Podocytes are highly specialized, terminally differentiated cells in the glomerulus of the kidney and these cells play a central role in blood filtration. In this review, we comprehensively describe the cell biology of podocytes under healthy conditions and in glomerular disorders wherein podocyte injury is a major pathological mechanism. First, the molecular mechanisms that maintain podocyte actin cytoskeleton structure, permanent cell cycle exit, and metabolism under healthy conditions are described. Secondly, the mechanisms of podocyte injury, including genetic alterations and external insults that ultimately disrupt podocyte actin cytoskeleton dynamics or interrupt podocyte quiescence and mitochondrial metabolism are discussed. This understanding forms the basis of described potential therapeutic agents that act by modulating dysregulated podocyte cytoskeleton organization, prevent or reverse cell cycle re-entry, and re-establish normal mitochondrial energy production. Lastly, the application of modern techniques such as single cell RNA sequencing, super resolution microscopy, atomic force microscopy, and glomerular organoids is improving the resolution of mechanistic podocytopathy knowledge. Taken together, our review provides critical insights into the cellular and molecular mechanisms leading to podocyte loss, necessary for the advancement of therapeutic development in glomerular diseases.

Kidney genetic services are being created worldwide, revolutionising the way in which we manage families with suspected monogenic kidney disease. There is potential to learn from one another, whether one is just embarking on this journey or within an established kidney genetics service model with aspirations to optimise it further. This concluding portion of our two-part educational review explores the global efforts to integrate genomics into nephrology. We discuss key considerations for establishing kidney genetics services and share insights from successful implementation in Australia, India, the United Kingdom (UK) and the United States (US), through case studies. Widespread integration of genomics within nephrology still faces barriers including limited genomics education among clinicians, high costs and ethical concerns. Educational strategies including workshop-based, online resources and clinical decision tools are aiming to address the genomic literacy gap among nephrologists. Multidisciplinary kidney genetics clinic models comprising nephrologists, geneticists, clinical scientists and counsellors are proving to be an effective model of delivering this diagnostic tool. Data of how kidney genetics clinics can foster collaboration with registries to facilitate research and shared learning to optimise care for patients are becoming evident. We also explore the importance of equitable access to genomics services across diverse populations, advocating for policies that address disparities in access to healthcare and genetic data representation. We hope to highlight the importance of upskilling the nephrology workforce to fully leverage the advances in genomic medicine and ensure comprehensive, accessible and personalised care for patients with genetic kidney diseases.

The increasing incidence of kidney diseases has highlighted the need for in vitro experimental models to mimic disease development and to test new therapeutic approaches. Traditional two-dimensional in vitro experimental models are not fully able to recapitulate renal diseases. Instead, kidney organoids represent three-dimensional models that better mimic the human organ from both structural and functional points of view. Human pluripotent stem cells (PSCs), both embryonic and induced, are ideal sources for generating renal organoids. These organoids contain all renal cell types and the protocols to differentiate PSCs into renal organoids consist of three different stages that recapitulate embryonic development: mesodermal induction, nephron progenitor formation, and nephron differentiation. Recently it has been establish a renal organoid model where collecting ducts are also present. In this case, the presence of ureteric bud progenitor cells is essential. Renal organoids are particularly useful for studying genetic diseases, by introducing the specific mutations in PSCs by genome editing or generating organoids from patient-derived PSCs. Moreover, renal organoids represent promising models in toxicology studies and testing new therapeutic approaches. Renal organoids can be established also from adult stem cells. This type of organoid, named tubuloid, is composed only of epithelial cells and recapitulates the tissue repair process. The tubuloids can be generated from adult stem or progenitor cells, obtained from renal biopsies or urine, and are promising in vitro models for studying tubular functions, diseases, and regeneration. Tubuloids can be derived from patients and permit the study of genetic diseases, performing personalized drug screening and modeling renal pathologies.

Genetic kidney disease (GKD) significantly affects the community and is responsible for a notable portion of adult kidney disease cases and about half of cases in paediatric patients. It substantially impacts the quality of life and life expectancy for affected children and adults across all stages of kidney disease. Precise genetic diagnosis in GKD promises to improve patient outcomes, provide access to targeted treatments, and reduce the disease burden for individuals, families, and healthcare systems. Genetic investigations are increasingly used in nephrology practice; however, many patients who undergo testing still lack a definitive diagnosis.

