Highly parallel production of designer organoids by mosaic patterning of progenitors.

Organoids derived from human stem cells are a promising approach for disease modeling, regenerative medicine, and fundamental research. However, organoid variability and limited control over morphological outcomes remain as challenges. One open question is the extent to which engineering control over culture conditions can guide organoids to specific compositions. Here, we extend a DNA "velcro" cell patterning approach, precisely controlling the number and ratio of human induced pluripotent stem cell-derived progenitors contributing to nephron progenitor (NP) organoids and mosaic NP/ureteric bud (UB) tip cell organoids within arrays of microwells. We demonstrate long-term control over organoid size and morphology, decoupled from geometric constraints. We then show emergent trends in organoid tissue proportions that depend on initial progenitor cell composition. These include higher nephron and stromal cell representation in mosaic NP/UB organoids vs. NP-only organoids and a "goldilocks" initial cell ratio in mosaic organoids that optimizes the formation of proximal tubule structures.

Expression pattern of Runt-related transcription factor (RUNX) family members and the role of RUNX1 during kidney development.

Runt-related transcription factor (RUNX) family members play critical roles in the development of multiple organs. Mammalian RUNX family members, consisting of RUNX1, RUNX2, and RUNX3, have distinct tissue-specific expression and function. In this study, we examined the spatiotemporal expression patterns of RUNX family members in developing kidneys and analyzed the role of RUNX1 during kidney development. In the developing mouse kidney, RUNX1 protein was strongly expressed in the ureteric bud (UB) tip and weakly expressed in the distal segment of the renal vesicle (RV), comma-shaped body (CSB), and S-shaped body (SSB). In contrast, RUNX2 protein was restricted to the stroma, and RUNX3 protein was only expressed in immune cells. We also analyzed the expression of RUNX family members in the cynomolgus monkey kidney. We found that expression patterns of RUNX2 and RUNX3 were conserved between rodents and primates, whereas RUNX1 was only expressed in the UB tip, not in the RV, CSB, or SSB of cynomolgus monkeys, suggesting a species differences. We further evaluated the roles of RUNX1 using two different conditional knockout mice: Runx1f/f:HoxB7-Cre and Runx1f/f:R26-CreERT2 and found no abnormalities in the kidney. Our findings showed that RUNX1, which is mainly expressed in the UB tip, is not essential for kidney development.

Case report: Ureteric bud intestinal-type adenocarcinoma involving the cervix was misdiagnosed as a large cervical fibroid.

Background: Malignant tumors of the ureteric bud are not common, and cervical involvement is even rarer. So far, there have been no such cases in the literature. Case summary: A 50-year-old woman developed intermittent light bleeding in the past 7 months and lower abdominal pain in the past 2 months. The human papillomavirus 16 (HPV) DNA, P16 chemical staining, thinPrep cytology test (TCT), and cervical and cervical canal tissue biopsy were all negative. Pelvic color Doppler ultrasound exhibited incomplete mediastinal uterus and heterogeneous echo from the cervical canal to the posterior wall of the cervix. Pelvic contrast-enhanced CT showed left cervical mass, left retroperitoneal mass, absence of the left kidney, and mediastinal uterus. An increase in human epididymal protein 4 (HE4) (133.6 pmol/L) was detected, while other tumor markers were at normal levels. Based on these examination results, a diagnosis of "cervical fibroids, left retroperitoneal mass, incomplete mediastinal uterus, left kidney deficiency"[SIC] was conducted, and expanded hysterectomy, right adnexectomy, and left retroperitoneal mass resection were performed. Through intraoperative rapid pathological diagnosis, postoperative pathological diagnosis combined with the re-evaluation of laboratory, and imaging and intraoperative examination results, the patient was diagnosed with ureteric bud intestinal-type adenocarcinoma involving the cervix. The patient has been tracked and followed up for approximately 11 months. She underwent six courses of chemotherapy. At present, the medication has been discontinued for 4 months, and there is no recurrence, metastasis, or deterioration of the tumor. Conclusion: For large masses of the cervix, it is feasible for the operation to be performed, improving the prognosis. There were a few limitations. A preoperative aspiration biopsy of masses was not performed to differentiate benign from malignant. Preoperative urography was not performed to clarify the function of the malformed urinary system structure. Partial cystectomy should be performed simultaneously with the resection of the ureteric bud for intestinal-type adenocarcinoma. In this case, a partial cystectomy was not performed, which can only be compensated with postoperative chemotherapy. Moreover, this patient did not undergo genetic screening, and it is currently unclear whether there are any genetic mutations associated with ureteric bud intestinal adenocarcinoma.

