Endogenous Wnt Signaling Research Articles

Small molecule valproic acid enhances ventral patterning of human neural tube organoids by regulating Wnt and Shh signalling.

Valproic acid (VPA), a clinically approved small molecule, has been reported to activate Wnt signalling that is critical for dorsal-ventral (DV) patterning of neural tube. However, little is known about the impact of VPA on DV patterning process. Here, we show that even though VPA has a negative impact on the early formation of human neural tube organoids (hNTOs), it significantly enhances the efficiency of ventrally patterned hNTOs, when VPA is added during the entire differentiation process. RNA sequencing and RT-qPCR analysis demonstrates VPA activates endogenous Wnt signalling in hNTOs. Surprisingly, transcriptome analysis also identifies upregulation of genes for degradation of GLI2 and GLI3 proteins, whose truncated fragment are transcriptional repressors of Shh signalling. The Western-blot analysis confirms the increase of GLI3R proteins after VPA treatment. Thus, VPA might enhance ventral patterning of hNTOs through both activating Wnt, which can antagonise Shh signalling by inducing GLI3 expression, and/or inhibiting Shh signalling by inducing GLI protein degradation. We further obtain results to show that VPA still increases patterning efficiency of hNTOs with a weak influence on their early formation when the initiation time of VPA is delayed and its duration is reduced. Taken together, this study demonstrates that VPA enhances the generation of more reproducible hNTOs with ventral patterning, opening the avenues for the applications of hNTOs in developmental biology and regenerative medicine.

Wnt-Independent SARS-CoV-2 Infection in Pulmonary Epithelial Cells.

The Wnt signaling pathway within host cells regulates infections by several pathogenic bacteria and viruses. Recent studies suggested that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection depends on β-catenin and can be inhibited by the antileprotic drug clofazimine. Since clofazimine has been identified by us as a specific inhibitor of Wnt/β-catenin signaling, these works could indicate a potential role of the Wnt pathway in SARS-CoV-2 infection. Here, we show that the Wnt pathway is active in pulmonary epithelial cells. However, we find that in multiple assays, SARS-CoV-2 infection is insensitive to Wnt inhibitors, including clofazimine, acting at different levels within the pathway. Our findings assert that endogenous Wnt signaling in the lung is unlikely required or involved in the SARS-CoV-2 infection and that pharmacological inhibition of this pathway with clofazimine or other compounds is not a universal way to develop treatments against the SARS-CoV-2 infection. IMPORTANCE The development of inhibitors of the SARS-CoV-2 infection remains a need of utmost importance. The Wnt signaling pathway in host cells is often implicated in infections by bacteria and viruses. In this work, we show that, despite previous indications, pharmacological modulation of the Wnt pathway does not represent a promising strategy to control SARS-CoV-2 infection in lung epithelia.

Sustained therapeutic effect of an anti-inflammatory peptide encapsulated in nanoparticles on ocular vascular leakage in diabetic retinopathy.

Pigment epithelium-derived factor (PEDF), an endogenous Wnt signaling inhibitor in the serine proteinase inhibitors (SERPIN) super family, is present in multiple organs, including the vitreous. Significantly low levels of PEDF in the vitreous are found to associate with pathological retinal vascular leakage and inflammation in diabetic retinopathy (DR). Intravitreal delivery of PEDF represents a promising therapeutic approach for DR. However, PEDF has a short half-life after intravitreal injection, which represents a major hurdle for the long-term treatment. Here we report the prolonged therapeutic effects of a 34-mer peptide of the PEDF N-terminus, encapsulated in poly (lactic-co-glycolic acid) (PLGA) nanoparticles (PEDF34-NP), on DR. PEDF34-NP inhibited hypoxia-induced expression of vascular endothelial growth factor and reduced levels of intercellular adhesion molecule 1 (ICAM-1) in cultured retinal cells. In addition, PEDF34-NP significantly ameliorated ischemia-induced retinal neovascularization in the oxygen-induced retinopathy rat model, and significantly reduced retinal vascular leakage and inflammation in streptozotocin-induced diabetic rats up to 4weeks after intravitreal injection, as compared to PLGA-NP control. Intravitreal injection of PEDF34-NP did not display any detectable toxicities to retinal structure and function. Our findings suggest that PEDF34-NP can confer sustained therapeutic effects on retinal inflammation and vascular leakage, having considerable potential to provide long-term treatment options for DR.

