osr1 couples intermediate mesoderm cell fate with temporal dynamics of vessel progenitor cell differentiation.

Transcriptional regulatory networks refine gene expression boundaries to define the dimensions of organ progenitor territories. Kidney progenitors originate within the intermediate mesoderm (IM), but the pathways that establish the boundary between the IM and neighboring vessel progenitors are poorly understood. Here, we delineate roles for the zinc-finger transcription factor Osr1 in kidney and vessel progenitor development. Zebrafish osr1 mutants display decreased IM formation and premature emergence of lateral vessel progenitors (LVPs). These phenotypes contrast with the increased IM and absent LVPs observed with loss of the bHLH transcription factor Hand2, and loss of hand2 partially suppresses osr1 mutant phenotypes. hand2 and osr1 are expressed together in the posterior mesoderm, but osr1 expression decreases dramatically prior to LVP emergence. Overexpressing osr1 during this timeframe inhibits LVP development while enhancing IM formation, and can rescue the osr1 mutant phenotype. Together, our data demonstrate that osr1 modulates the extent of IM formation and the temporal dynamics of LVP development, suggesting that a balance between levels of osr1 and hand2 expression is essential to demarcate the kidney and vessel progenitor territories.

Embryonic developmental process and clinical anatomy of the preperitoneal fascia and its clinical significance.

PURPOSE: Many researchers have different views on the origin and anatomy of the preperitoneal fascia. The purpose of this study is to review studies on the anatomy related to the preperitoneal fascia and to investigate the origin, structure, and clinical significance of the preperitoneal fascia in conjunction with previous anatomical findings of the genitourinary fascia, using the embryogenesis of the genitourinary system as a guide. METHODS: Publications on the preperitoneal and genitourinary fascia are reviewed, with emphasis on the anatomy of the preperitoneal fascia and its relationship to the embryonic development of the genitourinary organs. We also describe previous anatomical studies of the genitourinary fascia in the inguinal region through the fixation of formalin-fixed cadavers. RESULTS: Published literature on the origin, structure, and distribution of the preperitoneal fascia is sometimes inconsistent. However, studies on the urogenital fascia provide more than sufficient evidence that the formation of the preperitoneal fascia is closely related to the embryonic development of the urogenital fascia and its tegument. Combined with previous anatomical studies of the genitourinary fascia in the inguinal region of formalin-fixed cadavers showed that there is a complete fascial system. This fascial system moves from the retroperitoneum to the anterior peritoneum as the preperitoneal fascia. CONCLUSIONS: We can assume that the preperitoneal fascia (PPF) is continuous with the retroperitoneal renal fascia, ureter and its accessory vessels, lymphatic vessels, peritoneum of the bladder, internal spermatic fascia, and other peritoneal and pelvic urogenital organ surfaces, which means that the urogenital fascia (UGF) is a complete fascial system, which migrates into PPF in the preperitoneal space and the internal spermatic fascia in the inguinal canal.

Turning mesoderm into kidney.

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.

Programming of ES cells and reprogramming of fibroblasts into renal lineage-like cells.

This study aims to prepare intermediate mesoderm-like cells from mouse embryonic fibroblasts (MEFs). In the first step, intermediate mesoderm-like cells (IMLCs) and renal epithelial-like cells (RELCs) were extracted from mouse embryonic stem cells (mESCs) in a specified media that contained two small molecules, CHIR99021 and TTNPB, along with growth factors, FGF9and BMP7. Then, MEFs were directly converted into IM by genes for the pluripotency factors, which encode the transcription factors; Oct4, Sox2, Klf4, and c-Myc (OSKM). These unstable intermediate cells were quickly encouraged to form IM with the assistance of CHIR99021 and TTNPB. The results showed that exogenous expression of OSKM factors for four days was adequate to generate partially reprogrammed cells (SSEA1+/Nanog-). Real-time PCR and immunocytochemistry analysis confirmed the presence of the MEF-derived IMs. This study introduced a method for mESCs differentiation to RELCs followed by MEF conversion in an attempt to generate IM by circumventing pluripotency.

Wnt8a expands the pool of embryonic kidney progenitors in zebrafish.

