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Cystic renal-epithelial derived induced pluripotent stem cells from polycystic kidney disease patients.

ABSTRACT: Autosomal-dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease, leading to kidney failure in most patients. In approximately 85% of cases, the disease is caused by mutations in PKD1. How dysregulation of PKD1 leads to cyst formation on a molecular level is unknown. Induced pluripotent stem cells (iPSCs) are a powerful tool for in vitro modeling of genetic disorders. Here, we established ADPKD patient-specific iPSCs to study the function of PKD1 in kidney development and cyst formation in vitro. Somatic mutations are proposed to be the initiating event of cyst formation, and therefore, iPSCs were derived from cystic renal epithelial cells rather than fibroblasts. Mutation analysis of the ADPKD iPSCs revealed germline mutations in PKD1 but no additional somatic mutations in PKD1/PKD2. Although several somatic mutations in other genes implicated in ADPKD were identified in cystic renal epithelial cells, only few of these mutations were present in iPSCs, indicating a heterogeneous mutational landscape, and possibly in vitro cell selection before and during the reprogramming process. Whole-genome DNA methylation analysis indicated that iPSCs derived from renal epithelial cells maintain a kidney-specific DNA methylation memory. In addition, comparison of PKD1+/- and control iPSCs revealed differences in DNA methylation associated with the disease history. In conclusion, we generated and characterized iPSCs derived from cystic and healthy control renal epithelial cells, which can be used for in vitro modeling of kidney development in general and cystogenesis in particular.

SUBMITTER: Kenter AT

PROVIDER: S-EPMC7103626 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Cystic renal-epithelial derived induced pluripotent stem cells from polycystic kidney disease patients.

Kenter Annegien T AT Rentmeester Eveline E van Riet Job J Boers Ruben R Boers Joachim J Ghazvini Mehrnaz M Xavier Vanessa J VJ van Leenders Geert J L H GJLH Verhagen Paul C M S PCMS van Til Marjan E ME Eussen Bert B Losekoot Monique M de Klein Annelies A Peters Dorien J M DJM van IJcken Wilfred F J WFJ van de Werken Harmen J G HJG Zietse Robert R Hoorn Ewout J EJ Jansen Gert G Gribnau Joost H JH

Stem cells translational medicine 20200312 4

Autosomal-dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease, leading to kidney failure in most patients. In approximately 85% of cases, the disease is caused by mutations in PKD1. How dysregulation of PKD1 leads to cyst formation on a molecular level is unknown. Induced pluripotent stem cells (iPSCs) are a powerful tool for in vitro modeling of genetic disorders. Here, we established ADPKD patient-specific iPSCs to study the function of PKD1 in kidney develop ...[more]

PMID: 32163234

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Project description:UnlabelledAcute kidney injury (AKI) is defined as a rapid loss of renal function resulting from various etiologies, with a mortality rate exceeding 60% among intensive care patients. Because conventional treatments have failed to alleviate this condition, the development of regenerative therapies using human induced pluripotent stem cells (hiPSCs) presents a promising new therapeutic option for AKI. We describe our methodology for generating renal progenitors from hiPSCs that show potential in ameliorating AKI. We established a multistep differentiation protocol for inducing hiPSCs into OSR1+SIX2+ renal progenitors capable of reconstituting three-dimensional proximal renal tubule-like structures in vitro and in vivo. Moreover, we found that renal subcapsular transplantation of hiPSC-derived renal progenitors ameliorated the AKI in mice induced by ischemia/reperfusion injury, significantly suppressing the elevation of blood urea nitrogen and serum creatinine levels and attenuating histopathological changes, such as tubular necrosis, tubule dilatation with casts, and interstitial fibrosis. To our knowledge, few reports demonstrating the therapeutic efficacy of cell therapy with renal lineage cells generated from hiPSCs have been published. Our results suggest that regenerative medicine strategies for kidney diseases could be developed using hiPSC-derived renal cells.SignificanceThis report is the first to demonstrate that the transplantation of renal progenitor cells differentiated from human induced pluripotent stem (iPS) cells has therapeutic effectiveness in mouse models of acute kidney injury induced by ischemia/reperfusion injury. In addition, this report clearly demonstrates that the therapeutic benefits come from trophic effects by the renal progenitor cells, and it identifies the renoprotective factors secreted by the progenitors. The results of this study indicate the feasibility of developing regenerative medicine strategy using iPS cells against renal diseases.

