Kidney Organoids Research Articles

In Situ Detection of Kidney Organoid Generation From Stem Cells Using a Simple Electrochemical Method.

Organoids that mimic the structural and cellular characteristics of kidneys in vitro have recently emerged as a promising source for biomedical research. However, uncontrollable cellular heterogeneity after differentiation often results in the generation of off‐target cells, one of the most challenging issues in organoid research. This study proposes a new method that enables the real‐time assessment of kidney organoids derived from stem cells. When placed on a conductive surface, these organoids generate unique electrochemical signals at ≈0.3 V with intensities proportional to the amount of kidney‐specific cell types. Off‐target cells (i.e., non‐kidney cells) produce an electrical signature at 0 V that is distinguishable from other surrounding cell types, enabling non‐destructive assessment of both the differentiation, and maturation levels of kidney organoids. The developed platform can be applied to other types of organoids and is thus highly promising as a tool for organoid‐based drug screening, toxicity assessment, and therapeutics.

MO059: Trajectory Analysis of the Kidney Organoid Proteome Extends its Modelling Potential of Disease

Abstract BACKGROUND AND AIMS Kidney organoids are a valuable and innovative model to understand genetic diseases, kidney development and transcriptomic dynamics. However, their proteome has not been analyzed so far. It is unclear how their proteome changes during differentiation, and if more complex disease processes such as inflammatory tissue responses could be modelled with this approach. METHOD Here, we used proteomics to compare organoids with existing model systems such as native glomeruli and cultured cells. We characterize the trajectory of organoid differentiation and delineate innate immune responses in organoids to expand its scope as a model system in nephrology. We also compared our proteomics with bulk and single cell transcriptomic data. RESULTS Genes involved in podocytopathies and cystic kidney diseases were abundantly expressed on protein level, distinguishing organoids from almost every available cell culture model. On their pathway to terminal differentiation, organoids developed increased deposition of extracellular matrix. Single cell transcriptomic analysis suggests that most changes locate to podocytes and early podocyte progenitors. This matrix deposition is different from commonly used animal models of glomerular disease. A novel signaling system discovered was the TNFα system, a system also available in podocytes. Incubation of organoids with high concentrations of TNFα led to an activation of NF-kB signaling, and secretion of cytokines and complement components, alongside with extracellular matrix components. CONCLUSION Interestingly, this signaling system directly links inflammatory signaling, production of cytokines and complement, and production of extracellular matrix. Thus, we provide a repository of human kidney organoid proteins that revealed the potential to model pathophysiological pathways beyond genetic diseases.

Potential Strategies for Kidney Regeneration With Stem Cells: An Overview.

Kidney diseases are a major health problem worldwide. Despite advances in drug therapies, they are only capable of slowing the progression of kidney diseases. Accordingly, potential kidney regeneration strategies with stem cells have begun to be explored. There are two different directions for regenerative strategies, de novo whole kidney fabrication with stem cells, and stem cell therapy. De novo whole kidney strategies include: 1) decellularized scaffold technology, 2) 3D bioprinting based on engineering technology, 3) kidney organoid fabrication, 4) blastocyst complementation with chimeric technology, and 5) the organogenic niche method. Meanwhile, stem cell therapy strategies include 1) injection of stem cells, including mesenchymal stem cells, nephron progenitor cells, adult kidney stem cells and multi-lineage differentiating stress enduring cells, and 2) injection of protective factors secreted from these stem cells, including growth factors, chemokines, and extracellular vesicles containing microRNAs, mRNAs and proteins. Over the past few decades, there have been remarkable step-by-step developments in these strategies. Here, we review the current advances in the potential strategies for kidney regeneration using stem cells, along with their challenges for possible clinical use in the future.

DGAT2 Inhibition Potentiates Lipid Droplet Formation To Reduce Cytotoxicity in APOL1 Kidney Risk Variants.

