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Vascularization plays a critical role in organ maturation and cell type development. Drug discovery, organ mimicry, and ultimately transplantation in a clinical setting thereby hinges on achieving robust vascularization of in vitro engineered organs. Here, focusing on human kidney organoids, we overcome this hurdle by combining an inducible ETS translocation variant 2 (ETV2) human induced pluripotent stem cell (iPSC) line, which directs endothelial fate, with a non-transgenic iPSC line in suspension organoid culture. The resulting human kidney organoids show extensive vascularization by endothelial cells with an identity most closely related to endogenous kidney endothelia. Vascularized organoids also show increased maturation of nephron structures including more mature podocytes with improved marker expression, foot process interdigitation, an associated fenestrated endothelium, and the presence of renin+ 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. Furthermore, this approach is orthogonal to native tissue differentiation paths, hence readily adaptable to other organoid systems and thus has the potential for a broad impact on basic and translational organoid studies.

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

We aimed to generate human induced pluripotent stem cell (iPSC) lines from New Zealand donors. These lines are the first to be generated in New Zealand. Human dermal fibroblasts were collected from two individual donors and reprogrammed with the human OSKM transcription factors using the Sendai virus system. Emerging iPSC colonies were picked, expanded and karyotyped. Clones with normal karyotype were characterised for pluripotency marker expression, p53 mutational status and trilineage differentiation potential. The MANZ-2-2 and MANZ-4-37 iPSC lines showed normal karyotype and expressed pluripotency markers at RNA and protein levels without detectable transgene expression. Both lines differentiated into the three germ layers in vitro and passed the hPSC Scorecard assay for pluripotency and trilineage differentiation. Furthermore, both lines were susceptible to cell apoptosis mediated by nutlin-3a indicative of their wildtype p53 status. This study presents the successful derivation and characterisation of iPSC lines derived from New Zealand donors. These lines will facilitate iPSC-based research in New Zealand and beyond.

Acute kidney injury (AKI) is a major clinical and economic problem worldwide and currently there are no effective treatments. Due to the numerous renal insults that lead to AKI and the complexity of injury, many pathways are dysregulated leading to maladaptive repair, which can lead to chronic kidney disease. The focus of this chapter is to illustrate the pathways that play a role in AKI and how small molecules for therapeutic intervention can be utilized to modulate their activity and enhance productive repair. Given the large number of pathways that could be therapeutic targets, we will focus on only a limited number that have shown promising results in the treatment of AKI.

Background Hemolysis occurs in many injury settings and can trigger disease processes. In the kidney, extracellular hemoglobin can induce damage via several mechanisms. These include oxidative stress, mitochondrial dysfunction, and inflammation, which promote fibrosis and chronic kidney disease. Understanding the pathophysiology of these injury pathways offers opportunities to develop new therapeutic strategies. Methods To model hemolysis-induced kidney injury, human kidney organoids were treated with hemin, an iron-containing porphyrin, that generates reactive oxygen species. In addition, we developed an induced pluripotent stem cell line expressing the biosensor, CytochromeC-GFP (CytoC-GFP), which provides a real-time readout of mitochondrial morphology, health, and early apoptotic events. Results We found that hemin-treated kidney organoids show oxidative damage, increased expression of injury markers, impaired functionality of organic anion and cation transport and undergo fibrosis. Injury could be detected in live CytoC-GFP organoids by cytoplasmic localization of fluorescence. Finally, we show that 4-(phenylthio)butanoic acid, an HDAC inhibitor with anti-fibrotic effects in vivo, reduces hemin-induced human kidney organoid fibrosis. Conclusion This work establishes a hemin-induced model of kidney organoid injury. This platform provides a new tool to study the injury and repair response pathways in human kidney tissue and will assist in the development of new therapeutics.

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

Summary Kidney organoids represent a physiologically advanced model for studying the mechanisms of kidney development and disease. Here, we describe a simple two-step protocol for the differentiation of human pluripotent stem cells into kidney organoids. Our approach involves suspension culture that allows for rapid and cost-effective bulk production of organoids, which is well suited for large-scale assays such as drug screening. The organoids correspond to fetal human kidney tissue and may be of limited use for modeling adult kidney function. For complete details on the use and execution of this protocol, please refer to Przepiorski et al. (2018)., Graphical Abstract, Highlights • Generation of kidney organoids from hPSCs in two simple steps • The method is cost-efficient and amendable for large-scale production • Organoid differentiation is optimal between day 12 and day 20 • Organoid nephrons correspond to human fetal nephrons, Kidney organoids represent a physiologically advanced model for studying the mechanisms of kidney development and disease. Here, we describe a simple two-step protocol for the differentiation of human pluripotent stem cells into kidney organoids. Our approach involves suspension culture that allows for rapid and cost-effective bulk production of organoids, which is well suited for large-scale assays such as drug screening. The organoids correspond to fetal human kidney tissue and may be of limited use for modeling adult kidney function.