During zebrafish embryogenesis the pronephric kidney arises from a small population of posterior mesoderm cells that then undergo expansion during early stages of renal organogenesis. While wnt8 is required for posterior mesoderm formation during gastrulation, it is also transiently expressed in the post-gastrula embryo in the intermediate mesoderm, the precursor to the pronephros and some blood/vascular lineages. Here, we show that knockdown of wnt8a, using a low dose of morpholino that does not disrupt early mesoderm patterning, reduces the number of kidney and blood cells. For the kidney, wnt8a deficiency decreases renal progenitor growth during early somitogenesis, as detected by EdU incorporation, but has no effect on apoptosis. The depletion of the renal progenitor pool in wnt8a knockdown embryos leads to cellular deficits in the pronephros at 24 hpf that are characterised by a shortened distal-most segment and stretched proximal tubule cells. A pulse of the canonical Wnt pathway agonist BIO during early somitogenesis is sufficient to rescue the size of the renal progenitor pool while longer treatment expands the number of kidney cells. Taken together, these observations indicate that Wnt8, in addition to its well-established role in posterior mesoderm patterning, also plays a later role as a factor that expands the renal progenitor pool prior to kidney morphogenesis.

Generation of branching ureteric bud tissues from human pluripotent stem cells.

Recent progress in kidney regeneration research is noteworthy. However, the selective and robust differentiation of the ureteric bud (UB), an embryonic renal progenitor, from human pluripotent stem cells (hPSCs) remains to be established. The present study aimed to establish a robust induction method for branching UB tissue from hPSCs towards the creation of renal disease models. Here, we found that anterior intermediate mesoderm (IM) differentiates from anterior primitive streak, which allowed us to successfully develop an efficient two-dimensional differentiation method of hPSCs into Wolffian duct (WD) cells. We also established a simplified procedure to generate three-dimensional WD epithelial structures that can form branching UB tissues. This system may contribute to hPSC-based regenerative therapies and disease models for intractable disorders arising in the kidney and lower urinary tract.

Pronephric tubule morphogenesis in zebrafish depends on Mnx mediated repression of irx1b within the intermediate mesoderm.

Mutations in the homeobox transcription factor MNX1 are the major cause of dominantly inherited sacral agenesis. Studies in model organisms revealed conserved mnx gene requirements in neuronal and pancreatic development while Mnx activities that could explain the caudal mesoderm specific agenesis phenotype remain elusive. Here we use the zebrafish pronephros as a simple yet genetically conserved model for kidney formation to uncover a novel role of Mnx factors in nephron morphogenesis. Pronephros formation can formally be divided in four stages, the specification of nephric mesoderm from the intermediate mesoderm (IM), growth and epithelialisation, segmentation and formation of the glomerular capillary tuft. Two of the three mnx genes in zebrafish are dynamically transcribed in caudal IM in a time window that proceeds segmentation. We show that expression of one mnx gene, mnx2b, is restricted to the pronephric lineage and that mnx2b knock-down causes proximal pronephric tubule dilation and impaired pronephric excretion. Using expression profiling of embryos transgenic for conditional activation and repression of Mnx regulated genes, we further identified irx1b as a direct target of Mnx factors. Consistent with a repression of irx1b by Mnx factors, the transcripts of irx1b and mnx genes are found in mutual exclusive regions in the IM, and blocking of Mnx functions results in a caudal expansion of the IM-specific irx1b expression. Finally, we find that knock-down of irx1b is sufficient to rescue proximal pronephric tubule dilation and impaired nephron function in mnx-morpholino injected embryos. Our data revealed a first caudal mesoderm specific requirement of Mnx factors in a non-human system and they demonstrate that Mnx-dependent restriction of IM-specific irx1b activation is required for the morphogenesis and function of the zebrafish pronephros.

Evidence for intermediate mesoderm and kidney progenitor cell specification by Pax2 and PTIP dependent mechanisms.