Two variants in the gene encoding apolipoprotein L1 (APOL1) that are highly associated with African ancestry are major contributors to the large racial disparity in rates of human kidney disease. We previously demonstrated that recruitment of APOL1 risk variants G1 and G2 from the endoplasmic reticulum to lipid droplets leads to reduced APOL1-mediated cytotoxicity in human podocytes. We used CRISPR-Cas9 gene editing of induced pluripotent stem cells to develop human-derived APOL1G0/G0 and APOL1G2/G2 kidney organoids on an isogenic background, and performed bulk RNA sequencing of organoids before and after treatment with IFN-γ. We examined the number and distribution of lipid droplets in response to treatment with inhibitors of diacylglycerol O-acyltransferases 1 and 2 (DGAT1 and DGAT2) in kidney cells and organoids. APOL1 was highly upregulated in response to IFN-γ in human kidney organoids, with greater increases in organoids of high-risk G1 and G2 genotypes compared with wild-type (G0) organoids. RNA sequencing of organoids revealed that high-risk APOL1G2/G2 organoids exhibited downregulation of a number of genes involved in lipogenesis and lipid droplet biogenesis, as well as upregulation of genes involved in fatty acid oxidation. There were fewer lipid droplets in unstimulated high-risk APOL1G2/G2 kidney organoids than in wild-type APOL1G0/G0 organoids. Whereas DGAT1 inhibition reduced kidney organoid lipid droplet number, DGAT2 inhibition unexpectedly increased organoid lipid droplet number. DGAT2 inhibition promoted the recruitment of APOL1 to lipid droplets, with associated reduction in cytotoxicity. Lipogenesis and lipid droplet formation are important modulators of APOL1-associated cytotoxicity. Inhibition of DGAT2 may offer a potential therapeutic strategy to attenuate cytotoxic effects of APOL1 risk variants.

Gelatin Methacryloyl (GelMA) as a Suitable Three‐Dimensional Extracellular Environment for the Derivation of hiPSC‐Derived Kidney Organoids

The generation of human induced pluripotent stem cell (hiPSC)‐derived kidney organoids have facilitated novel insights into renal developmental processes and have the potential to provide personalised treatment strategies for patients with end‐stage renal disease. Traditional protocols for the directed differentiation of hiPSC‐derived kidney organoids have predominantly focused on biochemical cues to specify hiPSCs towards a mesonephric lineage, but have not considered the influence of the surrounding biophysical properties of the extracellular environment on organoid formation. We hypothesised that Gelatin methacryloyl (GelMA), a derivative of collagen with mechanically amendable hydrogel stiffness profiles, could be used to investigate the influence of extracellular matrix stiffness on kidney organoid specification. hiPSC‐derived kidney organoids were differentiated within photo‐crosslinked GelMA hydrogels of defined mechanical strengths. Enrichment of renal cell types in response to the various mechanical microenvironments was subsequently investigated using immunocytochemistry, qRT‐PCR, transmission electron microscopy (TEM) and histological staining methods. Rheological analysis of formulated hydrogels confirmed the generation of matrices with distinct stiffness (Young’s Modulus, G’) profiles. Hydrogels comparable to the stiffness of the gastrulation‐stage embryo (G’ = 400 Pa) and adult human kidney tissue (G’ = 5‐8 kPa) were generated. Importantly, the mechanical properties of the hydrogels showed remarkable stability even with prolonged time in culture. PCNA proliferation and cleaved caspase‐3 apoptotic staining of organoids embedded within scaffolds demonstrated high cell proliferation and viability in the hydrogel conditions by day 24 of differentiation. The formation of glomerular, proximal tubular and distal tubular structures, supported by basement membrane and interstitial cells were confirmed in conditions using immunofluorescent imaging. Interestingly, qRT‐PCR analysis revealed significant upregulation of nephron‐associated genes (including PAX8, NPHS2, NPHS1, SLC3A1 and AQP1) in organoids differentiated within extracellular environments that approximated adult human kidney tissue, when compared to organoids differentiated within the much softer microenvironments. Our results illustrate the influence of the extracellular environment on appropriate cell fate determination and propose GelMA hydrogels as faithful extracellular supports for the specification of hiPSC‐derived kidney organoids.

