Improvement of Protein Expression Profile in Three-Dimensional Renal Proximal Tubular Epithelial Cell Spheroids Selected Based on OAT1 Gene Expression: A Potential In Vitro Tool for Evaluating Human Renal Proximal Tubular Toxicity and Drug Disposition.

The proximal tubule plays an important role in the kidney and is a major site of drug interaction and toxicity. Analysis of kidney toxicity via in vitro assays is challenging, because only a few assays that reflect functions of drug transporters in renal proximal tubular epithelial cells (RPTECs) are available. In this study, we aimed to develop a simple and reproducible method for culturing RPTECs by monitoring organic anion transporter 1 (OAT1) as a selection marker. Culturing RPTECs in spherical cellular aggregates increased OAT1 protein expression, which was low in the conventional two-dimensional (2D) culture, to a level similar to that in human renal cortices. By proteome analysis, it was revealed that the expression of representative two proximal tubule markers was maintained and 3D spheroid culture improved the protein expression of approximately 7% of the 139 transporter proteins detected, and the expression of 2.3% of the 4,800 proteins detected increased by approximately fivefold that in human renal cortices. Furthermore, the expression levels of approximately 4,800 proteins in three-dimensional (3D) RPTEC spheroids (for 12 days) were maintained for over 20 days. Cisplatin and adefovir exhibited transporter-dependent ATP decreases in 3D RPTEC spheroids. These results indicate that the 3D RPTEC spheroids developed by monitoring OAT1 gene expression are a simple and reproducible in vitro experimental system with improved gene and protein expressions compared with 2D RPTECs and were more similar to that in human kidney cortices. Therefore, it can potentially be used for evaluating human renal proximal tubular toxicity and drug disposition. SIGNIFICANCE STATEMENT: This study developed a simple and reproducible spheroidal culture method with acceptable throughput using commercially available RPTECs by monitoring OAT1 gene expression. RPTECs cultured using this new method showed improved mRNA/protein expression profiles to those in 2D RPTECs and were more similar to those of human kidney cortices. This study provides a potential in vitro proximal tubule system for pharmacokinetic and toxicological evaluations during drug development.

A novel ADPKD model using kidney organoids derived from disease-specific human iPSCs.

Autosomal dominant polycystic kidney disease (ADPKD) is a hereditary disorder which manifests progressive renal cyst formation and leads to end-stage kidney disease. Around 85% of cases are caused by PKD1 heterozygous mutations, exhibiting relatively poorer renal outcomes than those with mutations in other causative gene PKD2. Although many disease models have been proposed for ADPKD, the pre-symptomatic pathology of the human disease remains unknown. To unveil the mechanisms of early cytogenesis, robust and genetically relevant human models are needed. Here, we report a novel ADPKD model using kidney organoids derived from disease-specific human induced pluripotent stem cells (hiPSCs). Importantly, we found that kidney organoids differentiated from gene-edited heterozygous PKD1-mutant as well as ADPKD patient-derived hiPSCs can reproduce renal cysts. Further, we demonstrated the possibility of ADPKD kidney organoids serving as drug screening platforms. This newly developed model will contribute to identifying novel therapeutic targets, extending the field of ADPKD research.

Generation of branching ureteric bud tissues from human pluripotent stem cells.

Recent progress in kidney regeneration research is noteworthy. However, the selective and robust differentiation of the ureteric bud (UB), an embryonic renal progenitor, from human pluripotent stem cells (hPSCs) remains to be established. The present study aimed to establish a robust induction method for branching UB tissue from hPSCs towards the creation of renal disease models. Here, we found that anterior intermediate mesoderm (IM) differentiates from anterior primitive streak, which allowed us to successfully develop an efficient two-dimensional differentiation method of hPSCs into Wolffian duct (WD) cells. We also established a simplified procedure to generate three-dimensional WD epithelial structures that can form branching UB tissues. This system may contribute to hPSC-based regenerative therapies and disease models for intractable disorders arising in the kidney and lower urinary tract.

Redefining definitive endoderm subtypes by robust induction of human induced pluripotent stem cells.

