Morphology and Transcriptome Analysis of Intestinal Organoids in Bovine fetus

Abstract

BackgroundIntestinal health is an important guarantee for the healthy development of mammals. Therefore, the research on intestinal development and function continues to be a hot spot. And intestinal organoids are a hopeful alternative for the intestine, especially in large animals. In this study, we selected intestine of fetus calves as a target to study the differences in intestinal development and the development and functions of organoids in vitro, which were less reported in the past. The fetus intestine tissue is considered a preferred source for obtaining organoid because of its sterility and excellent developmental potential. MethodsWe compared the morphology of crypts and epithelial cells from duodenum, jejunum, ileum and colon at 4-6 month age fetus calves by HE staining, immunohistochemistry and transmission electron microscopy. And we cultured organoids from jejunum tissue and analyzed the structure by optical and electronic microscopy, immunological methods. Subsequently, through RNA sequencing, we confirmed the characteristics of gene expression between organoids and crypts. ResultsThe results showed the villus of jejunum and ileum was significantly higher than others, but the depth of the duodenum crypt was significantly deeper than the other two (P<0.001). And a large number of fissions were observed in duodenum crypts. In addition, intestinal epithelial stem cell markers LGR5 and CTNNB1 were highly expressed in the epithelium, but there was significant difference in villus and crypt of different intestinal segments. And By electron microscopy, we noticed that crypt stem cells were typically long and arranged in single and multiple layers closely, with a big nucleus and a large number of microvilli at the top of the cells. Each cell contained a large number of organelles, such as mitochondria and ribosomes. There are more free ribosomes in jejunum, ileum and colon crypt. Some Paneth cells and goblet cells were interspersed among crypt cells. There are typical tight junctions and adherent junctions (fingers cross pattern) between crypt cells. In addition, we obtained jejunum organoids by tissue culture possessing similar traits, such as microvillus and cell tight junctions. CTNNB1, PCNA and Lyso were highly expressed in the crypt cells around the surface of the organoids which hinted at the prominent capacity of cell proliferation. Furthermore, according to RNA sequencing, we found metabolism-related genes were significantly higher expression in organoids than in crypt stem cells (Q<0.001). On the contrary, immune and disease related genes were significant higher in crypt stem cells (Q<0.001). Finally, we found metabolic genes existed significantly differential (Q<0.001): crypt stem cells were biased towards energy metabolism (fructose and mannose) and vitamin metabolism(retinol), and organoids were biased towards amino acid process(glutathione metabolic process) and cholesterol process. This also implied that enteral nutrition absorption needed to be further processed in myofibroblasts except the crypt epithelium. ConclusionsIn short, different intestinal epithelial morphology, structure and development were different and related to function and organoids from jejunum. The organoids from jejunum were recommended as an ideal model for studying nutrient metabolism in cattle.