neuronal Differentiation (Days 10-16) Days 10-16
  1. a Verify rosette formation by immunostaining (optional quality control)
  2. b Continue culture or initiate dissociation culture for neuronal differentiation
  3. c Culture dissociated neurons for maturation
Phase 5 · Regional Specification by Signaling Manipulation (Days 7-12, optional) Days 7-12
  1. a Sort Bf1+/Venus+ cells by FACS (optional, for regional specification experiments)
  2. b Quickly reaggregate sorted cells and treat with patterning factors
Phase 6 · Temporal Control of Layer-Specific Neurogenesis (Days 8-16, optional) Days 8-16
  1. a Perform early timepoint FACS and Notch inhibition for Reelin+ cell enrichment
  2. b Perform late timepoint FACS and Notch inhibition for Ctip2+ cell enrichment
Phase 7 · Human ESC Adaptation (Days 0-46, for hESCs only) Days 0-46
  1. a Prepare differentiation medium for hESCs
  2. b Add ROCK inhibitor to dissociation and reaggregation steps
  3. c Follow standard SFEBq protocol thereafter

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Attribution

This SOP was written by Organthis based on the published method in Eiraku et al., 2008. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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