The KidGen National Kidney Genomics Study aims to increase diagnostic yield for those with suspected monogenic kidney disease without a diagnosis after standard diagnostic genetic testing. The program will seek to enrol up to 200 families from KidGen Collaborative kidney genetics clinics across Australia who have yet to receive conclusive diagnoses despite prior testing. Participants will undergo a personalised pathway of research genomic investigations. These include re-analysing existing data and/or undergoing advanced genomic testing methods, including short and long-read whole-genome sequencing, RNA sequencing, and functional genomics strategies using mouse modelling or kidney organoids.

The KidGen National Kidney Genomics Study is a coordinated, multidisciplinary extension of previous research projects that aims to assess the diagnostic yield of advanced genomic approaches. The study's evidence will drive changes to current diagnostic pathways, including identifying which chronic kidney disease patients are most likely to benefit from a more comprehensive genomic approach to diagnosis.

Developing disease models and screening for effective drugs are key areas of modern medical research. Traditional methodologies frequently fall short in precisely replicating the intricate architecture of bodily tissues and organs. Nevertheless, recent advancements in biomaterial-assisted organoid technology have ushered in a paradigm shift in biomedical research. This innovative approach enables the cultivation of three-dimensional cellular structures in vitro that closely emulate the structural and functional attributes of organs, offering physiologically superior models compared to conventional techniques. The evolution of biomaterials plays a pivotal role in supporting the culture and development of organ tissues, thereby facilitating more accurate disease state modeling and the rigorous evaluation of drug efficacy and safety profiles. In this review, we will explore the roles that various biomaterials play in organoid development, examine the fundamental principles and advantages of utilizing these technologies in constructing disease models, and highlight recent advances and practical applications in drug screening using disease-specific organoid models. Additionally, the challenges and future directions of organoid technology are discussed. Through continued research and innovation, we aim to make remarkable strides in disease treatment and drug development, ultimately enhancing patient quality of life.

Predicting the risks of progression to chronic kidney disease (CKD) stage 5 in idiopathic nephrotic syndrome (NS) and recurrence of the disease (rNS) following kidney transplantation (KT) is a key assessment to provide essential management information. NS has been categorized etiologically as genetic and immune-based. A genetic cause can be identified in ~ 30% of children with steroid-resistant NS (SRNS), a finding associated with a very low risk of rNS following KT. In immune-based NS, clinical overlap is observed among steroid-sensitive NS, secondary-resistant NS, and SRNS not associated with disease-causing genetic variants (non-monogenic SRNS). While ~ 50% of SRNS patients with no identified monogenic disease respond to intensified immunosuppressive treatments, the ones that do not respond to this therapy have a high risk of progression to CKD stage 5 and post-KT rNS. Secondary-resistant patients who progress to CKD stage 5 display the highest risk of post-KT rNS. The proposed shared underlying mechanism of the immune-based NS associated with post-KT rNS is based on a systemic circulating factor (CF) that affects glomerular permeability by inducing foot process effacement and focal segmental glomerulosclerosis. However, identifying patients without a detected genetic form who will recur post-KT is a major challenge. Extensive efforts, therefore, have been made to identify CFs and biomarkers potentially capable of predicting the risk of progression to CKD stage 5 and post-KT rNS. This review discusses the in vitro and in vivo approaches employed to date to identify and characterize potential CFs and CF-induced biomarkers of recurrent NS and offers an assessment of their potential to improve outcomes of KT in this patient population.

Organoids are three-dimensional (3D) cell cultures derived from human pluripotent stem cells or adult stem cells that recapitulate the cellular heterogeneity, structure, and function of human organs. These microstructures are invaluable for biomedical research due to their ability to closely mimic the complexity of native tissues while retaining human genetic material. This fidelity to native organ systems positions organoids as a powerful tool for advancing our understanding of human biology and for enhancing preclinical drug testing. Recent advancements have led to the successful development of a variety of organoid types, reflecting a broad range of human organs and tissues. This progress has expanded their application across several domains, including regenerative medicine, where organoids offer potential for tissue replacement and repair; disease modeling, which allows for the study of disease mechanisms and progression in a controlled environment; drug discovery and evaluation, where organoids provide a more accurate platform for testing drug efficacy and safety; and microecological research, where they contribute to understanding the interactions between microbes and host tissues. This review provides a comprehensive overview of the historical development of organoid technology, highlights the key achievements and ongoing challenges in the field, and discusses the current and emerging applications of organoids in both laboratory research and clinical practice.

