PATIENT DERIVED INTESTINAL ORGANOIDS REPRESENT AN EPITHELIAL MODEL TO STUDY PEDIATRIC CROHNS DISEASE

Abstract Background Although therapeutics in Crohn’s disease (CD) have dramatically improved, current approaches targeting the immune system have limited success at a population level. Thus, there is a need for future therapeutics to target different pathways/cell types. Recently, patient derived stem cells have shown promise in helping to better understand the molecular basis of disease. We used patient derived organoids (PDOs), produced from both CD and healthy patients’ pinch biopsies, to determine the ability of PDOs to functionally represent the human intestinal epithelium, and to better understand the role of the epithelium in CD. Methods Mucosal biopsies from the terminal ileum were obtained from pediatric subjects (CD = 16, healthy controls = 13) by colonoscopy. We performed bulk RNA-seq on the freshly isolated ileal crypts (IC), and on the corresponding PDOs grown from the IC stem cells. The sequencing was performed on an Illumina HiSeq system in a single batch. The raw FastQ files were aligned with reference genome Hg38 using STAR package. Differential expression analysis was performed on the read counts using DEseq package. We compared the organoid gene expression to that of the IC for each patient, along with the differential analysis between groups. Results On average, 7.5M read counts were obtained for each IC and organoid sample. Of the 19,900 genes tested against Hg38, we found 15,554 genes expressed in either IC or organoids. Of those, 14,149 (91%) were expressed in both IC and organoids, 1,091 (7%) were expressed only in IC, and 314 (2%) expressed only in organoids. We compared PDOs from healthy controls to CD cases and the differentially expressed genes were identified using cutoff of log2FC > ±1.2 and statistically significant threshold p < 0.05. Compared to healthy controls, CCL5 and CXCL5 genes were up-regulated in CD, whereas PDGFB, IL-33 and ILDR2 genes were down-regulated. Conclusion We demonstrate that PDOs are 91% similar in gene expression to in vivo epithelium and thus are an excellent patient-specific in vitro epithelial model system. We have further used this model to show the CD specific transcriptional defects in the epithelium. Our data suggests that the epithelium is responsible, in part, in promoting neutrophil recruitment and inflammation through increases in CCL5 and CXCL5 expression, and changes in the Th1/Th2 profile through downregulation of IL-33 expression. Dysregulation of the cytoskeleton and barrier formation are both likely affected by down regulation of PDGFB and ILDR2, respectively, resulting in a negative effect on intestinal barrier function and restitution.