Boosting Neurogenesis in the Adult Hippocampus Using Antidepressants and Mesenchymal Stem Cells.

The hippocampus is one of the few privileged regions (neural stem cell niche) of the brain, where neural stem cells differentiate into new neurons throughout adulthood. However, dysregulation of hippocampal neurogenesis with aging, injury, depression and neurodegenerative disease leads to debilitating cognitive impacts. These debilitating symptoms deteriorate the quality of life in the afflicted individuals. Impaired hippocampal neurogenesis is especially difficult to rescue with increasing age and neurodegeneration. However, the potential to boost endogenous Wnt signaling by influencing pathway modulators such as receptors, agonists, and antagonists through drug and cell therapy-based interventions offers hope. Restoration and augmentation of hampered Wnt signaling to facilitate increased hippocampal neurogenesis would serve as an endogenous repair mechanism and contribute to hippocampal structural and functional plasticity. This review focuses on the possible interaction between neurogenesis and Wnt signaling under the control of antidepressants and mesenchymal stem cells (MSCs) to overcome debilitating symptoms caused by age, diseases, or environmental factors such as stress. It will also address some current limitations hindering the direct extrapolation of research from animal models to human application, and the technical challenges associated with the MSCs and their cellular products as potential therapeutic solutions.

Chimpanzee and pig-tailed macaque iPSCs: Improved culture and generation of primate cross-species embryos.

As our closest living relatives, non-human primates uniquely enable explorations of human health, disease, development, and evolution. Considerable effort has thus been devoted to generating induced pluripotent stem cells (iPSCs) from multiple non-human primate species. Here, we establish improved culture methods for chimpanzee (Pan troglodytes) and pig-tailed macaque (Macaca nemestrina) iPSCs. Such iPSCs spontaneously differentiate in conventional culture conditions, but can be readily propagated by inhibiting endogenous WNT signaling. As a unique functional test of these iPSCs, we injected them into the pre-implantation embryos of another non-human species, rhesus macaques (Macaca mulatta). Ectopic expression of gene BCL2 enhances the survival and proliferation of chimpanzee and pig-tailed macaque iPSCs within the pre-implantation embryo, although the identity and long-term contribution of the transplanted cells warrants further investigation. In summary, we disclose transcriptomic and proteomic data, cell lines, and cell culture resources that may be broadly enabling for non-human primate iPSCs research.

The effect of disease‐causing DVL1 mutations on chondrogenesis in chicken limb mesenchyme

The Wingless (WNT) signaling pathway is a key regulator of skeletal development. Perturbations in WNT signaling are associated with several skeletal disorders, including Robinow syndrome (RS). RS is a rare skeletal dysplasia characterized by deficiencies in facial and limb skeletogenesis. All identified pathogenic variants of RS occur in genes of the WNT signaling pathway. The majority of autosomal dominant RS patients (DRS) cases are attributed to mutations in Dishevelled‐1 (DVL1) (OMIM#1807000) and Dishevelled‐3 (DVL3) (OMIM#616894), a cytoplasmic phosphoprotein. All RS DVL1variants have frameshift mutation in the penultimate exon that substitutes an abnormal peptide for the C‐terminus of the protein. Previous research in our lab has shown the expression of avian‐ specific retroviruses containing human wild‐type DVL1 and RS DVL1 variants in the limb of the chicken embryo in vivo disrupts cartilage formation. In order to examine the possible role of WNT signaling in the dysregulation of chondrogenesis caused by DVL1, high‐density cultures of chicken limb mesenchyme were treated with DVL1 viruses and either TCS JNK6o, which antagonizes non‐canonical, JNK signaling or XAV939, a canonical pathway antagonist. Cultures were grown for eight‐days stained with Alcian blue for cartilage. The area occupied by cartilage was analyzed using two‐way ANOVA with Tukey’s post‐hoc test (Graphpad Prism v9.3). In the absence of drug treatment, wtDVL1 and RS DVL11519ΔTvariant significantly increased chondrogenesis compared to GFP controls (Fig. 1A,B). Cultures treated with TCS6o (Fig. 1A) did not significantly increase cartilage compared to non‐treated controls culture. The combination of wtDVL1 with TCS6o synergistically increased chondrogenesis. This suggests 1) that endogenous non‐canonical WNT signaling is not required for chondrogenesis, 2) that wtDVL1 has a degree of JNK signaling that when antagonized increases chondrogenesis and 3) that the variants are not interacting with JNK signaling. When canonical signaling was antagonized (Fig. 1B), chondrogenesis was significantly increased for all virus types compared to GFPcontrols. However, the effect of XAV939 on the DVL11519ΔT variant‐infected cultures did not further increase chondrogenesis over the virus alone. This suggest that endogenous canonical WNT signaling represses chondrogenesis and but the variant may have altered ability to respond to a decrease in canonical signaling. Misregulation of canonical and non‐canonical WNT signaling is tied to abnormal chondrogenesis and could be contributing to the shortened limbs observed in RS patients.