Vascularization plays a critical role in organ maturation and cell-type development. Drug discovery, organ mimicry, and ultimately transplantation hinge on achieving robust vascularization of in vitro engineered organs. Here, focusing on human kidney organoids, we overcame this hurdle by combining a human induced pluripotent stem cell (iPSC) line containing an inducible ETS translocation variant 2 (ETV2) (a transcription factor playing a role in endothelial cell development) that directs endothelial differentiation in vitro, with a non-transgenic iPSC line in suspension organoid culture. The resulting human kidney organoids show extensive endothelialization with a cellular identity most closely related to human kidney endothelia. Endothelialized kidney organoids also show increased maturation of nephron structures, an associated fenestrated endothelium with de novo formation of glomerular and venous subtypes, and the emergence of drug-responsive renin expressing cells. The creation of an engineered vascular niche capable of improving kidney organoid maturation and cell type complexity is a significant step forward in the path to clinical translation. Thus, incorporation of an engineered endothelial niche into a previously published kidney organoid protocol allowed the orthogonal differentiation of endothelial and parenchymal cell types, demonstrating the potential for applicability to other basic and translational organoid studies.

Inherited kidney diseases (IKDs) in children pose unique diagnostic and therapeutic challenges. IKD significantly impact patient quality of life, morbidity, mortality, and cost to the healthcare system. With over 150 genetic abnormalities, they account for approximately 30% of cases requiring renal replacement therapy. There is an urgent need to advance both diagnosis and treatment strategies. In this review, we present recent advances in diagnosis and treatment for facilitating personalized treatment approaches.

The diagnostic landscape for IKDs have evolved significantly, emphasizing precise genetic identification and classification of these disorders. Recent advancements include the refinement of genetic testing techniques, such as whole exome sequencing, which has improved the accuracy of diagnosing specific diseases and facilitated early intervention strategies. Additionally, this review categorizes IKDs based on genetic abnormalities and clinical manifestations, enhancing understanding and management approaches. Finally, it summarizes the corresponding treatment, and lists the application of emerging therapeutic options such as gene therapy and organoids, which show promise in transforming treatment outcomes.

This review summarizes the common types of IKDs in children, including their diagnosis and treatment advances, and provides an update on the status of gene therapy development for these disorders.

Heterozygosity for inverted formin-2 (INF2) mutations causes focal segmental glomerulosclerosis (FSGS) with or without Charcot-Marie-Tooth disease. A key question is whether the disease is caused by gain-of-function effects on INF2 or loss of function (haploinsufficiency). Despite established roles in multiple cellular processes, neither INF2 knockout mice nor mice with a disease-associated point mutation display an evident kidney or neurologic phenotype. Here, we compared responses to puromycin aminonucleoside (PAN)-induced kidney injury between INF2 R218Q and INF2 knockout mice. R218Q INF2 mice are susceptible to glomerular disease, in contrast to INF2 knockout mice. Colocalization, coimmunoprecipitation analyses, and cellular actin measurements showed that INF2 R218Q confers a gain-of-function effect on the actin cytoskeleton. RNA expression analysis showed that adhesion and mitochondria-related pathways were enriched in the PAN-treated R218Q mice. Both podocytes from INF2 R218Q mice and human kidney organoids with an INF2 mutation (S186P) recapitulate adhesion and mitochondrial phenotypes. Thus, gain-of-function mechanisms drive INF2-related FSGS and explain this disease's autosomal dominant inheritance.

Drug-induced nephrotoxicity is a leading cause of acute kidney injury (AKI). A major obstacle in predicting AKI is the lack of a comprehensive experimental model that mimics stable and physiologically relevant kidney functions and accurately reflects the changes a drug induces. Organoids derived from human-induced pluripotent stem cells (iPSCs) are promising models because of their reproducibility and similarity to the in vivo conditions. In this study, Esculentoside A, the triterpene saponin with the highest concentration isolated from the root of Phytolacca acinose Roxb., was used to induce kidney injury models in vivo and kidney organoids. Esculentoside A induced AKI in mice, together with pathological changes and enhanced apoptosis. Moreover, Esculentoside A damaged podocytes and proximal tubular endothelial cells in kidney organoids in a similar way as in vivo. We also found that treatment with 60 μM Esculentoside A induced the known biomarkers of kidney damage and inflammatory cytokines (such as kidney injury molecule (KIM-1), β2-microglobulin (β2-M), and cystatin C (CysC)) in the organoids, in which activation of Cleaved Caspase-3 was involved, possibly due to lowered mitochondrial membrane potential. In summary, this study strongly suggests using kidney organoids as a reliable platform to assess Chinese medicine-induced nephrotoxicity.

