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OTHER Publication-derived

Functional anterior pituitary generated in self-organizing culture of human embryonic stem cells

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

👤 Chikafumi Ozone, Hidetaka Suga, Mototsugu Eiraku, Taisuke Kadoshima, Shigenobu Yonemura, Nozomu Takata, Yutaka Oiso, Takashi Tsuji, Yoshiki Sasai ⏱ 100 days 📋 5 phases 🧫 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

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

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Ozone et al., 2016. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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