Kidney Organoids Research Articles

Rapid target validation in a Cas9-inducible hiPSC derived kidney model

Recent advances in induced pluripotent stem cells (iPSCs), genome editing technologies and 3D organoid model systems highlight opportunities to develop new in vitro human disease models to serve drug discovery programs. An ideal disease model would accurately recapitulate the relevant disease phenotype and provide a scalable platform for drug and genetic screening studies. Kidney organoids offer a high cellular complexity that may provide greater insights than conventional single-cell type cell culture models. However, genetic manipulation of the kidney organoids requires prior generation of genetically modified clonal lines, which is a time and labor consuming procedure. Here, we present a methodology for direct differentiation of the CRISPR-targeted cell pools, using a doxycycline-inducible Cas9 expressing hiPSC line for high efficiency editing to eliminate the laborious clonal line generation steps. We demonstrate the versatile use of genetically engineered kidney organoids by targeting the autosomal dominant polycystic kidney disease (ADPKD) genes: PKD1 and PKD2. Direct differentiation of the respective knockout pool populations into kidney organoids resulted in the formation of cyst-like structures in the tubular compartment. Our findings demonstrated that we can achieve > 80% editing efficiency in the iPSC pool population which resulted in a reliable 3D organoid model of ADPKD. The described methodology may provide a platform for rapid target validation in the context of disease modeling.

Strategies for vascularization in kidney organoids

The establishment of protocols for differentiating kidney organoids from human pluripotent stem cells (hPSCs) has potential for the application of kidney organoids in regenerative medicine. However, the primary obstacle to the regenerative application of hPSC-derived kidney organoids is precise vascularization due to the lack of vasculature in hPSC-derived kidney organoids. In this article, we review the recent methodologies for developing vasculature of kidney organoids to overcome this limitation of kidney organoids, together with a discussion of their clinical applications.

The New Generation hDHODH Inhibitor MEDS433 Hinders the In Vitro Replication of SARS-CoV-2 and Other Human Coronaviruses

Although coronaviruses (CoVs) have long been predicted to cause zoonotic diseases and pandemics with high probability, the lack of effective anti-pan-CoVs drugs rapidly usable against the emerging SARS-CoV-2 actually prevented a promptly therapeutic intervention for COVID-19. Development of host-targeting antivirals could be an alternative strategy for the control of emerging CoVs infections, as they could be quickly repositioned from one pandemic event to another. To contribute to these pandemic preparedness efforts, here we report on the broad-spectrum CoVs antiviral activity of MEDS433, a new inhibitor of the human dihydroorotate dehydrogenase (hDHODH), a key cellular enzyme of the de novo pyrimidine biosynthesis pathway. MEDS433 inhibited the in vitro replication of hCoV-OC43 and hCoV-229E, as well as of SARS-CoV-2, at low nanomolar range. Notably, the anti-SARS-CoV-2 activity of MEDS433 against SARS-CoV-2 was also observed in kidney organoids generated from human embryonic stem cells. Then, the antiviral activity of MEDS433 was reversed by the addition of exogenous uridine or the product of hDHODH, the orotate, thus confirming hDHODH as the specific target of MEDS433 in hCoVs-infected cells. Taken together, these findings suggest MEDS433 as a potential candidate to develop novel drugs for COVID-19, as well as broad-spectrum antiviral agents exploitable for future CoVs threats.

Kidney organoids generated from erythroid progenitors cells of patients with autosomal dominant polycystic kidney disease.

