Porcine Intestinal Organoids as Models of Regional Gut and Animal Identities: a Transcriptomic Approach

Organoids are emerging in vitro systems that are expected to bridge the gap between knowledge at the cellular, tissue, and whole-animal levels, with ethical benefits for animal science (3Rs). They offer promise for genotype-to-phenotype research; however, efforts are needed to assess their effective ability and reliability in reflecting the phenotypes of the original tissues from which they are derived. We generated RNA-seq profiles from intestinal organoids and matched tissues from the duodenum, jejunum, ileum, and colon of four pigs at slaughter age. Although organoids were globally distinct from tissues, they retained segment specificity--clearly separating large from small intestine and, to a lesser extent, discriminating among small-intestinal regions. Epithelial programmes remained regionally patterned, whereas innate immune signatures were reduced and less spatially resolved in vitro. Developmental mapping of ileum samples indicated that organoids only partially recapitulate native tissue programmes, consistent with a comparatively immature state. Across gut segments, organoids preserved key transcriptional and functional hallmarks, including immune regulation, metabolism, and developmental pathways. Inter-individual variability was detectable but modest relative to segment-driven differences. Notably, organoids maintained animal-specific epithelial specialisations, including glycosylation pathways involving FUT2 and B4GALNT2. In summary, intestinal organoids retain native tissue identity while displaying immature epithelial and innate immune signatures. They preserve principal segment-defined and animal-specific molecular programmes. These features support further complexification with stromal components and luminal microbiota, underscoring the utility of organoids as a model for genotype-to-phenotype research.