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With the prevalence of end-stage renal disease rising 8% per annum globally, there is an urgent need for renal regenerative strategies. The kidney is a mesodermal organ that differentiates from the intermediate mesoderm (IM) through the formation of a ureteric bud (UB) and the interaction between this bud and the adjacent IM-derived metanephric mesenchyme (MM). The nephrons arise from a nephron progenitor population derived from the MM (ref. ). The IM itself is derived from the posterior primitive streak. Although the developmental origin of the kidney is well understood, nephron formation in the human kidney is completed before birth. Hence, there is no postnatal stem cell able to replace lost nephrons. In this study, we have successfully directed the differentiation of human embryonic stem cells (hESCs) through posterior primitive streak and IM under fully chemically defined monolayer culture conditions using growth factors used during normal embryogenesis. This differentiation protocol results in the synchronous induction of UB and MM that forms a self-organizing structure, including nephron formation, in vitro. Such hESC-derived components show broad renal potential ex vivo, illustrating the potential for pluripotent-stem-cell-based renal regeneration. [ABSTRACT FROM AUTHOR]

While epidemiological and experimental studies have demonstrated kidney-protective effects of estrogen and female sex in adulthood, some epidemiological data showed deterioration of kidney function during puberty when estrogen production increases. However, molecular mechanisms explaining these conflicting phenomena remain unknown. Here, we showed that the pubertal sex hormone surge in female mice increases susceptibility to kidney ischemia reperfusion injury partly via downregulation of insulin-like growth factor 1 receptor (IGF-1R) expression in proximal tubules. Adult mice ovariectomized pre-pubertally (at postnatal day 21) showed strong tolerance to kidney ischemia, which was partly reversed by the administration of 17β-estradiol, while adult mice ovariectomized post-pubertally (at 8 weeks of age) were vulnerable to kidney ischemia. Kidney tubular IGF-1R protein expression decreased during postnatal growth but was highly expressed in adult mice ovariectomized pre-pubertally and in infant mice, which might be partly explained by different expression of an E3 ligase (MDM2) of IGF-1R. Mice deficient of Igf-1r in proximal tubules (iIGF-1RKO mice) during postnatal kidney growth showed increased susceptibility to ischemic injury. RNA-seq and western blotting analysis using proximal tubular cells from pre-pubertally ovariectomized iIGF-1RKO and control mice revealed altered expression of cell cycle-associated molecules such as cyclin D1. These results suggest that Igf-1r deletion during postnatal growth renders proximal tubular cells susceptible to ischemia possibly via altered cell cycle regulation. Thus, our findings provide evidence that exposure to pubertal sex hormones leads to increased susceptibility to kidney ischemia, which is partly mediated by modulation of IGF-1R signaling., (Copyright © 2024 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Alport syndrome (AS) is a hereditary disease caused by mutations in the COL4A5 gene and leads to chronic kidney disease. Currently, no specific treatment has been developed. However, a recent study using AS-model mice demonstrated that the exon skipping method could partially rescue the symptoms. In this study, we evaluated the effects of the exon skipping method using kidney organoids generated from AS-patient-derived induced pluripotent stem cells (AS-iPSCs). We generated kidney organoids from AS-iPSCs, which exhibited nephron structures. As expected, the C-terminus of COL4A5 was not expressed in AS-organoids. Interestingly, anti-sense oligonucleotides restored the expression of the C-terminus of COL4A5 in vitro. Next, we transplanted AS-organoids into mice and evaluated glomerular basement membrane formation in vivo. We found that AS-organoids formed a lower slit diaphragm ratio compared to control organoids. Finally, we assessed the effects of exon skipping on transplanted organoids but observed minimum effects. These studies suggest that AS-iPSCs can generate kidney organoids lacking the C-terminus of COL4A5, and that exon skipping can induce its expression in vitro., (© 2024 The Author(s). Genes to Cells published by Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

Renal transporters play critical roles in predicting potential drug-drug interactions. However, current in vitro models often fail to adequately express these transporters, particularly solute carrier proteins, including organic anion transporters (OAT1/3), and organic cation transporter 2 (OCT2). Here, we developed a hiPSC-derived kidney organoids-based proximal tubule-on-chip (OPTC) model that emulates in vivo renal physiology to assess transporter function. Compared to chips based on immortalized cells, OPTC derived from the two most commonly used differentiation protocols exhibited significant improvement in expression level and polarity of OAT1/3 and OCT2. Hence, the OPTC demonstrates enhanced functionality in efflux and uptake assessments, and nephrotoxicity. Furthermore, these functionalities are diminished upon adding inhibitors during substrate-inhibitor interactions, which were closer to in vivo observations. Overall, these results support that OPTC can reliably assess the role of renal transporters in drug transport and nephrotoxicity, paving the way for personalized models to assess renal transport and disease modeling., Competing Interests: R.Y., C.M., R.B.S., K.F., T.A., M.T., and R.N. are inventors on JP patent application no. 2024–73489 “Construction of proximal tubules micro-physiological system”., (© 2024 The Author(s).)