Project description:Background: Chronic kidney disease (CKD) is a global public health problem. Cell therapy using pluripotent stem cells represents an attractive therapeutic approach for the treatment of CKD.Methods: We transplanted mitomycin C (MMC)-treated human induced pluripotent stem cells (hiPSCs) and renal progenitor cells (RPCs) into a CKD rat model system. The RPC and hiPSC cells were characterized by immunofluorescence and qRT-PCR. Untreated 5/6 nephrectomized rats were compared to CKD animals receiving the same amount of MMC-treated hiPSCs or RPCs. Renal function, histology, and immunohistochemistry were evaluated 45?days post-surgery.Results: We successfully generated hiPSCs from peripheral blood and differentiated them into RPCs expressing renal progenitor genes (PAX2, WT1, SIX2, and SALL1) and podocyte-related genes (SYNPO, NPHS1). RPCs also exhibited reduced OCT4 expression, confirming the loss of pluripotency. After cell transplantation into CKD rats, the body weight change was significantly increased in both hiPSC and RPC groups, in comparison with the control group. Creatinine clearance (CCr) was preserved only in the hiPSC group. Similarly, the number of macrophages in the kidneys of the hiPSC group reached a statistically significant reduction, when compared to control rats. Both treatments reduced positive staining for the marker ?-smooth muscle actin. Histological features showed decreased tubulointerstitial damage (interstitial fibrosis and tubular atrophy) as well as a reduction in glomerulosclerosis in both iPSC and RPC groups.Conclusions: In conclusion, we describe that both MMC-treated hiPSCs and RPCs exert beneficial effects in attenuating CKD progression. Both cell types were equally efficient to reduce histological damage and weight loss caused by CKD. hiPSCs seem to be more efficient than RPCs, possibly due to a paracrine effect triggered by hiPSCs. These results demonstrate that the use of MMC-treated hiPSCs and RPCs improves clinical and histological CKD parameters, avoided tumor formation, and therefore may be a promising cell therapy strategy for CKD.

Project description:BackgroundIn diabetic kidney disease, high glucose damages specialized cells called podocytes that filter blood in the glomerulus. In vitro culture of podocytes is crucial for modeling of diabetic nephropathy and genetic podocytopathies and to complement animal studies. Recently, several methods have been published to derive podocytes from human-induced pluripotent stem cells (iPSCs) by directed differentiation. However, these methods have major variations in media composition and have not been compared.MethodsWe characterized our accelerated protocol by guiding the cells through differentiation with four different medias into MIXL1+ primitive streak cells with Activin A and CHIR for Wnt activation, intermediate mesoderm PAX8+ cells via increasing the CHIR concentration, nephron progenitors with FGF9 and Heparin for stabilization, and finally into differentiated podocytes with Activin A, BMP-7, VEGF, reduced CHIR, and retinoic acid. The podocyte morphology was characterized by scanning and transmission electron microscopy and by flow cytometry analysis for podocyte markers. To confirm cellular identity and niche localization, we performed cell recombination assays combining iPSC-podocytes with dissociated mouse embryonic kidney cells. Finally, to test iPSC-derived podocytes for the modeling of diabetic kidney disease, human podocytes were exposed to high glucose.ResultsPodocyte markers were expressed at similar or higher levels for our accelerated protocol as compared to previously published protocols that require longer periods of tissue culture. We confirmed that the human podocytes derived from induced pluripotent stem cells in twelve days integrated into murine glomerular structures formed following seven days of culture of cellular recombinations. We found that the high glucose-treated human podocytes displayed actin rearrangement, increased cytotoxicity, and decreased viability.ConclusionsWe found that our accelerated 12-day method for the differentiation of podocytes from human-induced pluripotent stem cells yields podocytes with comparable marker expression to longer podocytes. We also demonstrated that podocytes created with this protocol have typical morphology by electron microscopy. The podocytes have utility for diabetes modeling as evidenced by lower viability and increased cytotoxicity when treated with high glucose. We found that multiple, diverse methods may be utilized to create iPSC-podocytes, but closely mimicking developmental cues shortened the time frame required for differentiation.

Dataset Information

Cystic renal-epithelial derived induced pluripotent stem cells from polycystic kidney disease patients.

Publications

Cystic renal-epithelial derived induced pluripotent stem cells from polycystic kidney disease patients.

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