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3-D Human Renal Tubular Organoids Generated from Urine-Derived Stem Cells for Nephrotoxicity Screening.

ABSTRACT: The development of human cell-based systems to replace the use of rodents or the two-dimensional culture of cells for studying nephrotoxicity is urgently needed. Human urine-derived stem cells were differentiated into renal tubular epithelial cells in three-dimensional (3-D) culture after being induced by a kidney extracellular matrix. Levels of CYP2E1 and KIM-1 in 3-D organoids were significantly increased in response to acetone and cisplatin. This 3-D culture system provides an alternative tool for nephrotoxicity screening and research.

SUBMITTER: Guo H

PROVIDER: S-EPMC8118570 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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3-D Human Renal Tubular Organoids Generated from Urine-Derived Stem Cells for Nephrotoxicity Screening.

Guo Haibin H Deng Nan N Dou Lei L Ding Huifen H Criswell Tracy T Atala Anthony A Furdui Cristina M CM Zhang Yuanyuan Y

ACS biomaterials science & engineering 20201113 12

The development of human cell-based systems to replace the use of rodents or the two-dimensional culture of cells for studying nephrotoxicity is urgently needed. Human urine-derived stem cells were differentiated into renal tubular epithelial cells in three-dimensional (3-D) culture after being induced by a kidney extracellular matrix. Levels of CYP2E1 and KIM-1 in 3-D organoids were significantly increased in response to acetone and cisplatin. This 3-D culture system provides an alternative too ...[more]

PMID: 33320634

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Project description:BackgroundOrganoids 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.MethodsUSCs 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.ResultsThe 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.ConclusionsUSC-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.

Project description:Purpose: Drug-induced nephrotoxicity can occur in patients with pre-existing renal dysfunction or renal ischemia, potentially leading to chronic kidney disease (CKD) and end-stage renal disease (ESRD). Prompt treatment of CKD and the related side effects is critical in preventing progression to ESRD. The goal of this study was to demonstrate the therapeutic potential of urine-derived stem cells (USC) to treat chronic kidney disease-induced by nephrotoxic drugs and renal ischemia. Materials and methods: Human USC were collected, expanded and characterized by flow cytometry. A CKD model was induced by creating an ischemia-reperfusion injury and gentamicin administration. Twenty-eight adult immunodeficient rats were divided into three groups: PBS-treated group (n=9), USC-treated group (n=9), and sham group with age-matched control animals (n=10). Cell suspension of USC (5 x 106 / 100µl / kidney) or PBS was injected bilaterally into the renal parenchyma 9 weeks after CKD model creation. Renal function was evaluated by collection blood and urine samples to measure serum creatinine and glomerulus filtration rate. The kidneys were harvested 12 weeks after cell injection. Histologically, the extent of glomerulosclerosis and tubular atrophy, the amount of collagen deposition, interstitial fibrosis, inflammatory monocyte infiltration, and expression of transforming growth factor beta 1 (TGF-ß1), and superoxide dismutase 1 (SOD-1) were examined. Results: USC expressed renal parietal epithelial cells (CD24, CD29 and CD44). Renal function, measured by GFR and serum Cr in USC-treated group were significantly improved compared to PBS-treated animals (p<0.05). The degree of glomerular sclerosis and atrophic renal tubules, the amount of fibrosis, and monocyte infiltration significantly decreased in USC-treated group compared to the PBS group (p<0.05). The level of TGF-ß1 expression in renal tissues was also significantly lower in the PBS group, while the level of SOD-1 expression was significantly elevated in the USC group, compared to PBS group (p<0.05). Conclusions: The present study demonstrates the nephron-protective effect of USC on renal function via anti-inflammatory, anti-oxidative stress, and anti-fibrotic activity in a dual-injury CKD rat model. This provides an alternative treatment for CKD in certain clinical situations, such as instances where CKD is due to drug-induced nephrotoxicity and renal ischemia.

Dataset Information

3-D Human Renal Tubular Organoids Generated from Urine-Derived Stem Cells for Nephrotoxicity Screening.

Publications

3-D Human Renal Tubular Organoids Generated from Urine-Derived Stem Cells for Nephrotoxicity Screening.

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