Activation of the Pax2 gene marks the intermediate mesoderm shortly after gastrulation, as the mesoderm becomes compartmentalized into paraxial, intermediate, and lateral plate. Using an EGFP knock-in allele of Pax2 to identify and sort cells of the intermediate mesodermal lineage, we compared gene expression patterns in EGFP positive cells that were heterozygous or homozygous null for Pax2. Thus, we identified critical regulators of intermediate mesoderm and kidney development whose expression depended on Pax2 function. In cell culture models, Pax2 is thought to recruit epigenetic modifying complex to imprint activating histone methylation marks through interactions with the adaptor protein PTIP. In kidney organ culture, conditional PTIP deletion showed that many Pax2 target genes, which were activated early in renal progenitor cells, remained on once activated, whereas Pax2 target genes expressed later in kidney development were unable to be fully activated without PTIP. In Pax2 mutants, we also identified a set of genes whose expression was up-regulated in EGFP positive cells and whose expression was consistent with a cell fate transformation to paraxial mesoderm and its derivatives. These data provide evidence that Pax2 specifies the intermediate mesoderm and renal epithelial cells through epigenetic mechanisms and in part by repressing paraxial mesodermal fate.

Midline-derived Shh regulates mesonephric tubule formation through the paraxial mesoderm.

During organogenesis, Sonic hedgehog (Shh) possesses dual functions: Shh emanating from midline structures regulates the positioning of bilateral structures at early stages, whereas organ-specific Shh locally regulates organ morphogenesis at later stages. The mesonephros is a transient embryonic kidney in amniote, whereas it becomes definitive adult kidney in some anamniotes. Thus, elucidating the regulation of mesonephros formation has important implications for our understanding of kidney development and evolution. In Shh knockout (KO) mutant mice, the mesonephros was displaced towards the midline and ectopic mesonephric tubules (MTs) were present in the caudal mesonephros. Mesonephros-specific ablation of Shh in Hoxb7-Cre;Shh(flox/-) and Sall1(CreERT2/+);Shh(flox/-) mice embryos indicated that Shh expressed in the mesonephros was not required for either the development of the mesonephros or the differentiation of the male reproductive tract. Moreover, stage-specific ablation of Shh in Shh(CreERT2/flox) mice showed that notochord- and/or floor plate-derived Shh were essential for the regulation of the number and position of MTs. Lineage analysis of hedgehog (Hh)-responsive cells, and analysis of gene expression in Shh KO embryos suggested that Shh regulated nephrogenic gene expression indirectly, possibly through effects on the paraxial mesoderm. These data demonstrate the essential role of midline-derived Shh in local tissue morphogenesis and differentiation.

Zebrafish nephrogenesis is regulated by interactions between retinoic acid, mecom, and Notch signaling.

The zebrafish pronephros provides a conserved model to study kidney development, in particular to delineate the poorly understood processes of how nephron segment pattern and cell type choice are established. Zebrafish nephrons are divided into distinct epithelial regions that include a series of proximal and distal tubule segments, which are comprised of intercalated transporting epithelial cells and multiciliated cells (MCC). Previous studies have shown that retinoic acid (RA) regionalizes the renal progenitor field into proximal and distal domains and that Notch signaling later represses MCC differentiation, but further understanding of these pathways has remained unknown. The transcription factor mecom (mds1/evi1 complex) is broadly expressed in renal progenitors, and then subsequently marks the distal tubule. Here, we show that mecom is necessary to form the distal tubule and to restrict both proximal tubule formation and MCC fate choice. We found that mecom and RA have opposing roles in patterning discrete proximal and distal segments. Further, we discovered that RA is required for MCC formation, and that one mechanism by which RA promotes MCC fate choice is to inhibit mecom. Next, we determined the epistatic relationship between mecom and Notch signaling, which limits MCC fate choice by lateral inhibition. Abrogation of Notch signaling with the γ-secretase inhibitor DAPT revealed that Notch and mecom did not have additive effects in blocking MCC formation, suggesting that they function in the same pathway. Ectopic expression of the Notch signaling effector, Notch intracellular domain (NICD), rescued the expansion of MCCs in mecom morphants, indicating that mecom acts upstream to induce Notch signaling. These findings suggest a model in which mecom and RA arbitrate proximodistal segment domains, while MCC fate is modulated by a complex interplay in which RA inhibition of mecom, and mecom promotion of Notch, titrates MCC number. Taken together, our studies have revealed several essential and novel mechanisms that control pronephros development in the zebrafish.

Zebrafish Tbx16 regulates intermediate mesoderm cell fate by attenuating Fgf activity.

