An expression profile analysis of ES cell-derived definitive endodermal cells and Pdx1-expressing cells

<p>Abstract</p> <p>Background</p> <p>We developed an efficient <it>in vitro </it>method to differentiate mouse ES cells into the definitive endoderm (DE) and then Pdx1-expressing pancreatic lineages using mesodermal-derived supporting cells, M15. Using this method, resulting ES cell-derived DE and Pdx1-expressing cells were isolated by cell sorting, and their gene expression profiles were investigated with DNA microarray. Genes that were specifically expressed in DE and/or in Pdx1-expressing cells were extracted and their expression patterns in normal embryonic development were studied.</p> <p>Results</p> <p>Genes whose expression increased in DE and Pdx1 positive cells compared to the undifferentiated ES cells were chosen and <it>in situ </it>hybridizations were performed. Out of 54 genes examined, 27 were expressed in the DE of E8.5 mouse embryos and 15 genes were expressed in distinct domains in the pancreatic buds of E14.5 embryos. Among those genes expressed were <it>Foxq1, CpM, Foxp4, Pcdh1, and Zmiz1</it>, which were previously reported in other endodermal tissues. Genes, such as <it>Parm1, Tmem184a, Hipk2 </it>and <it>Sox4 </it>were reported to be expressed during early pancreatic development. <it>Nptx2, C2cd4b, Tcf7l2 and Kiss1r </it>were reported to be associated with beta cell or pancreatic functions in the adult. <it>Akr1c19, Aebp2, Pbxip1 </it>and <it>Creb3l1</it>, were novel and have not been described as being expressed either in DE or the pancreas.</p> <p>Conclusions</p> <p>We identified 27 genes, including 4 novel genes expressed in DE and pancreatic progenitor cells during normal development using an ES cell <it>in vitro </it>differentiation system. These results showed that DE cells and Pdx1/GFP-expressing cells obtained from our M15 based differentiation method mimic cells during the normal developmental processes. Additionally, ES cells are an excellent model for studies of early developmental processes.</p

Methionine Metabolism Regulates Maintenance and Differentiation of Human Pluripotent Stem Cells

SummaryMouse embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are in a high-flux metabolic state, with a high dependence on threonine catabolism. However, little is known regarding amino acid metabolism in human ESCs/iPSCs. We show that human ESCs/iPSCs require high amounts of methionine (Met) and express high levels of enzymes involved in Met metabolism. Met deprivation results in a rapid decrease in intracellular S-adenosylmethionine (SAM), triggering the activation of p53-p38 signaling, reducing NANOG expression, and poising human iPSC/ESCs for differentiation, follow by potentiated differentiation into all three germ layers. However, when exposed to prolonged Met deprivation, the cells undergo apoptosis. We also show that human ESCs/iPSCs have regulatory systems to maintain constant intracellular Met and SAM levels. Our findings show that SAM is a key regulator for maintaining undifferentiated pluripotent stem cells and regulating their differentiation

Development of detection device for dugong calls

December 15-17, 2007, Royal Phuket City Hotel, Phuket, ThailandAn acoustical approach for research on marine mammals has been a very active research method in recent years. Dugong (Dugong dugon) is one of the highly endangered species, which are strictly-marine herbivorous and mainly inhabit coastal areas. In order to detect dugong calls from recorded data, several algorithms have been adapted by researchers in the analyzing process. However, the number of misses in the detection is still non-zero. The sound of snapping shrimp recorded in a wide range (2-300 kHz) is one of the main background noises that makes the detection of dugong calls difficult in warm shallow waters. Impulse elimination was employed in the system to get rid of the snapping shrimp noise. In order to improve the performance of the detection system by increasing the detection rate and decreasing the number of misses, two new algorithms were tested in the experiment. The experimental results for the new algorithms including impulse elimination and the cepstrum method are presented in this paper

DNA Methylation Profiling of Embryonic Stem Cell Differentiation into the Three Germ Layers

Embryogenesis is tightly regulated by multiple levels of epigenetic regulation such as DNA methylation, histone modification, and chromatin remodeling. DNA methylation patterns are erased in primordial germ cells and in the interval immediately following fertilization. Subsequent developmental reprogramming occurs by de novo methylation and demethylation. Variance in DNA methylation patterns between different cell types is not well understood. Here, using methylated DNA immunoprecipitation and tiling array technology, we have comprehensively analyzed DNA methylation patterns at proximal promoter regions in mouse embryonic stem (ES) cells, ES cell-derived early germ layers (ectoderm, endoderm and mesoderm) and four adult tissues (brain, liver, skeletal muscle and sperm). Most of the methylated regions are methylated across all three germ layers and in the three adult somatic tissues. This commonly methylated gene set is enriched in germ cell-associated genes that are generally transcriptionally inactive in somatic cells. We also compared DNA methylation patterns by global mapping of histone H3 lysine 4/27 trimethylation, and found that gain of DNA methylation correlates with loss of histone H3 lysine 4 trimethylation. Our combined findings indicate that differentiation of ES cells into the three germ layers is accompanied by an increased number of commonly methylated DNA regions and that these tissue-specific alterations in methylation occur for only a small number of genes. DNA methylation at the proximal promoter regions of commonly methylated genes thus appears to be an irreversible mark which functions to fix somatic lineage by repressing the transcription of germ cell-specific genes

Albumin gene targeting in human embryonic stem cells and induced pluripotent stem cells with helper-dependent adenoviral vector to monitor hepatic differentiation

AbstractAlthough progresses in developing differentiation procedures have been achieved, it remains challenging to generate hES/iPS cell-derived mature hepatocytes. We performed knock-in of a monomeric Kusabira orange (mKO1) cassette in the albumin (ALB) gene, in human embryonic stem (hES) cells and induced pluripotent stem (hiPS) cells, with the use of the helper-dependent adenovirus vector (HDAdV). Upon induction into the hepatic lineages, these knock-in hES/iPS cells differentiated into cells that displayed several known hepatic functions. The mKO1 knock-in (ALB/mKo1) hES/hiPS cells were used to visualize hepatic differentiation in vitro. mKO1 reporter expression recapitulated endogenous ALB transcriptional activity. ALB/mKo1 [Hi] population isolated by flow cytometry was confirmed to be enriched with ALB mRNA. Expression profile analyses revealed that characteristic hepatocyte genes and genes related to drug metabolism and many aspects of liver function were highly enriched in the ALB/mKo1 [Hi] population. Our data demonstrate that ALB/mKo1 knock-in hES/iPS cells are valuable resources for monitoring in vitro hepatic differentiation, isolation and analyses of hES and hiPS cells-derived hepatic cells that actively transcribing ALB. These knock-in hES/iPS cell lines could provide further insights into the mechanism of hepatic differentiation and molecular signatures of the hepatic cells derived from hES/iPS cells