The Role of the ADAMTS18 Gene-Induced Immune Microenvironment in Mouse Kidney Development.

The aim of this study is to investigate the role of the ADAMTS18 gene in regulating the renal development of mice. PAS staining was used to observe the kidney development of E12.5-E17.5 mice, while immunofluorescence staining and RT-PCR were used to observe the expression of ADAMTS18. Ureteric bud (UB) branches were observed using immunofluorescence staining using the UB marker E-cadherin, and the apoptosis and proliferation of posterior renal mesenchymal cells were analyzed using TUNEL and PH3 fluorescence staining. Flow cytometry was used to analyze the immune cell infiltration, and western blotting (WB) was used to analyze the expression of PD-1/PD-L1 and CTLA-4. As a result, the ADAMTS18 gene expression gradually increased as the kidney continued to mature during embryonic development. Compared with that in the control and vector groups, UB branching was significantly reduced in the ADAMTS18 deletion group (p < 0.05), but that deletion of ADAMTS18 did not affect posterior renal mesenchymal cell proliferation or apoptosis (p > 0.05). Compared with those in the control and vector groups, the proportion of embryonic kidney B cells and the proportion of CD8+ cells were significantly greater after ADAMTS18 was knocked down (p < 0.05), but the difference in neutrophil counts was not significant (p > 0.05). The WB analysis revealed that the PD-1/PD-L1 and CTLA-4 expression was significantly increased after ADAMTS18 was knocked down (p < 0.05). In conclusion, the ADAMTS18 gene may be involved in mice kidney development by regulating the immune microenvironment and activating immune checkpoints. Deletion of the ADAMTS18 gene may be unfavorable for kidney development.

Human iPSC-derived renal collecting duct organoid model cystogenesis in ADPKD.

In autosomal dominant polycystic kidney disease (ADPKD), renal cyst lesions predominantly arise from collecting ducts (CDs). However, relevant CD cyst models using human cells are lacking. Although previous reports have generated in vitro renal tubule cyst models from human induced pluripotent stem cells (hiPSCs), therapeutic drug candidates for ADPKD have not been identified. Here, by establishing expansion cultures of hiPSC-derived ureteric bud tip cells, an embryonic precursor that gives rise to CDs, we succeed in advancing the developmental stage of CD organoids and show that all CD organoids derived from PKD1-/- hiPSCs spontaneously develop multiple cysts, clarifying the initiation mechanisms of cystogenesis. Moreover, we identify retinoic acid receptor (RAR) agonists as candidate drugs that suppress in vitro cystogenesis and confirm the therapeutic effects on an ADPKD mouse model in vivo. Therefore, our in vitro CD cyst model contributes to understanding disease mechanisms and drug discovery for ADPKD.

Advances and challenges toward developing kidney organoids for clinical applications.

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.

Unilateral multicystic dysplastic kidney disease associated with ipsilateral ureteric bud remnant and contralateral duplex collecting system.

Congenital anomalies of the kidney and urinary tract are among the most common developmental malformations. The heterogeneity of these anomalies is very high, some of them are rarely discussed in the literature. Herein, we present a case of a 5-year-old male who was found to have a combination of unilateral multicystic dysplastic kidney associated with ipsilateral ureteric bud remnant and contralateral duplex collecting system.

Novel concept of Wilms' tumor development: involvement of pluripotential cells of ureteric bud.

It is acknowledged that nephron develops after bilateral induction of the metanephric mesenchyma and branching ureteric bud (UB), and that nephrogenic rest and Wilms' tumor (nephroblastoma) arises from impaired differentiation of metanephric blastema. The aim of this study was to obtain more information on the involvement of UB derivatives in nephrogenic rest and Wilms' tumor. We applied immunohistochemistry to analyze nephrogenic rests and Wilms' tumors with mixed histology, including regressive and blastemal types. We used antibodies recognizing UB tip cells (ROBO1, SLIT2, RET), principal cells (AQP2), α- and ß-intercalated cells (SLC26A4, SLC4A1, ATP6V1B1, ATP6V0D2), and their precursors (CA2). Tubules surrounded by tumorous blastemal cells resembling UB tip were positive for RET, ROBO1, and SLIT2 in Wilms' tumor. Moreover, CA2-positive tubular structures and ATP6V1B1- and ATP6V0D2-positive immature non-α- and non-ß-intercalated cells were detected in nephrogenic rest and Wilms' tumor. We suggest that Wilms' tumor is more than nephroblastoma and propose a definition that Wilms tumor is a malignant embryonal neoplasm derived from pluripotential cells of nephrogenic blastema and of ureteric bud tip.

