Advances in Applications of Metabolomics in Pluripotent Stem Cell Research.

Stem cells undergo extensive metabolic rewiring during reprogramming, proliferation and differentiation, and numerous studies have demonstrated a significant role of metabolism in controlling stem cell fates. Recent applications of metabolomics, the study of concentrations and fluxes of small molecules in cells, have advanced efforts to characterize and maturate stem cell fates, assess drug toxicity in stem cell tissue models, identify biomarkers, and study the effects of environment on metabolic pathways in stem cells and their progeny. Looking to the future, combining metabolomics with other -omics approaches will provide a deeper understanding of the complex regulatory mechanisms of stem cells

Human pluripotent stem cell-derived epicardial progenitors can differentiate to endocardial-like endothelial cells.

During heart development, epicardial progenitors contribute various cardiac lineages including smooth muscle cells, cardiac fibroblasts, and endothelial cells. However, their specific contribution to the human endothelium has not yet been resolved, at least in part due to the inability to expand and maintain human primary or pluripotent stem cell (hPSC)-derived epicardial cells. Here we first generated CDH5-2A-eGFP knock-in hPSC lines and differentiated them into self-renewing WT1+ epicardial cells, which gave rise to endothelial cells upon VEGF treatment in vitro. In addition, we found that the percentage of endothelial cells correlated with WT1 expression in a WT1-2A-eGFP reporter line. The resulting endothelial cells displayed many endocardium-like endothelial cell properties, including high expression levels of endocardial-specific markers, nutrient transporters and well-organized tight junctions. These findings suggest that human epicardial progenitors may have the capacity to form endocardial endothelium during development and have implications for heart regeneration and cardiac tissue engineering

Astaxanthin promotes mitochondrial biogenesis and antioxidant capacity in chronic high-intensity interval training

Purpose: Reactive oxygen and nitrogen species are required for exercise-induced molecular adaptations; however, excessive exercise may cause cellular oxidative distress. We postulate that astaxanthin (ASX) can neutralize oxidative distress and stimulate mitochondrial biogenesis in high-intensity exercise-trained mice. Methods: Six-week-old mice (n = 8/group) were treated with ASX (10 mg/kg BW) or placebo. Training groups participated in 30 min/day high-intensity interval training (HIIT) for 6 weeks. Gastrocnemius muscle was collected and assayed following the exercise training period. Results: Compared to the HIIT control mice, the ASX-treated HIIT mice reduced malonaldehyde levels and upregulated the expression of Nrf2 and FOXO3a. Meanwhile, the genes NQO1 and GCLC, modulated by Nrf2, and SOD2, regulated by FOXO3a, and GPx4, were transcriptionally upregulated in the ASX-treated HIIT group. Meanwhile, the expression of energy sensors, AMPK, SIRT1, and SIRT3, increased in the ASX-treated HIIT group compared to the HIIT control group. Additionally, PGC-1α, regulated by AMPK and SIRT1, was upregulated in the ASX-treated HIIT group. Further, the increased PGC-1α stimulated the transcript of NRF1 and Tfam and mitochondrial proteins IDH2 and ATP50. Finally, the ASX-treated HIIT mice had upregulations in the transcript level of mitochondrial fusion factors, including Mfn1, Mfn2, and OPA1. However, the protein level of AMPK, SIRT1, and FOXO3a, and the transcript level of Nrf2, NQO1, PGC-1α, NRF1, Mfn1, Mfn2, and OPA1 decreased in the HIIT control group compared to the sedentary control group. Conclusion: Supplementation with ASX can reduce oxidative stress and promote antioxidant capacity and mitochondrial biogenesis during strenuous HIIT exercise in mice.</p

Fluorescent squaramides as anion receptors and transmembrane anion transporters

A series of squaramide-based anion transporters functionalised with the 1,8-naphthalimide fluorophore has been developed for improved ionophoric activity and fluorescent imaging in cells.112Ysciescopu

Chemically-defined albumin-free differentiation of human pluripotent stem cells to endothelial progenitor cells.

