Explore the vast range of titles available.

Diligent side-by-side comparisons of how different methodologies affect growth efficiency and quality of intestinal colonoids have not been performed leaving a gap in our current knowledge. Here, we summarize our efforts to optimize culture conditions for improved growth and functional differentiation of mouse and human colon organoids. Mouse and human colon organoids were grown in four different media. Media-dependent long-term growth was measured by quantifying surviving organoids imaging and a cell viability readout over five passages. The impact of diverse media on differentiation was assessed by quantifying the number of epithelial cell types using markers for enterocytes, stem cells, Goblet cells, and enteroendocrine cells by qPCR and histology upon removal of growth factors. In contrast to Wnt3a-conditioned media, media supplemented with recombinant Wnt3a alone did not support long-term survival of human or mouse colon organoids. Mechanistically, this observation can be attributed to the fact that recombinant Wnt3a did not support stem cell survival or proliferation as demonstrated by decreased LGR5 and Ki67 expression. When monitoring expression of markers for epithelial cell types, the highest level of organoid differentiation was observed after combined removal of Wnt3a, Noggin, and R-spondin from Wnta3a-conditioned media cultures. Our study defined Wnt3a-containing conditioned media as optimal for growth and survival of human and mouse organoids. Furthermore, we established that the combined removal of Wnt3a, Noggin, and R-spondin results in optimal differentiation. This study provides a step forward in optimizing conditions for intestinal organoid growth to improve standardization and reproducibility of this model platform.

Extrinsic molecules such as morphogens can regulate timed mRNA translation events in developing neurons. In particular, Wingless-type MMTV integration site family, member 3 (Wnt3), was shown to regulate the translation of Foxp2 mRNA encoding a Forkhead transcription factor P2 in the neocortex. However, the Wnt receptor that possibly mediates these translation events remains unknown. Here, we report Frizzled member 7 (Fzd7) as the Wnt3 receptor that lays downstream in Wnt3-regulated mRNA translation. Fzd7 proteins co-localize with Wnt3 ligands in developing neocortices. In addition, the Fzd7 proteins overlap in layer-specific neuronal subpopulations expressing different transcription factors, Foxp1 and Foxp2. When Fzd7 was silenced, we found decreased Foxp2 protein expression and increased Foxp1 protein expression, respectively. The Fzd7 silencing also disrupted the migration of neocortical glutamatergic neurons. In contrast, Fzd7 overexpression reversed the pattern of migratory defects and Foxp protein expression that we found in the Fzd7 silencing. We further discovered that Fzd7 is required for Wnt3-induced Foxp2 mRNA translation. Surprisingly, we also determined that the Fzd7 suppression of Foxp1 protein expression is not Wnt3 dependent. In conclusion, it is exhibited that the interaction between Wnt3 and Fzd7 regulates neuronal identity and the Fzd7 receptor functions as a downstream factor in ligand Wnt3 signaling for mRNA translation. In particular, the Wnt3-Fzd7 signaling axis determines the deep layer Foxp2-expressing neurons of developing neocortices. Our findings also suggest that Fzd7 controls the balance of the expression for Foxp transcription factors in developing neocortical neurons. These discoveries are presented in our manuscript within a larger framework of this review on the role of extrinsic factors in regulating mRNA translation.

Background/Objective: Alzheimer’s disease (AD) is a neurodegenerative condition characterized by oxidative stress, neuroinflammation, amyloidogenesis, and tau-related pathology. This study investigated the macronutrient and phytochemical composition of strawberry (S), lemon (L), and lemon juice-assisted strawberry (S/L) extracts and evaluated their neuroprotective efficacy relative to selenium (Se) in an aluminum chloride (AlCl3)-induced rat model of AD. Methods: Macronutrients and phenolics were quantified in S, L, and S/L, and the extracts were profiled using high-performance liquid chromatography and electrospray ionization tandem mass-spectrometry. Male Sprague–Dawley rats received AlCl3 with or without S, L, S/L, or Se, and their cognitive performance was assessed using the Morris water maze, Y-maze, and conditioned avoidance tests. Markers of oxidative status, inflammation, cholinergic function, apoptotic signaling, and Wnt3/β-catenin pathway activity were quantified in the brain tissue, and cortico-hippocampal morphology was examined. Results: The S/L extract showed the highest carbohydrate, protein, and lipid content. The total phenolic content was highest in S/L (60.46 mg gallic acid equivalents/g), followed by L (55.08) and S (44.75), with S/L also being the richest in gallic, ellagic, and chlorogenic acids. S/L attenuated AlCl3-induced cognitive deficits, restored antioxidant status, suppressed neuroinflammation, improved cholinergic indices, modulated apoptotic signaling, and downregulated amyloidogenic and NLRP3 inflammasome markers, consistent with histological evidence of neuronal preservation. Conclusions: Lemon juice-assisted extraction enhanced the macronutrient and phenolic richness and multitarget neuroprotection of strawberries. S/L co-extracts represent promising functional food–derived adjuvants for AD management and support integrative compositional–mechanistic profiling to optimize natural product–based interventions.