Morphometry of the ureteric bud branching in the developing mouse kidney / 解剖学报

[Abstract] Objective To investigate the branching pattern of the ureteric bud and the number of the nephron induced by each ureteric bud tip, through the three-dimensional tracing of the ureteric tree, combined with the morphological analysis and measurement of the ureteric tree. Methods The kidneys were obtained from three mice at various developing time points and prepared for paraffin and epoxy sections. Then the microscopic images were digitized and aligned from these sections. Based on the computer-assisted tracing and visualization of ureteric tree, the number of branches and the nephron induced by each ureteric bud tip were obtained by counting. In addition, paraffin sections were stained with HE staining for morphological observation of nephrogenic zone and ureteric bud, while in order to reflect the density of the ureteric bud tips at nephrogenic zone, the distance between two neighboring ureteric bud tips was measured aided with the Claudin-7 immunohistochemical staining. Results The ureteric bud branching tree revealed that the initial bifid iterative branching formed the framework of renal medulla, the branching became complicated and dense in cortex and nephrogenic zone, while the distance between ureteric bud tips were also decreasing. The number of the nephron induced by each ureteric bud tip increased from one (E14. 5) to two (E17. 5), and occasionally to three. Conclusion Threedimeasional Visualization of ureteric bud branching tree reveals regional complication, suggesting molecules in different regions drive different branching patterns; While the density of the ureteric bud tips at nephrogenic zone increases corresponding to decreasing of thickness of the nephrogenic zone, and the disappearance of the ureteric bud tips after birth is also consistent with the gradual consumption of nephron progenitor cells.

Toward the renal vesicle: Ultrastructural investigation of the cap mesenchyme splitting process in the developing kidney.

Background: A complex sequence of morphogenetic events leads to the development of the adult mouse kidney. In the present study, we investigated the morphological events that characterize the early stages of the mesenchymal-to-epithelial transition of cap mesenchymal cells, analyzing in depth the relationship between cap mesenchymal induction and ureteric bud (UB) branching. Design and methods: Normal kidneys of newborn non-obese diabetic (NOD) mice were excised and prepared for light and electron microscopic examination. Results: Nephrogenesis was evident in the outer portion of the renal cortex of all examined samples. This process was mainly due to the interaction of two primordial derivatives, the ureteric bud and the metanephric mesenchyme. Early renal developmental stages were initially characterized by the formation of a continuous layer of condensed mesenchymal cells around the tips of the ureteric buds. These caps of mesenchymal cells affected the epithelial cells of the underlying ureteric bud, possibly inducing their growth and branching. Conclusions: The present study provides morphological evidence of the reciprocal induction between the ureteric bud and the metanephric mesenchyme showing that the ureteric buds convert mesenchyme to epithelium that in turn stimulates the growth and the branching of the ureteric bud.

Bim Expression Modulates Branching Morphogenesis of the Epithelium and Endothelium.

Branching morphogenesis is a key developmental process during organogenesis, such that its disruption frequently leads to long-term consequences. The kidney and eye share many etiologies, perhaps, due to similar use of developmental branching morphogenesis and signaling pathways including cell death. Tipping the apoptotic balance towards apoptosis imparts a ureteric bud and retinal vascular branching phenotype similar to one that occurs in papillorenal syndrome. Here, to compare ureteric bud and retinal vascular branching in the context of decreased apoptosis, we investigated the impact of Bim, Bcl-2's rival force. In the metanephros, lack of Bim expression enhanced ureteric bud branching with increases in ureteric bud length, branch points, and branch end points. Unfortunately, enhanced ureteric bud branching also came with increased branching defects and other undesirable consequences. Although we did see increased nephron number and renal mass, we observed glomeruli collapse. Retinal vascular branching in the absence of Bim expression had similarities with the ureteric bud including increased vascular length, branching length, segment length, and branching interval. Thus, our studies emphasize the impact appropriate Bim expression has on the overall length and branching in both the ureteric bud and retinal vasculature.

A transgenic Alx4-CreER mouse to analyze anterior limb and nephric duct development.

