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

Abstract

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 USCorganoids with kECM.

Use of standard U-bottom and V-bottom well plates to generate neuroepithelial embryoid bodies

“…The EB diameter is influenced by the seeding concentration [26]. In our experiments, an increase in the diameter of the EBs during 6 days of culture was also a function of the initial cell density.…”

“…Many organoid cultures begin with circular EBs, including those related to kidney [26], liver [39], heart [40], optic cup [1], and other organs and tissues [38,41]. The methods provided here could potentially facilitate and streamline organoid cultures in a diverse spectrum of labs spanning different fields.…”

Emerging Roles of Urine-Derived Components for the Management of Bladder Cancer: One Man’s Trash Is Another Man’s Treasure

“…Organoids can be propagated from bladder cancer cells derived from urine [ 36 , 37 , 38 , 39 ] or bladder biopsies [ 40 ]. Urine and biopsy-derived organoids have been shown to recapitulate molecular subtypes and heterogeneity [ 36 , 37 , 38 , 39 ]. Specifically, steady expression of urothelial cell markers (e.g., CK7, CK20, UPK3A, and CD44) has been reported on the luminal side of UBC organoids along with that of the proliferation marker Ki67 [ 36 , 40 ].…”

Drug-Induced Nephrotoxicity Assessment in 3D Cellular Models

“…Gentamicin [17][18][19][20] Citrinin [17,21] Cisplatin [17][18][19][22][23][24][25][26][27][28][29] Rifampicin [17,30] Acetone [25,28,31] Aspirin [24,27,32] Penicillin G [24,27,33] Tenofovir [26,34] Cyclosporin A [26,35,36] Adriamycin [19,25,37] 4-aminophenol (PAP) [25,38] Colchicine [25,39] Cadmium chloride [40,41] Brush border membrane of the proximal tubules S2 proximal tubular segment Basolateral membrane of proximal tubules Apical membrane of renal proximal tubules S1 and S2 proximal tubular segment Loop of Henle Brush border membrane of the proximal tubules Basolateral mem-brane of proximal tubules Brush border membrane of the proximal tubules/Thick ascending limb of the loop of Henle Brush border membrane of the proximal tubules Loop of Henle S3 proximal tubular segment S1 proximal tubular segment Doxorubicin [17,42,…”