Pathogenic Role of Diabetes-Induced Overexpression of Kallistatin in Corneal Wound Healing Deficiency Through Inhibition of Canonical Wnt Signaling.

It was reported previously that circulation levels of kallistatin, an endogenous Wnt signaling inhibitor, are increased in patients with diabetes. The current study was conducted to determine the role of kallistatin in delayed wound healing in diabetic corneas. Immunostaining and Western blot analysis showed kallistatin levels were upregulated in corneas from humans and rodents with diabetes. In murine corneal wound healing models, the canonical Wnt signaling was activated in nondiabetic corneas and suppressed in diabetic corneas, correlating with delayed wound healing. Transgenic expression of kallistatin suppressed the activation of Wnt signaling in the cornea and delayed wound healing. Local inhibition of Wnt signaling in the cornea by kallistatin, an LRP6-blocking antibody, or the soluble VLDL receptor ectodomain (an endogenous Wnt signaling inhibitor) delayed wound healing. In contrast, ablation of the VLDL receptor resulted in overactivation of Wnt/β-catenin signaling and accelerated corneal wound healing. Activation of Wnt signaling in the cornea accelerated wound healing. Activation of Wnt signaling promoted human corneal epithelial cell migration and proliferation, which was attenuated by kallistatin. Our findings suggested that diabetes-induced overexpression of kallistatin contributes to delayed corneal wound healing by inhibiting the canonical Wnt signaling. Thus, kallistatin and Wnt/β-catenin signaling in the cornea could be potential therapeutic targets for diabetic corneal complications.

Soluble very low-density lipoprotein receptor (sVLDLR) inhibits fibrosis in neovascular age-related macular degeneration.

Subretinal fibrosis is a key pathological feature in neovascular age‐related macular degeneration (nAMD). Previously, we identified soluble very low‐density lipoprotein receptor (sVLDLR) as an endogenous Wnt signaling inhibitor. This study investigates whether sVLDLR plays an anti‐fibrogenic role in nAMD models, including Vldlr −/− mice and laser‐induced choroidal neovascularization (CNV). We found that fibrosis factors including P‐Smad2/3, α‐SMA, and CTGF were upregulated in the subretinal area of Vldlr −/− mice and the laser‐induced CNV model. The antibody blocking Wnt co‐receptor LRP6 significantly attenuated the overexpression of fibrotic factors in these two models. Moreover, there was a significant reduction of sVLDLR in the interphotoreceptor matrix (IPM) in the laser‐induced CNV model. A transgenic strain (sVLDLR‐Tg) with sVLDLR overexpression in the IPM was generated. Overexpression of sVLDLR ameliorated the profibrotic changes in the subretinal area of the laser‐induced CNV model. In addition, Wnt and TGF‐β signaling synergistically promoted fibrogenesis in human primary retinal pigment epithelium (RPE) cells. CRISPR/Cas9‐mediated LRP6 gene knockout (KO) attenuated this synergistic effect. The disruption of VLDLR expression promoted, while the overexpression of sVLDLR inhibited TGF‐β‐induced fibrosis. These findings suggest that overactivated Wnt signaling enhances the TGF‐β pathway in subretinal fibrosis. sVLDLR confers an antifibrotic effect, at least partially, through the inhibition of Wnt signaling and thus, has therapeutic potential for fibrosis.