Many reports have described methods that induce definitive endoderm (DE) cells from human pluripotent stem cells (hPSCs). However, it is unclear whether the differentiation propensity of these DE cells is uniform. This uncertainty is due to the different developmental stages that give rise to anterior and posterior DE from anterior primitive streak (APS). Therefore, these DE cell populations might be generated from the different stages of APS cells, which affect the DE cell differentiation potential. Here, we succeeded in selectively differentiating early and late APS cells from human induced pluripotent stem cells (hiPSCs) using different concentrations of CHIR99021, a small molecule Wnt/ß-catenin pathway activator. We also established novel differentiation systems from hiPSCs into three types of DE cells: anterior and posterior domains of anterior DE cells through early APS cells and posterior DE cells through late APS cells. These different DE cell populations could differentiate into distinct endodermal lineages in vitro, such as lung, liver or small intestine progenitors. These results indicate that different APS cells can produce distinct types of DE cells that have proper developmental potency and suggest a method to evaluate the quality of endodermal cell induction from hPSCs.

Transcriptional Analysis of Intravenous Immunoglobulin Resistance in Kawasaki Disease Using an Induced Pluripotent Stem Cell Disease Model.

BACKGROUND: Approximately 10-20% of Kawasaki disease (KD) patients are resistant to intravenous immunoglobulin (IVIG) treatment. Further, these patients are at a particularly high risk of having coronary artery abnormalities. The mechanisms of IVIG resistance in KD have been analyzed using patient leukocytes, but not patient vascular endothelial cells (ECs). The present study clarifies the mechanisms of IVIG resistance in KD using an induced pluripotent stem cell (iPSC) disease model.Methods and Results:Dermal fibroblasts or peripheral blood mononuclear cells from 2 IVIG-resistant and 2 IVIG-responsive KD patients were reprogrammed by the episomal vector-mediated transduction of 6 reprogramming factors. KD patient-derived iPSCs were differentiated into ECs (iPSC-ECs). The gene expression profiles of iPSC-ECs generated from IVIG-resistant and IVIG-responsive KD patients were compared by RNA-sequencing analyses. We found that the expression ofCXCL12was significantly upregulated in iPSC-ECs from IVIG-resistant KD patients. Additionally, Gene Set Enrichment Analysis (GSEA) revealed that gene sets involved in interleukin (IL)-6 signaling were also upregulated. CONCLUSIONS: The first iPSC-based model for KD is reported here. Our mechanistic analyses suggest thatCXCL12, which plays a role in leukocyte transmigration, is a key molecule candidate for IVIG resistance and KD severity. They also indicate that an upregulation of IL-6-related genes may be involved in this pathogenesis.

Kidney regeneration from human induced pluripotent stem cells.

PURPOSE OF REVIEW: Human induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) are potential unlimited cell sources for renal cells in regenerative medicine. This review highlights recent advance in the directed differentiation of human iPSCs into kidney lineages and discusses the remaining challenges to generate functional or mature renal cells from human iPSCs. RECENT FINDINGS: Recently, directed differentiation methods from human iPSCs/ESCs into embryonic renal progenitor cells, such as those included in metanephric mesenchyme and ureteric bud, that mimic embryonic development have been reported. These studies show the developmental potential of progenitor cells by forming renal tubule-like or glomerulus-like structures in vitro. However, it has not been verified whether the physiological functions of the induced progenitors are equivalent to their in-vivo counterparts. The establishment of definitive marker genes for kidney lineages and functional assay systems is essential for the verification. Such achievement is needed before kidney regeneration can provide cell replacement therapy, reliable disease models and elucidation of the mechanisms of kidney development. SUMMARY: In conclusion, this review outlines milestones in directed differentiation methods for functional renal cell types from human iPSCs toward clinical application and practical use.

Selective induction of human renal interstitial progenitor-like cell lineages from iPSCs reveals development of mesangial and EPO-producing cells.

