Establishment and Characterization of Patient-Derived Intestinal Organoids from Pediatric Crohn's Disease Patients

Abstract

Purpose: This study aimed to establish and characterize patient-derived intestinal organoids (PDOs) from children with Crohn's disease (CD).

Methods: To generate PDOs, endoscopic biopsy specimens were obtained from non-inflamed duodenal bulbs of normal controls and CD patients. To verify the presence of PDOs, histological staining and quantitative reverse transcription polymerase chain reaction (RT-qPCR) analyses were performed.

Results: PDOs were successfully established in normal controls (n=2) and CD patients (n=2). Hematoxylin and eosin staining of formalin-fixed, paraffin-embedded PDO sections revealed crypt and villus structures, whereas immunofluorescence staining with EpCAM and DAPI confirmed the epithelial-specific architecture of the PDOs. RT-qPCR results revealed a significant increase in Lgr5, Si, and Chga gene expression and a decrease in Olfm4 and Muc2 expression in CD patients compared to normal controls, suggesting altered stem cell activity and mucosal barrier function (p<0.05).

Conclusion: We successfully established and characterized PDOs in children with CD, providing a valuable tool for understanding the pathophysiology of the disease and evaluating potential therapeutic approaches. The differential gene expression of PDOs in CD patients might be caused by the complex interplay between epithelial adaptation and inflammation in the intestinal epithelium.

Keywords: Biopsy; Child; Crohn disease; Endoscopy; Organoids.

Fig. 1. Process of intestinal organoid growth from days one to six in fourth week passage in control group.

Fig. 2. Distinct crypt and villus structures in normal (A) and CD (B) samples visualized by hematoxylin and eosin staining from paraffin-embedded sections. Scale bars 50 µm. CD: Crohn’s disease.

Fig. 3. Structural analysis of intestinal epithelial cells in human duodenal 3-dimensional organoids from control (A) and CD (B) patients using EpCAM immunofluorescence staining. Representative images show epithelial cell marker with EpCAM (green) and nuclei (blue) with DAPI. Scale bars 60 µm. CD: Crohn’s disease.

Fig. 4. mRNA expression of intestine-specific genes between normal and CD patients in human duodenal 3-dimensional organoids. mRNA expression level of stem cell-associated genes (A) Lgr5 and (B) Olfm4, as well as intestinal epithelial maturation-associated genes (C) Si, (D) Muc2, (E) Chga, and (F) Lyz measured using quantitative reverse transcription polymerase chain reaction analyses. Gene expressions presented using 2^–ΔΔCt method. Statistical significance differences between groups analyzed by Student’s t-test. ns: not significant, CD: Crohn’s disease. ^**p<0.01, ^***p<0.001, ^****p<0.0001.