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GASTRIC Publication-derived

Gastric Organoids for Drug Screening

Full SOP with Academic or a plan

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

Authors: Kyle W. McCracken, Emily M. Catá, 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 6 phases Cell source: Human ESC (H1, H9), Human iPSC (from neonatal foreskin fibroblasts)

Abstract

This protocol describes the directed differentiation of human pluripotent stem cells (hPSCs) into three-dimensional human gastric organoids (hGOs) through sequential manipulation of FGF, WNT, BMP, retinoic acid, and EGF signaling pathways. The resulting organoids recapitulate normal antral stomach development and contain multiple cell types including surface and gland mucous cells, proliferative progenitors (LGR5+), and diverse gastric endocrine cells (gastrin, ghrelin, somatostatin, and serotonin-expressing). hGOs provide a human-specific in vitro model for studying stomach organogenesis, testing signaling mechanisms in early endoderm patterning, and modeling pathological responses to H. pylori infection.

Cell source
Human ESC (H1, H9), Human iPSC (from neonatal foreskin fibroblasts)
Application
Disease modeling (H. pylori infection pathogenesis), developmental study, drug discovery

Protocol overview

14 steps across 6 phases

Phase 1 · Pluripotent Stem Cell Maintenance and Plating d0 (preparation)
  1. a Culture and passage hESC/iPSC lines
  2. b Dissociate and plate hPSCs as single cells
Phase 2 · Definitive Endoderm Induction d1-d3
  1. a Induce definitive endoderm (DE)
Phase 3 · Foregut Specification and Patterning (Spheroid Generation) d3-d6
  1. a Pattern endoderm to foregut with WNT/FGF/Noggin
  2. b Posteriorize foregut endoderm with retinoic acid
Phase 4 · Antral Specification and Initial 3D Culture d6-d9
  1. a Transfer spheroids to 3D Matrigel culture
  2. b Culture in gut media with RA and Noggin
Phase 5 · Gastric Organoid Expansion and Morphogenesis d9-d34
  1. a Continue 3D culture with gut media and EGF
  2. b Re-plate organoids at day 20
  3. c Optional: reduce EGF for endocrine cell differentiation (if desired)
Phase 6 · H. pylori Infection Assay (Optional) d34 (or later)
  1. a Prepare H. pylori bacterial suspension
  2. b Remove antibiotics from organoid culture media
  3. c Microinject H. pylori into organoid lumen
  4. d Culture infected organoids for 24 hours

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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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