Human Embryonic Stem Cells and Embryonal Carcinoma Cells Have Overlapping and Distinct Metabolic Signatures

While human embryonic stem cells (hESCs) and human embryonal carcinoma cells (hECCs) have been studied extensively at the levels of the genome, transcriptome, proteome and epigenome our knowledge of their corresponding metabolomes is limited. Here, we present the metabolic signatures of hESCs and hESCs obtained by untargeted gas chromatography coupled to mass spectrometry (GC-MS). Whilst some metabolites are common to both cell types, representing the self-renewal and house-keeping signatures, others were either higher (e.g., octadecenoic acid, glycerol-3-phosphate, 4-hydroxyproline) or lower (e.g., glutamic acid, mannitol, malic acid, GABA) in hESCs (H9) compared to hECCs (NTERA2), these represent cell type specific signatures. Further, our combined results of GC-MS and microarray based gene expression profiling of undifferentiated and OCT4-depleted hESCs are consistent with the Warburg effect which is increased glycolysis in embryonic cells and tumor cells in the presence of O2 while oxidative phosphorylation (OXPHOS) is impaired or even shut down. RNAi-based OCT4 knock down mediated differentiation resulted in the activation of the poised OXPHOS machinery by expressing missing key proteins such as NDUFC1, UQCRB and COX, increase in TCA cycle activity and decreased lactate metabolism. These results shed light on the metabolite layer of pluripotent stem cells and could potentially establish novel metabolic markers of self renewal and pluripotency

Transcriptional Signature and Memory Retention of Human-Induced Pluripotent Stem Cells

Genetic reprogramming of somatic cells to a pluripotent state (induced pluripotent stem cells or iPSCs) by over-expression of specific genes has been accomplished using mouse and human cells. However, it is still unclear how similar human iPSCs are to human Embryonic Stem Cells (hESCs). Here, we describe the transcriptional profile of human iPSCs generated without viral vectors or genomic insertions, revealing that these cells are in general similar to hESCs but with significant differences. For the generation of human iPSCs without viral vectors or genomic insertions, pluripotent factors Oct4 and Nanog were cloned in episomal vectors and transfected into human fetal neural progenitor cells. The transient expression of these two factors, or from Oct4 alone, resulted in efficient generation of human iPSCs. The reprogramming strategy described here revealed a potential transcriptional signature for human iPSCs yet retaining the gene expression of donor cells in human reprogrammed cells free of viral and transgene interference. Moreover, the episomal reprogramming strategy represents a safe way to generate human iPSCs for clinical purposes and basic research

The Bacterium Endosymbiont of Crithidia deanei Undergoes Coordinated Division with the Host Cell Nucleus

In trypanosomatids, cell division involves morphological changes and requires coordinated replication and segregation of the nucleus, kinetoplast and flagellum. In endosymbiont-containing trypanosomatids, like Crithidia deanei, this process is more complex, as each daughter cell contains only a single symbiotic bacterium, indicating that the prokaryote must replicate synchronically with the host protozoan. In this study, we used light and electron microscopy combined with three-dimensional reconstruction approaches to observe the endosymbiont shape and division during C. deanei cell cycle. We found that the bacterium replicates before the basal body and kinetoplast segregations and that the nucleus is the last organelle to divide, before cytokinesis. In addition, the endosymbiont is usually found close to the host cell nucleus, presenting different shapes during the protozoan cell cycle. Considering that the endosymbiosis in trypanosomatids is a mutualistic relationship, which resembles organelle acquisition during evolution, these findings establish an excellent model for the understanding of mechanisms related with the establishment of organelles in eukaryotic cells

Aberrant epigenetic changes and gene expression in cloned cattle dying around birth

<p>Abstract</p> <p>Background</p> <p>Aberrant reprogramming of donor somatic cell nuclei may result in many severe problems in animal cloning. To assess the extent of abnormal epigenetic modifications and gene expression in clones, we simultaneously examined DNA methylation, histone H4 acetylation and expression of six genes (<it>β-actin</it>, <it>VEGF</it>, <it>oct4</it>, <it>TERT</it>, <it>H19 </it>and <it>Igf2</it>) and a repetitive sequence (<it>art2</it>) in five organs (heart, liver, spleen, lung and kidney) from two cloned cattle groups that had died at different stages. In the ED group (early death, n = 3), the cloned cattle died in the perinatal period. The cattle in the LD group (late death, n = 3) died after the perinatal period. Normally reproduced cattle served as a control group (n = 3).</p> <p>Results</p> <p>Aberrant DNA methylation, histone H4 acetylation and gene expression were observed in both cloned groups. The ED group showed relatively fewer severe DNA methylation abnormalities (p < 0.05) but more abnormal histone H4 acetylations (p < 0.05) and more abnormal expression (p < 0.05) of the selected genes compared to the LD group. However, our data also suggest no widespread gene expression abnormalities in the organs of the dead clones.</p> <p>Conclusion</p> <p>Deaths of clones may be ascribed to abnormal expression of a very limited number of genes.</p

Human embryonic stem cell-derived neurons as a tool for studying neuroprotection and neurodegeneration.

