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OTHER Publication-derived

Organoids for Regeneration and Disease Modelling

Full SOP with Academic or a plan

Source Ozone et al., 2016 · RIKEN Center for Developmental Biology · 10.1038/ncomms10351

Authors: Chikafumi Ozone, Hidetaka Suga, Mototsugu Eiraku, Taisuke Kadoshima, Shigenobu Yonemura, Nozomu Takata, Yutaka Oiso, Takashi Tsuji, Yoshiki Sasai Duration: 100 days 5 phases Cell source: Human ESC

Abstract

This protocol describes a three-dimensional self-organizing culture method (SFEBq) for differentiating human embryonic stem cells into functional anterior pituitary tissue capable of producing multiple hormone-producing cell types, including corticotrophs and somatotrophs that respond to physiological releasing and feedback signals. The generated pituitary progenitors differentiate into mature hormone-producing cells with demonstrated functional responses, and transplantation of in vitro-generated corticotrophs rescues hypopituitary mice.

Cell source
Human ESC
Application
Disease modeling; Developmental study; Regenerative medicine

Protocol overview

21 steps across 5 phases

Phase 1 · hESC Maintenance and Initial Aggregation Day -∞ to Day 0
  1. a Maintain undifferentiated hESCs on feeder layer
  2. b Prepare hESCs for SFEBq aggregation
  3. c Add medium on day 3
Phase 2 · Hypothalamic and Oral Ectoderm Co-induction Day 6-27
  1. a Initiate hedgehog signalling and BMP4 treatment
  2. b Promote Rathke's pouch formation with FGF2
  3. c Switch to suspension culture and increase KSR
Phase 3 · Maturation of Hormone-Producing Cells Day 27-98
  1. a Support corticotroph development
  2. b Induce somatotroph differentiation with dexamethasone
  3. c Induce gonadotroph differentiation with Notch inhibitor
Phase 4 · Functional Testing of Hormone-Producing Cells Day 80-100
  1. a Test corticotroph CRH responsiveness
  2. b Test glucocorticoid feedback suppression
  3. c Test somatotroph GHRH responsiveness
  4. d Test somatostatin-mediated GH suppression
Phase 5 · In Vivo Transplantation and Functional Assessment Day 71-82 (tissue harvest) and Week 0-16 (post-transplantation)
  1. a Prepare tissue for transplantation
  2. b Perform surgical hypophysectomy on SCID mice
  3. c Transplant pituitary tissue into kidney subcapsule
  4. d Perform CRH loading test in vivo
  5. e Measure corticosterone response
  6. f Assess spontaneous locomotor activity
  7. g Monitor body weight and survival
  8. h Assess long-term graft integration and vascularization

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Attribution

This SOP was written by Organthis based on the published method in Ozone et al., 2016. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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