BackgroundKidney organoids have been broadly obtained from commercially available induced pluripotent stem cells (iPSCs); however, it has been a great challenge to efficiently produce renal organoid models from patients with autosomal dominant polycystic kidney disease (ADPKD) that recapitulate both embryogenesis and the mechanisms of cystogenesis.MethodsBlood erythroid progenitors (EPs) from two ADPKD patients and one healthy donor (HC) was used as a comparative control to normalize the many technical steps for reprogramming EPs and for the organoids generation. EPs were reprogrammed by an episomal vector into iPSCs, which were differentiated into renal tubular organoids and then stimulated by forskolin to induce cysts formation.ResultsiPSCs derived from EPs exhibited all characteristics of pluripotency and were able to differentiate into all three germ layers. 3D tubular organoids were generated from single cells after 28 days in Matrigel. HC and ADPKD organoids did not spontaneously form cysts, but upon forskolin stimulation, cysts-like structures were observed in the ADPKD organoids but not in the HC-derived organoids.ConclusionThe findings of this study showed that kidney organoids were successfully generated from the blood EP cells of ADPKD patients and a healthy control donor. This approach should contribute as a powerful tool for embryonic kidney development model, which is able to recapitulate the very early pathophysiological mechanisms involved in cytogenesis.

Kidney Organoid and Microphysiological Kidney Chip Models to Accelerate Drug Development and Reduce Animal Testing.

Kidneys are critical for the elimination of many drugs and metabolites via the urine, filtering waste and maintaining proper fluid and electrolyte balance. Emerging technologies incorporating engineered three-dimensional (3D) in vitro cell culture models, such as organoids and microphysiological systems (MPS) culture platforms, have been developed to replicate nephron function, leading to enhanced efficacy, safety, and toxicity evaluation of new drugs and environmental exposures. Organoids are tiny, self-organized three-dimensional tissue cultures derived from stem cells that can include dozens of cell types to replicate the complexity of an organ. In contrast, MPS are highly controlled fluidic culture systems consisting of isolated cell type(s) that can be used to deconvolute mechanism and pathophysiology. Both systems, having their own unique benefits and disadvantages, have exciting applications in the field of kidney disease modeling and therapeutic discovery and toxicology. In this review, we discuss current uses of both hPSC-derived organoids and MPS as pre-clinical models for studying kidney diseases and drug induced nephrotoxicity. Examples such as the use of organoids to model autosomal dominant polycystic kidney disease, and the use of MPS to predict renal clearance and nephrotoxic concentrations of novel drugs are briefly discussed. Taken together, these novel platforms allow investigators to elaborate critical scientific questions. While much work needs to be done, utility of these 3D cell culture technologies has an optimistic outlook and the potential to accelerate drug development while reducing the use of animal testing.

Autonomous Calcium Signaling in Human and Zebrafish Podocytes Controls Kidney Filtration Barrier Morphogenesis.

Podocytes are critical to maintaining the glomerular filtration barrier, and mutations in nephrotic syndrome genes are known to affect podocyte calcium signaling. However, the role of calcium signaling during podocyte development remains unknown. We undertook live imaging of calcium signaling in developing podocytes, using zebrafish larvae and human kidney organoids. To evaluate calcium signaling during development and in response to channel blockers and genetic defects, the calcium biosensor GCaMP6s was expressed in zebrafish podocytes. We used electron microscopy to evaluate filtration barrier formation in zebrafish, and Fluo-4 to detect calcium signals in differentiating podocytes in human kidney organoids. Immature zebrafish podocytes (2.5 days postfertilization) generated calcium transients that correlated with interactions with forming glomerular capillaries. Calcium transients persisted until 4 days postfertilization, and were absent after glomerular barrier formation was complete. We detected similar calcium transients in maturing human organoid glomeruli, suggesting a conserved mechanism. In both models, inhibitors of SERCA or IP3 receptor calcium-release channels blocked calcium transients in podocytes, whereas lanthanum was ineffective, indicating the calcium source is from intracellular podocyte endoplasmic-reticulum stores. Calcium transients were not affected by blocking heartbeat or by blocking development of endothelium or endoderm, and they persisted in isolated glomeruli, suggesting podocyte-autonomous calcium release. Inhibition of expression of phospholipase C-γ1, but not nephrin or phospholipase C-ε1, led to significantly decreased calcium activity. Finally, blocking calcium release affected glomerular shape and podocyte foot process formation, supporting the critical role of calcium signaling in glomerular morphogenesis. These findings establish podocyte cell-autonomous calcium signaling as a prominent and evolutionarily conserved feature of podocyte differentiation and demonstrate its requirement for podocyte foot process formation.

