Slc25a36 modulates pluripotency of mouse embryonic stem cells by regulating mitochondrial function and glutathione level.

Mitochondria play a central role in the maintenance of the naive state of embryonic stem cells. Many details of the mechanism remain to be fully elucidated. Solute carrier family 25 member 36 (Slc25a36) might regulate mitochondrial function through transporting pyrimidine nucleotides for mtDNA/RNA synthesis. Its physical role in this process remains unknown; however, Slc25a36 was recently found to be highly expressed in naive mouse embryonic stem cells (mESCs). Here, the function of Slc25a36 was characterized as a maintenance factor of mESCs pluripotency. Slc25a36 deficiency (via knockdown) has been demonstrated to result in mitochondrial dysfunction, which induces the differentiation of mESCs. The expression of key pluripotency markers (Pou5f1, Sox2, Nanog, and Utf1) decreased, while that of key TE genes (Cdx2, Gata3, and Hand1) increased. Cdx2-positive cells emerged in Slc25a36-deficient colonies under trophoblast stem cell culture conditions. As a result of Slc25a36 deficiency, mtDNA of knockdown cells declined, leading to impaired mitochondria with swollen morphology, decreased mitochondrial membrane potential, and low numbers. The key transcription regulators of mitochondrial biogenesis also decreased. These results indicate that mitochondrial dysfunction leads to an inability to support the pluripotency maintenance. Moreover, down-regulated glutathione metabolism and up-regulated focal adhesion reinforced and stabilized the process of differentiation by separately enhancing OCT4 degradation and promoting cell spread. This study improves the understanding of the function of Slc25a36, as well as the relationship of mitochondrial function with naive pluripotency maintenance and stem cell fate decision.

Zearalenone causes embryotoxicity and induces oxidative stress and apoptosis in differentiated human embryonic stem cells.

In this study, murine embryonic stem cell test (mEST) and human embryonic stem cell test (hEST) models were developed to evaluate the embryonic toxicity of Zearalenone (ZEN) according to the methods established in our laboratory. The embryotoxicity of ZEN was described by comparing the three functions calculated based on three endpoints, that is, 50% inhibitory proliferation concentration (IC50) of embryonic stem cells (ESCs) and 3T3 cells and 50% inhibition of cardiomyocyte differentiation (ID50) of ESCs determined in the EST model. Moreover, differentiation of human embryonic stem cell (hESC) was initiated by embryoid bodies (EBs) formation; EBs were exposed to different concentrations of ZEN for 24 h to detect cellular reactive oxygen species (ROS) generation, the mitochondrial transmembrane potential (MMP), apoptosis, cell cycle, and the related protein expression. Based on the results of the three endpoints and functions of ZEN in mEST and hEST, ZEN was evaluated to have strong embryonic toxicity both by two models. The increases in cellular ROS and loss of MMP were observed at 2 and 4 µg/ml concentrations. Flow cytometry showed that ZEN induced cell cycle arrest and apoptosis. The upregulation of p53, caspase-9, caspase-3, and the ratio of Bax/Bcl-2 were observed at 2 and 4 µg/ml concentrations. Collectively these results demonstrate that ZEN has strong embryonic toxicity and induces oxidative stress and apoptosis in differentiated human ESCs.

MicroRNA regulation of endothelial homeostasis and commitment-implications for vascular regeneration strategies using stem cell therapies.

Human embryonic (hESC) and induced pluripotent (hiPSC) stem cells have broad therapeutic potential in the treatment of a range of diseases, including those of the vascular system. Both hESCs and hiPSCs have the capacity for indefinite self-renewal, in addition to their ability to differentiate into any adult cell type. These cells could provide a potentially unlimited source of cells for transplantation and, therefore, provide novel treatments, e.g. in the production of endothelial cells for vascular regeneration. MicroRNAs are short, noncoding RNAs that act posttranscriptionally to control gene expression and thereby exert influence over a wide range of cellular processes, including maintenance of pluripotency and differentiation. Expression patterns of these small RNAs are tissue specific, and changes in microRNA levels have often been associated with disease states in humans, including vascular pathologies. Here, we review the roles of microRNAs in endothelial cell function and vascular disease, as well as their role in the differentiation of pluripotent stem cells to the vascular endothelial lineage. Furthermore, we discuss the therapeutic potential of stem cells and how knowledge and manipulation of microRNAs in stem cells may enhance their capacity for vascular regeneration.

Single embryoid body formation in a multi-well plate

Embryoid bodies (EB) formed from murine embryonic stem (ES) cells recapitulate many aspects of a developing embryo. Of specific importance, synchronous differentiation of EB recapitulates organ-specific development and is achieved in culture by formation of uniformly sized EB. The method described here demonstrates a simple and cost-effective way of generating EB from murine ES cells. Single EB are formed in a multi-well plate format and large numbers of EB are generated using a 96-well multi-well plate. Uniform single-sized EB formed in the multi-well are an ideal system for screening compounds and determining differentiation effects. Since EB contain all three germ layers, they are appropriate for studying small molecule effects on differentiation of ES such as is performed in high-throughput screening protocols