Organoids

Organoids: applications Organoids are three-dimensional (3D) multicellular stem-cell-derived systems comprising organ-specific cell types, that exhibit key structural and functional properties of a variety of specific organs [1]; this technology has been chosen by Nature Methods as Method of the Year 2017 for the fascinating potential as tool to probe human biology and disease. Organoids can be grown from pluripotent stem cells, embryonic stem cells, induced pluripotent stem cells and from organ-restricted adult stem cells. These cells differentiate in a controlled fashion in specific growth factors and culture media, with the support of a 3D matrix which allows cell growth, orientation and stratification. The resulting organoids replicate the development, metabolism, architecture and physiology of the real organ [2]. In fact, unlike in vitro traditional 2D cultures, organoids are formed by genomic stable, self-renewing and self-organizing stem cell-derived populations of differentiated cells that exhibit similar composition (they comprise all major cell lineages at frequencies similar to those in living tissue) and similar organ architecture and functionality as the tissue of origin. The technique allows expansions of tissue-specific stem cells and their differentiated progeny. Starting from very small amounts of material (such as biopsies); organoids can be expanded indefinitely, cryopreserved as biobanks and easily manipulated using techniques similar to those used for traditional 2D cell cultures [3]. Organoids are of course more physiologically relevant than traditional cell culture models and are easier to manipulate and study than in vivo mouse/human models. Gene-editing technologies can be used to produce and even self-transplant repaired, healthy organoids from patients with genetic defects. In the last years, human organoids have been established for a wide range of organs, including salivary gland, esophagus, stomach, pancreas, liver, intestine, colon, kidney, prostate, lung, retina and brain. Using organoids, many types of human diseases have been modeled, ranging from Barrett's esophagus to Helicobacter pylori infection, cystic fibrosis, inflammatory bowel disease, bacterial or viral infections and even autism [3]. The luminal epithelia of human gastric organoids are efficiently colonized by Helicobacter pylori (the bacterium identified as the major cause of gastritis and one of the major agents of gastric cancer onset) and relevant physiological changes were demonstrated in this setting, including an increase in proliferation due to oncogenic CagA and increased β-catenin signaling [4]. Further, renal organoids have been used for the study of infections caused by Shiga-toxin-producing Escherichia coli [5]; these types of studies are yielding important contributions in understanding the pathogenic mechanisms of infections and are used to improve the strategies of treatment. Many cancer models have been studied using organoids from glioblastoma, colorectal, gastrointestinal, pancreatic, prostate, breast, bladder and liver cancers [6]. Large patient-derived tumor organoid biobank collections from biopsy samples have been obtained, profiled and exploited for drug development and personalized cancer treatment purposes, and to analyze cancer-related processes and signaling pathways. Moreover, CRISPR/Cas9-mediated gene editing is used in organoids to evaluate candidate gene function in pathologies and tumorigenesis. Organoids technology is also promising a boost to regenerative medicine. Transplantation of isogenic organoids from small patient biopsies or from allogenic but HLA-matched tissues represents a novel, exciting alternative to Future Med. Chem. (Epub ahead of print)

Liver disease is one of the leading causes of death worldwide, leading to the death of approximately 2 million people per year. Current therapies include orthotopic liver transplantation, however, donor organ shortage remains a great challenge. In addition, the development of novel therapeutics has been limited due to the lack of in vitro models that mimic in vivo liver physiology. Accordingly, hepatic cell lineages derived from human pluripotent stem cells (hPSCs) represent a promising cell source for liver cell therapy, disease modelling, and drug discovery. Moreover, the development of new culture systems bringing together the multiple liver-specific hepatic cell types triggered the development of hPSC-derived liver organoids. Therefore, these human liver-based platforms hold great potential for clinical applications. In this review, the production of the different hepatic cell lineages from hPSCs, including hepatocytes, as well as the emerging strategies to generate hPSC-derived liver organoids will be assessed, while current biomedical applications will be highlighted.

Three-dimensional (3D) organoid culture holds great promises in cancer precision medicine. However, Matrigel and stem cell-stimulating supplements are necessary for culturing 3D organoid cells. It costs a lot of money and consumes more time and effort compared with 2D cultured cells. Therefore, the establishment of cheaper and Matrigel-free organoid culture that can maintain the characteristics of a part of 3D organoids is demanded. In the previous study, we established a dog bladder cancer (BC) 3D organoid culture system by using their urine samples. Here, we successfully isolated cells named "2.5D organoid" from multiple strains of dog BC 3D organoids using 2.5 organoid media. The cell proliferation speed of 2.5D organoids was faster than parental 3D organoid cells. The expression pattern of stem cell markers was close to 3D organoids. Injection of 2.5D organoid cells into immunodeficient mice formed tumors and showed the histopathological characteristics of urothelial carcinoma similar to the injection of dog BC 3D organoids. The 2.5D organoids had a similar sensitivity profile for anti-cancer drug treatment to their parental 3D organoids. These data suggest that our established 2.5D organoid culture method might become a reasonable and useful tool instead of 3D organoids in dog BC research and therapy. Bladder cancer (BC), also known as transitional cell carcinoma (TCC) or urothelial carcinoma, is the most frequent form of urinary BC in dogs, affecting thousands of dogs annually worldwide 1. It constitutes approximately 1-2% of all naturally occurring cancers in dogs, a nearly similar rate to humans 2,3. The majority of dog BC is high grade invasive urothelial carcinoma 2,4,5. In addition to the late diagnosis of dog BC, it is very difficult to treat them 6,7. Although several experimental models of BC by using mouse and rat were developed 8-10 , they hardly reflect the characteristics of invasive or metastatic human BC 11 and the mechanism of its arising and development

SUMMARY Pancreatic duct epithelial cells have been suggested as a source of progenitors for pancreatic growth and regeneration. However, genetic lineage-tracing experiments with pancreatic duct-specific Cre expression have given conflicting results. Using immunofluorescence and flow cytometry, we show heterogeneous expression of both HNF1b and SOX9 in adult human and murine ductal epithelium. Their expression was dynamic and diminished significantly after induced replication. Purified pancreatic duct cells formed organoid structures in 3D culture, and heterogeneity of expression of Hnf1b and Sox9 was maintained even after passaging. Using antibodies against a second cell surface molecule CD51 (human) or CD24 (mouse), we could isolate living subpopulations of duct cells enriched for high or low expression of HNF1b and SOX9. Only the CD24 high (Hnfb high /Sox9 high) subpopulation was able to form organoids.