Skip to content
Back to browse
BRAIN Publication-derived

Brain Organoids for Disease Modelling (2011)

Full SOP with Academic or a plan

Source Suga et al., 2011 · RIKEN Center for Developmental Biology, Kobe, Japan · 10.1038/nature10637

Authors: Hidetaka Suga, Taisuke Kadoshima, Maki Minaguchi, Masatoshi Ohgushi, Mika Soen, Tokushige Nakano, Nozomu Takata, Takafumi Wataya, Keiko Muguruma, Hiroyuki Miyoshi, Shigenobu Yonemura, Yutaka Oiso, Yoshiki Sasai 7 phases Cell source: Mouse embryonic stem cells (ES cells)

Abstract

This protocol describes the generation of functional three-dimensional adenohypophysis (anterior pituitary) tissue from mouse embryonic stem cells in culture. Through controlled spatial arrangement of rostral head ectoderm and hypothalamic neuroectoderm, combined with hedgehog signaling, Rathke's-pouch-like structures self-form and differentiate into hormone-producing endocrine cells including corticotrophs and somatotrophs. The resulting tissue exhibits functional hormone secretion and can rescue glucocorticoid levels in hypopituitary mice.

Cell source
Mouse embryonic stem cells (ES cells)
Application
Disease modeling; developmental study of pituitary organogenesis

Protocol overview

27 steps across 7 phases

Phase 1 · ES Cell Maintenance and Preparation Prior to day 0
  1. a Maintain mouse ES cells in maintenance medium
  2. b Dissociate ES cells to single cells
Phase 2 · Large Cell Aggregation (LCA) Culture Initiation Day 0
  1. a Prepare growth-factor-free chemically defined medium (gfCDM)
  2. b Reaggregate dissociated ES cells at 10,000 cells per well
  3. c Add Smoothened agonist (SAG) to culture medium
Phase 3 · Rathke's Pouch Formation (Days 0-13) Days 0-13
  1. a Culture aggregates in LCA-SAG conditions (days 0-10)
  2. b Observe morphogenesis of Lim3+ structures (days 6-13)
  3. c Raise oxygen concentration to 40% from day 10 onwards
Phase 4 · Corticotroph Differentiation (Days 13-21) Days 13-21
  1. a Prepare Notch inhibitor (DAPT) treatment
  2. b Treat aggregates with DAPT on days 18-19
  3. c Continue culture to day 21 for ACTH+ cell accumulation
Phase 5 · Non-Corticotroph Lineage Differentiation (Days 13-33) Days 13-33
  1. a Prepare BIO (Wnt agonist) treatment for Pit1+ intermediate precursors
  2. b Treat with BIO on days 16-18 for Pit1 induction
  3. c Differentiate GH+ somatotrophs with glucocorticoid and insulin
  4. d Differentiate prolactin+ lactotrophs with oestradiol and insulin
  5. e Optional: Culture with PA6 stromal cell-conditioned medium for gonadotroph and TSH differentiation
Phase 6 · Functional Analysis: In Vitro ACTH Secretion Assay Days 21-23
  1. a Collect SAG+DAPT-treated aggregates for secretion assay
  2. b Rinse aggregates and pre-incubate
  3. c Stimulate with Corticotropin-Releasing Hormone (CRH)
  4. d Collect supernatant and measure ACTH by ELISA
  5. e Optional: Test glucocorticoid-mediated feedback inhibition
Phase 7 · Transplantation and In Vivo Function (Optional) Days 21-35 post-transplant
  1. a Perform surgical hypophysectomy in ICR mice
  2. b Prepare graft aggregates and dexamethasone supplementation
  3. c Perform ectopic transplantation under kidney capsule
  4. d Assess CRH-stimulated ACTH release at 7 days post-transplant
  5. e Measure basal ACTH and corticosterone levels
  6. f Perform behavioral and survival assessments

Full SOP

Full SOPs need an account and a plan

Create an account, then get free Academic access (for verified academics) or choose a plan. Plans include step-by-step procedures, reagent concentrations, QC checklists and batch record templates.

Create an account

Already registered? Log in

Attribution

This SOP was written by Organthis based on the published method in Suga et al., 2011. Once the originating laboratory has confirmed it, the SOP is marked Verified.

Something wrong with this entry? Report an issue with this protocol

Need a commercial licence?
Use this protocol in your therapeutic or diagnostic pipeline.
Request Commercial Licence