An efficient method to generate kidney organoids at the air-liquid interface.

The prevalence of kidney dysfunction continues to increase worldwide, driving the need to develop transplantable renal tissues. The kidney develops from four major renal progenitor populations: nephron epithelial, ureteric epithelial, interstitial and endothelial progenitors. Methods have been developed to generate kidney organoids but few or dispersed tubular clusters within the organoids hamper its use in regenerative applications. Here, we describe a detailed protocol of asynchronous mixing of kidney progenitors using organotypic culture conditions to generate kidney organoids tightly packed with tubular clusters and major renal structures including endothelial network and functional proximal tubules. This protocol provides guidance in the culture of human embryonic stem cells from a National Institute of Health-approved line and their directed differentiation into kidney organoids. Our 18-day protocol provides a rapid method to generate kidney organoids that facilitate the study of different nephrological events including in vitro tissue development, disease modeling and chemical screening. However, further studies are required to optimize the protocol to generate additional renal-specific cell types, interconnected nephron segments and physiologically functional renal tissues.

Thiol-ene cross-linked alginate hydrogel encapsulation modulates the extracellular matrix of kidney organoids by reducing abnormal type 1a1 collagen deposition

Differentiated kidney organoids from induced pluripotent stem cells hold promise as a treatment for patients with kidney diseases. Before these organoids can be translated to the clinic, shortcomings regarding their cellular and extracellular compositions, and their developmental plateau need to be overcome. We performed a proteomic analysis on kidney organoids cultured for a prolonged culture time and we found a specific change in the extracellular matrix composition with increased expression of types 1a1, 2 and 6a1 collagen. Such an excessive accumulation of specific collagen types is a hallmark of renal fibrosis that causes a life-threatening pathological condition by compromising key functions of the human kidney. Here we hypothesized the need for a three-dimensional environment to grow the kidney organoids, which could better mimic the in vivo surroundings of the developing kidney than standard culture on an air–liquid interface. Encapsulating organoids for four days in a soft, thiol-ene cross-linked alginate hydrogel resulted in decreased type 1a1 collagen expression. Furthermore, the encapsulation did not result in any changes of organoid structural morphology. Using a biomaterial to modulate collagen expression allows for a prolonged kidney organoid culture in vitro and a reduction of abnormal type 1a1 collagen expression bringing kidney organoids closer to clinical application.

Enhancing Kidney Vasculature in Tissue Engineering-Current Trends and Approaches: A Review.

Chronic kidney diseases are a leading cause of fatalities around the world. As the most sought-after organ for transplantation, the kidney is of immense importance in the field of tissue engineering. The primary obstacle to the development of clinically relevant tissue engineered kidneys is precise vascularization due to the organ’s large size and complexity. Current attempts at whole-kidney tissue engineering include the repopulation of decellularized kidney extracellular matrices or vascular corrosion casts, but these approaches do not eliminate the need for a donor organ. Stem cell-based approaches, such as kidney organoids vascularized in microphysiological systems, aim to construct a kidney without the need for organ donation. These organ-on-a-chip models show complex, functioning kidney structures, albeit at a small scale. Novel methodologies for developing engineered scaffolds will allow for improved differentiation of kidney stem cells and organoids into larger kidney grafts with clinical applications. While currently, kidney tissue engineering remains mostly limited to individual renal structures or small organoids, further developments in vascularization techniques, with technologies such as organoids in microfluidic systems, could potentially open doors for a large-scale growth of whole engineered kidneys for transplantation.