Human pluripotent stem cell (hPSC)-derived endothelial cells and their progenitors are important for vascular research and therapeutic revascularization. Here, we report a completely defined endothelial progenitor differentiation platform that uses a minimalistic medium consisting of Dulbecco's modified eagle medium and ascorbic acid, lacking of albumin and growth factors. Following hPSC treatment with a GSK-3β inhibitor and culture in this medium, this protocol generates more than 30% multipotent CD34+ CD31+ endothelial progenitors that can be purified to >95% CD34+ cells via magnetic activated cell sorting (MACS). These CD34+ progenitors are capable of differentiating into endothelial cells in serum-free inductive media. These hPSC-derived endothelial cells express key endothelial markers including CD31, VE-cadherin, and von Willebrand factor (vWF), exhibit endothelial-specific phenotypes and functions including tube formation and acetylated low-density lipoprotein (Ac-LDL) uptake. This fully defined platform should facilitate production of proliferative, xeno-free endothelial progenitor cells for both research and clinical applications

Efficient Differentiation of Human Pluripotent Stem Cells to Endothelial Progenitors via Small-Molecule Activation of WNT Signaling

SummaryHuman pluripotent stem cell (hPSC)-derived endothelial cells and their progenitors may provide the means for vascularization of tissue-engineered constructs and can serve as models to study vascular development and disease. Here, we report a method to efficiently produce endothelial cells from hPSCs via GSK3 inhibition and culture in defined media to direct hPSC differentiation to CD34+CD31+ endothelial progenitors. Exogenous vascular endothelial growth factor (VEGF) treatment was dispensable, and endothelial progenitor differentiation was β-catenin dependent. Furthermore, by clonal analysis, we showed that CD34+CD31+CD117+TIE-2+ endothelial progenitors were multipotent, capable of differentiating into calponin-expressing smooth muscle cells and CD31+CD144+vWF+I-CAM1+ endothelial cells. These endothelial cells were capable of 20 population doublings, formed tube-like structures, imported acetylated low-density lipoprotein, and maintained a dynamic barrier function. This study provides a rapid and efficient method for production of hPSC-derived endothelial progenitors and endothelial cells and identifies WNT/β-catenin signaling as a primary regulator for generating vascular cells from hPSCs

High level soluble expression, one-step purification and characterization of HIV-1 p24 protein

<p>Abstract</p> <p>Background</p> <p>P24 protein is the major core protein of HIV virus particle and has been suggested as a specific target for antiviral strategies. Recombinant p24 protein with natural antigenic activity would be useful for various studies, such as diagnostic reagents and multi-component HIV vaccine development. The aim of this study was to express and purify the p24 protein in soluble form in <it>E.coli</it>.</p> <p>Results</p> <p>According to the sequence of the p24 gene, a pair of primers was designed, and the target sequence of 700 bp was amplified using PCR. The PCR product was cloned into pQE30 vector, generating the recombinant plasmid pQE30-p24. SDS-PAGE analysis showed that the His-tagged recombinant p24 protein was highly expressed in soluble form after induction in <it>E. coli </it>strain BL21. The recombinant protein was purified by nickel affinity chromatography and used to react with HIV infected sera. The results showed that the recombinant p24 protein could specifically react with the HIV infected sera. To study the immunogenicity of this soluble recombinant p24 protein, it was used to immunize mice for the preparation of polyclonal antibody. Subsequent ELISA and Western-Blot analysis demonstrated that the p24 protein had proper immunogenicity in inducing mice to produce HIV p24 specific antibodies.</p> <p>Conclusion</p> <p>In this work, we report the high level soluble expression of HIV-1 p24 protein in <it>E. coli</it>. This soluble recombinant p24 protein specifically react with HIV infected sera and elicit HIV p24 specific antibodies in mice, indicating this soluble recombinant p24 protein could be a promising reagent for HIV diagnosis.</p