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GASTRIC Publication-derived

Gastric Organoids for Disease Modelling (2014)

Full SOP with Academic or a plan

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

Authors: 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 Duration: 34 days 5 phases Cell source: 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

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

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Attribution

This SOP was written by Organthis based on the published method in McCracken et al., 2014. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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