Progenitors of the zebrafish pronephros, red blood and trunk endothelium all originate from the ventral mesoderm and often share lineage with one another, suggesting that their initial patterning is linked. Previous studies have shown that spadetail (spt) mutant embryos, defective in tbx16 gene function, fail to produce red blood cells, but retain the normal number of endothelial and pronephric cells. We report here that spt mutants are deficient in all the types of early blood, have fewer endothelial cells as well as far more pronephric cells compared to wildtype. In vivo cell tracing experiments reveal that blood and endothelium originate in spt mutants almost exclusive from the dorsal mesoderm whereas, pronephros and tail originate from both dorsal and ventral mesoderm. Together these findings suggest possible defects in posterior patterning. In accord with this, gene expression analysis shows that mesodermal derivatives within the trunk and tail of spt mutants have acquired more posterior identity. Secreted signaling molecules belonging to the Fgf, Wnt and Bmp families have been implicated as patterning factors of the posterior mesoderm. Further investigation demonstrates that Fgf and Wnt signaling are elevated throughout the nonaxial region of the spt gastrula. By manipulating Fgf signaling we show that Fgfs both promote pronephric fate and repress blood and endothelial fate. We conclude that Tbx16 plays an important role in regulating the balance of intermediate mesoderm fates by attenuating Fgf activity.

Development of somites, muscle, and skeleton is independent of signals from the Wolffian duct.

BACKGROUND: In the vertebrate embryo, skeletal muscle and the axial skeleton arise from the somites. Patterning of the somites into the respective somite compartments, namely dermomyotome, myotome, and sclerotome, depends on molecular signals from neighboring structures, including surface ectoderm, neural tube, notochord, and lateral plate mesoderm. A potential role of the intermediate mesoderm, notably the Wolffian or nephric duct, in somite development is poorly understood. RESULTS: We studied somite compartmentalization as well as muscular and skeletal development after surgical ablation of the early Wolffian duct anlage, which lead to loss of the Wolffian duct and absence of the mesonephros, whereas Pax2 expression in the nephrogenic mesenchyme was temporarily maintained. We show that somite compartments, as well as the somite derivatives, skeletal muscle and the cartilaginous skeleton, develop normally in the absence of the Wolffian duct. CONCLUSIONS: Our results indicate that development of the musculoskeletal system is independent of the Wolffian duct as a signaling center.

Optimizing Nodal, Wnt and BMP signaling pathways for robust and efficient differentiation of human induced pluripotent stem cells to intermediate mesoderm cells.

Several differentiation protocols have enabled the generation of intermediate mesoderm (IM)-derived cells from human pluripotent stem cells (hPSC). However, the substantial variability between existing protocols for generating IM cells compromises their efficiency, reproducibility, and overall success, potentially hindering the utility of urogenital system organoids. Here, we examined the role of high levels of Nodal signaling and BMP activity, as well as WNT signaling in the specification of IM cells derived from a UCSD167i-99-1 human induced pluripotent stem cells (hiPSC) line. We demonstrate that precise modulation of WNT and BMP signaling significantly enhances IM differentiation efficiency. Treatment of hPSC with 3 µM CHIR99021 induced TBXT+/MIXL1+ mesoderm progenitor (MP) cells after 48 h of differentiation. Further treatment with a combination of 3 µM CHIR99021 and 4 ng/mL BMP4 resulted in the generation of OSR1+/GATA3+/PAX2+ IM cells within a subsequent 48 h period. Molecular characterization of differentiated cells was confirmed through immunofluorescence staining and RT-qPCR. Hence, this study establishes a consistent and reproducible protocol for differentiating hiPSC into IM cells that faithfully recapitulates the molecular signatures of IM development. This protocol holds promise for improving the success of protocols designed to generate urogenital system organoids in vitro, with potential applications in regenerative medicine, drug discovery, and disease modeling.

Kidney tissue regeneration using bioactive scaffolds incorporated with differentiating extracellular vesicles and intermediate mesoderm cells.