The use of differentiating human induced pluripotent stem cells (hiPSCs) in mini-tissue organoids provides an invaluable resource for regenerative medicine applications, particularly in the field of disease modeling. However, most studies using a kidney organoid model, focused solely on the transcriptomics and did not explore mechanisms of regulating kidney organoids related to metabolic effects and maturational phenotype. Here, we applied metabolomics coupled with transcriptomics to investigate the metabolic dynamics and function during kidney organoid differentiation. Not only did we validate the dominant metabolic alteration from glycolysis to oxidative phosphorylation in the iPSC differentiation process but we also showed that glycine, serine, and threonine metabolism had a regulatory role during kidney organoid formation and lineage maturation. Notably, serine had a role in regulating S-adenosylmethionine (SAM) to facilitate kidney organoid formation by altering DNA methylation. Our data revealed that analysis of metabolic characterization broadens our ability to understand phenotype regulation. The utilization of this comparative omics approach, in studying kidney organoid formation, can aid in deciphering unique knowledge about the biological and physiological processes involved in organoid-based disease modeling or drug screening.

The formation of three-dimensional kidney tissue (organoids) from human pluripotent stem cell lines provides a valuable tool to examine kidney function in an in vitro model and could be used for regenerative medicine approaches. Kidney organoids have the potential to model kidney diseases and congenital defects, be used for drug development, and to further our understanding of acute kidney injury, fibrosis, and chronic kidney disease. In this review, we examine the current stage of pluripotent stem cell-derived kidney organoid technology, challenges, shortcomings, and regenerative potential of kidney organoids in the future.

BACKGROUND: Mutations in CTNS-a gene encoding the cystine transporter cystinosin-cause the rare, autosomal, recessive, lysosomal-storage disease cystinosis. Research has also implicated cystinosin in modulating the mTORC1 pathway, which serves as a core regulator of cellular metabolism, proliferation, survival, and autophagy. In its severest form, cystinosis is characterized by cystine accumulation, renal proximal tubule dysfunction, and kidney failure. Because treatment with the cystine-depleting drug cysteamine only slows disease progression, there is an urgent need for better treatments. METHODS: To address a lack of good human-based cell culture models for studying cystinosis, we generated the first human induced pluripotent stem cell (iPSC) and kidney organoid models of the disorder. We used a variety of techniques to examine hallmarks of cystinosis-including cystine accumulation, lysosome size, the autophagy pathway, and apoptosis-and performed RNA sequencing on isogenic lines to identify differentially expressed genes in the cystinosis models compared with controls. RESULTS: Compared with controls, these cystinosis models exhibit elevated cystine levels, increased apoptosis, and defective basal autophagy. Cysteamine treatment ameliorates this phenotype, except for abnormalities in apoptosis and basal autophagy. We found that treatment with everolimus, an inhibitor of the mTOR pathway, reduces the number of large lysosomes, decreases apoptosis, and activates autophagy, but it does not rescue the defect in cystine loading. However, dual treatment of cystinotic iPSCs or kidney organoids with cysteamine and everolimus corrects all of the observed phenotypic abnormalities. CONCLUSIONS: These observations suggest that combination therapy with a cystine-depleting drug such as cysteamine and an mTOR pathway inhibitor such as everolimus has potential to improve treatment of cystinosis.

Acute kidney injury (AKI) remains a major global healthcare problem, and there is a need to develop human-based models to study AKI in vitro. Toward this goal, we have characterized induced pluripotent stem cell-derived human kidney organoids and their response to cisplatin, a chemotherapeutic drug that induces AKI and preferentially damages the proximal tubule. We found that a single treatment with 50 µM cisplatin induces hepatitis A virus cellular receptor 1 ( HAVCR1) and C-X-C motif chemokine ligand 8 ( CXCL8) expression, DNA damage (γH2AX), and cell death in the organoids but greatly impairs organoid viability. DNA damage was not specific to the proximal tubule but also affected the distal tubule and interstitial cell populations. This lack of specificity correlated with low expression of proximal tubule-specific SLC22A2/organic cation transporter 2 ( OCT2) for cisplatin. To improve viability, we developed a repeated low-dose regimen of 4 × 5 µM cisplatin over 7 days and found this caused less toxicity while still inducing a robust injury response that included secretion of known AKI biomarkers and inflammatory cytokines. This work validates the use of human kidney organoids to model aspects of cisplatin-induced injury, with the potential to identify new AKI biomarkers and develop better therapies.

