Stem Cell-derived Kidney Organoids Research Articles

Advancements in Research on Genetic Kidney Diseases Using Human-Induced Pluripotent Stem Cell-Derived Kidney Organoids.

Genetic or hereditary kidney disease stands as a pivotal cause of chronic kidney disease (CKD). The proliferation and widespread utilization of DNA testing in clinical settings have notably eased the diagnosis of genetic kidney diseases, which were once elusive but are now increasingly identified in cases previously deemed CKD of unknown etiology. However, despite these diagnostic strides, research into disease pathogenesis and novel drug development faces significant hurdles, chiefly due to the dearth of appropriate animal models and the challenges posed by limited patient cohorts in clinical studies. Conversely, the advent and utilization of human-induced pluripotent stem cells (hiPSCs) offer a promising avenue for genetic kidney disease research. Particularly, the development of hiPSC-derived kidney organoid systems presents a novel platform for investigating various forms of genetic kidney diseases. Moreover, the integration of the CRISPR/Cas9 technique into this system holds immense potential for efficient research on genetic kidney diseases. This review aims to explore the applications of in vitro kidney organoids generated from hiPSCs in the study of diverse genetic kidney diseases. Additionally, it will delve into the limitations of this research platform and outline future perspectives for advancing research in this crucial area.

Stepwise developmental mimicry generates proximal-biased kidney organoids.

The kidney maintains body fluid homeostasis by reabsorbing essential compounds and excreting waste. Proximal tubule cells, crucial for renal reabsorption of a range of sugars, ions, and amino acids, are highly susceptible to damage, leading to pathologies necessitating dialysis and kidney transplants. While human pluripotent stem cell-derived kidney organoids are used for modeling renal development, disease, and injury, the formation of proximal nephron cells in these 3D structures is incomplete. Here, we describe how to drive the development of proximal tubule precursors in kidney organoids by following a blueprint of in vivo human nephrogenesis. Transient manipulation of the PI3K signaling pathway activates Notch signaling in the early nephron and drives nephrons toward a proximal precursor state. These "proximal-biased" (PB) organoid nephrons proceed to generate proximal nephron precursor cells. Single-cell transcriptional analyses across the organoid nephron differentiation, comparing control and PB types, confirm the requirement of transient Notch signaling for proximal development. Indicative of functional maturity, PB organoids demonstrate dextran and albumin uptake, akin to in vivo proximal tubules. Moreover, PB organoids are highly sensitive to nephrotoxic agents, display an injury response, and drive expression of HAVCR1 / KIM1 , an early proximal-specific marker of kidney injury. Injured PB organoids show evidence of collapsed tubules, DNA damage, and upregulate the injury-response marker SOX9 . The PB organoid model therefore has functional relevance and potential for modeling mechanisms underpinning nephron injury. These advances improve the use of iPSC-derived kidney organoids as tools to understand developmental nephrology, model disease, test novel therapeutics, and for understanding human renal physiology.

Human pluripotent stem cell-derived kidney organoids reveal tubular epithelial pathobiology of heterozygous HNF1B-associated dysplastic kidney malformations

Hepatocyte nuclear factor 1B (HNF1B) encodes a transcription factor expressed in developing human kidney epithelia. Heterozygous HNF1B mutations are the commonest monogenic cause of dysplastic kidney malformations (DKMs). To understand their pathobiology, we generated heterozygous HNF1B mutant kidney organoids from CRISPR-Cas9 gene-edited human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) reprogrammed from a family with HNF1B-associated DKMs. Mutant organoids contained enlarged malformed tubules displaying deregulated cell turnover. Numerous genes implicated in Mendelian kidney tubulopathies were downregulated, and mutant tubules resisted the cyclic AMP (cAMP)-mediated dilatation seen in controls. Bulk and single-cell RNA sequencing (scRNA-seq) analyses indicated abnormal Wingless/Integrated (WNT), calcium, and glutamatergic pathways, the latter hitherto unstudied in developing kidneys. Glutamate ionotropic receptor kainate type subunit 3 (GRIK3) was upregulated in malformed mutant nephron tubules and prominent in HNF1B mutant fetal human dysplastic kidney epithelia. These results reveal morphological, molecular, and physiological roles for HNF1B in human kidney tubule differentiation and morphogenesis illuminating the developmental origin of mutant-HNF1B-causing kidney disease.

Interactions of the Immune System with Human Kidney Organoids.

