Benjamin Dekel

Mammalian body plan formation arises from the self-organization of pluripotent cells through conserved morphogenetic processes that are difficult to study in vivo. Stem cell–based embryo models (SEMs) offer accessible three-dimensional systems to investigate these events but are currently limited to mouse and human cells and largely recapitulate posterior embryonic structures. In addition, no in-vitro models exist for post-gastrulation development in ungulate species, whose early development differs from that of rodents and primates. Here, we establish SEMs for two common ungulates, sheep and pig, using pluripotent stem cell–derived aggregates. We generate ovine and porcine gastruloids that recapitulate key features of gastrulation, including germ layer specification, symmetry breaking, and axial elongation. We further develop ovine trunk-like structures (oTLSs) that robustly model post-gastrulation trunk development, exhibiting sustained elongation, neuromesodermal progenitor maintenance, segmented somite formation, and a central neural tube–like axis. Time-resolved single-cell RNA sequencing combined with immunostaining reveals coordinated emergence of neural, mesodermal, and intermediate mesodermal lineages arranged along an anteroposterior axis. Notably, oTLSs generate dorsal neural derivatives, anterior neuronal populations, and renal primordia, representing an expansion in the lineage repertoire reported for existing trunk models. Together, this work extends SEMs to livestock species and establishes a platform for comparative mammalian developmental studies, with potential applications in fundamental research, veterinary toxicology, and agricultural biotechnology.

Background: The nephron progenitor cells generate approximately one million nephrons during human nephrogenesis. At 34-36 weeks of human genstation, silencing of the key kidney progenitor genes results in depletion of this progenitor pool, limiting the regeneration capacity of the mature kidney. Concurrently, the increasing incidence of end-stage kidney disease underscores the urgent need for innovative regenerative strategies.Methods: We employed lentiviral vectors to ectopically induce two key kidney progenitor genes OSR1 and SIX2 individually or together in primary human adult kidney (hAK) cells. We then analyzed the cellular and molecular consequences through morphological assessments, functional assays, in vivo transplantation studies, and comprehensive transcriptional profiling.Results: OSR1 and SIX2 induced distinct reprogramming processes with differential functional outcomes; SIX2 overexpression was found to maintain epithelial morphology while significantly enhancing proliferation and clonogenic efficiency. Transcriptionally, SIX2 established epithelialization and cell-cycle networks by downregulating proximal tubule markers while upregulating distal nephron markers and proliferation genes. In vivo, SIX2-expressing cells formed organized tubular structures with a distinct luminal architecture in a proof-of-concept model. In contrast, OSR1 overexpression was found to induce morphological changes and activate developmental morphogenetic pathways, including epithelial tube morphogenesis and canonical Wnt signaling; however, it did not enhance proliferation and showed minimal tubulogenic capacity in vivo. Unexpectedly, OSR1 overexpression led to malignant transformation in one clone and exhibited Wilms’-tumor-like features, including expression of kidney developmental markers (i.e., SIX2, NCAM1, and WT1) and blastemal phenotype.Conclusion: Our findings suggest that SIX2 overexpression in primary hAK cells functionally confers enhanced self-renewal and tubulogenic capacity while transcriptionally inducing a proximal-to-distal tubular cell diversion with maintained proliferative programs. In contrast, OSR1 activates the broader developmental morphogenetic networks but poses potential oncogenic risks. The malignant transformation observed with OSR1 overexpression provides insights into the potential cellular origins of Wilms’ tumor and raises important safety considerations for regenerative medicine approaches involving developmental gene induction in adult kidney cells.

Frizzled 7 (FZD7), a Wnt receptor that activates canonical Wnt/β-catenin signaling, has been implicated in multiple cancers, including Wilms tumor (WT), the most common pediatric kidney malignancy. We previously identified FZD7 as a marker of the WT cancer stem cell population and a potential therapeutic target. To evaluate this, we generated a panel of monoclonal anti-FZD7 antibodies using epitope mapping of the receptor and assessed their functional activity in primary WT cells and xenograft models. Among the panel, clone 288.1 induced significant cell death in primary Wilms tumor cells and inhibited cell proliferation and migration. This effect correlated with canonical Wnt signaling inhibition, a reduction in activated β-catenin and downregulation of Wnt/β-catenin target genes concomitant with diminished Wilms tumor cancer stem cell (CSC) markers. In vivo, treatment with anti-FZD7-288.1 significantly inhibited WT xenograft growth, resulting in reduced tumor volume. These findings demonstrate that FZD7 is a critical driver of Wilms tumor progression and support antibody-mediated FZD7 blockade as a promising therapeutic strategy.