Summary Kidney organoids made from pluripotent stem cells have the potential to revolutionize how kidney development, disease, and injury are studied. Current protocols are technically complex, suffer from poor reproducibility, and have high reagent costs that restrict scalability. To overcome some of these issues, we have established a simple, inexpensive, and robust method to grow kidney organoids in bulk from human induced pluripotent stem cells. Our organoids develop tubular structures by day 8 and show optimal tissue morphology at day 14. A comparison with fetal human kidneys suggests that day-14 organoid tissue most closely resembles late capillary loop stage nephrons. We show that deletion of HNF1B, a transcription factor linked to congenital kidney defects, interferes with tubulogenesis, validating our experimental system for studying renal developmental biology. Taken together, our protocol provides a fast, efficient, and cost-effective method for generating large quantities of human fetal kidney tissue, enabling the study of normal and aberrant kidney development., Graphical Abstract, Highlights • Technically simple and cost-efficient protocol for kidney organoid generation • Tubular organoids are obtained rapidly, with high efficiency, yield, and robustness • Organoids contain nephrons that correspond to human fetal nephrons • The applicability to model congenital kidney defects is presented, In this study, Przepiorski et al. present a technically simple and robust protocol to derive kidney organoids from human induced pluripotent stem cells. This technique is inexpensive and efficient, allowing kidney organoids to be generated in bulk. Organoids are obtained rapidly (within 8 days) and, based on their segmentation pattern, correspond to “late capillary loop” stage fetal human nephrons.

Acute kidney injury (AKI) remains a major global healthcare problem and there is a need to develop human-based models to study AKI in vitro. Towards this goal, we have characterized induced pluripotent stem cell-derived human kidney organoids and their response to cisplatin, a chemotherapeutic drug that induces AKI and preferentially damages the proximal tubule. We found that a single treatment with 50 µM cisplatin induces HAVCR1 and CXCL8 expression, DNA damage (γH2AX) and cell death in the organoids in a dose-dependent manner but greatly impairs organoid viability. DNA damage was not specific to the proximal tubule but also affected the distal tubule and interstitial populations. This lack of specificity correlated with low expression of the proximal tubule-specific SLC22A2/OCT2 transporter for cisplatin. To improve viability, we developed a repeated low-dose regimen of 4x 5 µM cisplatin over 7 days and found this causing less toxicity while still inducing a robust AKI response that included secretion of known AKI biomarkers and inflammatory cytokines. This work validates the use of human kidney organoids to model aspects of AKI in vitro, with the potential to identify new AKI biomarkers and develop better therapies.

Cystinosis is a lysosomal storage disease caused by mutations inCTNS, encoding a cystine transporter, and in its severest form is characterized by cystine accumulation, renal proximal tubule dysfunction and kidney failure. Cystinosis is treated with the cystine-depleting drug cysteamine, however this only slows progression of the disease and there is an urgent need for better treatments. Here, we have generated and characterized the first human induced pluripotent stem cell (iPSC) and kidney organoid models of cystinosis. These models exhibit elevated cystine and cysteine levels, enlarged lysosomes and a block in basal autophagy flux. Cysteamine treatment ameliorates this phenotype except for the basal autophagy flux defect. We found that treatment with Everolimus, an inhibitor of the mTOR pathway, reduces the number of large lysosomes and activates autophagy but does not rescue the cystine/cysteine loading defect. However, dual treatment of cystinotic iPSCs or kidney organoids with cysteamine and Everolimus corrects all of the observed phenotypes indicating that a combination therapy has therapeutic potential to improve the treatment of cystinosis.

The intermediate mesoderm is located between the somites and the lateral plate mesoderm and gives rise to renal progenitors that contribute to the three mammalian kidney types (pronephros, mesonephros and metanephros). In this review, focusing largely on murine kidney development, we examine how the intermediate mesoderm forms during gastrulation/axis elongation and how it progressively gives rise to distinct renal progenitors along the rostro-caudal axis. We highlight some of the potential signalling cues and core transcription factor circuits that direct these processes, up to the point of early metanephric kidney formation.