Kidney organoids are an innovative tool in transplantation research. The aim of the present study was to investigate whether kidney organoids are susceptible for allo-immune attack and whether they can be used as a model to study allo-immunity in kidney transplantation. Human induced pluripotent stem cell-derived kidney organoids were co-cultured with human peripheral blood mononuclear cells (PBMC), which resulted in invasion of allogeneic T-cells around nephron structures and macrophages in the stromal cell compartment of the organoids. This process was associated with the induction of fibrosis. Subcutaneous implantation of kidney organoids in immune-deficient mice followed by adoptive transfer of human PBMC led to the invasion of diverse T-cell subsets. Single cell transcriptomic analysis revealed that stromal cells in the organoids upregulated expression of immune response genes upon immune cell invasion. Moreover, immune regulatory PD-L1 protein was elevated in epithelial cells while genes related to nephron differentiation and function were downregulated. This study characterized the interaction between immune cells and kidney organoids, which will advance the use of kidney organoids for transplantation research.

Human pluripotent stem cell-derived kidney organoids: Current progress and challenges.

Human pluripotent stem cell (hPSC)-derived kidney organoids share similarities with the fetal kidney. However, the current hPSC-derived kidney organoids have some limitations, including the inability to perform nephrogenesis and lack of a corticomedullary definition, uniform vascular system, and coordinated exit pathway for urinary filtrate. Therefore, further studies are required to produce hPSC-derived kidney organoids that accurately mimic human kidneys to facilitate research on kidney development, regeneration, disease modeling, and drug screening. In this review, we discussed recent advances in the generation of hPSC-derived kidney organoids, how these organoids contribute to the understanding of human kidney development and research in disease modeling. Additionally, the limitations, future research focus, and applications of hPSC-derived kidney organoids were highlighted.

3D Hydrogel Encapsulation Regulates Nephrogenesis in Kidney Organoids.

Stem cell-derived kidney organoids contain nephron segments that recapitulate morphological and functional aspects of the human kidney. However, directed differentiation protocols for kidney organoids are largely conducted using biochemical signals to control differentiation. Here, the hypothesis that mechanical signals regulate nephrogenesis is investigated in 3D culture by encapsulating kidney organoids within viscoelastic alginate hydrogels with varying rates of stress relaxation. Tubular nephron segments are significantly more convoluted in kidney organoids differentiated in encapsulating hydrogels when compared with those in suspension culture. Hydrogel viscoelasticity regulates the spatial distribution of nephron segments within the differentiating kidney organoids. Consistent with these observations, a particle-based computational model predicts that the extent of deformation of the hydrogel-organoid interface regulates the morphology of nephron segments. Elevated extracellular calcium levels in the culture medium, which can be impacted by the hydrogels, decrease the glomerulus-to-tubule ratio of nephron segments. These findings reveal that hydrogel encapsulation regulates nephron patterning and morphology and suggest that the mechanical microenvironment is an important design variable for kidney regenerative medicine.

Kidney organoid models reveal cilium-autophagy metabolic axis as a therapeutic target for PKD both in vitro and in vivo

Human pluripotent stem cell-derived kidney organoids offer unprecedented opportunities for studying polycystic kidney disease (PKD), which still has no effective cure. Here, we developed both invitro and invivo organoid models of PKD that manifested tubular injury and aberrant upregulation of renin-angiotensin aldosterone system. Single-cell analysis revealed that a myriad of metabolic changes occurred during cystogenesis, including defective autophagy. Experimental activation of autophagy via ATG5 overexpression or primary cilia ablation significantly inhibited cystogenesis in PKD kidney organoids. Employing the organoid xenograft model of PKD, which spontaneously developed tubular cysts, we demonstrate that minoxidil, a potent autophagy activator and an FDA-approved drug, effectively attenuated cyst formation invivo. This invivo organoid model of PKD will enhance our capability to discover novel disease mechanisms and validate candidate drugs for clinical translation.

