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Several Wnt proteins are expressed in the embryonic kidney during various stages of development. Gene knockout models and ex vivo studies have provided strong evidence that Wnt-mediated signals are essential in renal ontogeny. Perhaps the most critical factors, Wnt9b and Wnt4, function during the early phase when the cap mesenchyme is induced to undergo morphogenesis into a nephron. Wnt11 controls early ureteric bud branching and contributes to the final kidney size. In addition to its inductive role, later on Wnt9b plays a significant role in the convergent extension of the tubular epithelial cells, while Wnt4 signaling controls smooth muscle cell fates in the medulla. Wnt7b has a specific function together with its likely antagonist Dkk1 in controlling the morphogenesis of the renal medulla. The signal-transduction mechanisms of the Wnts in kidney ontogeny have not been resolved, but studies characterizing the downstream signaling pathways are emerging. Aberrant Wnt signaling may lead to kidney diseases ranging from fatal kidney agenesis to more benign phenotypes. Wnt-mediated signaling regulates several critical aspects of kidney development from the early inductive stages to later steps of tubular epithelial maturation.

Extracellular vesicles (EVs) have recently attracted a great deal of interest as they may represent a new biosignaling paradigm. According to the mode of biogenesis, size and composition, two broad categories of EVs have been described, exosomes and microvesicles. EVs have been shown to carry cargoes of signaling proteins, RNA species, DNA and lipids. Once released, their content is selectively taken up by near or distant target cells, influencing their behavior. Exosomes are involved in cell–cell communication in a wide range of embryonic developmental processes and in fetal–maternal communication. In the present review, an outline of the role of EVs in neural development, regeneration and diseases is presented. EVs can act as regulators of normal homeostasis, but they can also promote either neuroinflammation/degeneration or tissue repair in pathological conditions, depending on their content. Since EV molecular cargo constitutes a representation of the origin cell status, EVs can be exploited in the diagnosis of several diseases. Due to their capability to cross the blood–brain barrier (BBB), EVs not only have been suggested for the diagnosis of central nervous system disorders by means of minimally invasive procedures, i.e., “liquid biopsies”, but they are also considered attractive tools for targeted drug delivery across the BBB. From the therapeutic perspective, mesenchymal stem cells (MSCs) represent one of the most promising sources of EVs. In particular, the neuroprotective properties of MSCs derived from the dental pulp are here discussed.

Background The human sweat is a mixture of secretions from three types of glands: eccrine, apocrine, and sebaceous. Eccrine glands open directly on the skin surface and produce high amounts of water-based fluid in response to heat, emotion, and physical activity, whereas the other glands produce oily fluids and waxy sebum. While most body fluids have been shown to contain nucleic acids, both as ribonucleoprotein complexes and associated with extracellular vesicles (EVs), these have not been investigated in sweat. In this study we aimed to explore and characterize the nucleic acids associated with sweat particles. Results We used next generation sequencing (NGS) to characterize DNA and RNA in pooled and individual samples of EV-enriched sweat collected from volunteers performing rigorous exercise. In all sequenced samples, we identified DNA originating from all human chromosomes, but only the mitochondrial chromosome was highly represented with 100% coverage. Most of the DNA mapped to unannotated regions of the human genome with some regions highly represented in all samples. Approximately 5 % of the reads were found to map to other genomes: including bacteria (83%), archaea (3%), and virus (13%), identified bacteria species were consistent with those commonly colonizing the human upper body and arm skin. Small RNA-seq from EV-enriched pooled sweat RNA resulted in 74% of the trimmed reads mapped to the human genome, with 29% corresponding to unannotated regions. Over 70% of the RNA reads mapping to an annotated region were tRNA, while misc. RNA (18,5%), protein coding RNA (5%) and miRNA (1,85%) were much less represented. RNA-seq from individually processed EV-enriched sweat collection generally resulted in fewer percentage of reads mapping to the human genome (7–45%), with 50–60% of those reads mapping to unannotated region of the genome and 30–55% being tRNAs, and lower percentage of reads being rRNA, LincRNA, misc. RNA, and protein coding RNA. Conclusions Our data demonstrates that sweat, as all other body fluids, contains a wealth of nucleic acids, including DNA and RNA of human and microbial origin, opening a possibility to investigate sweat as a source for biomarkers for specific health parameters.