Recent regenerative studies using human pluripotent stem cells (hPSCs) have developed multiple kidney-lineage cells and organoids. However, to further form functional segments of the kidney, interactions of epithelial and interstitial cells are required. Here we describe a selective differentiation of renal interstitial progenitor-like cells (IPLCs) from human induced pluripotent stem cells (hiPSCs) by modifying our previous induction method for nephron progenitor cells (NPCs) and analyzing mouse embryonic interstitial progenitor cell (IPC) development. Our IPLCs combined with hiPSC-derived NPCs and nephric duct cells form nephrogenic niche- and mesangium-like structures in vitro. Furthermore, we successfully induce hiPSC-derived IPLCs to differentiate into mesangial and erythropoietin-producing cell lineages in vitro by screening differentiation-inducing factors and confirm that p38 MAPK, hypoxia, and VEGF signaling pathways are involved in the differentiation of mesangial-lineage cells. These findings indicate that our IPC-lineage induction method contributes to kidney regeneration and developmental research.

Human iPSC-derived renal collecting duct organoid model cystogenesis in ADPKD.

In autosomal dominant polycystic kidney disease (ADPKD), renal cyst lesions predominantly arise from collecting ducts (CDs). However, relevant CD cyst models using human cells are lacking. Although previous reports have generated in vitro renal tubule cyst models from human induced pluripotent stem cells (hiPSCs), therapeutic drug candidates for ADPKD have not been identified. Here, by establishing expansion cultures of hiPSC-derived ureteric bud tip cells, an embryonic precursor that gives rise to CDs, we succeed in advancing the developmental stage of CD organoids and show that all CD organoids derived from PKD1-/- hiPSCs spontaneously develop multiple cysts, clarifying the initiation mechanisms of cystogenesis. Moreover, we identify retinoic acid receptor (RAR) agonists as candidate drugs that suppress in vitro cystogenesis and confirm the therapeutic effects on an ADPKD mouse model in vivo. Therefore, our in vitro CD cyst model contributes to understanding disease mechanisms and drug discovery for ADPKD.

Elucidation of HHEX in pancreatic endoderm differentiation using a human iPSC differentiation model.

For pluripotent stem cell (PSC)-based regenerative therapy against diabetes, the differentiation efficiency to pancreatic lineage cells needs to be improved based on the mechanistic understanding of pancreatic differentiation. Here, we aimed to elucidate the molecular mechanisms underlying pancreatic endoderm differentiation by searching for factors that regulate a crucial pancreatic endoderm marker gene, NKX6.1. Unbiasedly screening an siRNA knockdown library, we identified a candidate transcription factor, HHEX. HHEX knockdown suppressed the expression of another pancreatic endoderm marker gene, PTF1A, as well as NKX6.1, independently of PDX1, a known regulator of NKX6.1 expression. In contrast, the overexpression of HHEX upregulated the expressions of NKX6.1 and PTF1A. RNA-seq analysis showed decreased expressions of several genes related to pancreatic development, such as NKX6.1, PTF1A, ONECUT1 and ONECUT3, in HHEX knockdown pancreatic endoderm. These results suggest that HHEX plays a key role in pancreatic endoderm differentiation.

iPSC-derived type IV collagen α5-expressing kidney organoids model Alport syndrome.

Alport syndrome (AS) is a hereditary glomerulonephritis caused by COL4A3, COL4A4 or COL4A5 gene mutations and characterized by abnormalities of glomerular basement membranes (GBMs). Due to a lack of curative treatments, the condition proceeds to end-stage renal disease even in adolescents. Hampering drug discovery is the absence of effective in vitro methods for testing the restoration of normal GBMs. Here, we aimed to develop kidney organoid models from AS patient iPSCs for this purpose. We established iPSC-derived collagen α5(IV)-expressing kidney organoids and confirmed that kidney organoids from COL4A5 mutation-corrected iPSCs restore collagen α5(IV) protein expression. Importantly, our model recapitulates the differences in collagen composition between iPSC-derived kidney organoids from mild and severe AS cases. Furthermore, we demonstrate that a chemical chaperone, 4-phenyl butyric acid, has the potential to correct GBM abnormalities in kidney organoids showing mild AS phenotypes. This iPSC-derived kidney organoid model will contribute to drug discovery for AS.

Protocol for the generation and expansion of human iPS cell-derived ureteric bud organoids.