Generation of renal tubular organoids from adult SOX9+ kidney progenitor cells

Abstract The pathogenesis of several kidney diseases results in the eventual destruction of the renal tubular system, which can progress to end-stage renal disease. Previous studies have demonstrated the involvement of a population of SOX9-positive cells in kidney regeneration and repair process following kidney injury. However, the ability of these cells to autonomously generate kidney organoids has never been investigated. Here, we isolated SOX9+ kidney progenitor cells (KPCs) from both mice and humans and tested their differentiation potential in vitro. The data showed that the human SOX9+ KPC could self-assemble into organoids with kidney-like morphology. We also used single-cell RNA sequencing to characterize the organoid cell populations and identified four distinct types of renal tubular cells. Compared to the induced pluripotent stem cell-derived kidney organoids, KPC demonstrated more tubular differentiation potential but failed to differentiate into glomerular cells. KPC-derived organoid formation involved the expression of genes related to metanephric development and followed a similar mechanism to renal injury repair in acute kidney injury patients. Altogether, our study provided a potentially useful approach to generating kidney tubular organoids for future application.

T-Cell Mediated Immune Rejection of Beta-2-Microglobulin Knockout Induced Pluripotent Stem Cell-Derived Kidney Organoids.

Immune evasive induced pluripotent stem cell (iPSC)-derived kidney organoids, known as "stealth" organoids, hold promise for clinical transplantation. To address immune rejection, we investigated the impact of genetically modifying human leukocyte antigen (HLA) class I in kidney organoids prior to transplantation. By using CRISPR-Cas9, we successfully knocked out beta-2-microglobulin (B2M), resulting in iPSCs devoid of HLA class I surface expression. In vitro, the B2M knockout protected kidney organoids derived from these iPSCs against T-cell rejection. To assess in vivo protection, unmodified (control) and B2M-/- kidney organoids were transplanted into humanized mice engrafted with human peripheral blood mononuclear cells (PBMCs). Successful engraftment of human PBMCs was confirmed, and after 4 weeks, we observed no discernible difference in the infiltration rate, proliferation, or cytotoxicity of CD4+ and CD8+ T cells between control and B2M-/- organoids. Both groups of organoids showed compromised tissue integrity, displaying tubulitis and loss of tubule integrity. Notably, while B2M-/- organoids failed to express HLA class I on their cell surface, there was preexisting expression of HLA class II in both control and B2M-/- organoids transplanted into mice with human PBMCs. HLA class II expression was not limited to antigen-presenting cells but also evident in epithelial cells of the kidney organoid, posing an additional immunological challenge to its transplantation. Consequently, we conclude that B2M knockout alone is insufficient to protect iPSC-derived kidney organoids from T-cell-mediated immune rejection. Additionally, our findings suggest that modulating HLA class II signaling will be necessary to prevent rejection following transplantation.

Kidney Organoid Derived from Human Pluripotent and Adult Stem Cells for Disease Modeling.

Kidney disease affects a significant portion of the global population, yet effective therapies are lacking despite advancements in identifying genetic causes. This limitation can be attributed to the absence of adequate in vitro models that accurately mimic human kidney disease, hindering targeted therapeutic development. However, the emergence of human induced pluripotent stem cells (PSCs) and the development of organoids using them have opened up a way to model kidney development and disease in humans, as well as validate the effects of new drugs. To fully leverage their capabilities in these fields, it is crucial for kidney organoids to closely resemble the structure and functionality of adult human kidneys. In this review, we aim to discuss the potential of using human PSCs or adult kidney stem cell-derived kidney organoids to model genetic kidney disease and renal cancer.

Cells sorted off hiPSC-derived kidney organoids coupled with immortalized cells reliably model the proximal tubule

Of late, numerous microphysiological systems have been employed to model the renal proximal tubule. Yet there is lack of research on refining the functions of the proximal tubule epithelial layer—selective filtration and reabsorption. In this report, pseudo proximal tubule cells extracted from human-induced pluripotent stem cell-derived kidney organoids are combined and cultured with immortalized proximal tubule cells. It is shown that the cocultured tissue is an impervious epithelium that offers improved levels of certain transporters, extracellular matrix proteins collagen and laminin, and superior glucose transport and P-glycoprotein activity. mRNA expression levels higher than those obtained from each cell type were detected, suggesting an anomalous synergistic crosstalk between the two. Alongside, the improvements in morphological characteristics and performance of the immortalized proximal tubule tissue layer exposed, upon maturation, to human umbilical vein endothelial cells are thoroughly quantified and compared. Glucose and albumin reabsorption, as well as xenobiotic efflux rates through P-glycoprotein were all improved. The data presented abreast highlight the advantages of the cocultured epithelial layer and the non-iPSC-based bilayer. The in vitro models presented herein can be helpful in personalized nephrotoxicity studies.