Acute kidney injury (AKI) and chronic kidney disease (CKD) are significant public health issues associated with a long-term increase in mortality risk, resulting from various etiologies including renal ischemia, sepsis, drug toxicity, and diabetes mellitus. Numerous preclinical models have been developed to deepen our understanding of the pathophysiological mechanisms and therapeutic approaches for kidney diseases. Among these, rodent models have proven to be powerful tools in the discovery of novel therapeutics, while the development of kidney organoids has emerged as a promising advancement in the field. This review provides a comprehensive analysis of the construction methodologies, underlying biological mechanisms, and recent therapeutic developments across different AKI and CKD models. Additionally, this review summarizes the advantages, limitations, and challenges inherent in these preclinical models, thereby contributing robust evidence to support the development of effective therapeutic strategies.

Over the past decade, the induction protocols for the two types of kidney organoids (nephron organoids and ureteric bud organoids) from pluripotent stem cells (PSCs) have been established based on the knowledge gained in developmental nephrology. Kidney organoids are now used for disease modeling and drug screening, but they also have potential as tools for clinical transplantation therapy. One of the options to achieve this goal would be to assemble multiple renal progenitor cells (nephron progenitor, ureteric bud, stromal progenitor) to reproduce the organotypic kidney structure from PSCs. At least from mouse PSCs, all the three progenitors have been induced and assembled into such "higher order" kidney organoids. We will provide an overview of the developmental nephrology required for the induction of renal progenitors and discuss recent advances and remaining challenges of kidney organoids for clinical transplantation therapy.

Biobanks of human biosamples and cell lines are indispensable for biomedical research on human health and disease and for drug development projects. Many human cell line biobanks worldwide hold collections of lymphoblastoid cell lines (LCLs), representing thousands of affected and control donors from diverse ethnic/ancestry groups. In recent years, induced human pluripotent stem cells (iPSCs) and differentiated human cells derived from these iPSCs have become indispensable for applied biomedical research. Establishing iPSCs remains a laborious and costly step towards generating differentiated human cells. To address this research need, several non-profit and commercial biobanks have established iPSC collections for distribution to researchers, thereby serving as a resource for generating differentiated human cells. The most common starting materials for generation of iPSCs are a skin biopsy for harvesting fibroblasts, or a blood sample for collection of peripheral blood mononuclear cells. However untapped resources include the large established collections of biobanked human LCLs which can be reprogrammed to iPSCs using a variety of published protocols including the use of non-integrating episomal vectors. Many biobanks curate LCLs from diverse ethnic/ancestry populations, an aspect largely absent in most established iPSC biobanks which tend to primarily reflect populations from developed countries. Here, we call upon researchers across the breadth of iPSC research to tap the unique resource of existing and diverse human LCL collections for establishing biobanked iPSC panels that better represent the varied human ethnic (and hence genomic) diversity, thereby benefiting precision medicine and drug development research on a global scale.

Kidney disease is a global health crisis affecting more than 850 million people worldwide. In the United States, annual Medicare expenditures for kidney disease and organ failure exceed $81 billion. Efforts to develop targeted therapeutics are limited by a poor understanding of the molecular mechanisms underlying human kidney disease onset and progression. Additionally, 90% of drug candidates fail in human clinical trials, often due to toxicity and efficacy not accurately predicted in animal models. The advent of ex vivo kidney models, such as those engineered from induced pluripotent stem (iPS) cells and organ-on-a-chip (organ-chip) systems, has garnered considerable interest owing to their ability to more accurately model tissue development and patient-specific responses and drug toxicity. This review describes recent advances in developing kidney organoids and organ-chips by harnessing iPS cell biology to model human-specific kidney functions and disease states. We also discuss challenges that must be overcome to realize the potential of organoids and organ-chips as dynamic and functional conduits of the human kidney. Achieving these technological advances could revolutionize personalized medicine applications and therapeutic discovery for kidney disease.