Pluripotent stem cell (PSC)–derived kidney organoids are used to model human renal development and disease; however, accessible models of human fetal development to benchmark PSC-derived organoids remain underdeveloped. Here, we establish a chemically defined, serum-free protocol for prolonged culture of human fetal kidney-derived organoids (hFKOs) in vitro. hFKOs self-organize into polarized renal epithelium, reinitiate from NCAM1 ⁺ progenitors, and recapitulate nephrogenic and ureteric bud lineages. Bulk transcriptomics, single-cell RNA sequencing, pseudotime analysis, and immunostaining revealed diverse renal tissue cell populations, with a preserved epithelial progenitor pool and tubular differentiation axis. hFKOs were enriched for Notch signaling genes, enabling single-cell analysis of pharmacological Notch inhibition. This revealed a maturation block with increased nephron progenitors and a shift toward distal over early proximal tubule fates. We also identified a novel prominin-1-expressing cell state that evades Notch inhibition to generate both proximal and distal tubules. Overall, hFKOs provide a faithful model to gain insights into human kidney development, advancing the fields of stem cell biology and regenerative medicine.

Wilms’ tumors are pediatric malignancies that are thought to arise from faulty kidney development. They contain a wide range of poorly differentiated cell states resembling various distorted developmental stages of the fetal kidney, and as a result, differ between patients in a continuous manner that is not well understood. Here, we used three computational approaches to characterize this continuous heterogeneity in high-risk blastemal-type Wilms’ tumors. Using Pareto task inference, we show that the tumors form a triangle-shaped continuum in latent space that is bounded by three tumor archetypes with “stromal”, “blastemal”, and “epithelial” characteristics, which resemble the un-induced mesenchyme, the cap mesenchyme, and early epithelial structures of the fetal kidney. By fitting a generative probabilistic “grade of membership” model, we show that each tumor can be represented as a unique mixture of three hidden “topics” with blastemal, stromal, and epithelial characteristics. Likewise, cellular deconvolution allows us to represent each tumor in the continuum as a unique combination of fetal kidney-like cell states. These results highlight the relationship between Wilms’ tumors and kidney development, and we anticipate that they will pave the way for more quantitative strategies for tumor stratification and classification.

Significance Statement: Coronavirus disease 2019 (COVID-19) commonly results in AKI, but it is unknown whether AKI in patients with COVID-19 stems from direct kidney infection or indirect mechanisms. The authors explored the effects of infection with the causative virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), both in monolayers of primary human kidney cells and in kidney spheroids, a three-dimensional model recapitulating human kidneys in cell composition and function. They demonstrated that the virus effectively infects and replicates in human tubular epithelial cells, but does not cause cytopathic effects. They also provide molecular evidence that activation of a type 1 IFN response may be the underlying mechanism of resistance to SARS-CoV-2–related cytopathic damage. In all, the findings indicate minimal—if any—direct tubular damage by SARS-CoV-2. Background: Although coronavirus disease 2019 (COVID-19) causes significan t morbidity, mainly from pulmonary involvement, extrapulmonary symptoms are also major componen ts of the disease. Kidney disease, usually presenting as AKI, is particularly severe among patients with COVID-19. It is unknown, however, whether such injury results from direct kidney infection with COVID-19’s causative virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), or from indirect mechanisms. Methods: Using ex vivo cell models, we sought to analyze SARS-CoV-2 interactions with kidney tubular cells and assess direct tubular injury. These models comprised primary human kidney epithelial cells (derived from nephrectomies) and grown as either proliferating monolayers or quiescent three-dimensional kidney spheroids. Results: We demonstrated that viral entry molecules and high baseline levels of type 1 IFN–related molecules were present in monolayers and kidney spheroids. Although both models support viral infection and replication, they did not exhibit a cytopathic effect and cell death, outcomes that were strongly present in SARS-CoV-2–infected controls (African green monkey kidney clone E6 [Vero E6] cultures). A comparison of monolayer and spheroid cultures demonstrated higher infectivity and replication of SARS-CoV-2 in actively proliferating monolayers, although the spheroid cultures exhibited high er levels of ACE2. Monolayers exhibited elevation of some tubular injury molecules—including molecules related to fibrosis (COL1A1 and STAT6) and dedifferentiation (SNAI2)—and a loss of cell identity, evident by reduction in megalin (LRP2). The three-dimensional spheroids were less prone to such injury. Conclusions: SARS-CoV-2 can infect kidney cells without a cytopathic effect. AKI-induced cellular proliferation may potentially intensify infectivity and tubular damage by SARS-CoV-2, suggesting that early intervention in AKI is warranted to help minimize kidney infection.

The worldwide rise in prevalence of chronic kidney disease (CKD) demands innovative bio-medical solutions for millions of kidney patients. Kidney regenerative medicine aims to replenish tissue which is lost due to a common pathological pathway of fibrosis/inflammation and rejuvenate remaining tissue to maintain sufficient kidney function. To this end, cellular therapy strategies devised so far utilize kidney tissue-forming cells (KTFCs) from various cell sources, fetal, adult, and pluripotent stem-cells (PSCs). However, to increase engraftment and potency of the transplanted cells in a harsh hypoxic diseased environment, it is of importance to co-transplant KTFCs with vessel forming cells (VFCs). VFCs, consisting of endothelial cells (ECs) and mesenchymal stem-cells (MSCs), synergize to generate stable blood vessels, facilitating the vascularization of self-organizing KTFCs into renovascular units. In this paper, we review the different sources of KTFCs and VFCs which can be mixed, and report recent advances made in the field of kidney regeneration with emphasis on generation of vascularized kidney tissue by cell transplantation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.