RECOVERING A LOST IDENTITY: EVALUATING DISEASE-SPECIFIC TRANSCRIPTIONAL PROGRAMS BETWEEN PERIANAL CROHN’S DISEASE PATIENT-DERIVED ORGANOIDS AND CORRESPONDING MUCOSAL EPITHELIUM

Background: Perianal Crohn’s disease (CD) induces extensive mucosal damage that is proposed to pathologically alter epithelium and mesenchyme. Here, we tested the ability of patient derived intestinal organoids to maintain the disease characteristics of perianal CD.
Methods: Rectal mucosal biopsies were obtained from patients (n=31, 12 inflamed, 19 non-inflamed) at Children’s Healthcare of Atlanta and immediately processed for single cell RNA sequencing, and for a subset of those patients (n=13), organoids were established and sequenced after 3 passages. Organoids and mucosal epithelial cells were bioinformatically compared using phenotypic and unsupervised categorization.
Results: Mucosal epithelial cells (n=96,392) and cells from paired patient-derived organoids (n=77,044) revealed significant differences at the cell subtype and transcriptomic levels. While mucosal epithelial cells displayed 18 subtypes, organoids comprised 10. Organoid subtypes were transcriptionally distinct from mucosal subtypes, with only partial indication of early subtype lineages. Unlike mucosal epithelial cells where variation by principal components corresponded to patients’ phenotypic inflammation, organoids diverged into two groups based on their proliferative activity. The mucosal epithelial cell type distribution (17% undifferentiated, 62% absorptive, 18% secretory, 17% other) was not retained by the patients’ organoids (63% undifferentiated, 19% early absorptive, 17% early secretory, 1% other). Further investigation found that organoids do not express transcription factors critical to transitioning into mature secretory (goblet: ATOH1/SPDEF, tuft: POU2F3, enteroendocrine: NEUROD1) and select absorptive cell types (BEST4+: OTOP2/SPIB) but constitutively express HES1 that promotes the absorptive lineage in mucosal epithelial cells. Hierarchical clustering conveyed that transcription profiles of patient-derived organoids shared similar composition, regardless of patient phenotype, than to their mucosal counterparts. Microfold-like (CCL20+) cells detected in organoids appeared to be the only epithelial subtype most closely resembling a mucosal counterpart, but lacked SPIB expression, a transcription factor required for full microfold differentiation. Nonetheless, some remnants of disease were retained in vitro, including altered PPAR signaling, lipid synthesis, and epithelial barrier function.
Conclusion: Culturing organoids in high Wnt media without a crypt-axis gradient of chemical cues from the mesenchyme prohibits their ability to fully recapitulate the normal epithelial subtypes or disease phenotypes. However, changes in epithelial metabolic and barrier function in perianal CD persist.