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BRAIN Publication-derived

Hypothalamic-Pituitary Functional Unit Generation from Human iPS Cells via 3D Culture

Source Kasai et al., 2020 · Nagoya University Graduate School of Medicine · 10.1016/j.celrep.2019.12.009

👤 Takatoshi Kasai, Hidetaka Suga, Mayu Sakakibara, Chikafumi Ozone, Ryusaku Matsumoto, Mayuko Kano, Kazuki Mitsumoto, Koichiro Ogawa, Yu Kodani, Hiroshi Nagasaki, Naoko Inoshita, Mariko Sugiyama, Takeshi Onoue, Taku Tsunekawa, Yoshihiro Ito, Hiroshi Takagi, Daisuke Hagiwara, Shintaro Iwama, Motomitsu Goto, Ryoichi Banno, Jun Takahashi, Hiroshi Arima ⏱ 500 days 📋 10 phases 🧫 Human iPSC

Abstract

A protocol for generating functional hypothalamic-pituitary units from human induced pluripotent stem cells using 3D floating culture (SFEBq method). The induced pituitary cells achieve ACTH secretion capacity comparable to adult mouse anterior pituitary cells, and the hybrid organoids demonstrate responsiveness to environmental stimuli (low glucose) via the CRH-ACTH pathway.

Cell source
Human iPSC
Application
Disease modeling; Developmental study

Protocol overview

60 steps across 10 phases

iPS Cell Seeding and Initial Differentiation (SFEBq) Day 0–30
  1. 1 Prepare gfCDM Base Medium
  2. 2 Dissociate iPS Cells to Single Cells
  3. 3 Seed Cells in V-Bottom 96-Well Plate
  4. 4 Day 3 Medium Supplementation
  5. 5 Day 6–27 Medium Changes with Growth Factor Addition
  6. 6 Day 18 Oxygen Environment Adjustment
  7. 7 Day 30 Transfer to EZ Sphere
  8. 8 Day 30 Onward: Medium Transition to 10% KSR
  9. 9 Day 33 Onward: Complete Medium Replacement
  10. 10 Day 51 Onward: Final Medium Transition to 20% KSR
Modified Condition Optimization for Enhanced ACTH Maturation Day 0–100
  1. 1 Optimize Initial Cell Seeding Density
  2. 2 Increase BMP4 Concentration
  3. 3 Maintain SAG Concentration at 2 μM
  4. 4 Day 6–30 Medium Replacement with 10% KSR
  5. 5 Maintain High Oxygen Concentration (40%) from Day 18
Long-Term Culture for Hypothalamic-Pituitary Co-maturation Day 51–500
  1. 1 Maintain gfCDM + 20% KSR Medium
  2. 2 Monitor Morphology and Marker Expression
  3. 3 Measure ACTH Secretion in Culture Supernatant
  4. 4 Modified Hypothalamic Differentiation Medium (Optional, from Day 51)
  5. 5 Continue Culture Until Day 300–500
Functional Assays: CRH Stimulation Test Day 113–152
  1. 1 Prepare Aggregates for Assay
  2. 2 Baseline ACTH Measurement (Control Condition)
  3. 3 CRH Stimulation
  4. 4 Measure ACTH in CRH-Stimulated Condition
  5. 5 Optional: Verify CRH-R1 Expression
Functional Assays: Dexamethasone Suppression Test Day 141–229
  1. 1 Prepare Aggregates and Baseline Measurement
  2. 2 Dexamethasone Treatment
  3. 3 Measure ACTH in Dexamethasone-Treated Condition
Functional Assays: CRH Receptor Inhibitor Tests Day 163–273
  1. 1 Antalarmin Loading Test—Baseline Measurement
  2. 2 Antalarmin Treatment
  3. 3 Measure ACTH After Antalarmin Treatment
  4. 4 NBI27914 Loading Test—Baseline Measurement
  5. 5 NBI27914 Treatment
  6. 6 Measure ACTH After NBI27914 Treatment
Functional Assays: Low-Glucose Stimulation Test Day 192–240
  1. 1 Prepare Aggregates for Low-Glucose Assay
  2. 2 Prepare Low-Glucose Medium
  3. 3 Prepare Normal-Glucose Medium
  4. 4 Baseline ACTH Measurement in Normal Glucose
  5. 5 Low-Glucose Stimulation
  6. 6 Compare ACTH Levels
Functional Assays: Low-Glucose + CRH-R Inhibitor Test Day 222–270
  1. 1 Prepare Aggregates
  2. 2 Confirm ACTH Response to Low Glucose (Positive Control)
  3. 3 Overnight Pretreatment with CRH-R Inhibitors
  4. 4 Low-Glucose Stimulation in Presence of CRH-R Inhibitors
  5. 5 Compare ACTH Responses
Electron Microscopy for Structural Characterization Day 500 (or endpoint)
  1. 1 Fix Aggregates for Electron Microscopy
  2. 2 Post-Fix in Osmium Tetroxide
  3. 3 Dehydration in Graded Alcohol Series
  4. 4 Embed in Epon 812 Resin
  5. 5 Prepare Semi-Thin Sections
  6. 6 Prepare Ultrathin Sections
  7. 7 Stain Ultrathin Sections
  8. 8 Examine Under Transmission Electron Microscope
Immuno-Electron Microscopy for ACTH Localization Day 500 (or endpoint)
  1. 1 Prepare Ultrathin Sections on EM Grids
  2. 2 Pre-Treatment with Sodium Periodate and Hydrogen Peroxide
  3. 3 Incubate with Mouse Anti-ACTH Primary Antibody
  4. 4 Wash Sections in PBS
  5. 5 Incubate with Gold-Conjugated Secondary Antibody
  6. 6 Final Wash and Air-Dry
  7. 7 Image by Transmission Electron Microscopy

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Kasai et al., 2020. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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