Generation of Differentiating and Long-Living Intestinal Organoids Reflecting the Cellular Diversity of Canine Intestine

Abstract

Functional intestinal disorders constitute major, potentially lethal health problems in humans. Consequently, research focuses on elucidating the underlying pathobiological mechanisms and establishing therapeutic strategies. In this context, intestinal organoids have emerged as a potent in vitro model as they faithfully recapitulate the structure and function of the intestinal segment they represent. Interestingly, human-like intestinal diseases also affect dogs, making canine intestinal organoids a promising tool for canine and comparative research. Therefore, we generated organoids from canine duodenum, jejunum and colon, and focused on simultaneous long-term expansion and cell differentiation to maximize applicability. Following their establishment, canine intestinal organoids were grown under various culture conditions and then analyzed with respect to cell viability/apoptosis and multi-lineage differentiation by transcription profiling, proliferation assay, cell staining, and transmission electron microscopy. Standard expansion medium supported long-term expansion of organoids irrespective of their origin, but inhibited cell differentiation. Conversely, transfer of organoids to differentiation medium promoted goblet cell and enteroendocrine cell development, but simultaneously induced apoptosis. Unimpeded stem cell renewal and concurrent differentiation was achieved by culturing organoids in the presence of tyrosine kinase ligands. Our findings unambiguously highlight the characteristic cellular diversity of canine duodenum, jejunum and colon as fundamental prerequisite for accurate in vitro modelling.