The ureteric bud (UB) is a kidney precursor tissue that repeats branching morphogenesis and gives rise to the collecting ducts (CDs) and lower urinary tract. Here, we describe protocols to generate iUB organoids from human iPSCs; iUB organoids repeat branching morphogenesis. We describe how to expand iUB-organoid-derived tip colonies and how to induce CD progenitors from iUB organoids. These organoids can be used to study CD development and potentially as a model of kidney and urinary tract diseases. For complete details on the use and execution of this protocol, please refer to Mae et al. (2020).

Retinoic acid regulates erythropoietin production cooperatively with hypoxia-inducible factors in human iPSC-derived erythropoietin-producing cells.

Erythropoietin (EPO) is a crucial hormone for erythropoiesis and produced by adult kidneys. Insufficient EPO production in chronic kidney disease (CKD) can cause renal anemia. Although hypoxia-inducible factors (HIFs) are known as a main regulator, the mechanisms of EPO production have not been fully elucidated. In this study, we aimed to examine the roles of retinoic acid (RA) in EPO production using EPO-producing cells derived from human induced pluripotent stem cells (hiPSC-EPO cells) that we previously established. RA augmented EPO production by hiPSC-EPO cells under hypoxia or by treatment with prolyl hydroxylase domain-containing protein (PHD) inhibitors that upregulate HIF signals. Combination treatment with RA and a PHD inhibitor improved renal anemia in vitamin A-depleted CKD model mice. Our findings using hiPSC-EPO cells and CKD model mice may contribute to clarifying the EPO production mechanism and developing efficient therapies for renal anemia.

Combination of small molecules enhances differentiation of mouse embryonic stem cells into intermediate mesoderm through BMP7-positive cells.

Embryonic stem cells (ESCs) are potentially powerful tools for regenerative medicine and establishment of disease models. The recent progress in ESC technologies is noteworthy, but ESC differentiation into renal lineages is relatively less established. The present study aims to differentiate mouse ESCs (mESCs) into a renal progenitor pool, the intermediate mesoderm (IM), without addition of exogenous cytokines and embryoid formation. First, we treated mESCs with a combination of small molecules (Janus-associated tyrosine kinase inhibitor 1, LY294002, and CCG1423) and differentiated them into BMP7-positive cells, BMP7 being the presumed inducing factor for IM. When these cells were cultured with adding retinoic acid, expression of odd-skipped related 1 (Osr1), which is essential to IM differentiation, was enhanced. To simplify the differentiation protocol, the abovementioned four small molecules (including retinoic acid) were combined and added to the culture. Under this condition, more than one-half of the cells were positive for Osr1, and at the same time, Pax2 (another IM marker) was detected by real-time PCR. Expressions of ectodermal marker and endodermal marker were not enhanced, while mesodermal marker changed. Moreover, expression of genes indispensable to kidney development, i.e., Lim1 and WT1, was detected by RT-PCR. These results indicate the establishment of a specific, effective method for differentiation of the ESC monolayer into IM using a combination of small molecules, resulting in an attractive cell source that could be experimentally differentiated to understand nephrogenic mechanisms and cell-to-cell interactions in embryogenesis.

Expansion of Human iPSC-Derived Ureteric Bud Organoids with Repeated Branching Potential.

Ureteric bud (UB) is the embryonic kidney progenitor tissue that gives rise to the collecting duct and lower urinary tract. UB-like structures generated from human pluripotent stem cells by previously reported methods show limited developmental ability and limited branching. Here we report a method to generate UB organoids that possess epithelial polarity and tubular lumen and repeat branching morphogenesis. We also succeed in monitoring UB tip cells by utilizing the ability of tip cells to uptake very-low-density lipoprotein, cryopreserving UB progenitor cells, and expanding UB tip cells that can reconstitute the organoids and differentiate into collecting duct progenitors. Moreover, we successfully reproduce some phenotypes of multicystic dysplastic kidney (MCDK) using the UB organoids. These methods will help elucidate the developmental mechanisms of UB branching and develop a selective differentiation method for collecting duct cells, contributing to the creation of disease models for congenital renal abnormalities.

A Modular Differentiation System Maps Multiple Human Kidney Lineages from Pluripotent Stem Cells.