BACKGROUND: To overcome the limitations of current alternative therapies for chronic kidney disease (CKD), tissue engineering-mediated regeneration strategies have demonstrated the possibilities for complete kidney tissue regeneration. Given the challenges associated with the reproducibility of renal basal cells, the incorporation of intermediate mesoderm (IM) cells and bioactive materials to control bioactivities of cells with supported scaffolds should be considered as a viable approach to enable the regeneration of the complex kidney structure via renal differentiation. METHODS: We developed PMEZ scaffolds by combining crucial bioactive components, such as ricinoleic acid-grafted Mg(OH)2 (M), extracellular matrix (E), and alpha lipoic acid-conjugated ZnO (Z) integrated into biodegradable porous PLGA (P) platform. Additionally, we utilized differentiating extracellular vesicles (dEV) isolated during intermediate mesoderm differentiation into kidney progenitor cells, and IM cells were serially incorporated to facilitate kidney tissue regeneration through their differentiation into kidney progenitor cells in the 3/4 nephrectomy mouse model. RESULTS: The use of differentiating extracellular vesicles facilitated IM differentiation into kidney progenitor cells without additional differentiation factors. This led to improvements in various regeneration-related bioactivities including tubule and podocyte regeneration, anti-fibrosis, angiogenesis, and anti-inflammation. Finally, implanting PMEZ/dEV/IM scaffolds in mouse injury model resulted in the restoration of kidney function. CONCLUSIONS: Our study has demonstrated that utilizing biodegradable PLGA-based scaffolds, which include multipotent cells capable of differentiating into various kidney progenitor cells along with supporting components, can facilitate kidney tissue regeneration in the mouse model that simulates CKD through 3/4 nephrectomy.

Generation of mitochondria-rich kidney organoids from expandable intermediate mesoderm progenitors reprogrammed from human urine cells under defined medium.

BACKGROUND: The kidneys require vast amounts of mitochondria to provide ample energy to reabsorb nutrients and regulate electrolyte, fluid, and blood pressure homeostasis. The lack of the human model hinders the investigation of mitochondria homeostasis related to kidney physiology and disease. RESULTS: Here, we report the generation of mitochondria-rich kidney organoids via partial reprogramming of human urine cells (hUCs) under the defined medium. First, we reprogrammed mitochondria-rich hUCs into expandable intermediate mesoderm progenitor like cells (U-iIMPLCs), which in turn generated nephron progenitors and formed kidney organoids in both 2D and 3D cultures. Cell fate transitions were confirmed at each stage by marker expressions at the RNA and protein levels, along with chromatin accessibility dynamics. Single cell RNA-seq revealed hUCs-induced kidney organoids (U-iKOs) consist of podocytes, tubules, and mesenchyme cells with 2D dominated with mesenchyme and 3D with tubule and enriched specific mitochondria function associated genes. Specific cell types, such as podocytes and proximal tubules, loop of Henle, and distal tubules, were readily identified. Consistent with these cell types, 3D organoids exhibited the functional and structural features of the kidney, as indicated by dextran uptake and transmission electron microscopy. These organoids can be further matured in the chick chorioallantoic membrane. Finally, cisplatin, gentamicin, and forskolin treatment led to anatomical abnormalities typical of kidney injury and altered mitochondria homeostasis respectively. CONCLUSIONS: Our study demonstrates that U-iKOs recapitulate the structural and functional characteristics of the kidneys, providing a promising model to study mitochondria-related kidney physiology and disease in a personalized manner.

Reconstitution of human adrenocortical specification and steroidogenesis using induced pluripotent stem cells.

The mechanisms leading to adrenal cortex development and steroid synthesis in humans remain poorly understood due to the paucity of model systems. Herein, we recapitulate human fetal adrenal cortex specification processes through stepwise induction of human-induced pluripotent stem cells through posterior intermediate mesoderm-like and adrenocortical progenitor-like states to ultimately generate fetal zone adrenal-cortex-like cells (FZLCs), as evidenced by histomorphological, ultrastructural, and transcriptome features and adrenocorticotropic hormone (ACTH)-independent Δ5 steroid biosynthesis. Furthermore, FZLC generation is promoted by SHH and inhibited by NOTCH, ACTIVIN, and WNT signaling, and steroid synthesis is amplified by ACTH/PKA signaling and blocked by inhibitors of Δ5 steroid synthesis enzymes. Finally, NR5A1 promotes FZLC survival and steroidogenesis. Together, these findings provide a framework for understanding and reconstituting human adrenocortical development in vitro, paving the way for cell-based therapies of adrenal insufficiency.