Inverted formin-2 (INF2) gene mutations are among the most common causes of genetic focal segmental glomerulosclerosis (FSGS) with or without Charcot-Marie-Tooth (CMT) disease. Recent studies suggest that INF2, through its effects on actin and microtubule arrangement, can regulate processes including vesicle trafficking, cell adhesion, mitochondrial calcium uptake, mitochondrial fission, and T-cell polarization. Despite roles for INF2 in multiple cellular processes, neither the human pathogenic R218Q INF2 point mutation nor the INF2 knock-out allele is sufficient to cause disease in mice. This discrepancy challenges our efforts to explain the disease mechanism, as the link between INF2-related processes, podocyte structure, disease inheritance pattern, and their clinical presentation remains enigmatic. Here, we compared the kidney responses to puromycin aminonucleoside (PAN) induced injury between R218Q INF2 point mutant knock-in and INF2 knock-out mouse models and show that R218Q INF2 mice are susceptible to developing proteinuria and FSGS. This contrasts with INF2 knock-out mice, which show only a minimal kidney phenotype. Co-localization and co-immunoprecipitation analysis of wild-type and mutant INF2 coupled with measurements of cellular actin content revealed that the R218Q INF2 point mutation confers a gain-of-function effect by altering the actin cytoskeleton, facilitated in part by alterations in INF2 localization. Differential analysis of RNA expression in PAN-stressed heterozygous R218Q INF2 point-mutant and heterozygous INF2 knock-out mouse glomeruli showed that the adhesion and mitochondria-related pathways were significantly enriched in the disease condition. Mouse podocytes with R218Q INF2, and an INF2-mutant human patient's kidney organoid-derived podocytes with an S186P INF2 mutation, recapitulate the defective adhesion and mitochondria phenotypes. These results link INF2-regulated cellular processes to the onset and progression of glomerular disease. Thus, our data demonstrate that gain-of-function mechanisms drive INF2-related FSGS and explain the autosomal dominant inheritance pattern of this disease.

Human induced pluripotent stem cells (iPSC) have the potential to produce desired target cell types in vitro and allow for the high-throughput screening of drugs/chemicals at population level thereby minimising the cost of drug discovery and drug withdrawals after clinical trials. There is a substantial need for the characterisation of the iPSC derived models to better understand and utilise them for toxicological relevant applications. In our study, iPSC (SBAD2 or SBAD3 lines obtained from StemBANCC project) were differentiated towards toxicologically relevant cell types: alveolar macrophages, brain capillary endothelial cells, brain cells, endothelial cells, hepatocytes, lung airway epithelium, monocytes, podocytes and renal proximal tubular cells. A targeted transcriptomic approach was employed to understand the effects of differentiation protocols on these cell types. Pearson correlation and principal component analysis (PCA) separated most of the intended target cell types and undifferentiated iPSC models as distinct groups with a high correlation among replicates from the same model. Based on PCA, the intended target cell types could also be separated into the three germ layer groups (ectoderm, endoderm and mesoderm). Differential expression analysis (DESeq2) presented the upregulated genes in each intended target cell types that allowed the evaluation of the differentiation to certain degree and the selection of key differentiation markers. In conclusion, these data confirm the versatile use of iPSC differentiated cell types as standardizable and relevant model systems for in vitro toxicology.

Podocytes form the backbone of the glomerular filtration barrier and are exposed to various mechanical forces throughout the lifetime of an individual. The highly dynamic biomechanical environment of the glomerular capillaries greatly influences the cell biology of podocytes and their pathophysiology. Throughout the past two decades, a holistic picture of podocyte cell biology has emerged, highlighting mechanobiological signalling pathways, cytoskeletal dynamics and cellular adhesion as key determinants of biomechanical resilience in podocytes. This biomechanical resilience is essential for the physiological function of podocytes, including the formation and maintenance of the glomerular filtration barrier. Podocytes integrate diverse biomechanical stimuli from their environment and adapt their biophysical properties accordingly. However, perturbations in biomechanical cues or the underlying podocyte mechanobiology can lead to glomerular dysfunction with severe clinical consequences, including proteinuria and glomerulosclerosis. As our mechanistic understanding of podocyte mechanobiology and its role in the pathogenesis of glomerular disease increases, new targets for podocyte-specific therapeutics will emerge. Treating glomerular diseases by targeting podocyte mechanobiology might improve therapeutic precision and efficacy, with potential to reduce the burden of chronic kidney disease on individuals and health-care systems alike.

Induced pluripotent stem (iPS) cell technology has transformed biomedical research. New opportunities now exist to create new organoids, microtissues, and body-on-a-chip systems for basic biology investigations and clinical translations.

We discuss the utility of proteomics for attaining an unbiased view into protein expression changes during iPS cell differentiation, cell maturation, and tissue generation. The ability to discover cell-type specific protein markers during the differentiation and maturation of iPS-derived cells has led to new strategies to improve cell production yield and fidelity. In parallel, proteomic characterization of iPS-derived organoids is helping to realize the goal of bridging in vitro and in vivo systems.

We discuss some current challenges of proteomics in iPS cell research and future directions, including the integration of proteomic and transcriptomic data for systems-level analysis.

