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.

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.

OBJECTIVE: 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.

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.

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.

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.

Sertoli cells (SCs) in the mammalian testes are well known as supporting cells of spermatogenesis, but have recently become an attractive source of cell therapy because of their capacity for immune modulation and trophic effects. In order to increase their applicable efficacy, we demonstrate a novel differentiation method for mouse embryonic stem cell (ESC)-derived Sertoli-like cells (SLCs) via the intermediate mesoderm (IM). We show that IM derived from an induction of 6 days expressed markers such as Wt1, Lhx1, Pax2 and Osr1, and that a sequential induction of 6 days resulted in ESC-SLCs. The SLCs expressed their marker genes ( Sf1, Sox9, Gata4, Wt1, Fshr and Scf), but the pluripotency-marker gene Oct4 was decreased. After sorting by FSHR expression, high-purity (> 90%) SLCs were collected that showed distinct characteristics of SCs such as high phagocytic and immune modulation activities as well as the expression of immune-related genes. In addition, when transplanted into the seminiferous tubule of busulfan-treated mice, SLCs re-located and were maintained in the basal region of the tubule. These results demonstrated that our robust sequential differentiation system produced functional SLCs from mouse ESCs in vitro.

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.

The kidney is a complex organ whose excretory and regulatory functions are vital for maintaining homeostasis. Previous techniques used to study the kidney, including various animal models and 2D cell culture systems to investigate the mechanisms of renal development and regeneration have many benefits but also possess inherent shortcomings. Some of those limitations can be addressed using the emerging technology of 3D organoids. An organoid is a 3D cluster of differentiated cells that are developed ex vivo by addition of various growth factors that result in a miniature organ containing structures present in the tissue of origin. Here, we discuss renal organoids, their development, and how they can be employed to further understand kidney development and disease.

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.