In humans, uric acid is an end-product of purine metabolism. Urate excretion from the human kidney is tightly regulated by reabsorption and secretion. At least eleven genes have been identified as human renal urate transporters. However, it remains unclear whether all renal tubular cells express the same set of urate transporters. Here, we show renal tubular cells are divided into three distinct cell populations for urate handling. Analysis of healthy human kidneys at single-cell resolution revealed that not all tubular cells expressed the same set of urate transporters. Only 32% of tubular cells were related to both reabsorption and secretion, while the remaining tubular cells were related to either reabsorption or secretion at 5% and 63%, respectively. These results provide physiological insight into the molecular function of the transporters and renal urate handling on single-cell units. Our findings suggest that three different cell populations cooperate to regulate urate excretion from the human kidney, and our proposed framework is a step forward in broadening the view from the molecular to the cellular level of transport capacity., (© 2023. The Author(s).)

The prostate is a male reproductive gland which secretes prostatic fluid that enhances male fertility. During development and instigated by fetal testosterone, prostate cells arise caudal to the bladder at the urogenital sinus (UGS), when the urogenital mesenchyme (UGM) secretes signals to the urogenital epithelium (UGE). These initial mesenchymal signals induce prostate-specific gene expression in the UGE, after which epithelial progenitor cells form prostatic buds. Although many important factors for prostate development have been described using UGS organ cultures, those necessary and sufficient for prostate budding have not been clearly identified. This has been in part due to the difficulty to dissect the intricate signaling and feedback between epithelial and mesenchymal UGS cells. In this study, we separated the UGM from the UGE and tested candidate growth factors to show that when FGF10 is present, testosterone is not required for initiating prostate budding from the UGE. Moreover, in the presence of low levels of FGF10, canonical WNT signaling enhances the expression of several prostate progenitor markers in the UGE before budding of the prostate occurs. At the later budding stage, higher levels of FGF10 are required to increase budding and retinoic acid is indispensable for the upregulation of prostate-specific genes. Lastly, we show that under optimized conditions, female UGE can be instructed towards a prostatic fate, and in vitro generated prostate buds from male UGE can differentiate into a mature prostate epithelium after in vivo transplantation. Taken together, our results clarify the signals that can induce fetal prostate buds in the urogenital epithelium in the absence of the surrounding, instructive mesenchyme., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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., (© 2023. The Author(s).)

The neural crest (NC) is an important multipotent embryonic cell population and its impaired specification leads to various developmental defects, often in an anteroposterior (A-P) axial level-specific manner. The mechanisms underlying the correct A-P regionalisation of human NC cells remain elusive. Recent studies have indicated that trunk NC cells, the presumed precursors of childhood tumour neuroblastoma, are derived from neuromesodermal-potent progenitors of the postcranial body. Here we employ human embryonic stem cell differentiation to define how neuromesodermal progenitor (NMP)-derived NC cells acquire a posterior axial identity. We show that TBXT, a pro-mesodermal transcription factor, mediates early posterior NC/spinal cord regionalisation together with WNT signalling effectors. This occurs by TBXT-driven chromatin remodelling via its binding in key enhancers within HOX gene clusters and other posterior regulator-associated loci. This initial posteriorisation event is succeeded by a second phase of trunk HOX gene control that marks the differentiation of NMPs toward their TBXT-negative NC/spinal cord derivatives and relies predominantly on FGF signalling. Our work reveals a previously unknown role of TBXT in influencing posterior NC fate and points to the existence of temporally discrete, cell type-dependent modes of posterior axial identity control., Competing Interests: AG, CS, IG, FW, IM, MW, TF, MG, AB, CB, FH, MT, MG, AT No competing interests declared, (© 2022, Gogolou et al.)