The capacity to generate myriad differentiated cell types, including neurons, from human embryonic stem cell (hESC) lines offers great potential for developing cell-based therapies and also for increasing our understanding of human developmental mechanisms. In addition, the emerging development of this technology as an experimental tool represents a potential opportunity for neuroscientists interested in mechanisms of neuroprotection and neurodegeneration. Potentially unlimited generation of well-defined functional neurons from hES and patient specific induced pluripotent (iPS) cells offers new systems to study disease mechanisms, signalling pathways and receptor pharmacology within a human cellular environment. Such systems may help in overcoming interspecies differences. Far from replacing rodent in vivo and primary culture systems, hES and iPS cell-derived neurons offer a complementary resource to overcome issues of interspecies differences, accelerate drug discovery, study of disease mechanism as well as provide basic insight into human neuronal physiology

Cardiac commitment of primate embryonic stem cells

International audiencePrimate nonhuman and human embryonic stem (ES) cells provide a powerful model of early cardiogenesis. Furthermore, engineering of cardiac progenitors or cardiomyocytes from ES cells offers a tool for drug screening in toxicology or to search for molecules to improve and scale up the process of cardiac differentiation using high-throughput screening technology, as well as a source of cell therapy of heart failure. Spontaneous differentiation of ES cells into cardiomyocytes is however limited. Herein, we describe a simple protocol to commit both rhesus and human ES cells toward a cardiac lineage and to sort out early cardiac progenitors. Primate ES cells are challenged for 4 d with the cardiogenic morphogen bone morphogenetic protein 2 (BMP2) and sorted out using anti-SSEA-1 antibody-conjugated magnetic beads. Cardiac progenitor cells can be generated and isolated in 4 d using this protocol

Pontos de corte para a aptidão cardiorrespiratória e a triagem de fatores de risco para doenças cardiovasculares na infância Cardiorespiratory fitness cut offs points and cardiovascular risk factors screening at infancy

O principal objetivo deste estudo foi analisar a validade e propor novos pontos de corte para aptidão cardiorrespiratória de escolares de sete a 12 anos de idade. A amostra foi constituída por 1.413 escolares. A aptidão cardiorrespiratória foi medida através do teste de corrida/caminhada de nove minutos. O colesterol total e as pressões arterial sistólica e diastólica foram medidas e através delas foram criadas referências de fatores de risco para doenças cardiovasculares. Através da curva receiver operating characteristic entre a aptidão cardiorrespiratória e as referências de fatores de risco para doenças cardiovasculares foram identificados os valores dos pontos de corte. Adicionalmente foi utilizada a análise bivariada seguida do cálculo de razão de chances (odds ratio-OR) para identificar quanto os indivíduos que não atenderam os pontos de corte propostos tinham a mais de chance de apresentar fatores de risco para doenças cardiovasculares. Os resultados indicaram que, dentre as propostas analisadas, a que foi sugerida neste estudo apresentou melhores ajustamentos entre sensibilidade e especificidade. Os resultados da análise bivariada mostraram que indivíduos que não atenderam os pontos de corte propostos têm maiores chances de apresentarem fatores de risco para doenças cardiovasculares.<br>The main objective of the study was to analyze and propose news cardiorespiratory fitness cut offs points for 7 to 12-year old school students. The sample was composed of 1,413 students. Cardiorespiratory fitness was measured by the 9-minutes walking/running test. Total cholesterol and systolic and diastolic blood pressure were measured and with their results cardiovascular risk factors reference were designed. The Receiver Operating Characteristic curve between cardiorespiratory fitness and cardiovascular risk factors reference identified the cut offs point values. The bivariate analysis and odds ratio (OR) calculation was used to identify to what extent the individuals that did not reach the proposed cut offs points could show cardiovascular risk factors. The results showed that the cut offs points suggested in this study presented the best balance between sensibility and specificity. The bivariate analysis results showed increased chance of cardiovascular risk for the individuals that did not reach the cut offs points compared to those individuals who did