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BRAIN Publication-derived

Brain Organoids for Developmental Studies (2015)

Full SOP with Academic or a plan

Source Sakaguchi et al., 2015 · RIKEN Center for Developmental Biology · 10.1038/ncomms9896

Authors: Hideya Sakaguchi, Taisuke Kadoshima, Mika Soen, Nobuhiro Narii, Yoshihito Ishida, Masatoshi Ohgushi, Jun Takahashi, Mototsugu Eiraku, Yoshiki Sasai Duration: 197 days 5 phases Cell source: Human ESC

Abstract

This protocol generates functional hippocampal granule and pyramidal neurons from self-organizing human embryonic stem cell-derived dorsomedial telencephalic tissue. Through sequential treatment with bone morphogenetic protein and Wnt signaling, the method produces choroid plexus, cortical hem, and medial pallium tissues that mature into electrically active, network-forming hippocampal neurons in vitro, recapitulating human hippocampal development.

Cell source
Human ESC
Application
Developmental study

Protocol overview

15 steps across 5 phases

Phase 1 · hESC Maintenance and Initial Differentiation Day 0 to Day 18
  1. a Maintain hESCs on feeder layer
  2. b Prepare single-cell suspension for SFEBq
  3. c Add neural induction inhibitors
Phase 2 · Choroid Plexus Induction (Condition 2) Day 18 to Day 42
  1. a Transfer aggregates and initiate dorsomedial signaling
  2. b Maintain choroid plexus differentiation
Phase 3 · Medial Pallium Induction (Condition 3) Day 18 to Day 21
  1. a Transient CHIR and BMP4 exposure
  2. b Switch to patterning medium
  3. c Continue culture to day 27
Phase 4 · Long-Term Medial Pallium Culture and Hippocampal Primordium Formation Day 27 to Day 75
  1. a Switch to Neurobasal medium
  2. b Half-cut aggregates at day 35
  3. c Transfer to Lumox dishes at day 50
  4. d Maintain culture to day 75
Phase 5 · Neuronal Dissociation Culture and Maturation Day 73 to Day 197+
  1. a Dissociate neurons from aggregates
  2. b Change medium after 3 days
  3. c Continue long-term culture with half-medium changes

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Attribution

This SOP was written by Organthis based on the published method in Sakaguchi et al., 2015. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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