Flow Cytometry and Confocal Imaging Analysis of Low Wnt Expression in Axin2-mTurquoise2 Reporter Thymocytes.

Measuring Wnt expression levels is essential when trying to identify or test new Wnt therapeutic targets. Previous studies have shown that canonical Wnt signaling operates via a dosage-driven mechanism, motivating the need to study and measure Wnt signaling in various cell types. Although several reporter models have been proposed to represent physiological Wnt expression, either the genetic context or the reporter protein highly influenced the validity, accuracy, and flexibility of these tools. This paper describes methods for acquiring and analyzing data obtained with the Axin2-mTurquoise2 mouse Wnt reporter model, which contains a mutated Axin2em1Fstl allele. This model facilitates the study of endogenous canonical Wnt signaling in individual cells over a wide range of Wnt activity. This protocol describes how to fully appreciate Axin2-mTurquoise2 reporter activity using cell population analysis of the hematopoietic system, combined with cell surface markers or β-catenin intracellular staining. These procedures serve as a base for implementation and reproduction in other tissues or cells of interest. By combining fluorescence-activated cell sorting and confocal imaging, distinct canonical Wnt expression levels can be visualized. The recommended measurement and analysis strategies provide quantitative data on the fluorescent expression levels for precise assessment of canonical Wnt signaling. These methods will be useful for researchers who want to use the Axin2-mTurquise2 model for canonical Wnt expression patterns.

Striatal Synapse Degeneration and Dysfunction Are Reversed by Reactivation of Wnt Signaling.

Synapse degeneration in the striatum has been associated with the early stages of Parkinson’s and Huntington’s diseases (PD and HD). However, the molecular mechanisms that trigger synaptic dysfunction and loss are not fully understood. Increasing evidence suggests that deficiency in Wnt signaling triggers synapse degeneration in the adult brain and that this pathway is affected in neurodegenerative diseases. Here, we demonstrate that endogenous Wnt signaling is essential for the integrity of a subset of inhibitory synapses on striatal medium spiny neurons (MSNs). We found that inducible expression of the specific Wnt antagonist Dickkopf-1 (Dkk1) in the adult striatum leads to the loss of inhibitory synapses on MSNs and affects the synaptic transmission of D2-MSNs. We also discovered that re-activation of the Wnt pathway by turning off Dkk1 expression after substantial loss of synapses resulted in the complete recovery of GABAergic and dopamine synapse number. Our results also show that re-activation of the Wnt pathway leads to a recovery of amphetamine response and motor function. Our studies identify the Wnt signaling pathway as a potential therapeutic target for restoring neuronal circuits after synapse degeneration.

Human ES Cell Culture Conditions Fail to Preserve the Mouse Epiblast State.

Mouse embryonic stem cells (mESCs) and mouse epiblast stem cells (mEpiSCs) are the pluripotent stem cells (PSCs), derived from the inner cell mass (ICM) of preimplantation embryos at embryonic day 3.5 (E3.5) and postimplantation embryos at E5.5-E7.5, respectively. Depending on their environment, PSCs can exist in the so-called naïve (ESCs) or primed (EpiSCs) states. Exposure to EpiSC or human ESC (hESC) culture condition can convert mESCs towards an EpiSC-like state. Here, we show that the undifferentiated epiblast state is however not stabilized in a sustained manner when exposing mESCs to hESC or EpiSC culture condition. Rather, prolonged exposure to EpiSC condition promotes a transition to a primitive streak- (PS-) like state via an unbiased epiblast-like intermediate. We show that the Brachyury-positive PS-like state is likely promoted by endogenous WNT signaling, highlighting a possible species difference between mouse epiblast-like stem cells and human Embryonic Stem Cells.