Growth and differentiation of human induced pluripotent stem cell (hiPSC) derived kidney organoids using fully synthetic peptide hydrogels

Human induced pluripotent stem cell (hiPSC)‐derived kidney organoids have prospective applications ranging from basic disease modelling to personalised medicine. However, there remains a necessity to refine the biophysical and biochemical parameters that govern kidney organoid formation. Differentiation within fully‐controllable and physiologically relevant 3D growth environments will be critical to improving organoid reproducibility and maturation. Here, we matured hiPSC‐derived kidney organoids within fully synthetic self‐assembling peptide hydrogels (SAPHs) of variable stiffness (storage modulus, G′). The resulting organoids contained complex structures comparable to those differentiated within the animal‐derived matrix, Matrigel. Single‐cell RNA sequencing (scRNA‐seq) was then used to compare organoids matured within SAPHs to those grown within Matrigel or at the air‐liquid interface. A total of 13,179 cells were analysed, revealing 14 distinct clusters. Organoid compositional analysis revealed a larger proportion of nephron cell types within Transwell‐derived organoids, while SAPH derived organoids were enriched for stromal‐associated cell populations. Notably, differentiation within a higher G’ SAPH generated podocytes with more mature gene expression profiles. Additionally, maturation within a 3D microenvironment significantly reduced the derivation of offtarget cell types, which are a known limitation of current kidney organoid protocols. This work demonstrates the utility of synthetic peptide‐based hydrogels with a defined stiffness, as a minimally complex microenvironment for the selected differentiation of kidney organoids.

Integrated single cell RNA‐seq and ATAC‐seq of iPSC‐derived kidney organoids reveals the complexity of TGFβ signalling to chromatin during the origin of fibroblasts

Differential methylation patterns and changes in post‐translational histone modifications are observed in patients with or without progressive diabetic kidney disease (DKD). TGFβ1 plays a central regulatory role in the determination of renal cell fate and the progression of DKD. Using ChIP‐seq, we identified an association between SMAD3 and the histone methyltransferase, EZH2, in iPSCs and in iPSC derived nephron progenitors. Using the 10X Genomics platform, we performed single cell RNA‐seq and ‐ATAC‐seq on human iPSC‐derived kidney organoids treated with the EZH2 specific inhibitor, GSK343, prior to treatment with TGFβ1. Integration of single cell RNAseq and ‐ATACseq identified de novo ACTA2+ve/POSTN+ve myofibroblasts in TGFβ1‐treated kidney organoids, characterised by increased SMAD3‐dependent cis chromatin accessibility and the expression of genes associated with fibroblast activation, which are similarly overexpressed in DKD patients. This fibrosis‐associated regulon is characterised by enrichment of SMAD3 and AP‐1, corresponding to FOS/JUN motifs at accessible loci identified by scATACseq. Treatment with GSK343 blocked SMAD3‐dependent cis co‐accessibility and inhibited myofibroblast activation. We propose that that the enzymatic function of the polycomb repressive complex is necessary for TGFβ1 induced increase in chromatin accessibility and its subsequent gene regulatory functions. Manipulation of the association between SMAD3 and EZH2 may be a useful therapeutic strategy for the resolution of renal fibrosis.