A parathyroid hormone/salt-inducible kinase signaling axis controls renal vitamin D activation and organismal calcium homeostasis.

The renal actions of parathyroid hormone (PTH) promote 1,25-vitamin D generation; however, the signaling mechanisms that control PTH-dependent vitamin D activation remain unknown. Here, we demonstrated that salt-inducible kinases (SIKs) orchestrated renal 1,25-vitamin D production downstream of PTH signaling. PTH inhibited SIK cellular activity by cAMP-dependent PKA phosphorylation. Whole-tissue and single-cell transcriptomics demonstrated that both PTH and pharmacologic SIK inhibitors regulated a vitamin D gene module in the proximal tubule. SIK inhibitors increased 1,25-vitamin D production and renal Cyp27b1 mRNA expression in mice and in human embryonic stem cell-derived kidney organoids. Global- and kidney-specific Sik2/Sik3 mutant mice showed Cyp27b1 upregulation, elevated serum 1,25-vitamin D, and PTH-independent hypercalcemia. The SIK substrate CRTC2 showed PTH and SIK inhibitor-inducible binding to key Cyp27b1 regulatory enhancers in the kidney, which were also required for SIK inhibitors to increase Cyp27b1 in vivo. Finally, in a podocyte injury model of chronic kidney disease-mineral bone disorder (CKD-MBD), SIK inhibitor treatment stimulated renal Cyp27b1 expression and 1,25-vitamin D production. Together, these results demonstrated a PTH/SIK/CRTC signaling axis in the kidney that controls Cyp27b1 expression and 1,25-vitamin D synthesis. These findings indicate that SIK inhibitors might be helpful for stimulation of 1,25-vitamin D production in CKD-MBD.

Inhibition of complement activation by CD55 overexpression in human induced pluripotent stem cell derived kidney organoids.

End stage renal disease is an increasing problem worldwide driven by aging of the population and increased prevalence of metabolic disorders and cardiovascular disease. Currently, kidney transplantation is the only curative option, but donor organ shortages greatly limit its application. Regenerative medicine has the potential to solve the shortage by using stem cells to grow the desired tissues, like kidney tissue. Immune rejection poses a great threat towards the implementation of stem cell derived tissues and various strategies have been explored to limit the immune response towards these tissues. However, these studies are limited by targeting mainly T cell mediated immune rejection while the rejection process also involves innate and humoral immunity. In this study we investigate whether inhibition of the complement system in human induced pluripotent stem cells (iPSC) could provide protection from such immune injury. To this end we created knock-in iPSC lines of the membrane bound complement inhibitor CD55 to create a transplant-specific protection towards complement activation. CD55 inhibits the central driver of the complement cascade, C3 convertase, and we show that overexpression is able to decrease complement activation on both iPSCs as well as differentiated kidney organoids upon stimulation with anti-HLA antibodies to mimic the mechanism of humoral rejection.

Enhanced metanephric specification to functional proximal tubule enables toxicity screening and infectious disease modelling in kidney organoids

While pluripotent stem cell-derived kidney organoids are now being used to model renal disease, the proximal nephron remains immature with limited evidence for key functional solute channels. This may reflect early mispatterning of the nephrogenic mesenchyme and/or insufficient maturation. Here we show that enhanced specification to metanephric nephron progenitors results in elongated and radially aligned proximalised nephrons with distinct S1 - S3 proximal tubule cell types. Such PT-enhanced organoids possess improved albumin and organic cation uptake, appropriate KIM-1 upregulation in response to cisplatin, and improved expression of SARS-CoV-2 entry factors resulting in increased viral replication. The striking proximo-distal orientation of nephrons resulted from localized WNT antagonism originating from the organoid stromal core. PT-enhanced organoids represent an improved model to study inherited and acquired proximal tubular disease as well as drug and viral responses.

Vasculogenesis in kidney organoids upon transplantation

Human induced pluripotent stem cell-derived kidney organoids have potential for disease modeling and to be developed into clinically transplantable auxiliary tissue. However, they lack a functional vasculature, and the sparse endogenous endothelial cells (ECs) are lost upon prolonged culture in vitro, limiting maturation and applicability. Here, we use intracoelomic transplantation in chicken embryos followed by single-cell RNA sequencing and advanced imaging platforms to induce and study vasculogenesis in kidney organoids. We show expansion of human organoid-derived ECs that reorganize into perfused capillaries and form a chimeric vascular network with host-derived blood vessels. Ligand-receptor analysis infers extensive potential interactions of human ECs with perivascular cells upon transplantation, enabling vessel wall stabilization. Perfused glomeruli display maturation and morphogenesis to capillary loop stage. Our findings demonstrate the beneficial effect of vascularization on not only epithelial cell types, but also the mesenchymal compartment, inducing the expansion of ´on target´ perivascular stromal cells, which in turn are required for further maturation and stabilization of the neo-vasculature. The here described vasculogenic capacity of kidney organoids will have to be deployed to achieve meaningful glomerular maturation and kidney morphogenesis in vitro.