Organoids are being widely introduced as novel research models in multiple research fields. Human-induced pluripotent stem cells-derived kidney organoids became an indispensable tool to study human kidney development, model various diseases and infections leading to kidney damage, and offer a new route towards better drug development and validation, personalized drug screening, and regenerative medicine. In this review, we provide an update of the most recent developments in kidney organoid induction: their main goals, advantages, and shortcomings. We further discuss their current applications in providing modeling and treatment avenues to various kidney injuries, their use in genome-wide screening of kidney diseases, and the cell interactions occurring in these kidney structures., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Regenerative medicine relies on basic research outcomes that are only practical when cost effective. The human eyeball requires the retinal pigment epithelium (RPE) to interface the neural retina and the choroid at large. Millions of people suffer from age-related macular degeneration (AMD), a blinding multifactor genetic disease among RPE degradation pathologies. Recently, autologous pluripotent stem-cell-derived RPE cells were prohibitively expensive due to time; therefore, we developed a faster reprogramming system. We stably induced RPE-like cells (iRPE) from human fibroblasts (Fibs) by conditional overexpression of both broad plasticity and lineage-specific transcription factors (TFs). iRPE cells displayed critical RPE benchmarks and significant in vivo integration in transplanted retinas. Herein, we detail the iRPE system with comprehensive single-cell RNA sequencing (scRNA-seq) profiling to interpret and characterize its best cells. We anticipate that our system may enable robust retinal cell induction for basic research and affordable autologous human RPE tissue for regenerative cell therapy., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

An organoid, a self-organizing organ-like tissue developed from stem cells, can exhibit a miniaturized three-dimensional (3D) structure and part of the physiological functions of the original organ. Due to the reproducibility of tissue complexity and ease of handling, organoids have replaced real organs and animals for a variety of uses, such as investigations of the mechanisms of organogenesis and disease onset, and screening of drug effects and/or toxicity. The recent advent of tissue clearing and 3D imaging techniques have great potential contributions to organoid studies by allowing the collection and analysis of 3D images of whole organoids with a reasonable throughput and thus can expand the means of examining the 3D architecture, cellular components, and variability among organoids. Genetic and histological cell-labeling methods, together with organoid clearing, also allow visualization of critical structures and cellular components within organoids. The collected 3D data may enable image analysis to quantitatively assess structures within organoids and sensitively/effectively detect abnormalities caused by perturbations. These capabilities of tissue/organoid clearing and 3D imaging techniques not only extend the utility of organoids in basic biology but can also be applied for quality control of clinical organoid production and large-scale drug screening., Competing Interests: RIKEN and CUBICStars Co. have filed patents regarding this work, in which ES. was a co-inventor. ES was also a senior researcher employed by CUBICStars Co. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Susaki and Takasato.)

Recently, a new wave of synthetic embryo systems (SESs) has been established from cultured cells for efficient and ethical embryonic development research. We recently reported our epiblast stem cell (EPISC) reprogramming SES that generates numerous blastocyst (BC)-like hemispheres (BCLH) with pluripotent and extraembryonic cell features detected by microscopy. Here, we further explored the system over key time points with single-cell RNA-sequencing analysis. We found broad induction of the 2C-like reporter MERVL and RNA velocities diverging to three major cell populations with gene expression profiles resembling those of pluripotent epiblast, primitive endoderm, and trophectoderm. Enrichment of those three induced BC-like cell fates involved key gene-regulatory networks, zygotic genome activation-related genes, and specific RNA splicing, and many cells closely resembled in silico models. This analysis confirms the induction of extraembryonic cell populations during EPISC reprogramming. We anticipate that our unique BCLH SES and rich dataset may uncover new facets of cell potency, improve developmental biology, and advance biomedicine., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The excretion and reabsorption of uric acid both to and from urine are tightly regulated by uric acid transporters. Metabolic syndrome conditions, such as obesity, hypercholesterolemia, and insulin resistance, are believed to regulate the expression of uric acid transporters and decrease the excretion of uric acid. However, the mechanisms driving cholesterol impacts on uric acid transporters have been unknown. Here, we show that cholesterol metabolite 27-hydroxycholesterol (27HC) upregulates the uric acid reabsorption transporter URAT1 encoded by SLC22A12 via estrogen receptors (ER). Transcriptional motif analysis showed that the SLC22A12 gene promoter has more estrogen response elements (EREs) than other uric acid reabsorption transporters such as SLC22A11 and SLC22A13, and 27HC-activated SLC22A12 gene promoter via ER through EREs. Furthermore, 27HC increased SLC22A12 gene expression in human kidney organoids. Our results suggest that in hypercholesterolemic conditions, elevated levels of 27HC derived from cholesterol induce URAT1/SLC22A12 expression to increase uric acid reabsorption, and thereby, could increase serum uric acid levels., (© 2020 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Alport syndrome (AS) is a progressive inherited kidney disease characterized by hearing loss and ocular abnormalities. There are three forms of AS depending on inheritance mode: X-linked Alport syndrome (XLAS), autosomal recessive AS (ARAS), and autosomal dominant AS (ADAS). XLAS is caused by pathogenic variants in COL4A5, which encodes type IV collagen α5 chain, while ADAS and ARAS are caused by variants in COL4A3 or COL4A4, which encode type IV collagen α3 or α4 chain, respectively. In male XLAS or ARAS cases, end-stage kidney disease (ESKD) develops around a median age of 20 to 30 years old, while female XLAS or ADAS cases develop ESKD around a median age of 60 to 70 years old. The diagnosis of AS is dependent on either genetic or pathological findings. However, determining the pathogenicity of the variants detected by gene tests can be difficult. Recently, we applied the following molecular investigation tools to determine pathogenicity: 1) in silico and in vitro trimer formation assay of α345 chains to assess triple helix formation ability, 2) kidney organoids constructed from patients' induced pluripotent stem cells to identify α5 chain expression on the glomerular basement membrane, and 3) in vitro splicing assay to detect aberrant splicing to determine the pathogenicity of variants. In this review article, we discuss the genetic background and novel assays for determining the pathogenicity of variants. We also discuss the current treatment approaches and introduce exon skipping therapy as one potential treatment option.