Adult mouse kidney stem cells orchestrate the de novo assembly of a nephron via Sirt2‐ modulated canonical Wnt/β‐catenin signaling

End‐stage renal disease affects an estimated 2.4 million people annually worldwide. With cases increasing 8% every year, regenerative strategies are needed to help alleviate, and potentially replace, the increasing demand for kidney transplants. An important question remains whether stem cells reside in the adult mammalian kidney. Here we developed a methodology for isolation of kidney stem cells from adult mouse kidney by using Sca1 and Oct4 stem cell markers. Using these cells, we generated three types of kidney‐like structures. First, we observed the assembly of self‐organizing three‐dimensional kidney organoids. These kidney organoids show characteristic of kidney structures and contain podocytes and endothelial cells that form networks of capillary loop‐like structures. We also observed the de novogeneration of a self‐organizing nephron (and its associated collecting duct) from monolayer cultures. Comma‐ and S‐shaped bodies that expressed activated Notch1 were present during this formation, suggesting that Notch signaling played a key role in driving nephrogenesis. This is consistent with our current knowledge of nephron formation in the mouse embryo. Furthermore, we observed the differentiation of monolayer cells into functionally mature tubules and self‐organizing kidney‐shaped structures that selectively endocytose dextran. Finally, we demonstrated that expression of Sirt2 was upregulated, which modulated the dynamics of canonical Wnt/β‐catenin signaling during kidney organoid development. Altogether, our observations provide the first evidence for the existence of stem cells in the adult mouse kidney. These findings offer powerful tools for future applications, including drug screening, disease modelling, cell therapy, and kidney regeneration.

Human pluripotent stem cell-derived kidney organoids for personalized congenital and idiopathic nephrotic syndrome modeling

ABSTRACTNephrotic syndrome (NS) is characterized by severe proteinuria as a consequence of kidney glomerular injury due to podocyte damage. In vitro models mimicking in vivo podocyte characteristics are a prerequisite to resolve NS pathogenesis. The detailed characterization of organoid podocytes resulting from a hybrid culture protocol showed a podocyte population that resembles adult podocytes and was superior compared with 2D counterparts, based on single-cell RNA sequencing, super-resolution imaging and electron microscopy. In this study, these next-generation podocytes in kidney organoids enabled personalized idiopathic nephrotic syndrome modeling, as shown by activated slit diaphragm signaling and podocyte injury following protamine sulfate, puromycin aminonucleoside treatment and exposure to NS plasma containing pathogenic permeability factors. Organoids cultured from cells of a patient with heterozygous NPHS2 mutations showed poor NPHS2 expression and aberrant NPHS1 localization, which was reversible after genetic correction. Repaired organoids displayed increased VEGFA pathway activity and transcription factor activity known to be essential for podocyte physiology, as shown by RNA sequencing. This study shows that organoids are the preferred model of choice to study idiopathic and congenital podocytopathies.

Adult Mouse Kidney Stem Cells Orchestrate the De Novo Assembly of a Nephron via Sirt2‐Modulated Canonical Wnt/β‐Catenin Signaling (Adv. Sci. 15/2022)

De Novo Generation of Kidney Organoids Using Adult Kidney Stem Cells Generation of kidney organoids using autologous kidney stem cells represents an attractive strategy for treating and potentially replacing the failing kidneys. In article number 2104034, Xiaobin Han and Zhongjie Sun report a previously unidentified adult kidney Sca1+ Oct4+ stem cells. Interestingly, culturing these stem cells led to de novo generation of self-organizing nephrons and kidney-like structures. This finding provides the first evidence that kidney stem cells are capable of de novo generating kidney organoids. This finding also offers a new opportunity for studying mammalian kidney development and personalized kidney regeneration.

Physiology assays in human kidney organoids.

Kidney organoids derived from human pluripotent stem cells constitute a novel model of disease, development, and regenerative therapy. Organoids are human, experimentally accessible, high throughput, and enable reconstitution of tissue-scale biology in a petri dish. Although gene expression patterns in organoid cells have been analyzed extensively, less is known about the functionality of these structures. Here, we review assays of physiological function in human kidney organoids, including best practices for quality control, and future applications. Tubular structures in organoids accumulate specific molecules through active transport, including dextran and organic anions, and swell with fluid in response to cAMP stimulation. When engrafted into animal models in vivo, organoids form vascularized glomerulus-like structures capable of size-selective filtration. Organoids exhibit metabolic, endocrine, injury, and infection phenotypes, although their specificity is not yet fully clear. To properly interpret organoid physiology assays, it is important to incorporate appropriate negative and positive controls, statistical methods, data presentation, molecular mechanisms, and clinical data sets. Improvements in organoid perfusion, patterning, and maturation are needed to enable branching morphogenesis, urine production, and renal replacement. Reconstituting renal physiology with kidney organoids is a new field with potential to provide fresh insights into classical phenomena.

