Exploiting embryonic niche conditions to grow Wilms tumor blastema in culture.

Introduction: Wilms Tumor (WT), or nephroblastoma, is the most common pediatric kidney cancer. Most WTs display a "favorable" triphasic histology, in which the tumor is comprised of blastemal, stromal, and epithelial cell types. Blastemal predominance after neoadjuvant chemotherapy or diffuse anaplasia ("unfavorable" histology; 5-8%) portend a worse prognosis.

The International Society of Differentiation: Past, present, and future.

The International Society of Differentiation was born from the First International Conference on Cell Differentiation conceived by D.V. and held in Nice, France in 1971.

Fgf8 promotes survival of nephron progenitors by regulating BAX/BAK-mediated apoptosis.

Fibroblast growth factors (Fgfs) have long been implicated in processes critical to embryonic development, such as cell survival, migration, and differentiation. Several mouse models of organ development ascribe a prosurvival requirement specifically to FGF8. Here, we explore the potential role of prosurvival FGF8 signaling in kidney development.

Constitutive metanephric mesenchyme-specific expression of interferon-gamma causes renal dysplasia by regulating Sall1 expression.

Transplacental viral and parasitic infections have been shown to initiate an innate response in the mammalian embryo by increasing the expression of pro-inflammatory cytokines such as interferon-gamma (Ifng). However, the developmental consequences of an activated innate immunity and, in particular, the effects of induction of Ifng expression independent of infection have been largely overlooked. Here, we demonstrate in vivo that the conditional overexpression of Ifng in metanephric mesenchymal (MM) progenitors results in renal agenesis or hypoplasia.

Mesoderm-specific Stat3 deletion affects expression of Sox9 yielding Sox9-dependent phenotypes.

To date, mutations within the coding region and translocations around the SOX9 gene both constitute the majority of genetic lesions underpinning human campomelic dysplasia (CD). While pathological coding-region mutations typically result in a non-functional SOX9 protein, little is known about what mechanism(s) controls normal SOX9 expression, and subsequently, which signaling pathways may be interrupted by alterations occurring around the SOX9 gene. Here, we report the identification of Stat3 as a key modulator of Sox9 expression in nascent cartilage and developing chondrocytes.

Selective In Vitro Propagation of Nephron Progenitors Derived from Embryos and Pluripotent Stem Cells.

Nephron progenitors in the embryonic kidney propagate while generating differentiated nephrons. However, in mice, the progenitors terminally differentiate shortly after birth. Here, we report a method for selectively expanding nephron progenitors in vitro in an undifferentiated state.

Modeling renal progenitors - defining the niche.

Significant recent advances in methodologies for the differentiation of pluripotent stem cells to renal progenitors as well as the definition of niche conditions for sustaining those progenitors have dramatically enhanced our understanding of their biology and developmental programing, prerequisites for establishing viable approaches to renal regeneration. In this article, we review the evolution of culture techniques and models for the study of metanephric development, describe the signaling mechanisms likely to be driving progenitor self-renewal, and discuss current efforts to generate de novo functional tissues, providing in depth protocols and niche conditions for the stabilization of the nephronic Six2+progenitor.

Preferential Propagation of Competent SIX2+ Nephronic Progenitors by LIF/ROCKi Treatment of the Metanephric Mesenchyme.

Understanding the mechanisms responsible for nephrogenic stem cell preservation and commitment is fundamental to harnessing the potential of the metanephric mesenchyme (MM) for nephron regeneration. Accordingly, we established a culture model that preferentially expands the MM SIX2+ progenitor pool using leukemia inhibitory factor (LIF), a Rho kinase inhibitor (ROCKi), and extracellular matrix. Passaged MM cells express the key stem cell regulators Six2 and Pax2 and remain competent to respond to WNT4 induction and form mature tubular epithelia and glomeruli.

Continuous-flow sorting of stem cells and differentiation products based on dielectrophoresis.

This paper presents a continuous-flow microfluidic device for sorting stem cells and their differentiation progenies. The principle of the device is based on the accumulation of multiple dielectrophoresis (DEP) forces to deflect cells laterally in conjunction with the alternating on/off electric field to manipulate the cell trajectories. The microfluidic device containing a large array of oblique interdigitated electrodes was fabricated using a combination of standard and soft lithography techniques to generate a PDMS-gold electrode construct.

Non-canonical Wnt5a/Ror2 signaling regulates kidney morphogenesis by controlling intermediate mesoderm extension.

Congenital anomalies of the kidney and urinary tract (CAKUT) affect about 1 in 500 births and are a major cause of morbidity in infants. Duplex collecting systems rank among the most common abnormalities of CAKUT, but the molecular basis for this defect is poorly understood. In mice, conditional deletion of Wnt5a in mesoderm results in bilateral duplex kidney and ureter formation.

Sall1 maintains nephron progenitors and nascent nephrons by acting as both an activator and a repressor.