Precise base editing for the in vivo study of developmental signaling and human pathologies in zebrafish.

While zebrafish is emerging as a new model system to study human diseases, an efficient methodology to generate precise point mutations at high efficiency is still lacking. Here we show that base editors can generate C-to-T point mutations with high efficiencies without other unwanted on-target mutations. In addition, we established a new editor variant recognizing an NAA protospacer adjacent motif, expanding the base editing possibilities in zebrafish. Using these approaches, we first generated a base change in the ctnnb1 gene, mimicking oncogenic an mutation of the human gene known to result in constitutive activation of endogenous Wnt signaling. Additionally, we precisely targeted several cancer-associated genes including cbl. With this last target, we created a new zebrafish dwarfism model. Together our findings expand the potential of zebrafish as a model system allowing new approaches for the endogenous modulation of cell signaling pathways and the generation of precise models of human genetic disease-associated mutations.

R-Spondin 2 Induces Odontogenic Differentiation of Dental Pulp Stem/Progenitor Cells via Regulation of Wnt/β-Catenin Signaling.

Odontoblast cells generated from human dental pulp stem/progenitor cells (hDPSCs) secrete reparative dentin in responds to an injury. Endogenous Wnt signaling is also activated during this process, and these Wnt-activated cells are responsible for the following repair response. R-spondin 2 (Rspo2) is a potent stem cell growth factor, which strongly potentiates Wnt/β-catenin signaling and plays a vital role in cell differentiation and regeneration. However, the role of Rspo2 during odontoblast differentiation in hDPSCs has not yet been completely understood. This study investigated the effects of Rspo2 on hDPSCs to provide therapeutic insight into dentin regeneration and reparative dentin formation. HDPSCs were extracted from human molars or premolars. Immunofluorescence staining and flow cytometric analysis were used to detect the mesenchymal stem cell markers in hDPSCs. EdU assay and Cell Counting Kit-8 (CCK-8) were performed to explore cell proliferation. The odontogenic differentiation levels were determined by measuring the mRNA and protein expression of DSPP, DMP-1, ALP, and BSP. Immunofluorescence staining was performed to detect the localization of β-catenin. The biological effects of Rspo2 on hDPSCs was investigated using the Lentivirus-based Rspo2 shRNA and recombined human Rspo2 (rhRspo2). Recombined human DKK-1 (rhDKK-1) and recombined human Wnt3a (rhWnt3a) were used for further investigation. The cells generated from human dental pulp expressed mesenchymal stem cell markers Vimentin, Stro-1, Nestin, C-kit, CD90, and CD73, while were negative for CD3, CD31, and CD34. The mRNA expression levels of the odontogenic-related genes DSPP, DMP-1, ALP, and BSP were upregulated in the rhRspo2 treated cells. Silencing Rspo2 suppressed the proliferation and differentiation of the hDPSCs. Blockade of Wnt signaling with DKK-1 inhibited Rspo2-induced activation of Wnt/β-catenin signaling and cell differentiation. The combined use of rhWnt3a and rhRspo2 created a synergistic effect to improve the activation of Wnt/β-catenin signaling. Rspo2 promoted the proliferation and odontogenic differentiation of hDPSCs by regulating the Wnt/β-catenin signaling pathway.

Variable Outcomes in Neural Differentiation of Human PSCs Arise from Intrinsic Differences in Developmental Signaling Pathways

SummaryDirected differentiation of human pluripotent stem cells varies in specificity and efficiency. Stochastic, genetic, intracellular, and environmental factors affect maintenance of pluripotency and differentiation into early embryonic lineages. However, factors affecting variation in in vitro differentiation to defined cell types are not well understood. To address this, we focused on a well-established differentiation process to cerebral cortex neural progenitor cells and their neuronal progeny from human pluripotent stem cells. Analysis of 162 differentiation outcomes of 61 stem cell lines derived from 37 individuals showed that most variation occurs along gene expression axes reflecting dorsoventral and rostrocaudal spatial expression during in vivo brain development. Line-independent and line-dependent variations occur, with the latter driven largely by differences in endogenous Wnt signaling activity. Tuning Wnt signaling during a specific phase early in the differentiation process reduces variability, demonstrating that cell-line/genome-specific differentiation outcome biases can be corrected by controlling extracellular signaling.