Finding new strategies for the treatment of heart failures using stem cells has attracted a lot of attention. Meanwhile, nanotechnology-based approaches to regenerative medicine hypothesize a possible combination of stem cells and nanotechnology in the treatment of diseases. This study aims to investigate the in vitro effect of silver nanoparticles (Ag-NPs) on the cardiomyogenic differentiation of bone marrow-derived mesenchymal stem cells (BM-MSCs) through detection of cardiac markers. For this purpose, MSCs were isolated from bone marrow resident and differentiated to the cardiac cells using a dedicated medium with Ag-NPs. Also, the cardiomyogenic differentiation of BM-MSCs was confirmed using immunocytochemistry. Then, real-time PCR and western blotting assay were used for measuring absolute telomere length (TL) measurement, and gene and protein assessment of the cells, respectively. It was found that 2.5 µg/mL Ag-NPs caused elongation of the telomeres and altered VEGF, C-TnI, VWF, SMA, GATA-4, TERT, and cyclin D protein and gene expression in the cardiomyogenically differentiated BM-MSCs. Also, there was a significant increase in the protein and gene expression of Wnt3 and β-catenin as main components of pathways. We concluded that Ag-NPs could change the in vitro expression of cardiac markers of BM-MSCs via the Wnt3/β-catenin signaling pathway.

Hyperbaric oxygen (HBO) therapy may promote neurological recovery from hypoxic-ischemic encephalopathy (HIE). However, the therapeutic effects of HBO and its associated mechanisms remain unknown. The canonical Wnt/β-catenin signaling pathways and bone morphogenetic protein (BMP) play important roles in mammalian nervous system development. The present study examined whether HBO stimulates the differentiation of neural stem cells (NSCs) and its effect on Wnt3/β-catenin and BMP2 signaling pathways. We showed HBO treatment (2 ATA, 60 min) promoted differentiation of NSCs into neurons and oligodendrocytes in vitro. In addition, rat hypoxic-ischemic brain damage (HIBD) tissue extracts also promoted the differentiation of NSCs into neurons and oligodendrocytes, with the advantage of reducing the number of astrocytes. These effects were most pronounced when these two were combined together. In addition, the expression of Wnt3a, BMP2, and β-catenin nuclear proteins were increased after HBO treatment. However, blockade of Wnt/β-catenin or BMP signaling inhibited NSC differentiation and reduced the expression of Wnt3a, BMP2, and β-catenin nuclear proteins. In conclusion, HBO promotes differentiation of NSCs into neurons and oligodendrocytes and reduced the number of astrocytes in vitro possibly through regulation of Wnt3/β-catenin and BMP2 signaling pathways. HBO may serve as a potential therapeutic strategy for treating HIE.

Generation of an epitope-tagged, functional Wnt3 knock-in allele, the signal produced by Paneth cells to regulate intestinal stem cells. Wnt signalling in the intestinal crypt The Wnt signalling pathway is a key mediator of the intestinal stem-cell niche and is involved in maintaining the intestinal crypt structures in the epithelial lining of the small intestine in mice. Although Wnt3 is secreted by Paneth cells to act on intestinal stem cells, it has not been possible to follow its fate in vivo , so it is not clear if its propagation involves a gradient set by diffusion. Henner Farin et al . have generated a tagged Wnt3 and find that it binds to the basolateral membrane of intestinal stem cells in an organoid-based system, and is propagated through partition of cell membrane by cell division rather than through diffusion. Mammalian Wnt proteins are believed to act as short-range signals 1 , 2 , 3 , 4 , yet have not been previously visualized in vivo . Self-renewal, proliferation and differentiation are coordinated along a putative Wnt gradient in the intestinal crypt 5 . Wnt3 is produced specifically by Paneth cells 6 , 7 . Here we have generated an epitope-tagged, functional Wnt3 knock-in allele. Wnt3 covers basolateral membranes of neighbouring stem cells. In intestinal organoids, Wnt3-transfer involves direct contact between Paneth cells and stem cells. Plasma membrane localization requires surface expression of Frizzled receptors, which in turn is regulated by the transmembrane E3 ligases Rnf43/Znrf3 and their antagonists Lgr4-5/R-spondin. By manipulating Wnt3 secretion and by arresting stem-cell proliferation, we demonstrate that Wnt3 mainly travels away from its source in a cell-bound manner through cell division, and not through diffusion. We conclude that stem-cell membranes constitute a reservoir for Wnt proteins, while Frizzled receptor turnover and ‘plasma membrane dilution’ through cell division shape the epithelial Wnt3 gradient.

