Home › Cell Biology › Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens
Cell Biology JoVE (Open Access) Citable · DOI

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

DOI: 10.3791/59478-v
What you'll learn
  • ✓ Differentiate hiPSCs into functional intestinal organoids (iHOs) with epithelial architecture
  • ✓ Perform microinjection of pathogenic Salmonella into iHO lumens for infection studies
  • ✓ Assess epithelial invasion and protection mechanisms using organoid-based assays
  • ✓ Apply cytokine stimulation to modulate epithelial barrier function in vitro
Protocol

Biopharma Insights Human induced pluripotent stem cell (hiPSC)-derived intestinal organoids offer exciting opportunities to model enteric diseases in vitro. We demonstrate the differentiation of hiPSCs into intestinal organoids (iHOs), the stimulation of these iHOs with cytokines, and the microinjection of Salmonella Typhimurium into the iHO lumen, enabling the study of an epithelial invasion by this pathogen.

Difficulty
advanced
Total time
~4–6 weeks (differentiation + maintenance + infection assay)
Model organism
Human induced pluripotent stem cells (hiPSCs)
Biosafety
BSL-2

Steps

1
Differentiate iPSCs into hindgut endoderm

Culture hiPSCs and apply directed differentiation protocol to generate hindgut specification prior to organoid formation. This establishes the foundational cell type for intestinal organoid development.

▶ 00:38
2
Embed hindgut tissue in basement membrane matrix

Encapsulate differentiated hindgut cells in Matrigel or comparable extracellular matrix to promote 3D self-organization and maturation into polarized intestinal organoids.

▶ 01:10
3
Maintain, passage, and stimulate iHOs with rhIL-22

Culture intestinal organoids in maintenance media, passage at defined intervals, and expose to recombinant human IL-22 to enhance epithelial barrier function and innate immunity.

▶ 03:13
4
Microinject Salmonella into iHO lumen and perform invasion assays

Introduce Salmonella Typhimurium into the lumen of iHOs via microinjection, then quantify intracellular bacterial invasion and epithelial damage using standard cell biology techniques.

▶ 05:28
5
Analyze epithelial protection and pathogenic response outcomes

Evaluate organoid integrity, bacterial burden, and epithelial cell death to assess how cytokine pre-treatment and organoid maturation affect resistance to Salmonella invasion.

▶ 09:19
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