Authors

W AndersonD Price²; K Hildersley²; F Wang³; M Faber²; TN McNeilly²; D Smith²;  ¹ University of Glasgow , UK;  ² Moredun Research Insitute, UK;  ³ University of Michigan, United States

Discussion

Organoids are self-organizing, multicellular cell culture models that are representative of the tissue from which they are derived. The development of these three-dimensional culture systems is accelerating research that seeks to address specific questions tied to human and animal health.

Due to their multicellular composition, organoids represent valuable in vitro tools for modelling specific host:parasite interactions. However, some specialized cell types have a very low prevalence in organoids grown under normal growth conditions. Using differential transcriptomics and immunofluorescence microscopy analysis, we demonstrate the optimization of ruminant gastric and intestinal organoid cultures for the enrichment of specialized cell types. We used a single cell transcriptomic dataset of the ovine abomasum to establish cell type-specific markers and found a significant increase in multiple cell markers associated with paneth cells, tuft cells and goblet cells in our differential RNA-seq analysis of bovine and ovine gastric and intestinal organoids grown in differentiation conditions. These findings were supported by immunofluorescence analysis of organoids cultured in growth and differentiation conditions. Using the tuft cell transcription factor POU2F3 as an indicator of tuft cells and Ki67 as a proliferation factor, we found an increase in relative number of tuft cells and a decrease in proliferative stem cells when organoids were cultured in differentiation conditions. To facilitate the counting of cells across lots of organoids and samples, we also developed an imageJ macro for automated cell counting within organoids that represents a useful research tool. Specialized cell types play important roles during parasitic infection. For example, tuft cells are important parasite-sensing cells that trigger the host immune response through the release of IL-25. However, tuft cells are a rare cell type, representing <1% of cells in GI epithelia. This low abundancy makes studying specialized cell types difficult in vivo, even with the advance of single cell sequencing technology. Therefore, the ability to enrich for rare cell types in organoid cultures poses a valuable research development for studying their biology and more specifically, for determining the specific roles and responses of rare and specialized cell types during infection with different pathogens of medical and veterinary importance.