Polyps of the cnidarian Hydra maintain their adult anatomy through two developmental organizers, the head organizer located apically and the foot organizer basally. The head organizer is made of two antagonistic cross-reacting components, an activator, driving apical differentiation and an inhibitor, preventing ectopic head formation. Here we characterize the head inhibitor by comparing planarian genes down-regulated when β-catenin is silenced to Hydra genes displaying a graded apical-to-basal expression and an up-regulation during head regeneration. We identify Sp5 as a transcription factor that fulfills the head inhibitor properties: leading to a robust multiheaded phenotype when knocked-down in Hydra , acting as a transcriptional repressor of Wnt3 and positively regulated by Wnt/β-catenin signaling. Hydra and zebrafish Sp5 repress Wnt3 promoter activity while Hydra Sp5 also activates its own expression, likely via β-catenin/TCF interaction. This work identifies Sp5 as a potent feedback loop inhibitor of Wnt/β-catenin signaling, a function conserved across eumetazoan evolution. Hydra regenerate various body parts on amputation by activation of the appropriate organiser, but how head formation is controlled is unclear. Here, the authors identify the transcription factor Sp5 as restricting head formation, by being activated by beta-catenin and then acting as a repressor of Wnt3 .

Breaking of left–right symmetry in mouse embryos requires fluid flow at the node, but the precise action of the flow has remained unknown. Here we show that the left–right asymmetry of Cerl2 expression around the node, a target of the flow, is determined post-transcriptionally by decay of Cerl2 mRNA in a manner dependent on its 3′ untranslated region. Cerl2 mRNA is absent specifically from the apical region of crown cells on the left side of the node. Preferential decay of Cerl2 mRNA on the left is initiated by the leftward flow and further enhanced by the operation of Wnt-Cerl2 interlinked feedback loops, in which Wnt3 upregulates Wnt3 expression and promotes Cerl2 mRNA decay, whereas Cerl2 promotes Wnt degradation. Mathematical modelling and experimental data suggest that these feedback loops behave as a bistable switch that can amplify in a noise-resistant manner a small bias conferred by fluid flow. During embryonic development, midline fluid flow results in asymmetric nodal gene expression. Using genetic manipulations and mathematical modelling, Nakamura et al . find that expression of the nodal antagonist Cerl2 is regulated post-transcriptionally, and that asymmetry is maintained by Wnt-Cerl2 feedback loops.

Definitive endoderm differentiation is crucial for generating respiratory and gastrointestinal organs including pancreas and liver. However, whether epigenetic regulation contributes to this process is unknown. Here, we show that the H3K27me3 demethylases KDM6A and KDM6B play an important role in endoderm differentiation from human ESCs. Knockdown of KDM6A or KDM6B impairs endoderm differentiation, which can be rescued by sequential treatment with WNT agonist and antagonist. KDM6A and KDM6B contribute to the activation of WNT3 and DKK1 at different differentiation stages when WNT3 and DKK1 are required for mesendoderm and definitive en- doderm differentiation, respectively. Our study not only uncovers an important role of the H3K27me3 demethylases in definitive endoderm differentiation, but also reveals that they achieve this through modulating the WNT signaling pathway.

Lack of effective anti-metastatic drugs creates a major hurdle for metastatic lung cancer therapy. For successful lung cancer treatment, there is a strong need of newer therapeutics with metastasis-inhibitory potential. In the present study, we determined the anti-metastatic and anti-angiogenic potential of a natural plant triterpenoid, Cucurbitacin B (CuB) against non-small cell lung cancer (NSCLC) both in vitro and in vivo . CuB demonstrated a strong anti-migratory and anti-invasive ability against metastatic NSCLC at nanomolar concentrations. CuB also showed significant tumor angiogenesis-inhibitory effects as evidenced by the inhibition of migratory, invasive and tube-forming capacities of human umbilical vein endothelial cells. CuB-mediated inhibition of angiogenesis was validated by the inhibition of pre-existing vasculature in chick embryo chorio-allantoic membrane and matrigel plugs. Similarly, CuB inhibited the migratory behavior of TGF-β1-induced experimental EMT model. The CuB-mediated inhibition of metastasis and angiogenesis was attributable to the downregulation of Wnt/β-catenin signaling axis, validated by siRNA-knockdown of Wnt3 and Wnt3a . The CuB-mediated downregulation of Wnt/β-catenin signaling was also validated using 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis model in vivo . Collectively, our findings suggest that CuB inhibited the metastatic abilities of NSCLC through the inhibition of Wnt/β-catenin signaling axis.