Advances and Challenges in Kidney Organoids.

The advent of organoids has renewed researchers' interest in in vitro cell culture systems. A wide variety of protocols, primarily utilizing pluripotent stem cells, are under development to improve organoid generation to mimic organ development. The complexity of organoids generated is greatly influenced based on the method used. Understanding the process of kidney organoid formation gives developmental insights into how renal cells form, mature, and interact with the adjacent cells to form specific spatiotemporal structural patterns. This knowledge can bridge the gaps in understanding in vivo renal developmental processes. Evaluating genetic and epigenetic signatures in specialized cell types can help interpret the molecular mechanisms governing cell fate. In addition, development in single-cell RNA sequencing, 3D bioprinting and microfluidic technologies has led to better identification and understanding of a variety of cell types during differentiation and designing of complex structures to mimic the conditions in vivo. While several reviews have highlighted the application of kidney organoids, there is no comprehensive review of various methodologies specifically focusing on kidney organoids. This review summarizes the updated differentiation methodologies, applications, and challenges associated with kidney organoids. Here we have comprehensively collated all the different variables influencing the organoid generation.

Kidney Decellularized Extracellular Matrix Enhanced the Vascularization and Maturation of Human Kidney Organoids.

Kidney organoids derived from human pluripotent stem cells (hPSCs) have extensive potential for disease modelling and regenerative medicine. However, the limited vascularization and immaturity of kidney organoids have been still remained to overcome. Extracellular matrix (ECM) can provide mechanical support and a biochemical microenvironment for cell growth and differentiation. Here in vitro methods using a kidney decellularized extracellular matrix (dECM) hydrogel to culture hPSC‐derived kidney organoids, which have extensive vascular network and their own endothelial cells, are reported. Single‐cell transcriptomics reveal that the vascularized kidney organoids cultured using the kidney dECM have more mature patterns of glomerular development and higher similarity to human kidney than those cultured without the kidney dECM. Differentiation of α‐galactosidase A (GLA)‐knock‐out hPSCs generated using CRISPR/Cas9 into kidney organoids by the culture method using kidney dECM efficiently recapitulate Fabry nephropathy with vasculopathy. Transplantation of kidney organoids with kidney dECM into kidney of mouse accelerates the recruitment of endothelial cells from the host mouse kidney and maintains vascular integrity with the more organized slit diaphragm‐like structures than those without kidney dECM. The kidney dECM methodology for inducing extensive vascularization and maturation of kidney organoids can be applied to studies for kidney development, disease modeling, and regenerative medicine.

Adult Mouse Kidney Stem Cells Orchestrate the De Novo Assembly of a Nephron via Sirt2-Modulated Canonical Wnt/β-Catenin Signaling.

Generation of kidney organoids using autologous kidney stem cells represents an attractive strategy for treating and potentially replacing the failing kidneys. However, whether adult mammalian kidney stem cells have regenerative capacity remains unknown. Here, previously unidentified adult kidney Sca1+ Oct4+ stem/progenitor cells are isolated. Interestingly, culturing these cells leads to generation of kidney‐like structures. First, the assembly of self‐organizing 3D kidney‐like structures is observed. These kidney organoids contain podocytes, proximal tubules, and endothelial cells that form networks of capillary loop‐like structures. Second, the differentiation of kidney stem cells into functionally mature tubules and self‐organizing kidney‐shaped structures in monolayer culture that selectively endocytoses dextran, is shown. Finally, the de novo generation of an entire self‐organizing nephron from monolayer cultures is observed. Mechanistically, it is demonstrated that Sirt2‐mediated canonical Wnt/β‐catenin signaling is critical for the development of kidney organoids. Thus, the first evidence is provided that the adult mouse kidney stem cells are capable of de novo generating kidney organoids.