Currently an in vitro model that fully recapitulates the human embryonic gonad is lacking. Here we describe a fully defined feeder-free protocol to generate early testis-like cells with the ability to be cultured as an organoid, from human induced pluripotent stem cells. This stepwise approach uses small molecules to mimic embryonic development, with upregulation of bipotential gonad markers (LHX9, EMX2, GATA4, and WT1) at day 10 of culture, followed by induction of testis Sertoli cell markers (SOX9, WT1, and AMH) by day 15. Aggregation into 3D structures and extended culture on Transwell filters yielded organoids with defined tissue structures and distinct Sertoli cell marker expression. These studies provide insight into human gonadal development, suggesting that a population of precursor cells may originate from a more lateral region of the mesoderm. Our protocol represents a significant advance toward generating a much-needed human gonad organoid for studying disorders/differences of sex development., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Currently, there are no treatments for Alport syndrome, which is the second most commonly inherited kidney disease. Here we report the development of an exon-skipping therapy using an antisense-oligonucleotide (ASO) for severe male X-linked Alport syndrome (XLAS). We targeted truncating variants in exon 21 of the COL4A5 gene and conducted a type IV collagen α3/α4/α5 chain triple helix formation assay, and in vitro and in vivo treatment efficacy evaluation. We show that exon skipping enabled trimer formation, leading to remarkable clinical and pathological improvements including expression of the α5 chain on glomerular and the tubular basement membrane. In addition, the survival period was clearly prolonged in the ASO treated mice group. This data suggests that exon skipping may represent a promising therapeutic approach for treating severe male XLAS cases.

Juvenile nephronophthisis is an inherited renal ciliopathy, causing cystic kidney disease, renal fibrosis, and end-stage renal failure. Human induced pluripotent stem cell (hiPSC) lines, derived from two Juvenile nephronophthisis patients, were generated from peripheral blood mononuclear cells by episomal plasmid vectors. Generated hiPSC lines showed self-renewal and pluripotency and carried a large deletion in NPHP1 (Nephrocystin 1) gene. Since the molecular pathogenesis caused by NPHP1 dysfunction remains unclear, these cell resources provide useful tools to establish disease models and to develop new therapies for juvenile nephronophthisis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interestsor personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

The utility of human pluripotent stem cell-derived kidney organoids relies implicitly on the robustness and transferability of the protocol. Here we analyze the sources of transcriptional variation in a specific kidney organoid protocol. Although individual organoids within a differentiation batch showed strong transcriptional correlation, we noted significant variation between experimental batches, particularly in genes associated with temporal maturation. Single-cell profiling revealed shifts in nephron patterning and proportions of component cells. Distinct induced pluripotent stem cell clones showed congruent transcriptional programs, with interexperimental and interclonal variation also strongly associated with nephron patterning. Epithelial cells isolated from organoids aligned with total organoids at the same day of differentiation, again implicating relative maturation as a confounder. This understanding of experimental variation facilitated an optimized analysis of organoid-based disease modeling, thereby increasing the utility of kidney organoids for personalized medicine and functional genomics.

