Formation and optimization of three-dimensional organoids generated from urine-derived stem cells for renal function in vitro

Background Organoids play an important role in basic research, drug screening, and regenerative medicine. Here, we aimed to develop a novel kind of three-dimensional (3D) organoids generated from urine-derived stem cells (USCs) and to explore whether kidney-specific extracellular matrix (kECM) could enable such organoids for renal function in vitro. Methods USCs were isolated from human urine samples and cultured with kECM extraction to generate 3D organoids in vitro. Eight densities from 1000 to 8000 cells per organoids were prepared, and both ATP assay and Live/Dead staining were used to determine the optimal USC density in forming organoids and kECM additive concentration. The morphology and histology of as-made organoids were evaluated by hematoxylin and eosin (H.E.) staining, immunofluorescence staining and whole mount staining. Additionally, RT-qPCR was implemented to detect renal-related gene expression. Drug toxicity test was conducted to evaluate the potential application for drug screening. The renal organoids generated from whole adult kidney cells were used as a positive control in multiple assessments. Results The optimized cell density to generate ideal USC-derived organoids (USC-organoids) was 5000 cells/well, which was set as applying density in the following experiments. Besides, the optimal concentration of kECM was revealed to be 10%. On this condition, Live/Dead staining showed that USC-organoids were well self-organized without significant cell death. Moreover, H.E. staining showed that compact and viable organoids were generated without obvious necrosis inside organoids, which were very close to renal organoids morphologically. Furthermore, specific proximal tubule marker Aquaporin-1 (AQP1), kidney endocrine product erythropoietin (EPO), kidney glomerular markers Podocin and Synaptopodin were detected positively in USC-organoids with kECM. Nephrotoxicity testing showed that aspirin, penicillin G, and cisplatin could exert drug-induced toxicity on USC-organoids with kECM. Conclusions USC-organoids could be developed from USCs via an optimal procedure. Combining culture with kECM, USC-organoid properties including morphology, histology, and specific gene expression were identified to be similar with real renal organoids. Additionally, USC-organoids posed kECM in vitro showed the potential to be a drug screening tool which might take the place of renal organoids to some extent in the future. Keywords: Urine-derived stem cells, Organoids,