Renal cell carcinoma (RCC) accounts for 90% of all kidney cancers. Due to poor diagnosis, high resistance to the systemic therapies and the fact that most RCC cases occur sporadically, current research switched its focus on studying the molecular mechanisms underlying RCC. The aim is the discovery of new effective and less toxic anti-cancer drugs and novel diagnostic markers. Besides the PI3K/Akt/mTOR, HGF/Met and VHL/hypoxia cellular signaling pathways, the involvement of the Wnt/β-catenin pathway in RCC is commonly studied. Wnt signaling and its targeted genes are known to actively participate in different biological processes during embryonic development and renal cancer. Recently, studies have shown that targeting this pathway by alternating/inhibiting its intracellular signal transduction can reduce cancer cells viability and inhibit their growth. The targets and drugs identified show promising potential to serve as novel RCC therapeutics and prognostic markers. This review aims to summarize the current status quo regarding recent research on RCC focusing on the involvement of the Wnt/β-catenin pathway and how its understanding could facilitate the identification of potential therapeutic targets, new drugs and diagnostic biomarkers.

Extracellular vesicles (EVs) represent a diverse group of small membrane-encapsulated particles involved in cell–cell communication, but the technologies to characterize EVs are still limited. Hypoxia is a typical condition in solid tumors, and cancer-derived EVs support tumor growth and invasion of tissues by tumor cells. We found that exposure of renal adenocarcinoma cells to hypoxia induced EV secretion and led to notable changes in the EV protein cargo in comparison to normoxia. Proteomics analysis showed overrepresentation of proteins involved in adhesion, such as integrins, in hypoxic EV samples. We further assessed the efficacy of time-gated Raman spectroscopy (TG-RS) and surface-enhanced time-gated Raman spectroscopy (TG-SERS) to characterize EVs. While the conventional continuous wave excitation Raman spectroscopy did not provide a notable signal, prominent signals were obtained with the TG-RS that were further enhanced in the TG-SERS. The Raman signal showed characteristic changes in the amide regions due to alteration in the chemical bonds of the EV proteins. The results illustrate that the TG-RS and the TG-SERS are promising label free technologies to study cellular impact of external stimuli, such as oxygen deficiency, on EV production, as well as differences arising from distinct EV purification protocols.

The kidney is a complex organ that is comprised of thousands of nephrons developing through reciprocal inductive interactions between metanephric mesenchyme (MM) and ureteric bud (UB). The MM undergoes mesenchymal to epithelial transition (MET) in response to the signaling from the UB. The secreted protein Wnt4, one of the Wnt family members, is critical for nephrogenesis as mouse Wnt4 −/− mutants fail to form pretubular aggregates (PTA) and therefore lack functional nephrons. Here, we generated mouse embryonic stem cell (mESC) line lacking Wnt4 by applying the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated systems 9 (Cas9). We describe here, differentiation of the wild type and Wnt4 knockout mESCs into kidney progenitors, and such cells induced to undergo nephrogenesis by the mouse E11.5 UB mediated induction. The wild type three-dimensional (3D) self-organized organoids depict appropriately segmented nephron structures, while the Wnt4 -deficient organoids fail to undergo the MET, as is the case in the phenotype of the Wnt4 knockout mouse model in vivo . In summary, we have established a platform that combine CRISPR/Cas9 and kidney organoid technologies to model kidney development in vitro and confirmed that mutant organoids are able to present similar actions as in the in vivo studies.

Microfluidic technology has created new opportunities for developing innovative tools for biological applications. Given the significance of extracellular vesicles (EVs), extensive research has focused on developing microfluidic techniques for EV isolation. This research protocol presents electro‐viscoelastic microfluidics as a novel approach for manipulating EVs. The system leverages the viscoelasticity of the suspending medium along with an externally applied electric field to alter EV motion within a microchannel. These findings suggest that our electro‐viscoelastic microfluidic system has the potential for further development to be used for EV isolation. The electro‐viscoelastic manipulation as a potential method for separation of particles based on size. The particles introduced as a sheath flow migrate to the channel center under the influence of simultaneously applied electric field and pressure driven flow.