Despite advancements in treatment protocols, cancer is one of the leading cause of deaths worldwide. Therefore, there is a need to identify newer and personalized therapeutic targets along with screening technologies to combat cancer. With the advent of pan-omics technologies, such as genomics, transcriptomics, proteomics, metabolomics, and lipidomics, the scientific community has witnessed an improved molecular and metabolomic understanding of various diseases, including cancer. In addition, three-dimensional (3-D) disease models have been efficiently utilized for understanding disease pathophysiology and as screening tools in drug discovery. An integrated approach utilizing pan-omics technologies and 3-D in vitro tumor models has led to improved understanding of the intricate network encompassing various signalling pathways and molecular cross-talk in solid tumors. In the present review, we underscore the current trends in omics technologies and highlight their role in understanding genotypic-phenotypic co-relation in cancer with respect to 3-D in vitro tumor models. We further discuss the challenges associated with omics technologies and provide our outlook on the future applications of these technologies in drug discovery and precision medicine for improved management of cancer.

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.

The endogenous capacity of the kidney to repair is limited, and generation of new nephrons after injury for adequate function recovery remains a need. Discovery of factors that promote the endogenous regenerative capacity of the injured kidney or generation of transplantable kidney tissue represent promising therapeutic strategies. While several encouraging results are obtained after administration of stem or progenitor cells, stem cell secretome, or extracellular vesicles in experimental kidney injury models, very little data exist in the clinical setting to make conclusions about their efficacy. In this review, we provide an overview of the cutting-edge knowledge on kidney regeneration, including pre-clinical methodologies used to elucidate regenerative pathways and describe the perspectives of regenerative medicine for kidney patients.

During embryogenesis, the mammalian kidney arises because of reciprocal interactions between the ureteric bud (UB) and the metanephric mesenchyme (MM), driving UB branching and nephron induction. These morphogenetic processes involve a series of cellular rearrangements that are tightly controlled by gene regulatory networks and signaling cascades. Here, we discuss how kidney developmental studies have informed the definition of procedures to obtain kidney organoids from human pluripotent stem cells (hPSCs). Moreover, bioengineering techniques have emerged as potential solutions to externally impose controlled microenvironments for organoid generation from hPSCs. Next, we summarize some of these advances with major focus On recent works merging hPSC-derived kidney organoids (hPSC-kidney organoids) with organ-on-chip to develop robust models for drug discovery and disease modeling applications. We foresee that, in the near future, coupling of different organoid models through bioengineering approaches will help advancing to recreate organ-to-organ crosstalk to increase our understanding on kidney disease progression in the human context and search for new therapeutics.

Podocytes are of key interest for the prediction of nephrotoxicity as they are especially sensitive to toxic insults due to their central role in the glomerular filtration apparatus. However, currently, prediction of nephrotoxicity in humans remains insufficiently reliable, thus highlighting the need for advanced in vitro model systems using human cells with improved prediction capacity. Recent approaches for refining in vitro model systems focus on closely replicating physiological conditions as observed under the in vivo situation typical of the respective nephron section of interest. PODO/TERT256, a human immortalized podocyte cell line, were employed in a semi-static transwell system to evaluate its potential use as a human podocyte in vitro system for modelling potential human glomerular toxicity. Furthermore, the impact of routinely employed excessive oxygen tension (21 % - AtmOx), when compared to the physiological oxygen tensions (10 % - PhysOx) observed in vivo, was analyzed. Generally, cultured PODO/TERT256 formed a stable, contact-inhibited monolayer with typical podocyte morphology (large cell body, apical microvilli, finger-like cytoplasmic projections (reminiscent of foot processes), and interdigitating cell-cell junctions) and developed a size-selective filtration barrier. PhysOx, however, induced a more pronounced in vivo like phenotype, comprised of significantly larger cell bodies, significantly enhanced filtration barrier size-selectivity, and a remarkable re-localization of nephrin to the cell membrane, thus suggesting an improved in vitro replication of in vivo characteristics. Preliminary toxicity characterization with the known glomerulotoxin doxorubicin (DOX) suggested an increasing change in filtration permeability, already at the lowest DOX concentrations tested (0.01 μM) under PhysOx, whereas obvious changes under AtmOx were observed as of 0.16 μM and higher with a near all or nothing effect. The latter findings suggested that PODO/TERT256 could serve as an in vitro human podocyte model for studying glomerulotoxicity, whereby culturing at PhyOx tension appeared critical for an improved in vivo-like phenotype and functionality. Moreover, PODO/TERT256 could be incorporated into advanced human glomerulus systems in vitro, recapitulating microfluidic conditions and multiple cell types (endothelial and mesenchymal cells) that can even better predict human glomerular toxicity.