Canonical Wnt signaling promotes pacemaker cell specification of cardiac mesodermal cells derived from mouse and human embryonic stem cells.

Cardiac differentiation of embryonic stem cells (ESCs) can give rise to de novo chamber cardiomyocytes and nodal pacemaker cells. Compared with our understanding of direct differentiation toward atrial and ventricular myocytes, the mechanisms for nodal pacemaker cell commitment are not well understood. Taking a cue from the prominence of canonical Wnt signaling during cardiac pacemaker tissue development in chick embryos, we asked if modulations of Wnt signaling influence cardiac progenitors to bifurcate to either chamber cardiomyocytes or pacemaker cells. Omitting an exogenous Wnt inhibitor, which is routinely added to maximize cardiac myocyte yield during differentiation of mouse and human ESCs, led to increased yield of spontaneously beating cardiomyocytes with action potential properties similar to those of native sinoatrial node pacemaker cells. The pacemaker phenotype was accompanied by enhanced expression of genes and gene products that mark nodal pacemaker cells such as Hcn4, Tbx18, Tbx3, and Shox2. Addition of exogenous Wnt3a ligand, which activates canonical Wnt/β-catenin signaling, increased the yield of pacemaker-like myocytes while reducing cTNT-positive pan-cardiac differentiation. Conversely, addition of inhibitors of Wnt/β-catenin signaling led to increased chamber myocyte lineage development at the expense of pacemaker cell specification. The positive impact of canonical Wnt signaling on nodal pacemaker cell differentiation was evidenced in direct differentiation of two human ESC lines and human induced pluripotent stem cells. Our data identify the Wnt/β-catenin pathway as a critical determinant of cardiac myocyte subtype commitment during ESC differentiation: endogenous Wnt signaling favors the pacemaker lineage, whereas its suppression promotes the chamber cardiomyocyte lineage.

Infection-induced signals generated at the plasma membrane epigenetically regulate Wnt signaling in vitro and in vivo.

Wnt signaling regulates immunomodulatory functions during infection and inflammation. Employing NCCIT and HCT116 cells, having high endogenous Wnt signaling, we observed elevated levels of low-density lipoprotein receptor-related protein 5/6 (LRP5/6) and Frizzled class receptor 10 (FZD10) and increases in β-catenin, doublecortin-like kinase 1 (DCLK1), CD44 molecule (CD44), and aldehyde dehydrogenase 1 family member A1 (ALDH1A1). siRNA-induced knockdown of these receptors antagonized TOPflash reporter activity and spheroid growth in vitro and elevated Wnt-inhibitory factor 1 (WIF1) activity. Elevated mRNA and protein levels of LRP5/6 and FZD10 paralleled expression of WNT2b and WNT4 in colonic crypts at days 6 and 12 post-infection with Citrobacter rodentium (CR) and tended to decline at days 20-34. The CR mutant escV or the tankyrase inhibitor XAV939 attenuated these responses. A three-dimensional organoid assay in colonic crypts isolated from CR-infected mice revealed elevated levels of LRP5/6 and FZD10 and β-catenin co-localization with enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2). Co-immunoprecipitation in the membrane fraction revealed that axin associates with LRP5/6 in CR-infected crypts, and this association was correlated with increased β-catenin. Colon tumors from either CR-infected ApcPMin/+ or azoxymethane/dextran sodium sulfate (AOM/DSS)-treated mice had high LRP5/6 or FZD10 levels, and chronic Notch blockade through the γ-secretase inhibitor dibenzazepine down-regulated LRP5/6 and FZD10 expression. In CR-responsive CT-26 cells, siRNA-induced LRP5/6 or FZD10 knockdown antagonized TOPflash reporter activity. Elevated miR-153-3p levels correlated with LRP5/6 and FZD10, and miR-153-3p sequestration via a plasmid-based miR inhibitor system attenuated Wnt signaling. We conclude that infection-induced signals from the plasma membrane epigenetically regulate Wnt signaling.