Generation of patterned kidney organoids that recapitulate the adult kidney collecting duct system from expandable ureteric bud progenitors

Current kidney organoids model development and diseases of the nephron but not the contiguous epithelial network of the kidney’s collecting duct (CD) system. Here, we report the generation of an expandable, 3D branching ureteric bud (UB) organoid culture model that can be derived from primary UB progenitors from mouse and human fetal kidneys, or generated de novo from human pluripotent stem cells. In chemically-defined culture conditions, UB organoids generate CD organoids, with differentiated principal and intercalated cells adopting spatial assemblies reflective of the adult kidney’s collecting system. Aggregating 3D-cultured nephron progenitor cells with UB organoids in vitro results in a reiterative process of branching morphogenesis and nephron induction, similar to kidney development. Applying an efficient gene editing strategy to remove RET activity, we demonstrate genetically modified UB organoids can model congenital anomalies of kidney and urinary tract. Taken together, these platforms will facilitate an enhanced understanding of development, regeneration and diseases of the mammalian collecting duct system.

Detection of Hematopoietic Stem Cell Transcriptome in Human Fetal Kidneys and Kidney Organoids Derived From Human Induced Pluripotent Stem Cells.

BackgroundIn mammalians, hematopoietic stem cells (HSCs) arise in the dorsal aorta from the hemogenic endothelium, followed by their migration to the fetal liver and to the bone marrow. In zebrafish, the kidney is the site of primary hematopoiesis. In humans, the presence of HSCs in the fetal or adult kidney has not been established.MethodsWe analyzed the presence of HSC markers in the human fetal kidneys by analysis of single-cell datasets. We then analyzed in kidney organoids derived from induced pluripotent stem cells (iPSCs) the presence of hematopoietic markers using transcriptome analyses.ResultsTwelve clusters were identified as stromal, endothelial, and nephron cell type-specific markers in the two fetal stage (17 weeks) kidney datasets. Among these, the expression of hematopoietic cells in cluster 9 showed an expression of primitive markers. Moreover, whole transcriptome analysis of our iPSC-derived kidney organoids revealed induction of the primitive hematopoietic transcription factor RUNX1 as found in the human fetal kidney cortex.ConclusionThese finding support the presence of cells expressing HSC transcriptome in the human kidney. The mechanisms of the appearance of the cells with the same transcriptional features during iPSC-derived kidney organoid generation require further investigation.

Defense of COVID-19 by Human Organoids.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has created an immense menace to public health worldwide, exerting huge effects on global economic and political conditions. Understanding the biology and pathogenesis mechanisms of this novel virus, in large parts, relies on optimal physiological models that allow replication and propagation of SARS-CoV-2. Human organoids, derived from stem cells, are three-dimensional cell cultures that recapitulate the cellular organization, transcriptional and epigenetic signatures of their counterpart organs. Recent studies have indicated their great values as experimental virology platforms, making human organoid an ideal tool for investigating host–pathogen interactions. Here, we summarize research developments for SARS-CoV-2 infection of various human organoids involved in multiple systems, including lung, liver, brain, intestine, kidney and blood vessel organoids. These studies help us reveal the pathogenesis mechanism of COVID-19, and facilitate the development of effective vaccines and drugs as well as other therapeutic regimes.

Extracellular Vesicles as a Therapeutic Tool for Kidney Disease: Current Advances and Perspectives.

Extracellular vesicles (EVs) have been described as important mediators of cell communication, regulating several physiological processes, including tissue recovery and regeneration. In the kidneys, EVs derived from stem cells have been shown to support tissue recovery in diverse disease models and have been considered an interesting alternative to cell therapy. For this purpose, however, several challenges remain to be overcome, such as the requirement of a high number of EVs for human therapy and the need for optimization of techniques for their isolation and characterization. Moreover, the kidney’s complexity and the pathological process to be treated require that EVs present a heterogeneous group of molecules to be delivered. In this review, we discuss the recent advances in the use of EVs as a therapeutic tool for kidney diseases. Moreover, we give an overview of the new technologies applied to improve EVs’ efficacy, such as novel methods of EV production and isolation by means of bioreactors and microfluidics, bioengineering the EV content and the use of alternative cell sources, including kidney organoids, to support their transfer to clinical applications.

