A urine-dependent human urothelial organoid offers a potential alternative to rodent models of infection

Abstract

Murine models describe a defined host/pathogen interaction for urinary tract infection, but human cell studies are scant. Although recent human urothelial organoid models are promising, none demonstrate long-term tolerance to urine, the natural substrate of the tissue and of the uropathogens that live there. We developed a novel human organoid from progenitor cells which demonstrates key structural hallmarks and biomarkers of the urothelium. After three weeks of transwell culture with 100% urine at the apical interface, the organoid stratified into multiple layers. The apical surface differentiated into enlarged and flattened umbrella-like cells bearing characteristic tight junctions, structures resembling asymmetric unit membrane plaques, and a glycosaminoglycan layer. The apical cells also expressed cytokeratin-20, a spatial feature of the mammalian urothelium. Urine itself was necessary for full development, and undifferentiated cells were urine-tolerant despite the lack of membrane plaques and a glycosaminoglycan layer. Infection with Enterococcus faecalis revealed the expected invasive outcome, including urothelial sloughing and the formation of intracellular colonies similar to those previously observed in patient cells. This new biomimetic model could help illuminate invasive behaviours of uropathogens, and serve as a reproducible test bed for disease formation, treatment and resolution in patients.

A revalidation and critique of assumptions about urinary sample collection methods, specimen quality and contamination

“…Compared with CSU, the MSU technique provided a more substantial sample of the inflammatory sediment. UTI involves colonisation of the urothelial cells by pathogenic microbes [12,27,40]. The innate immune response to this microbial colonisation is increased urothelial cell shedding, which may be promoted by mast cells [41].…”

Organoids – New Models for Host–Helminth Interactions

“…In addition, the involvement of bacteria in adenocarcinoma formation in gallbladders has been studied using murine gallbladder organoids infected with Salmonella [4,5]. Infection with the uropathogen Enterococcus faecalis has been studied in human urothelial organoids [32]. More recently, Fallopian tube organoids have served as a model to study the long-term impact of Chlamydia trachomatis infections in the human epithelium that may contribute to the development of ovarian cancer [18].…”

Bladder Organoids and Spheroids: Potential Tools for Normal and Diseased Tissue Modelling

“…A majority of studies of normal bladder cells utilized normal human urothelium cells (12)(13)(14)(15)(16)(17)(18)(19)(20)(21). Because the urothelium is the predominant site for bladder cancer, using normal human urothelial cells in experimental models is particularly important for cancer development and tumor targeting studies.…”

“…Similar culture systems were used for establishing a malignant urothelium multilayer culture in two other studies (45,52). Exposing culture of urothelial cells to sterilized human urine, Horsley et al established normal urothelial organoids which precisely recapitulated spatial organization and features of native urothelium (21). Initially, progenitor urothelial cells were kept on Millicell transwells in nominal culture media.…”