Recent studies using human pluripotent stem cells (hPSCs) have developed protocols to induce kidney-lineage cells and reconstruct kidney organoids. However, the separate generation of metanephric nephron progenitors (NPs), mesonephric NPs, and ureteric bud (UB) cells, which constitute embryonic kidneys, in in vitro differentiation culture systems has not been fully investigated. Here, we create a culture system in which these mesoderm-like cell types and paraxial and lateral plate mesoderm-like cells are separately generated from hPSCs. We recapitulate nephrogenic niches from separately induced metanephric NP-like and UB-like cells, which are subsequently differentiated into glomeruli, renal tubules, and collecting ducts in vitro and further vascularized in vivo. Our selective differentiation protocols should contribute to understanding the mechanisms underlying human kidney development and disease and also supply cell sources for regenerative therapies.

Protocol to Generate Ureteric Bud Structures from Human iPS Cells.

The generation of ureteric bud (UB), which is the renal progenitor that gives rise to renal collecting ducts and lower urinary tract, from human-induced pluripotent stem cells (hiPSCs) provides a cell source for studying the development of UB and kidney disease. Here we describe a stepwise and efficient two-dimensional differentiation method of hiPSCs into Wolffian duct (WD) cells. We also describe how to generate three-dimensional WD epithelial structures that can differentiate into UB-like structures.

[Dissecting early development of the kidney by single cell transcriptomics].

Each of the billions of the cells in our body exhibits their identity with unique gene expression profile. Recent advances in single cell transcriptomics enable to conduct cell taxonomy identifying new cell types and to re-arrange cells in order of pseudo-time course describing differentiation status of each cell. Even though the cost is still high, the single cell transcriptomics now becomes one of the conventional assays. We have applied the single cell gene expression analysis to dissect human development. In this article, we show our recent progress on a study describing early development of the kidney using human iPS cells by the single cell transcriptomics.

Author Correction: Development of new method to enrich human iPSC-derived renal progenitors using cell surface markers.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Differentiation and isolation of iPSC-derived remodeling ductal plate-like cells by use of an AQP1-GFP reporter human iPSC line.

Cholangiocytes are the epithelial cells that line bile ducts, and ductal plate malformation is a developmental anomaly of bile ducts that causes severe congenital biliary disorders. However, because of a lack of specific marker genes, methods for the stepwise differentiation and isolation of human induced pluripotent stem cell (hiPSC)-derived cholangiocyte progenitors at ductal plate stages have not been established. We herein generated an AQP1-GFP reporter hiPSC line and developed a combination treatment with transforming growth factor (TGF) ß2 and epidermal growth factor (EGF) to induce hiPSC-derived hepatoblasts into AQP1+ cells in vitro. By confirming that the isolated AQP1+ cells showed similar gene expression patterns to cholangiocyte progenitors at the remodeling ductal plate stage around gestational week (GW) 20, we established a differentiation protocol from hiPSCs through SOX9+CK19+AQP1- ductal plate-like cells into SOX9+CK19+AQP1+ remodeling ductal plate-like cells. We further generated 3D bile duct-like structures from the induced ductal plate-like cells. These results suggest that AQP1 is a useful marker for the generation of remodeling ductal plate cells from hiPSCs. Our methods may contribute to elucidating the differentiation mechanisms of ductal plate cells and the pathogenesis of ductal plate malformation.

Development of new method to enrich human iPSC-derived renal progenitors using cell surface markers.

Cell therapy using renal progenitors differentiated from human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs) has the potential to significantly reduce the number of patients receiving dialysis therapy. However, the differentiation cultures may contain undifferentiated or undesired cell types that cause unwanted side effects, such as neoplastic formation, when transplanted into a body. Moreover, the hESCs/iPSCs are often genetically modified in order to isolate the derived renal progenitors, hampering clinical applications. To establish an isolation method for renal progenitors induced from hESCs/iPSCs without genetic modifications, we screened antibodies against cell surface markers. We identified the combination of four markers, CD9-CD140a+CD140b+CD271+, which could enrich OSR1+SIX2+ renal progenitors. Furthermore, these isolated cells ameliorated renal injury in an acute kidney injury (AKI) mouse model when used for cell therapy. These cells could contribute to the development of hiPSC-based cell therapy and disease modeling against kidney diseases.