Human pluripotent stem cell (hPSC)-derived kidney organoids may facilitate disease modeling and the generation of tissue for renal replacement. Long-term application, however, will require transferability between hPSC lines and significant improvements in organ maturation. A key question is whether time or a patent vasculature is required for ongoing morphogenesis. Here, we show that hPSC-derived kidney organoids, derived in fully defined medium conditions and in the absence of any exogenous vascular endothelial growth factor, develop host-derived vascularization. In vivo imaging of organoids under the kidney capsule confirms functional glomerular perfusion as well as connection to pre-existing vascular networks in the organoids. Wide-field electron microscopy demonstrates that transplantation results in formation of a glomerular basement membrane, fenestrated endothelial cells, and podocyte foot processes. Furthermore, compared with non-transplanted organoids, polarization and segmental specialization of tubular epithelium are observed. These data demonstrate that functional vascularization is required for progressive morphogenesis of human kidney organoids., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

An organoid can be defined as a three-dimensional organ-like structure formed from organ-specific progenitor cells. Organ progenitor cells were empirically found to self-organize three-dimensional tissues when they were aggregated and cultivated in vitro. While this nature power of progenitor cells has an amazing potential to recreate artificial organs in vitro, there had been difficulty to apply this technology to human organs due to the inaccessibility to human progenitor cells until human-induced pluripotent stem cell (hiPSC) was invented by Takahashi and Yamanaka in 2007. As embryonic stem cells do, hiPSCs also have pluripotency to give rise to any organs/tissues cell types, including the kidney, via directed differentiation. Here, we provide a detailed protocol for generating kidney organoids using human pluripotent stem cells. The protocol differentiates human pluripotent stem cells into the posterior primitive streak. This is followed by the simultaneous induction of posterior and anterior intermediate mesoderm that are subsequently aggregated and undergo self-organization into the kidney organoid. Such kidney organoids are comprised of all anticipated kidney cell types including nephrons segmented into the glomerulus, proximal tubule, loop of Henle, and distal tubule as well as the collecting duct, endothelial network, and renal interstitium.

Directed differentiation of human pluripotent stem cells (hPSCs) can provide us any required tissue/cell types by recapitulating the development in vitro. The kidney is one of the most challenging organs to generate from hPSCs as the kidney progenitors are composed of at least 4 different cell types, including nephron, collecting duct, endothelial and interstitium progenitors, that are developmentally distinguished populations. Although the actual developmental process of the kidney during human embryogenesis has not been clarified yet, studies using model animals accumulated knowledge about the origins of kidney progenitors. The implications of these findings for the directed differentiation of hPSCs into the kidney include the mechanism of the intermediate mesoderm specification and its patterning along with anteroposterior axis. Using this knowledge, we previously reported successful generation of hPSCs-derived kidney organoids that contained all renal components and modelled human kidney development in vitro. In this review, we explain the developmental basis of the strategy behind this differentiation protocol and compare strategies of studies that also recently reported the induction of kidney cells from hPSCs. We also discuss the characterization of such kidney organoids and limitations and future applications of this technology., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The treatment of renal failure has seen little change in the past 70 years. Patients with end-stage renal disease (ESRD) are treated with renal replacement therapy, including dialysis or organ transplantation. The growing imbalance between the availability of donor organs and prevalence of ESRD is pushing an increasing number of patients to undergo dialysis. Although the prospect of new treatment options for patients through regenerative medicine has long been suggested, advances in the generation of human kidney cell types through the directed differentiation of human pluripotent stem cells over the past 2 years have brought this prospect closer to delivery. These advances are the result of careful research into mammalian embryogenesis. By understanding the decision points made within the embryo to pattern the kidney, it is now possible to recreate self-organizing kidney tissues in vitro. In this Review, we describe the key decision points in kidney development and how these decisions have been mimicked experimentally. Recreation of human nephrons from human pluripotent stem cells opens the door to patient-derived disease models and personalized drug and toxicity screening. In the long term, we hope that these efforts will also result in the generation of bioengineered organs for the treatment of kidney disease.

The human kidney develops from four progenitor populations-nephron progenitors, ureteric epithelial progenitors, renal interstitial progenitors and endothelial progenitors-resulting in the formation of maximally 2 million nephrons. Until recently, the reported methods differentiated human pluripotent stem cells (hPSCs) into either nephron progenitor or ureteric epithelial progenitor cells, consequently forming only nephrons or collecting ducts, respectively. Here we detail a protocol that simultaneously induces all four progenitors to generate kidney organoids within which segmented nephrons are connected to collecting ducts and surrounded by renal interstitial cells and an endothelial network. As evidence of functional maturity, proximal tubules within organoids display megalin-mediated and cubilin-mediated endocytosis, and they respond to a nephrotoxicant to undergo apoptosis. This protocol consists of 7 d of monolayer culture for intermediate mesoderm induction, followed by 18 d of 3D culture to facilitate self-organizing renogenic events leading to organoid formation. Personnel experienced in culturing hPSCs are required to conduct this protocol.

