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GASTRIC Publication-derived

Modelling human development and disease in pluripotent stem-cell-derived gastric organoids

Source McCracken et al., 2014 · Cincinnati Children's Hospital Medical Center, Division of Developmental Biology · 10.1038/nature13863

👤 Kyle W. McCracken, Emily M. Cata, Calyn M. Crawford, Katie L. Sinagoga, Michael Schumacher, Briana E. Rockich, Yu-Hwai Tsai, Christopher N. Mayhew, Jason R. Spence, Yana Zavros, James M. Wells ⏱ 34 days 📋 5 phases 🧫 Human ESC (WA01/H1, WA09/H9) and Human iPSC (72.3)

Abstract

This protocol describes the directed differentiation of human pluripotent stem cells (ESCs and iPSCs) into three-dimensional human gastric organoids (hGOs) recapitulating antral development. hGOs contain surface mucous cells, antral gland cells, endocrine cells, and LGR5+ stem cells, and model physiological responses to Helicobacter pylori infection including CagA-dependent epithelial proliferation.

Cell source
Human ESC (WA01/H1, WA09/H9) and Human iPSC (72.3)
Application
Disease modeling and developmental study of human gastric organogenesis

Protocol overview

16 steps across 5 phases

Pluripotent Stem Cell Culture and Maintenance Ongoing until differentiation
  1. 1 Culture pluripotent stem cells in feeder-free conditions
Definitive Endoderm Induction Day 0–3
  1. 1 Plate pluripotent stem cells as single cells
  2. 2 Induce definitive endoderm
Endoderm Patterning and Foregut Spheroid Generation Day 3–6
  1. 1 Establish foregut patterning conditions
  2. 2 Add retinoic acid on day 5
  3. 3 Harvest foregut spheroids
Antral Specification and Three-Dimensional Organoid Growth Day 6–34
  1. 1 Continue retinoic acid treatment for antral specification
  2. 2 Transfer spheroids to three-dimensional Matrigel culture
  3. 3 Overlay with gut media containing EGF
  4. 4 Maintain organoid culture for maturation
  5. 5 Optional: Re-plate organoids at day 20
  6. 6 Optional: Reduce EGF for endocrine differentiation
H. pylori Infection Assay (Optional) Day 34
  1. 1 Prepare H. pylori inoculum
  2. 2 Microinject H. pylori into organoid lumen
  3. 3 Culture infected organoids for 24 hours
  4. 4 Harvest infected organoids for analysis

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in McCracken et al., 2014. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

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