SummaryKidney organoids generated from human pluripotent stem cells have the potential to revolutionize how kidney development and injury are studied. Current protocols are technically complex and suffer from poor reproducibility and high reagent costs restricting scalability. To overcome these issues, we have established a simple, inexpensive and robust method to grow kidney organoids in bulk from human induced pluripotent stem cells. Our organoids develop tubular structures by day (d) 8 and show optimal tissue morphology at d14. A comparison with fetal human kidney suggests that d14 organoid renal structures most closely resemble ‘capillary loop’ stage nephrons. We show that deletion ofHNF1B,a transcription factor linked to congenital kidney defects, interferes with tubulogenesis, validating our experimental system for studying renal developmental biology. Taken together, our protocol provides a fast, efficient and cost-effective method for generating large quantities of human fetal kidney tissue, enabling the study of normal and aberrant human renal development.

Nephrons comprise a blood filter and an epithelial tubule that is subdivided into proximal and distal segments, but what directs this patterning during kidney organogenesis is not well understood. Using zebrafish, we found that the HNF1β paralogues hnf1ba and hnf1bb, which encode homeodomain transcription factors, are essential for normal segmentation of nephrons. Embryos deficient in hnf1ba and hnf1bb did not express proximal and distal segment markers, yet still developed an epithelial tubule. Initiating hnf1ba/b expression required Pax2a and Pax8, but hnf1ba/b-deficient embryos did not exhibit the expected downregulation of pax2a and pax8 at later stages of development, suggesting complex regulatory loops involving these molecules. Embryos deficient in hnf1ba/b also did not express the irx3b transcription factor, which is responsible for differentiation of the first distal tubule segment. Reciprocally, embryos deficient in irx3b exhibited downregulation of hnf1ba/b transcripts in the distal early segment, suggesting a segment-specific regulatory circuit. Deficiency of hnf1ba/b also led to ectopic expansion of podocytes into the proximal tubule domain. Epistasis experiments showed that the formation of podocytes required wt1a, which encodes the Wilms’ tumor suppressor-1 transcription factor, and rbpj, which encodes a mediator of canonical Notch signaling, downstream or parallel to hnf1ba/b. Taken together, these results suggest that Hnf1β factors are essential for normal segmentation of nephrons during kidney organogenesis.

Background Tamoxifen is used in hormone therapy for estrogen-receptor (ER)-positive breast cancer, but also has chemopreventative effects against ER-negative breast cancers. This study sought to investigate whether oral iron-saturated bovine lactoferrin (Fe-Lf), a natural product which enhances chemotherapy, could improve the chemotherapeutic effects of tamoxifen in the treatment of ER-negative breast cancers. Methods In a model of breast cancer prevention, female Balb/c mice treated with tamoxifen (5 mg/Kg) were fed an Fe-Lf supplemented diet (5 g/Kg diet) or the base diet. At week 2, 4T1 mammary carcinoma cells were injected into an inguinal mammary fat pad. In a model of breast cancer treatment, tamoxifen treatment was not started until two weeks following tumor cell injection. Tumor growth, metastasis, body weight, and levels of interleukin 18 (IL-18) and interferon γ (IFN-γ) were analyzed. Results Tamoxifen weakly (IC50 ~ 8 μM) inhibited the proliferation of 4T1 cells at pharmacological concentrations in vitro. In the tumor prevention study, a Fe-Lf diet in combination with tamoxifen caused a 4 day delay in tumor formation, and significantly inhibited tumor growth and metastasis to the liver and lung by 48, 58, and 66% (all P P Conclusions The results indicate that Fe-Lf is a potent natural adjuvant capable of augmenting the chemotherapeutic activity of tamoxifen. It could have application in delaying relapse in tamoxifen-treated breast cancer patients who are at risk of developing ER-negative tumors.

Blood is collected during animal experimentation to measure haematological and metabolic parameters. It cannot be assumed that circulating blood has the same composition irrespective of its location, and indeed, differences in the composition of blood sampled from the arterial and venous compartments have been reported. Here we investigated whether blood collected by cardiac puncture (CP) versus that collected following removal of the distal 1 mm of the tail tip (TT) differs with respect to glucose and lipid profiles in male C57BL/6J mice at 4, 7, 20 and 28 weeks of age. Blood was first collected from the TT of unanaesthetized mice, which were then immediately anaesthetized using ketamine/xylazine, and a second blood sample was collected by CP. The CP glucose concentration was significantly higher than TT glucose by a positive bias averaging +80% ( P < 0.01), irrespective of the age of the mice. Conversely, the concentrations of the CP lipids, including total cholesterol, high-density lipoprotein cholesterol and triglyceride were lower than TT lipids by a negative bias averaging −25% ( P < 0.05). These observations highlight the difficulty in measuring and comparing metabolic parameters such as glucose and lipid between one blood compartment and another. They illustrate the need to standardize sampling sites, especially when repeated blood sampling is required.