The human kidney contains up to 2 million epithelial nephrons responsible for blood filtration. Regenerating the kidney requires the induction of the more than 20 distinct cell types required for excretion and the regulation of pH, and electrolyte and fluid balance. We have previously described the simultaneous induction of progenitors for both collecting duct and nephrons via the directed differentiation of human pluripotent stem cells. Paradoxically, although both are of intermediate mesoderm in origin, collecting duct and nephrons have distinct temporospatial origins. Here we identify the developmental mechanism regulating the preferential induction of collecting duct versus kidney mesenchyme progenitors. Using this knowledge, we have generated kidney organoids that contain nephrons associated with a collecting duct network surrounded by renal interstitium and endothelial cells. Within these organoids, individual nephrons segment into distal and proximal tubules, early loops of Henle, and glomeruli containing podocytes elaborating foot processes and undergoing vascularization. When transcription profiles of kidney organoids were compared to human fetal tissues, they showed highest congruence with first trimester human kidney. Furthermore, the proximal tubules endocytose dextran and differentially apoptose in response to cisplatin, a nephrotoxicant. Such kidney organoids represent powerful models of the human organ for future applications, including nephrotoxicity screening, disease modelling and as a source of cells for therapy.

Background: Genetic renal diseases (GRD) are a heterogeneous and incompletely understood group of disorders accounting for approximately 10 % of those diagnosed with kidney disease. The advent of Next Generation sequencing and new approaches to disease modelling may allow the identification and validation of novel genetic variants in patients with previously incompletely explained or understood GRD., Methods/design: This study will recruit participants in families/trios from a multidisciplinary sub-specialty Renal Genetics Clinic where known genetic causes of GRD have been excluded or where genetic testing is not available. After informed patient consent, whole exome and/or genome sequencing will be performed with bioinformatics analysis undertaken using a customised variant assessment tool. A rigorous process for participant data management will be undertaken. Novel genetic findings will be validated using patient-derived induced pluripotent stem cells via differentiation to renal and relevant extra-renal tissue phenotypes in vitro. A process for managing the risk of incidental findings and the return of study results to participants has been developed., Discussion: This investigator-initiated approach brings together experts in nephrology, clinical and molecular genetics, pathology and developmental biology to discover and validate novel genetic causes for patients in Australia affected by GRD without a known genetic aetiology or pathobiology.

The mammalian kidney, the metanephros, is a mesodermal organ classically regarded as arising from the intermediate mesoderm (IM). Indeed, both the ureteric bud (UB), which gives rise to the ureter and the collecting ducts, and the metanephric mesenchyme (MM), which forms the rest of the kidney, derive from the IM. Based on an understanding of the signalling molecules crucial for IM patterning and kidney morphogenesis, several studies have now generated UB or MM, or both, in vitro via the directed differentiation of human pluripotent stem cells. Although these results support the IM origin of the UB and the MM, they challenge the simplistic view of a common progenitor for these two populations, prompting a reanalysis of early patterning events within the IM. Here, we review our understanding of the origin of the UB and the MM in mouse, and discuss how this impacts on kidney regeneration strategies and furthers our understanding of human development., (© 2015. Published by The Company of Biologists Ltd.)

Purpose of Review: Recent studies on the directed differentiation of human pluripotent stem cells report tissue self-organization in vitro such that multiple component cell types arise in concert and arrange with respect to each, thereby recapitulating the morphogenetic events typical for that organ. Such self-organization has generated pituitary, optic cup, liver, brain, intestine, stomach and now kidney. Here, we will describe the cell types present within the self-organizing kidney, how these signal to each other to form a kidney organoid and the potential applications of kidney organoids., Recent Findings: Protocols for the directed differentiation of human pluripotent cells focus on recapitulating the developmental steps required during embryogenesis. In the case of the kidney, this has involved mesodermal differentiation through posterior primitive streak and intermediate mesoderm. Recent studies have observed the simultaneous formation of both ureteric epithelium and nephron progenitors in vitro. These component cell types signal to each other to initiate nephron formation as would occur during development., Summary: The generation of kidney organoids is a major advance in nephrology. Such organoids may be useful for disease modelling and drug screening. Ultimately, our capacity to generate organoids may extend to the development of tissues for transplantation.