BACKGROUND: Genetic tools to study gene function and the fate of cells in the anterior limb bud are very limited. RESULTS: We describe a transgenic mouse line expressing CreERT2 from the Aristaless-like 4 (Alx4) promoter that induces recombination in the anterior limb. Cre induction at embryonic day 8.5 revealed that Alx4-CreERT2 labeled cells using the mTmG Cre reporter contributed to anterior digits I to III as well as the radius of the forelimb. Cre activity is expanded further along the AP axis in the hindlimb than in the forelimb resulting in some Cre reporter cells contributing to digit IV. Induction at later time points labeled cells that become progressively restricted to more anterior digits and proximal structures. Comparison of Cre expression from the Alx4 promoter transgene with endogenous Alx4 expression reveals Cre expression is slightly expanded posteriorly relative to the endogenous Alx4 expression. Using Alx4-CreERT2 to induce loss of intraflagellar transport 88 (Ift88), a gene required for ciliogenesis, hedgehog signaling, and limb patterning, did not cause overt skeletal malformations. However, the efficiency of deletion, time needed for Ift88 protein turnover, and for cilia to regress may hinder using this approach to analyze cilia in the limb. Alx4-CreERT2 is also active in the mesonephros and nephric duct that contribute to the collecting tubules and ducts of the adult nephron. Embryonic activation of the Alx4-CreERT2 in the Ift88 conditional line results in cyst formation in the collecting tubules/ducts. CONCLUSION: Overall, the Alx4-CreERT2 line will be a new tool to assess cell fates and analyze gene function in the anterior limb, mesonephros, and nephric duct.

Kidney development to kidney organoids and back again.

Kidney organoid technology has led to a renaissance in kidney developmental biology. The complex underpinnings of mammalian kidney development have provided a framework for the generation of kidney cells and tissues from human pluripotent stem cells. Termed kidney organoids, these 3-dimensional structures contain kidney-specific cell types distributed similarly to in vivo architecture. The adult human kidney forms from the reciprocal induction of two disparate tissues, the metanephric mesenchyme (MM) and ureteric bud (UB), to form nephrons and collecting ducts, respectively. Although nephrons and collecting ducts are derived from the intermediate mesoderm (IM), their development deviates in time and space to impart distinctive inductive signaling for which separate differentiation protocols are required. Here we summarize the directed differentiation protocols which generate nephron kidney organoids and collecting duct kidney organoids, making note of similarities as much as differences. We discuss limitations of these present approaches and discuss future directions to improve kidney organoid technology, including a greater understanding of anterior IM and its derivatives to enable an improved differentiation protocol to collecting duct organoids for which historic and future developmental biology studies will be instrumental.

Unravelling the Role of PAX2 Mutation in Human Focal Segmental Glomerulosclerosis.

No effective treatments are available for familial steroid-resistant Focal Segmental Glomerulosclerosis (FSGS), characterized by proteinuria due to ultrastructural abnormalities in glomerular podocytes. Here, we studied a private PAX2 mutation identified in a patient who developed FSGS in adulthood. By generating adult podocytes using patient-specific induced pluripotent stem cells (iPSC), we developed an in vitro model to dissect the role of this mutation in the onset of FSGS. Despite the PAX2 mutation, patient iPSC properly differentiated into podocytes that exhibited a normal structure and function when compared to control podocytes. However, when exposed to an environmental trigger, patient podocytes were less viable and more susceptible to cell injury. Fixing the mutation improved their phenotype and functionality. Using a branching morphogenesis assay, we documented developmental defects in patient-derived ureteric bud-like tubules that were totally rescued by fixing the mutation. These data strongly support the hypothesis that the PAX2 mutation has a dual effect, first in renal organogenesis, which could account for a suboptimal nephron number at birth, and second in adult podocytes, which are more susceptible to cell death caused by environmental triggers. These abnormalities might translate into the development of proteinuria in vivo, with a progressive decline in renal function, leading to FSGS.

Possibility of culturing the early developing kidney cells by utilizing simulated microgravity environment.

In recent years, the successful construction of tissues derived from established iPSCs has been disclosed, but it has been reported that the constructed tissues encounter problems of internal necrosis when their size increases. To solve this problem, a simulated microgravity device is used. However, the culture of early developing kidney cells using this device has not yet been reported. This study investigated whether developing kidney cells cultured in a simulated microgravity environment can differentiate into glomerular cells and renal epithelial cells. The results showed that both mouse developing kidney cells cultured in simulated microgravity and static environment formed kidney spheroids. In static culture, ureteric bud and glomerular structures were not found. While ureteric buds, podocytes, PECAM-1 positive cell aggregates, and primordial vascular plexus were formed in the kidney spheroids in simulated microgravity culture. Moreover, the expression level of the PECAM-1 gene was significant in simulated microgravity culture as compared to that of static culture. These results indicate that simulated microgravity is effective for the differentiation of developing kidney cells.

