Evaluation of different media compositions promoting hepatocyte differentiation in the canine liver organoid model.

Organoids are 3-dimensional (3D) self-assembled structures capable of replicating the microanatomy and physiology of the epithelial components of their organ of origin. Adult stem cell (ASC) derived organoids from the liver have previously been shown to differentiate into primarily mature cholangiocytes, and their partial differentiation into functional hepatocytes can be promoted using specific media compositions. While full morphological differentiation of mature hepatocytes from ASCs has not yet been reported for any species, the functional differentiation can be approximated using various media compositions. Six differentiation media formulations from published studies on hepatic organoids were used for the differentiation protocol. Target species for these protocols were humans, mice, cats, and dogs, and encompassed various combinations and concentrations of four major hepatocyte media components: Bone morphogenetic protein 7 (BMP7), Fibroblast Growth Factor 19 (FGF19), Dexamethasone (Dex), and Gamma-Secretase Inhibitor IX (DAPT). Additionally, removing R-spondin from basic organoid media has previously been shown to drive the differentiation of ASC into mature hepatocytes. Differentiation media (N = 20) were designed to encompass combinations of the four major hepatocyte media components. The preferred differentiation of ASC-derived organoids from liver tissue into mature hepatocytes over cholangiocytes was confirmed by albumin production in the culture supernatant. Out of the twenty media compositions tested, six media resulted in the production of the highest amounts of albumin in the supernatant of the organoids. The cell lines cultured using these six media were further characterized via histological staining, transmission electron microscopy, RNA in situ hybridization, analysis of gene expression patterns, immunofluorescence, and label-free proteomics. The results indicate that preferential hepatocyte maturation from canine ADC-derived organoids from liver tissue is mainly driven by Dexamethasone and DAPT components. FGF19 did not enhance organoid differentiation but improved cell culture survival. Furthermore, we confirm that removing R-spondin from the media is crucial for establishing mature hepatic organoid cultures.

Establishment and characterization of turtle liver organoids provides a potential model to decode their unique adaptations.

Painted turtles are remarkable for their freeze tolerance and supercooling ability along with their associated resilience to hypoxia/anoxia and oxidative stress, rendering them an ideal biomedical model for hypoxia-induced injuries (including strokes), tissue cooling during surgeries, and organ cryopreservation. Yet, such research is hindered by their seasonal reproduction and slow maturation. Here we developed and characterized adult stem cell-derived turtle liver organoids (3D self-assembled in vitro structures) from painted, snapping, and spiny softshell turtles spanning ~175My of evolution, with a subset cryopreserved. This development is, to the best of our knowledge, a first for this vertebrate Order, and complements the only other non-avian reptile organoids from snake venom glands. Preliminary characterization, including morphological, transcriptomic, and proteomic analyses, revealed organoids enriched in cholangiocytes. Deriving organoids from distant turtles and life stages demonstrates that our techniques are broadly applicable to chelonians, permitting the development of functional genomic tools currently lacking in herpetological research. Such platform could potentially support studies including genome-to-phenome mapping, gene function, genome architecture, and adaptive responses to climate change, with implications for ecological, evolutionary, and biomedical research.

Adult Animal Stem Cell-Derived Organoids in Biomedical Research and the One Health Paradigm.

Preclinical biomedical research is limited by the predictiveness of in vivo and in vitro models. While in vivo models offer the most complex system for experimentation, they are also limited by ethical, financial, and experimental constraints. In vitro models are simplified models that do not offer the same complexity as living animals but do offer financial affordability and more experimental freedom; therefore, they are commonly used. Traditional 2D cell lines cannot fully simulate the complexity of the epithelium of healthy organs and limit scientific progress. The One Health Initiative was established to consolidate human, animal, and environmental health while also tackling complex and multifactorial medical problems. Reverse translational research allows for the sharing of knowledge between clinical research in veterinary and human medicine. Recently, organoid technology has been developed to mimic the original organ's epithelial microstructure and function more reliably. While human and murine organoids are available, numerous other organoids have been derived from traditional veterinary animals and exotic species in the last decade. With these additional organoid models, species previously excluded from in vitro research are becoming accessible, therefore unlocking potential translational and reverse translational applications of animals with unique adaptations that overcome common problems in veterinary and human medicine.

Changes of Enterocyte Morphology and Enterocyte: Goblet Cell Ratios in Dogs with Protein-Losing and Non-Protein-Losing Chronic Enteropathies.

This study aimed to assess the morphometry of enterocytes as well as the goblet cell-to-enterocyte ratio in different intestinal segments of dogs with chronic enteropathies (CE). Histopathological intestinal samples from 97 dogs were included in the study (19 healthy juveniles, 21 healthy adults, 24 dogs with protein-losing enteropathy (PLE), and 33 CE dogs without PLE). Healthy adult small intestinal enterocytes showed progressively reduced epithelial cell height in the aboral direction, while juvenile dogs showed progressively increased epithelial cell height in the aboral direction. CE dogs had increased epithelial cell height in the duodenum, while PLE dogs had decreased epithelial cell heights compared to healthy adult dogs. Both the CE and PLE dogs showed decreased enterocyte width in the duodenal segment, and the ileal and colonic enterocytes of CE dogs were narrower than those of healthy adult dogs. CE dogs had a lower goblet cell-to-enterocyte ratio in the colon segment compared to healthy dogs. This study provides valuable morphometric information on enterocytes during canine chronic enteropathies, highlighting significant morphological enterocyte alterations, particularly in the small intestine, as well as a reduced goblet cell-to-enterocyte ratio in the colon of CE cases compared to healthy adult dogs.