Recent years have challenged the view that adult somatic cells reach a state of terminal differentiation. Although the ultimate example of this, somatic cell nuclear transfer, has not proven feasible in human beings, dedifferentiation of mature cell types to a more primitive state, direct reprogramming from one mature state to another, and the reprogramming of any adult cell type to a pluripotent state via enforced expression of key transcription factors now all have been shown. The implications of these findings for kidney disease include the re-creation of key renal cell types from more readily available and expandable somatic cell sources. The feasibility of such an approach recently was shown with the dedifferentiation of proximal tubule cells to nephrogenic mesenchyme. In this review, we examine the technical and clinical challenges that remain to such an approach and how new reprogramming approaches also may be useful for kidney disease., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Access to human pluripotent cells theoretically provides a renewable source of cells that can give rise to any required cell type for use in cellular therapy or bioengineering. However, successfully directing this differentiation remains challenging for most desired endpoints cell type, including renal cells. This challenge is compounded by the difficulty in identifying the required cell type in vitro and the multitude of renal cell types required to build a kidney. Here we review our understanding of how the embryo goes about specifying the cells of the kidney and the progress to date in adapting this knowledge for the recreation of nephron progenitors and their mature derivatives from pluripotent cells.

Direct reprogramming involves the enforced re-expression of key transcription factors to redefine a cellular state. The nephron progenitor population of the embryonic kidney gives rise to all cells within the nephron other than the collecting duct through a mesenchyme-to-epithelial transition, but this population is exhausted around the time of birth. Here, we sought to identify the conditions under which adult proximal tubule cells could be directly transcriptionally reprogrammed to nephron progenitors. Using a combinatorial screen for lineage-instructive transcription factors, we identified a pool of six genes (SIX1, SIX2, OSR1, EYA1, HOXA11, and SNAI2) that activated a network of genes consistent with a cap mesenchyme/nephron progenitor phenotype in the adult proximal tubule (HK2) cell line. Consistent with these reprogrammed cells being nephron progenitors, we observed differential contribution of the reprogrammed population into the Six2(+) nephron progenitor fields of an embryonic kidney explant. Dereplication of the pool suggested that SNAI2 can suppress E-CADHERIN, presumably assisting in the epithelial-to-mesenchymal transition (EMT) required to form nephron progenitors. However, neither TGFβ-induced EMT nor SNAI2 overexpression alone was sufficient to create this phenotype, suggesting that additional factors are required. In conclusion, these results suggest that reinitiation of kidney development from a population of adult cells by generating embryonic progenitors may be feasible, opening the way for additional cellular and bioengineering approaches to renal repair and regeneration.

Renal lineages including kidney are derived from intermediate mesoderm, which are differentiated from a subset of caudal undifferentiated mesoderm. The inductive mechanisms of mammalian intermediate mesoderm and renal lineages are still poorly understood. Mouse embryonic stem cells (mESCs) can be a good in vitro model to reconstitute the developmental pathway of renal lineages and to analyze the mechanisms of the sequential differentiation. We examined the effects of Activin A and retinoic acid (RA) on the induction of intermediate mesoderm from mESCs under defined, serum-free, adherent, monolayer culture conditions. We measured the expression level of intermediate mesodermal marker genes and examined the developmental potential of the differentiated cells into kidney using an ex vivo transplantation assay. Adding Activin A followed by RA to mESC cultures induced the expression of marker genes and proteins for intermediate mesoderm, odd-skipped related 1 (Osr1) and Wilm’s Tumor 1 (Wt1). These differentiated cells integrated into laminin-positive tubular cells and Pax2-positive renal cells in cultured embryonic kidney explants. We demonstrated that intermediate mesodermal marker expression was also induced by RA receptor (RAR) agonist, but not by retinoid X receptor (RXR) agonists. Furthermore, the expression of these markers was decreased by RAR antagonists. We directed the differentiation of mESCs into intermediate mesoderm using Activin A and RA and revealed the role of RAR signaling in this differentiation. These methods and findings will improve our understanding of renal lineage development and could contribute to the regenerative medicine of kidney.

Glomeruli comprise an important filtering apparatus in the kidney and are derived from the metanephric mesenchyme. A nuclear protein, Sall1, is expressed in this mesenchyme, and we previously reported that Trb2, a mouse homolog of Drosophila tribbles, is expressed in the mesenchyme-derived tissues of the kidney by microarray analyses using Sall1-GFP knock-in mice. In the present report, we detected Trb2 expression in a variety of organs during gestation, including the kidneys, mesonephros, testes, heart, eyes, thymus, blood vessels, muscle, bones, tongue, spinal cord, and ganglions. In the developing kidney, Trb2 signals were detected in podocytes and the prospective mesangium of the glomeruli, as well as in ureteric bud tips. However, Trb2 mutant mice did not display any apparent phenotypes and no proteinuria was observed, indicating normal glomerular functions. These results suggest that Trb2 plays minimal roles during kidney and mouse development.