Ureteric Bud-derivatives in Wilms Tumor and Nephrogenic Rest.

BACKGROUND/AIM: Recent studies suggest that not only the nephrogenic blastema but also the ureteric bud is involved in oncogenesis of Wilms' tumor (WT). However, the occurrence of ureteric bud (UB) derivatives in nephrogenic rest is not yet known. The aim of our study was to find UB derivatives in WT. MATERIALS AND METHODS: Keratin 17 (KRT17) is expressed exclusively in UB in foetal kidneys. In this study KRT17 immunohistochemistry was used to detect UB-derivatives in 21 triphasic, 2 stromal and 3 epithelial predominant WTs and 9 nephrogenic rests. RESULTS: We have detected KRT17 positive tubular structures resembling UB in 3 of 9 nephrogenic rests and 15 of 26 WTs. CONCLUSION: Not only the metanephric blastema but also the UB is involved in the histogenesis of nephrogenic rest and WT.

Caspase-3 regulates ureteric branching in mice via cell migration.

Inhibition of caspase-3 (Casp3) reduces ureteric branching in organ culture but the mechanism remains unclear. Since Casp3 has non-apoptotic functions, we examined whether Casp3 regulates ureteric branching by promoting cell migration, using a ureteric bud (UB) cell line and Casp3-deficient (Casp3-/-) mice. Also, we examined whether Casp3 plays a role in the reduced ureteric branching of metanephroi from nutrient restricted mothers, in which Casp3 activity is suppressed. A Casp3 inhibitor Ac-DNLD-CHO reduced FGF2-induced cord formation of UB cells in 3D culture. UB cell migration assessed by Boyden chamber and wound healing assays was inhibited by Ac-DNLD-CHO. Glomerular number was reduced by ≈ 30%, and ureteric tip number was lower in Casp3-/- mice compared with controls. Maternal nutrient restriction decreased ureteric tip number in controls but not in Casp3-/-. In conclusion, Casp3 regulates ureteric branching by promoting UB cell migration. Inhibited ureteric branching by maternal nutrient restriction may be mediated by Casp3.

Development of the metanephric kidney.

The kidney plays an integral role in filtering the blood-removing metabolic by-products from the body and regulating blood pressure. This requires the establishment of large numbers of efficient and specialized blood filtering units (nephrons) that incorporate a system for vascular exchange and nutrient reabsorption as well as a collecting duct system to remove waste (urine) from the body. Kidney development is a dynamic process which generates these structures through a delicately balanced program of self-renewal and commitment of nephron progenitor cells that inhabit a constantly evolving cellular niche at the tips of a branching ureteric "tree." The former cells build the nephrons and the latter the collecting duct system. Maintaining these processes across fetal development is critical for establishing the normal "endowment" of nephrons in the kidney and perturbations to this process are associated both with mutations in integral genes and with alterations to the fetal environment.

iPSC technology-based regenerative medicine for kidney diseases.

With few curative treatments for kidney diseases, increasing attention has been paid to regenerative medicine as a new therapeutic option. Recent progress in kidney regeneration using human-induced pluripotent stem cells (hiPSCs) is noteworthy. Based on the knowledge of kidney development, the directed differentiation of hiPSCs into two embryonic kidney progenitors, nephron progenitor cells (NPCs) and ureteric bud (UB), has been established, enabling the generation of nephron and collecting duct organoids. Furthermore, human kidney tissues can be generated from these hiPSC-derived progenitors, in which NPC-derived glomeruli and renal tubules and UB-derived collecting ducts are interconnected. The induced kidney tissues are further vascularized when transplanted into immunodeficient mice. In addition to the kidney reconstruction for use in transplantation, it has been demonstrated that cell therapy using hiPSC-derived NPCs ameliorates acute kidney injury (AKI) in mice. Disease modeling and drug discovery research using disease-specific hiPSCs has also been vigorously conducted for intractable kidney disorders, such as autosomal dominant polycystic kidney disease (ADPKD). In an attempt to address the complications associated with kidney diseases, hiPSC-derived erythropoietin (EPO)-producing cells were successfully generated to discover drugs and develop cell therapy for renal anemia. This review summarizes the current status and future perspectives of developmental biology of kidney and iPSC technology-based regenerative medicine for kidney diseases.