High-throughput "read-on-ski" automated imaging and label-free detection system for toxicity screening of compounds using personalised human kidney organoids.

Organoid models are used to study kidney physiology, such as the assessment of nephrotoxicity and underlying disease processes. Personalized human pluripotent stem cell-derived kidney organoids are ideal models for compound toxicity studies, but there is a need to accelerate basic and translational research in the field. Here, we developed an automated continuous imaging setup with the "read-on-ski" law of control to maximize temporal resolution with minimum culture plate vibration. High-accuracy performance was achieved: organoid screening and imaging were performed at a spatial resolution of 1.1 μm for the entire multi-well plate under 3 min. We used the in-house developed multi-well spinning device and cisplatin-induced nephrotoxicity model to evaluate the toxicity in kidney organoids using this system. The acquired images were processed via machine learning-based classification and segmentation algorithms, and the toxicity in kidney organoids was determined with 95% accuracy. The results obtained by the automated "read-on-ski" imaging device, combined with label-free and non-invasive algorithms for detection, were verified using conventional biological procedures. Taking advantage of the close-to-in vivo-kidney organoid model, this new development opens the door for further application of scaled-up screening using organoids in basic research and drug discovery.

Enhanced Microvasculature Formation and Patterning in iPSC-Derived Kidney Organoids Cultured in Physiological Hypoxia.

Stem cell-derived kidney organoids have been shown to self-organize from induced pluripotent stem cells into most important renal structures. However, the structures remain immature in culture and contain endothelial networks with low connectivity and limited organoid invasion. Furthermore, the nephrons lose their phenotype after approximately 25days. To become applicable for future transplantation, further maturation in vitro is essential. Since kidneys in vivo develop in hypoxia, we studied the modulation of oxygen availability in culture. We hypothesized that introducing long-term culture at physiological hypoxia, rather than the normally applied non-physiological, hyperoxic 21% O2, could initiate angiogenesis, lead to enhanced growth factor expression and improve the endothelial patterning. We therefore cultured the kidney organoids at 7% O2 instead of 21% O2 for up to 25days and evaluated nephrogenesis, growth factor expression such as VEGF-A and vascularization. Whole mount imaging revealed a homogenous morphology of the endothelial network with enhanced sprouting and interconnectivity when the kidney organoids were cultured in hypoxia. Three-dimensional vessel quantification confirmed that the hypoxic culture led to an increased average vessel length, likely due to the observed upregulation of VEGFA-189 and VEGFA-121, and downregulation of the antiangiogenic protein VEGF-A165b measured in hypoxia. This research indicates the importance of optimization of oxygen availability in organoid systems and the potential of hypoxic culture conditions in improving the vascularization of organoids.

Soft, Dynamic Hydrogel Confinement Improves Kidney Organoid Lumen Morphology and Reduces Epithelial-Mesenchymal Transition in Culture.

Pluripotent stem cell‐derived kidney organoids offer a promising solution to renal failure, yet current organoid protocols often lead to off‐target cells and phenotypic alterations, preventing maturity. Here, various dynamic hydrogel architectures are created, conferring a controlled and biomimetic environment for organoid encapsulation. How hydrogel stiffness and stress relaxation affect renal phenotype and undesired fibrotic markers are investigated. The authors observe that stiff hydrogel encapsulation leads to an absence of certain renal cell types and signs of an epithelial–mesenchymal transition (EMT), whereas encapsulation in soft, stress‐relaxing hydrogels leads to all major renal segments, fewer fibrosis or EMT associated proteins, apical proximal tubule polarization, and primary cilia formation, representing a significant improvement over current approaches to culture kidney organoids. The findings show that engineering hydrogel mechanics and dynamics have a decided benefit for organoid culture. These structure–property–function relationships can enable the rational design of materials, bringing us closer to functional engraftments and disease‐modeling applications.

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.

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).