P157 Microbiome Analysis Reveals That Ralstonia is Responsible for Decreased Renal Function in Patients with Ulcerative Colitis

Abstract Background Inflammatory bowel diseases (IBDs), such as ulcerative colitis (UC), are characterized by a disturbance of the normal gut microbiota and contribute to the development of chronic kidney disease (CKD). The incidence of CKD is higher in individuals with UC, but the causal link is unknown. Therefore, we investigated the role of gut microbiota in decreasing renal function in patients with UC Methods We performed 16S ribosomal DNA sequencing using ileocecal mucosal samples from nine individuals with UC and CKD (UC+CKD), 29 individuals with UC only, and 12 healthy controls. We also analyzed the operational taxonomic units, microbial diversity, and correlation with renal function. Co-culture assay using kidney organoids and Caco-2 cells was performed. Figure 1. The taxonomic composition of the gut microbiota of the study population and comparison of the 10 most abundant genera and species in the UC + CKD, UC, and control groups. Results Bacterial species diversity was significantly decreased in the UC+CKD group compared to that in the other groups based on Shannon and inverse Simpson indexes. At the genus level, Ralstonia had significantly greater abundance in the UC+CKD and UC groups compared to the control group. At the species level, unclassified Ralstonia species and Citrobacter portucalensis showed higher abundance in the UC+CKD group compared to the other groups. The relative abundance of Ralstonia showed a negative correlation with the estimated glomerular filtration rate (eGFR), but a positive correlation with the serum uric acid level. Ralstonia pickettii represented a negative correlation with eGFR, and induced damaging changes to kidney organoids in co-culture assay. Figure 2. The correlation between the relative abundance of Ralstonia with renal function (indicated by eGFR) and serum uric acid level. Effects of Ralstonia pickettii on Caco-2 cells and kidney organoids. (A) mRNA expression of pro-inflammatory cytokine genes in Caco-2 cells treated with and without Ralstonia pickettii, reflecting the inflammation seen in ulcerative colitis. Conclusion Gut microbial community profiles of the UC+CKD group are different from those of the UC group. Furthermore, Ralstonia pickettii contributes in decreasing renal function in patients with UC.

Orphan nuclear receptor COUP-TFII enhances myofibroblast glycolysis leading to kidney fibrosis.

Recent studies demonstrate that metabolic disturbance, such as augmented glycolysis, contributes to fibrosis. The molecular regulation of this metabolic perturbation in fibrosis, however, has been elusive. COUP-TFII (also known as NR2F2) is an important regulator of glucose and lipid metabolism. Its contribution to organ fibrosis is undefined. Here, we found increased COUP-TFII expression in myofibroblasts in human fibrotic kidneys, lungs, kidney organoids, and mouse kidneys after injury. Genetic ablation of COUP-TFII in mice resulted in attenuation of injury-induced kidney fibrosis. A non-biased proteomic study revealed the suppression of fatty acid oxidation and the enhancement of glycolysis pathways in COUP-TFII overexpressing fibroblasts. Overexpression of COUP-TFII in fibroblasts also induced production of alpha-smooth muscle actin (αSMA) and collagen 1. Knockout of COUP-TFII decreased glycolysis and collagen 1 levels in fibroblasts. Chip-qPCR revealed the binding of COUP-TFII on the promoter of PGC1α. Overexpression of COUP-TFII reduced the cellular level of PGC1α. Targeting COUP-TFII serves as a novel treatment approach for mitigating fibrosis in chronic kidney disease and potentially fibrosis in other organs.