Wnt signaling in cutaneous wound healing

Human skin is an efficient barrier that protects the organism from noxious substances. Wounds destroy this barrier. Wound healing is a phased physiological regeneration of the destroyed tissue that ideally leads to occlusion of a wound, in particular by regeneration of connective tissue and capillaries. The Wnt signaling pathway is a highly conserved signal transduction cascade across the animal kingdom that controls basic cellular interactions in multicellular organisms. Accordingly, through the Wnt signaling path many processes, e. g. as the balance between proliferation and differentiation or apoptosis, coordinated. Wnt signaling is activated by a wound and participates in each subsequent phase of the healing process, beginning with inflammatory control and programmed cell death, to the mobilization of stem cells within the wound. Endogenous Wnt signaling is an attractive therapeutic approach to assist in the repair of skin wounds, as the complex mechanisms of the Wnt signaling pathway have become increasingly understood over the years. This review summarizes current data to clarify the role of Wnt signaling in the wound healing process of the skin.

Exploiting differential Wnt target gene expression to generate a molecular biomarker for colorectal cancer stratification

ObjectivePathological Wnt pathway activation is a conserved hallmark of colorectal cancer. Wnt-activating mutations can be divided into: i) ligand-independent (LI) alterations in intracellular signal transduction proteins (Adenomatous polyposis coli, β-catenin),...

A Correlation between Wnt/Beta-catenin Signaling and the Rate of Dentin Secretion

IntroductionOdontoblasts produce dentin throughout life and in response to trauma. The purpose of this study was to identify the roles of endogenous Wnt signaling in regulating the rate of dentin accumulation. MethodsHistology, immunohistochemistry, vital dye labeling, and histomorphometric assays were used to quantify the rate of dentin accumulation as a function of age. Two strains of Wnt reporter mice were used to identify and follow the distribution and number of Wnt-responsive odontoblasts as a function of age. To show a causal relationship between dentin secretion and Wnt signaling, dentin accumulation was monitored in a strain of mice in which Wnt signaling was aberrantly elevated. ResultsDentin deposition occurs throughout life, but the rate of accumulation slows with age. This decline in dentin secretion correlates with a decrease in endogenous Wnt signaling. In a genetically modified strain of mice, instead of tubular dentin, aberrantly elevated Wnt signaling resulted in accumulation of reparative dentin or osteodentin secreted from predontoblasts. ConclusionsWnt signaling regulates dentin secretion by odontoblasts, and the formation of reparative or osteodentin is the direct consequence of elevated Wnt signaling. These preclinical data have therapeutic implications for the development of a biologically based pulp capping medicant.

The Effect of Wnt Pathway Modulators on Human iPSC-Derived Pancreatic Beta Cell Maturation.

Current published protocols for targeted differentiation of human stem cells toward pancreatic β-cells fail to deliver sufficiently mature cells with functional properties comparable to human islet β-cells. We aimed to assess whether Wnt-modulation could promote the final protocol stages of β-cell maturation, building our hypothesis on our previous findings of Wnt activation in immature hiPSC-derived stage 7 (S7) cells compared to adult human islets and with recent data reporting a link between Wnt/PCP and in vitro β-cell maturation. In this study, we stimulated canonical and non-canonical Wnt signaling in hiPSC-derived S7 cells using syntetic proteins including WNT3A, WNT4, WNT5A and WNT5B, and we inhibited endogenous Wnt signaling with the Tankyrase inhibitor G007-LK (TKi). Whereas neither canonical nor non-canonical Wnt stimulation alone was able to mature hiPSC-derived S7 cells, WNT-inhibition with TKi increased the fraction of monohormonal cells and global proteomics of TKi-treated S7 cells showed a proteomic signature more similar to adult human islets, suggesting that inhibition of endogenous Wnt contributes toward final β-cell maturation.