Wnts are a family of secreted proteins that regulate multiple steps of neural development and stem cell differentiation. Two of them, Wnt1 and Wnt5a, activate distinct branches of Wnt signaling and individually regulate different aspects of midbrain dopaminergic (DA) neuron development. However, several of their functions and interactions remain to be elucidated. Here, we report that loss of Wnt1 results in loss of Lmx1a and Ngn2 expression, as well as agenesis of DA neurons in the midbrain floor plate. Remarkably, a few ectopic DA neurons still emerge in the basal plate of Wnt1 ⁻/⁻ mice, where Lmx1a is ectopically expressed. These results indicate that Wnt1 orchestrates DA specification and neurogenesis in vivo. Analysis of Wnt1 ⁻/⁻;Wnt5a ⁻/⁻ mice revealed a greater loss of Nurr1 ⁺ cells and DA neurons than in single mutants, indicating that Wnt1 and Wnt5a interact genetically and cooperate to promote midbrain DA neuron development in vivo. Our results unravel a functional interaction between Wnt1 and Wnt5a resulting in enhanced DA neurogenesis. Taking advantage of these findings, we have developed an application of Wnts to improve the generation of midbrain DA neurons from neural and embryonic stem cells. We thus show that coordinated Wnt actions promote DA neuron development in vivo and in stem cells and suggest that coordinated Wnt administration can be used to improve DA differentiation of stem cells and the development of stem cell-based therapies for Parkinson’s disease.

Astrocytes are central nervous system (CNS)-restricted glial cells involved in synaptic function and CNS blood flow regulation. Astrocyte extracellular vesicles (EVs) participate in neuronal regulation. EVs carry RNAs, either surface-bound or luminal, which can be transferred to recipient cells. We characterized the secreted EVs and RNA cargo of human astrocytes derived from an adult brain. EVs were isolated by serial centrifugation and characterized with nanoparticle tracking analysis (NTA), Exoview, and immuno-transmission electron microscopy (TEM). RNA from cells, EVs, and proteinase K/RNase-treated EVs was analyzed by miRNA-seq. Human adult astrocyte EVs ranged in sizes from 50 to 200 nm, with CD81 as the main tetraspanin marker and larger EVs positive for integrin β1. Comparison of the RNA between the cells and EVs identified RNA preferentially secreted in the EVs. In the case of miRNAs, enrichment analysis of their mRNA targets indicates that they are good candidates for mediating EV effects on recipient cells. The most abundant cellular miRNAs were also abundant in EVs, and the majority of their mRNA targets were found to be downregulated in mRNA-seq data, but the enrichment analysis lacked neuronal specificity. Proteinase K/RNase treatment of EV-enriched preparations identified RNAs secreted independently of EVs. Comparing the distribution of cellular and secreted RNA identifies the RNAs involved in intercellular communication via EVs.

Diabetes is a multifactorial disorder characterized by loss or dysfunction of pancreatic β-cells. β-cells are heterogeneous, exhibiting different glucose sensing, insulin secretion and gene expression. They communicate with other endocrine cell types via paracrine signals and between β-cells via gap junctions. Here, we identify the importance of signaling between β-cells via the extracellular signal WNT4. We show heterogeneity in Wnt4 expression, most strikingly in the postnatal maturation period, Wnt4 -positive cells, being more mature while Wnt4- negative cells are more proliferative. Knock-out in adult β-cells shows that WNT4 controls the activation of calcium signaling in response to a glucose challenge, as well as metabolic pathways converging to lower ATP/ADP ratios, thereby reducing insulin secretion. These results reveal that paracrine signaling between β-cells is important in addition to gap junctions in controling insulin secretion. Together with previous reports of WNT4 up-regulation in obesity our observations suggest an adaptive insulin response coordinating β-cells. Diabetes is characterized by loss or dysfunction of pancreatic β-cells. Here the authors show that the signalling protein WNT4 is heterogeneously activated by β-cells during their postnatal maturation with WNT4 positive cells being more mature, and that inactivating WNT4 in β-cells after maturation impairs beta-cell function.