Vascular deficiencies in renal organoids and ex vivo kidney organogenesis

Chronic kidney disease (CKD) and end stage renal disease (ESRD) are increasingly frequent and devastating conditions that have driven a surge in the need for kidney transplantation. A stark shortage of organs has fueled interest in generating viable replacement tissues ex vivo for transplantation. One promising approach has been self-organizing organoids, which mimic developmental processes and yield multicellular, organ-specific tissues. However, a recognized roadblock to this approach is that many organoid cell types fail to acquire full maturity and function. Here, we comprehensively assess the vasculature in two distinct kidney organoid models as well as in explanted embryonic kidneys. Using a variety of methods, we show that while organoids can develop a wide range of kidney cell types, as previously shown, endothelial cells (ECs) initially arise but then rapidly regress over time in culture. Vasculature of cultured embryonic kidneys exhibit similar regression. By contrast, engraftment of kidney organoids under the kidney capsule results in the formation of a stable, perfused vasculature that integrates into the organoid. This work demonstrates that kidney organoids offer a promising model system to define the complexities of vascular-nephron interactions, but the establishment and maintenance of a vascular network present unique challenges when grown ex vivo.

Human Amniotic Membrane as a Novel Scaffold for Inducible Pluripotent Stem Cell-derived Kidney Organoids.

Human Amniotic Membrane as a Novel Scaffold for Inducible Pluripotent Stem Cell-derived Kidney Organoids.

Elucidation of a novel mechanism for faulty protein retention and a therapeutic strategy for facilitated lysosomal removal

Intracellular accumulation of misfolded proteins causes toxic proteinopathies, diseases without targeted therapies. Mucin 1 kidney disease results from a frameshift mutation in the MUC1 gene (MUC1-fs). The main goal of this study is to investigate the cellular and molecular mechanism by which misfolded MUC1-fs accumulates and alters epithelial cell function, and to develop a mechanism-based therapy to reverse it. We found that the frameshift mutation results in toxic accumulation of the misfolded protein, through a previously unknown retention mechanism. The mutant protein was shown to accumulate in the early secretory pathway where it is trapped in TMED9 cargo receptor-enriched vesicles between the cis-Golgi and the ER. This accumulation induces ER stress, eventually leading to cell death. We have identified BRD4780, a small molecule that targets TMED9, releases MUC1-fs from early secretory compartments and reroutes it to the lysosome where it is degraded and removed from the cell. The compound cleared the mutant protein from patient cells, kidneys of knock-in mice and patient iPSC-derived kidney organoids. In summary, we have elucidated a novel molecular mechanism responsible for the retention of misfolded proteins, and discovered a promising small molecule as a therapeutic lead for toxic proteinopathies.

A Simplified Method for Generating Kidney Organoids from Human Pluripotent Stem Cells.

Kidney organoids generated from hPSCs have provided an unlimited source of renal tissue. Human kidney organoids are an invaluable tool for studying kidney disease and injury, developing cell-based therapies, and testing new therapeutics. For such applications, large numbers of uniform organoids and highly reproducible assays are needed. We have built upon our previously published kidney organoid protocol to improve the overall health of the organoids. This simple, robust 3D protocol involves the formation of uniform embryoid bodies in minimum component medium containing lipids, insulin-transferrin-selenium-ethanolamine supplement and polyvinyl alcohol with GSK3 inhibitor (CHIR99021) for 3 days, followed by culture in knock-out serum replacement (KOSR)-containing medium. In addition, agitating assays allows for reduction in clumping of the embryoid bodies and maintaining a uniform size, which is important for reducing variability between organoids. Overall, the protocol provides a fast, efficient, and cost-effective method for generating large quantities of kidney organoids.

A Simplified Method for Generating Kidney Organoids from Human Pluripotent Stem Cells

A Simplified Method for Generating Kidney Organoids from Human Pluripotent Stem Cells