Validation of HDAC8 Inhibitors as Drug Discovery Starting Points to Treat Acute Kidney Injury.

Acute kidney injury (AKI), a sudden loss of kidney function, is a common and serious condition for which there are no approved specific therapies. While there are multiple approaches to treat the underlying causes of AKI, no targets have been clinically validated. Here, we assessed a series of potent, selective competitive inhibitors of histone deacetylase 8 (HDAC8), a promising therapeutic target in an AKI setting. Using biochemical assays, zebrafish AKI phenotypic assays, and human kidney organoid assays, we show that selective HDAC8 inhibitors can lead to efficacy in increasingly stringent models. One of these, PCI-34051, was efficacious in a rodent model of AKI, further supporting the potential for HDAC8 inhibitors and, in particular, this scaffold as a therapeutic approach to AKI.

Studying Kidney Diseases Using Organoid Models.

The prevalence of chronic kidney disease (CKD) is rapidly increasing over the last few decades, owing to the global increase in diabetes, and cardiovascular diseases. Dialysis greatly compromises the life quality of patients, while demand for transplantable kidney cannot be met, underscoring the need to develop novel therapeutic approaches to stop or reverse CKD progression. Our understanding of kidney disease is primarily derived from studies using animal models and cell culture. While cross-species differences made it challenging to fully translate findings from animal models into clinical practice, primary patient cells quickly lose the original phenotypes during in vitro culture. Over the last decade, remarkable achievements have been made for generating 3-dimensional (3D) miniature organs (organoids) by exposing stem cells to culture conditions that mimic the signaling cues required for the development of a particular organ or tissue. 3D kidney organoids have been successfully generated from different types of source cells, including human pluripotent stem cells (hPSCs), adult/fetal renal tissues, and kidney cancer biopsy. Alongside gene editing tools, hPSC-derived kidney organoids are being harnessed to model genetic kidney diseases. In comparison, adult kidney-derived tubuloids and kidney cancer-derived tumoroids are still in their infancy. Herein, we first summarize the currently available kidney organoid models. Next, we discuss recent advances in kidney disease modelling using organoid models. Finally, we consider the major challenges that have hindered the application of kidney organoids in disease modelling and drug evaluation and propose prospective solutions.

Modeling injury and repair in kidney organoids reveals that homologous recombination governs tubular intrinsic repair.

Kidneys have the capacity for intrinsic repair, preserving kidney architecture with return to a basal state after tubular injury. When injury is overwhelming or repetitive, however, that capacity is exceeded and incomplete repair results in fibrotic tissue replacing normal kidney parenchyma. Loss of nephrons correlates with reduced kidney function, which defines chronic kidney disease (CKD) and confers substantial morbidity and mortality to the worldwide population. Despite the identification of pathways involved in intrinsic repair, limited treatments for CKD exist, partly because of the limited throughput and predictivity of animal studies. Here, we showed that kidney organoids can model the transition from intrinsic to incomplete repair. Single-nuclear RNA sequencing of kidney organoids after cisplatin exposure identified 159 differentially expressed genes and 29 signal pathways in tubular cells undergoing intrinsic repair. Homology-directed repair (HDR) genes including Fanconi anemia complementation group D2 (FANCD2) and RAD51 recombinase (RAD51) were transiently up-regulated during intrinsic repair but were down-regulated in incomplete repair. Single cellular transcriptomics in mouse models of obstructive and hemodynamic kidney injury and human kidney samples of immune-mediated injury validated HDR gene up-regulation during tubular repair. Kidney biopsy samples with tubular injury and varying degrees of fibrosis confirmed loss of FANCD2 during incomplete repair. Last, we performed targeted drug screening that identified the DNA ligase IV inhibitor, SCR7, as a therapeutic candidate that rescued FANCD2/RAD51-mediated repair to prevent the progression of CKD in the cisplatin-induced organoid injury model. Our findings demonstrate the translational utility of kidney organoids to identify pathologic pathways and potential therapies.