Nephrotoxicity is a significant concern during the development of new drugs or when assessing the safety of chemicals in consumer products. Traditional methods for testing nephrotoxicity involve animal models or 2D in vitro cell cultures, the latter of which lack the complexity and functionality of the human kidney. 3D in vitro models are created by culturing human primary kidney cells derived from urine in a 3D microenvironment that mimics the fluid shear stresses of the kidney. Thus, 3D in vitro models provide more accurate and reliable predictions of human nephrotoxicity compared to existing 2D models. In this review, we focus on precision nephrotoxicity testing using 3D in vitro models with human autologous urine-derived kidney cells as a promising approach for evaluating drug safety.

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.

With the advent of induced pluripotent stem cells and modern differentiation protocols, many advances in our understanding of disease have been made possible by in vitro disease modeling; in some cases, their use may have supplanted animal models. Yet in vitro models often rely on rigid cell culture substrates that could limit our ability to completely reproduce human disease in a dish. Nascent work, however, suggests that the combination of biomaterials and/or advanced microphysiological systems-which better recapitulate tissue properties-with stem cells expressing disease mimicking genetics, could substantially improve current disease modeling efforts where genetics alone is insufficient. This review will highlight such recent advances as well as review current challenges that the fields must overcome to create more personalized therapeutics in the future.

Recently, organoids have emerged as revolutionizing tools with the unprecedented potential to recreate organ-specific microanatomy in vitro. Upon their derivation from human pluripotent stem cells (hPSCs), organoids reveal the blueprints of human organogenesis, further allowing the faithful recapitulation of their physiology. Nevertheless, along with the evolution of this field, advanced research exposed the organoids' shortcomings, particularly regarding poor reproducibility rates and overall immatureness. To resolve these challenges, many studies have started to underscore the relevance of mechanical cues as a relevant source to induce and externally control hPSCs differentiation. Indeed, established organoid generation protocols from hPSCs have mainly relyed on the biochemical induction of fundamental signalling pathways present during kidney formation in mammals, whereas mechanical cues have largely been unexplored. This review aims to discuss the pertinence of (bio) physical cues within hPSCs-derived organoid cultures, while deciphering their effect on morphogenesis. Moreover, we will explore state-of-the-art mechanobiology techniques as revolutionizing means for understanding the underlying role of mechanical forces in biological processes in organoid model systems.

Multicellular spheroids, which mimic the natural organ counterparts, allow the prospect of drug screening and regenerative medicine. However, their application is hampered by low processing efficiency or limited scale. This study introduces an efficient method to drive rapid multicellular spheroid formation by a cellulose nanofibril matrix. This matrix enables the facilitated growth of spheroids (within 48 h) through multiple cell assembly into size-controllable aggregates with well-organized physiological microstructure. The efficiency, dimension, and conformation of the as-formed spheroids depend on the concentration of extracellular nanofibrils, the number of assembled cells, and the heterogeneity of cell types. The above strategy allows the robust formation mechanism of compacted tumoroids and hepatocyte spheroids.

HNF4 genes promote proximal tubule differentiation in mice, but their function in human nephrogenesis is not fully defined. This study uses human pluripotent stem cell (PSC)-derived kidney organoids as a model to investigate HNF4A and HNF4G functions. The loss of HNF4A , but not HNF4G , impaired reabsorption-related molecule expression and microvilli formation in human proximal tubules. Cleavage under targets and release using nuclease (CUT&RUN) sequencing and CRISPR-mediated transcriptional activation (CRISPRa) further confirm that HNF4A directly regulates its target genes. Human kidney organoids provide a good model for studying transcriptional regulation in human kidney development.

The proximal tubule plays a major role in electrolyte homeostasis. Previous studies have shown that HNF4A regulates reabsorption-related genes and promotes proximal tubule differentiation during murine kidney development. However, the functions and gene regulatory mechanisms of HNF4 family genes in human nephrogenesis have not yet been investigated.

We generated HNF4A -knock out (KO), HNF4G -KO, and HNF4A/4G -double KO human pluripotent stem cell lines, differentiated each into kidney organoids, and used immunofluorescence analysis, electron microscopy, and RNA-seq to analyze them. We probed HNF4A-binding sites genome-wide by cleavage under targets and release using nuclease sequencing in both human adult kidneys and kidney organoid-derived proximal tubular cells. Clustered Regularly Interspaced Short Palindromic Repeats-mediated transcriptional activation validated HNF4A and HNF4G function in proximal tubules during kidney organoid differentiation.