Mutations in SALL4, the human homolog of the Drosophila homeotic gene spalt (sal), cause the autosomal dominant disorder known as Okihiro syndrome. In this study, we show that a targeted null mutation in the mouse Sall4 gene leads to lethality during peri-implantation. Growth of the inner cell mass from the knockout blastocysts was reduced, and Sall4-null embryonic stem (ES) cells proliferated poorly with no aberrant differentiation. Furthermore, we demonstrated that anorectal and heart anomalies in Okihiro syndrome are caused by Sall4 haploinsufficiency and that Sall4/Sall1 heterozygotes exhibited an increased incidence of anorectal and heart anomalies, exencephaly and kidney agenesis. Sall4 and Sall1 formed heterodimers, and a truncated Sall1 caused mislocalization of Sall4 in the heterochromatin; thus, some symptoms of Townes-Brocks syndrome caused by SALL1 truncations could result from SALL4 inhibition.

Renal stem or progenitor cells with a multilineage differentiation potential remain to be isolated, and the differentiation mechanism of these cell types in kidney development or regeneration processes is unknown. In an attempt to resolve this issue, we set up an in vitro culture system using NIH3T3 cells stably expressing Wnt4 (3T3Wnt4) as a feeder layer, in which a single renal progenitor in the metanephric mesenchyme forms colonies consisting of several types of epithelial cells that exist in glomeruli and renal tubules. We found that only cells strongly expressing Sall1 (Sall1-GFP(high) cells), a zinc-finger nuclear factor essential for kidney development, form colonies, and that they reconstitute a three-dimensional kidney structure in an organ culture setting. We also found that Rac- and JNK-dependent planar cell polarity (PCP) pathways downstream of Wnt4 positively regulate the colony size, and that the JNK pathway is also involved in mesenchymal-to-epithelial transformation of colony-forming progenitors. Thus our colony-forming assay, which identifies multipotent progenitors in the embryonic mouse kidney, can be used for examining mechanisms of renal progenitor differentiation.

SALL1 is a mammalian homologue of the Drosophila region-specific homeotic gene spalt (sal) and heterozygous mutations in SALL1 in humans lead to Townes-Brocks syndrome. We isolated a mouse homologue of SALL1 (Sall1) and found that mice deficient in Sall1 die in the perinatal period with kidney agenesis. Sall1 is expressed in the metanephric mesenchyme surrounding ureteric bud and homozygous deletion of Sall1 results in an incomplete ureteric bud outgrowth. Therefore, Sall1 is essential for ureteric bud invasion, the initial key step for metanephros development. We also generated mice in which a green fluorescent protein (GFP) gene was inserted into the Sall1 locus and we isolated the GFP-positive population from embryonic kidneys of these mice by fluorescence-activated cell sorting (FACS). We then compared gene expression profiles in the GFP-positive and -negative population using microarray analysis, followed by in situ hybridization. We detected many genes known to be important for metanephros development, and genes expressed abundantly in the metanephric mesenchyme. We also found groups of genes which are not known to be expressed in the metanephric mesenchyme. Thus a combination of microarray technology and Sall1-GFP mice is useful for systematic identification of genes expressed in the developing kidney.

SALL1, a causative gene for Townes-Brocks syndrome, encodes a zinc finger protein, and its mouse homolog (Sall1) is essential for metanephros development, as noted during gene targeting. In the embryonic kidney, Sall1 is expressed abundantly in mesenchyme-derived structures from condensed mesenchyme, S-, comma-shaped bodies, to renal tubules and podocytes. We generated mice in which a green fluorescent protein (GFP) gene was inserted into the Sall1 locus and we isolated the GFP-positive population from embryonic kidneys of these mice by fluorescein-activated cell sorting. The GFP-positive population indeed expressed mesenchymal genes, while the negative population expressed genes in the ureteric bud. To systematically search for genes expressed in the mesenchyme-derived cells, we compared gene expression profiles in the GFP-positive and -negative populations using microarray analysis, followed by in situ hybridization. We detected many genes known to be important for metanephros development including Sall1, GDNF, Raldh2, Pax8 and FoxD1, and genes expressed abundantly in the metanephric mesenchyme such as Unc4.1, Six2, Osr-2 and PDGFc. We also found groups of genes including SSB-4, Smarcd3, micro-Crystallin, TRB-2, which are not known to be expressed in the metanephric mesenchyme. Therefore a combination of microarray technology and Sall1-GFP mice is useful for systematic identification of genes expressed in the developing kidney., (Copyright 2004 Elsevier Ireland Ltd.)