The embryonic ontogeny of the gonadal somatic cells in mice and monkeys.

In the early fetal stage, the gonads are bipotent and only later become the ovary or testis, depending on the genetic sex. Despite many studies examining how sex determination occurs from biopotential gonads, the spatial and temporal organization of bipotential gonads and their progenitors is poorly understood. Here, using lineage tracing in mice, we find that the gonads originate from a T+ primitive streak through WT1+ posterior intermediate mesoderm and appear to share origins anteriorly with the adrenal glands and posteriorly with the metanephric mesenchyme. Comparative single-cell transcriptomic analyses in mouse and cynomolgus monkey embryos reveal the convergence of the lineage trajectory and genetic programs accompanying the specification of biopotential gonadal progenitor cells. This process involves sustained expression of epithelial genes and upregulation of mesenchymal genes, thereby conferring an epithelial-mesenchymal hybrid state. Our study provides key resources for understanding early gonadogenesis in mice and primates.

Directed Differentiation of Human Pluripotent Stem Cells for the Generation of High-Order Kidney Organoids.

Our understanding in the inherent properties of human pluripotent stem cells (hPSCs) have made possible the development of differentiation procedures to generate three-dimensional tissue-like cultures, so-called organoids. Here we detail a stepwise methodology to generate kidney organoids from hPSCs. This is achieved through direct differentiation of hPSCs in two-dimensional monolayer culture toward the posterior primitive streak fate, followed by induction of intermediate mesoderm-committed cells, which are further aggregated and cultured in three-dimensions to generate kidney organoids containing segmented nephron-like structures in a process that lasts 20 days. We also provide a concise description on how to assess renal commitment during the time course of kidney organoid generation. This includes the use of flow cytometry and immunocytochemistry analyses for the detection of specific renal differentiation markers.

Protocol to Generate Ureteric Bud Structures from Human iPS Cells.

The generation of ureteric bud (UB), which is the renal progenitor that gives rise to renal collecting ducts and lower urinary tract, from human-induced pluripotent stem cells (hiPSCs) provides a cell source for studying the development of UB and kidney disease. Here we describe a stepwise and efficient two-dimensional differentiation method of hiPSCs into Wolffian duct (WD) cells. We also describe how to generate three-dimensional WD epithelial structures that can differentiate into UB-like structures.

Induced Intermediate Mesoderm Combined with Decellularized Kidney Scaffolds for Functional Engineering Kidney.

Background: Chronic kidney disease is a severe threat to human health with no ideal treatment strategy. Mature mammalian kidneys have a fixed number of nephrons, and regeneration is difficult once they are damaged. For this reason, developing an efficient approach to achieve kidney regeneration is necessary. The technology of the combination of decellularized kidney scaffolds with stem cells has emerged as a new strategy; however, in previous studies, the differentiation of stem cells in decellularized scaffolds was insufficient for functional kidney regeneration, and many problems remain. Methods: We used 0.5% sodium dodecyl sulfate (SDS) to produce rat kidney decellularized scaffolds, and induce adipose-derived stem cells (ADSCs) into intermediate mesoderm by adding Wnt agonist CHIR99021 and FGF9 in vitro. The characteristics of decellularized scaffolds and intermediate mesoderm induced from adipose-derived stem cells were identified. The scaffolds were recellularized with ADSCs and intermediate mesoderm cells through the renal artery and ureter. After cocultured for 10 days, cells adhesion and differentiation was evaluated. Results: Intermediate mesoderm cells were successfully induced from ADSCs and identified by immunofluorescence and Western blotting assays (OSR1 + , PAX2 +). Immunofluorescence showed that intermediate mesoderm cells differentiated into tubular-like (E-CAD + , GATA3 +) and podocyte-like (WT1 +) cells with higher differentiation efficiency than ADSCs in the decellularized scaffolds. Comparatively, this phenomenon was not observed in induced intermediate mesoderm cells cultured in vitro. Conclusion: In this study, we demonstrated that intermediate mesoderm cells could be induced from ADSCs and that they could differentiate well after cocultured with decellularized scaffolds.