Organoids lacking HNF4A , but not HNF4G , showed reduced expression of transport-related, endocytosis-related, and brush border-related genes, as well as disorganized brush border structure in the apical lumen of the organoid proximal tubule. Cleavage under targets and release using nuclease revealed that HNF4A primarily bound promoters and enhancers of genes that were downregulated in HNF4A -KO, suggesting direct regulation. Induced expression of HNF4A or HNF4G by CRISPR-mediated transcriptional activation drove increased expression of selected target genes during kidney organoid differentiation.

This study reveals regulatory mechanisms of HNF4A and HNF4G during human proximal tubule differentiation. The experimental strategy can be applied more broadly to investigate transcriptional regulation in human kidney development.

Brain organoids have been considered as an advanced platform for in vitro disease modeling and drug screening, but numerous roadblocks exist, such as lack of large-scale production technology and lengthy protocols with multiple manipulation steps, impeding the industrial translation of brain organoid technology. Here, we describe the high-speed and large-scale production of midbrain organoids using a high-throughput screening-compatible platform within 30 days. Micro midbrain organoids (µMOs) exhibit a highly uniform morphology and gene expression pattern with minimal variability. Notably, µMOs show dramatically accelerated maturation, resulting in the generation of functional µMOs within only 30 days of differentiation. Furthermore, individual µMOs display highly consistent responsiveness to neurotoxin, suggesting their usefulness as an in vitro high-throughput drug toxicity screening platform. Collectively, our data indicate that µMO technology could represent an advanced and robust platform for in vitro disease modeling and drug screening for human neuronal diseases.

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.

Kidney organoids are a promising model to study kidney disease, but their use is constrained by limited knowledge of their functional protein expression profile. Here, we define the organoid proteome and transcriptome trajectories over culture duration and upon exposure to TNFα, a cytokine stressor. Older organoids increase deposition of extracellular matrix but decrease expression of glomerular proteins. Single cell transcriptome integration reveals that most proteome changes localize to podocytes, tubular and stromal cells. TNFα treatment of organoids results in 322 differentially expressed proteins, including cytokines and complement components. Transcript expression of these 322 proteins is significantly higher in individuals with poorer clinical outcomes in proteinuric kidney disease. Key TNFα-associated protein (C3 and VCAM1) expression is increased in both human tubular and organoid kidney cell populations, highlighting the potential for organoids to advance biomarker development. By integrating kidney organoid omic layers, incorporating a disease-relevant cytokine stressor and comparing with human data, we provide crucial evidence for the functional relevance of the kidney organoid model to human kidney disease.

Organoids are established through in vitro 3D culture, and they can mimic the structure and physiological functions of organs or tissues in vivo. Organoids have attracted much attention in recent years. They can provide a reliable technology platform for cancer research and treatment and are a valuable preclinical model for academic research and personalized medicine. A number of studies have confirmed that organoids have great application prospects in new drug development, drug screening, tumour mechanism research, and precision medicine. In this review, we mainly focus on recent advances in the application of organoids in cancer research. We also discussed the opportunities and challenges facing organoids, hoping to indicate directions for the development of organoids in the future.

Kidney organoids have enabled modeling of human development and disease. While methods of generating the nephron lineage are well established, new protocols to induce another lineage, the ureteric bud/collecting duct, have been reported in the past 5 years. Many reports have described modeling of various hereditary kidney diseases, with polycystic kidney disease serving as the archetypal disease, by using patient-derived or genome-edited kidney organoids. The generation of more organotypic kidneys is also becoming feasible. In this review, I also discuss the significant challenges for more sophisticated disease modeling and for realizing the ambitious goal of generating transplantable synthetic kidneys.

Recreating human tissues and organs in the petri dish to establish models as tools in biomedical sciences has gained momentum. These models can provide insight into mechanisms of human physiology, disease onset, and progression, and improve drug target validation, as well as the development of new medical therapeutics. Transformative materials play an important role in this evolution, as they can be programmed to direct cell behavior and fate by controlling the activity of bioactive molecules and material properties. Using nature as an inspiration, scientists are creating materials that incorporate specific biological processes observed during human organogenesis and tissue regeneration. This article presents the reader with state-of-the-art developments in the field of in vitro tissue engineering and the challenges related to the design, production, and translation of these transformative materials. Advances regarding (stem) cell sources, expansion, and differentiation, and how novel responsive materials, automated and large-scale fabrication processes, culture conditions, in situ monitoring systems, and computer simulations are required to create functional human tissue models that are relevant and efficient for drug discovery, are described. This paper illustrates how these different technologies need to converge to generate in vitro life-like human tissue models that provide a platform to answer health-based scientific questions.