The balanced self-renewal and differentiation of nephron progenitors are critical for kidney development and controlled, in part, by the transcription factor Six2, which antagonizes canonical Wnt signaling-mediated differentiation. A nuclear factor, Sall1, is expressed in Six2-positive progenitors as well as differentiating nascent nephrons, and it is essential for kidney formation. However, the molecular functions and targets of Sall1, especially the functions and targets in the nephron progenitors, remain unknown.

CITED1 confers stemness to Wilms tumor and enhances tumorigenic responses when enriched in the nucleus.

Wilms tumor (WT) is the most common childhood kidney cancer and retains gene expression profiles reminiscent of the embryonic kidney. We have shown previously that CITED1, a transcriptional regulator that labels the self-renewing, multipotent nephron progenitor population of the developing kidney, is robustly expressed across all major WT disease and patient characteristics. In this malignant context, CITED1 becomes enriched in the nucleus, which deviates from its cytosolic predominance in embryonic nephron progenitors.

Protein/peptide transduction in metanephric explant culture.

While gene targeting methods have largely supplanted cell/explant culture models for studying developmental processes, they have not eliminated the need for or value of such approaches in the investigator's technical arsenal. Explant culture models, such as those devised for the metanephric kidney and its progenitors, remain invaluable as tools for screening regulatory factors involved in tissue induction or in the inhibition of progenitor specification. Thus, some factors capable of inducing tissue condensations or nephronic tubule formation in explants of metanephric mesenchyme have been identified through direct treatment of cultures rather than lengthy genetic engineering in animals.

Aberrant activation, nuclear localization, and phosphorylation of Yes-associated protein-1 in the embryonic kidney and Wilms tumor.

Background: The Yes-associated-protein-1 (YAP1) is a novel, direct regulator of stem cell genes both in development and cancer. FAT4 is an upstream regulator that induces YAP1 cytosolic sequestering by phosphorylation (p-Ser 127) and therefore inhibits YAP1-dependent cellular proliferation. We hypothesized that loss of FAT4 signaling would result in expansion of the nephron progenitor population in kidney development and that YAP1 subcellular localization would be dysregulated in Wilms tumor (WT), an embryonal malignancy that retains gene expression profiles and histologic features reminiscent of the embryonic kidney.

Stromal-epithelial crosstalk regulates kidney progenitor cell differentiation.

Present models suggest that the fate of the kidney epithelial progenitors is solely regulated by signals from the adjacent ureteric bud. The bud provides signals that regulate the survival, renewal and differentiation of these cells. Recent data suggest that Wnt9b, a ureteric-bud-derived factor, is sufficient for both progenitor cell renewal and differentiation.

CITED1 expression in liver development and hepatoblastoma.

Hepatoblastoma, the most common pediatric liver cancer, consists of epithelial mixed embryonal/fetal (EMEF) and pure fetal histologic subtypes, with the latter exhibiting a more favorable prognosis. Few embryonal histology markers that yield insight into the biologic basis for this prognostic discrepancy exist. CBP/P-300 interacting transactivator 1 (CITED1), a transcriptional co-activator, is expressed in the self-renewing nephron progenitor population of the developing kidney and broadly in its malignant analog, Wilms tumor (WT).

Wnt4 induces nephronic tubules in metanephric mesenchyme by a non-canonical mechanism.

Wnt4 and β-catenin are both required for nephrogenesis, but studies using TCF-reporter mice suggest that canonical Wnt signaling is not activated in metanephric mesenchyme (MM) during its conversion to the epithelia of the nephron. To better define the role of Wnt signaling, we treated rat metanephric mesenchymal progenitors directly with recombinant Wnt proteins. These studies revealed that Wnt4 protein, which is required for nephron formation, induces tubule formation and differentiation markers Lim1 and E-cadherin in MM cells, but does not activate a TCF reporter or up regulate expression of canonical Wnt target gene Axin-2 and has little effect on the stabilization of β-catenin or phosphorylation of disheveled-2.

Functional coupling between the extracellular matrix and nuclear lamina by Wnt signaling in progeria.

The segmental premature aging disease Hutchinson-Gilford Progeria (HGPS) is caused by a truncated and farnesylated form of Lamin A. In a mouse model for HGPS, a similar Lamin A variant causes the proliferative arrest and death of postnatal, but not embryonic, fibroblasts. Arrest is due to an inability to produce a functional extracellular matrix (ECM), because growth on normal ECM rescues proliferation.

STAT1 activation regulates proliferation and differentiation of renal progenitors.

We have shown previously that activation of STAT1 contributes to the pathogenesis of Wilms tumor. This neoplasm caricatures metanephric development and is believed to originate from embryonic renal mesenchymal progenitors that lose their ability to undergo mesenchymal-epithelial transition (MET). Therefore, we hypothesized that STAT1 is also activated and functional during metanephric development.

miR-23b targets proline oxidase, a novel tumor suppressor protein in renal cancer.

Proline oxidase (POX) is a novel mitochondrial tumor suppressor that can suppress proliferation and induce apoptosis through the generation of reactive oxygen species (ROS) and decreasing hypoxia-inducible factor (HIF) signaling. Recent studies have shown the absence of expression of POX in human cancer tissues, including renal cancer. However, the mechanism for the loss of POX remains obscure.