Tissue Culture Models of AKI: From Tubule Cells to Human Kidney Organoids.

AKI affects approximately 13.3 million people around the world each year, causing CKD and/or mortality. The mammalian kidney cannot generate new nephrons after postnatal renal damage and regenerative therapies for AKI are not available. Human kidney tissue culture systems can complement animal models of AKI and/or address some of their limitations. Donor-derived somatic cells, such as renal tubule epithelial cells or cell lines (RPTEC/hTERT, ciPTEC, HK-2, Nki-2, and CIHP-1), have been used for decades to permit drug toxicity screening and studies into potential AKI mechanisms. However, tubule cell lines do not fully recapitulate tubular epithelial cell properties in situ when grown under classic tissue culture conditions. Improving tissue culture models of AKI would increase our understanding of the mechanisms, leading to new therapeutics. Human pluripotent stem cells (hPSCs) can be differentiated into kidney organoids and various renal cell types. Injury to human kidney organoids results in renal cell-type crosstalk and upregulation of kidney injury biomarkers that are difficult to induce in primary tubule cell cultures. However, current protocols produce kidney organoids that are not mature and contain off-target cell types. Promising bioengineering techniques, such as bioprinting and "kidney-on-a-chip" methods, as applied to kidney nephrotoxicity modeling advantages and limitations are discussed. This review explores the mechanisms and detection of AKI in tissue culture, with an emphasis on bioengineered approaches such as human kidney organoid models.

SARS-CoV-2 pirates the kidneys: A scar(y) story

SARS-CoV-2 can cause diverse severe and lasting damage to the kidneys. In the latest issue of Cell Stem Cell, Jansen et al. utilized data gleaned from human kidney autopsies and human induced pluripotent stem cell-derived kidney organoids to investigate the direct effects of SARS-CoV-2 infection on kidney cells. They found that such infections resulted in renal scarring (notably, tubulointerstitial fibrosis).

Kidney Organoids Are Capable of Forming Tumors, but Not Teratomas.

Induced pluripotent stem cell (iPSC)-derived kidney organoids are a potential tool for the regeneration of kidney tissue. They represent an early stage of nephrogenesis and have been shown to successfsully vascularize and mature further in vivo. However, there are concerns regarding the long-term safety and stability of iPSC derivatives. Specifically, the potential for tumorigenesis may impede the road to clinical application. To study safety and stability of kidney organoids, we analyzed their potential for malignant transformation in a teratoma assay and following long-term subcutaneous implantation in an immune-deficient mouse model. We did not detect fully functional residual iPSCs in the kidney organoids as analyzed by gene expression analysis, single-cell sequencing and immunohistochemistry. Accordingly, kidney organoids failed to form teratoma. Upon long-term subcutaneous implantation of whole organoids in immunodeficient IL2Ry−/−RAG2−/− mice, we observed tumor formation in 5 out of 103 implanted kidney organoids. These tumors were composed of WT1+CD56+ immature blastemal cells and showed histological resemblance with Wilms tumor. No genetic changes were identified that contributed to the occurrence of tumorigenic cells within the kidney organoids. However, assessment of epigenetic changes revealed a unique cluster of differentially methylated genes that were also present in undifferentiated iPSCs. We discovered that kidney organoids have the capacity to form tumors upon long-term implantation. The presence of epigenetic modifications combined with the lack of environmental cues may have caused an arrest in terminal differentiation. Our results indicate that the safe implementation of kidney organoids should exclude the presence of pro-tumorigenic methylation in kidney organoids.