Reprogramming somatic cells to a pluripotent state drastically reconfigures the cellular anabolic requirements, thus potentially inducing cancer-like metabolic transformation. Accordingly, we and others previously showed that somatic mitochondria and bioenergetics are extensively remodeled upon derivation of induced pluripotent stem cells (iPSCs), as the cells transit from oxidative to glycolytic metabolism. In the attempt to identify possible regulatory mechanisms underlying this metabolic restructuring, we investigated the contributing role of hypoxia-inducible factor 1 {alpha} (HIF1{alpha}), a master regulator of energy metabolism, in the induction and maintenance of pluripotency. We discovered that the ablation of HIF1{alpha} function in dermal fibroblasts dramatically hampers reprogramming efficiency, while small molecule-based activation of HIF1{alpha} significantly improves cell fate conversion. Transcriptional and bioenergetic analysis during reprogramming initiation indicated that the transduction of the four factors is sufficient to up-regulate the HIF1{alpha} target pyruvate dehydrogenase kinase (PDK) 1 and set in motion the glycolytic shift. However, additional HIF1{alpha} activation appears critical in the early up-regulation of other HIF1{alpha}-associated metabolic regulators, including PDK3 and pyruvate kinase (PK) isoform M2 (PKM2), resulting in increased glycolysis and enhanced reprogramming. Accordingly, elevated levels of PDK1, PDK3, and PKM2 and reduced PK activity could be observed in iPSCs and human embryonic stem cells (hESCs) in the undifferentiated state. Overall, the findings suggest that the early induction of HIF1{alpha} targets may be instrumental in iPSC derivation via the activation of a glycolytic program. These findings implicate the HIF1{alpha} pathway as an enabling regulator of cellular reprogramming.

Teratoma formation in mice is today the most stringent test for pluripotency that is available for human pluripotent cells, as chimera formation and tetraploid complementation cannot be performed with human cells. The teratoma assay could also be applied for assessing the safety of human pluripotent cell-derived cell populations intended for therapeutic applications. In our study we examined the spontaneous differentiation behaviour of human embryonic stem cells (hESCs) in a perfused 3D multi-compartment bioreactor system and compared it with differentiation of hESCs and human induced pluripotent cells (hiPSCs) cultured in vitro as embryoid bodies and in vivo in an experimental mouse model of teratoma formation. Results from biochemical, histological/immunohistological and ultrastuctural analyses revealed that hESCs cultured in bioreactors formed tissue-like structures containing derivatives of all three germ layers. Comparison with embryoid bodies and the teratomas revealed a high degree of similarity of the tissues formed in the bioreactor to these in the teratomas at the histological as well as transcriptional level, as detected by comparative whole-genome RNA expression profiling. The 3D culture system represents a novel in vitro model that permits stable long-term cultivation, spontaneous multi-lineage differentiation and tissue formation of pluripotent cells that is comparable to in vivo differentiation. Such a model is of interest, e.g. for the development of novel cell differentiation strategies. In addition, the 3D in vitro model could be used for teratoma studies and pluripotency assays in a fully defined, controlled environment, alternatively to in vivo mouse models. Copyright (c) 2012 John Wiley & Sons, Ltd.

Quantitative real-time polymerase chain reaction (QRT-PCR) has become an extensively applied technique. It enables quantitative analyses of gene expression applicable to basic molecular biology, medicine, and diagnostics. Nowadays, it is broadly used to describe messenger RNA (mRNA) expression patterns and to compare the relative levels of mRNA within distinct biological samples. The scope of the QRT-PCR technique makes it applicable across a wide range of experimental conditions and allows experimental comparison between normal and abnormal tissue. Most importantly, this technique enables additional independent confirmation of microarray or next generation sequencing (NGS)-based results. An inherent advantage of QRT-PCR is the large dynamic range, remarkable sensitivity, and sequence-specificity. We provide a detailed step by step guide to the principles underlying a successful QRT-PCR experiment.

Atrial fibrillation (AF) affects millions of individuals worldwide. The genome-wide association studies have identified robust genetic associations with AF.We genotyped 5461 participants of Japanese ancestry for 11 AF-related loci and determined the effects of carrying different numbers of risk alleles on disease development and age at disease onset. The weighted genetic risk score (GRS) was calculated, and its ability to predict AF was determined.Six single-nucleotide polymorphisms—rs593479 (1q24 in PRRX1), rs1906617 (4q25 near PITX2), rs11773845 (7q31 in CAV1), rs6584555 (10q25 in NEURL), rs6490029 (12q24 in CUX2), and rs12932445 (16q22 in ZFHX3) (P < 1.9 × 10−5)—were confirmed as being associated with AF. Patients with a high total number of risk alleles (9-12) had a younger median age at onset of AF (58 years; 95% confidence interval [CI], 55-60 years) than those with a low total number (1-4) (63 years; 95% CI, 61-64 years) (P = 0.0015). We observed a 4.38-fold (95% CI, 3.69-5.19) difference in risk of AF between individuals with scores in the top and bottom quartiles of the GRS. Receiver operating characteristic analysis indicated an area under the curve of 0.641 (95% CI, 0.628-0.653; P < 0.0001).Six loci were validated as associated with AF in a Japanese population. This study suggests that a combination of common genetic markers modestly facilitates discrimination of AF. This is the first report, to our knowledge, to demonstrate that the age of onset of AF is affected by common risk alleles.La fibrillation auriculaire (FA) touche des millions d’individus dans le monde. Les études d’association sur tout le génome ont permis de cerner des associations génétiques fiables avec la FA.Nous avons établi le génotype de 5461 participants d’ascendance japonaise pour 11 locus liés à la FA et déterminé les effets d’être porteurs d’un nombre différent d’allèles du risque sur le développement de la maladie et l’âge à l’apparition de la maladie. Nous avons calculé le score de risque génétique (SRG) pondéré et déterminé sa capacité à prédire la FA.Nous avons confirmé l’association de 6 polymorphismes de nucléotide simple à la FA, soit le rs593479 (1q24 en PRRX1), le rs1906617 (4q25 près de PITX2), le rs11773845 (7q31 en CAV1), le rs6584555 (10q25 en NEURL), le rs6490029 (12q24 en CUX2) et le rs12932445 (16q22 en ZFHX3) (P < 1,9 × 10−5). Les patients porteurs d’un nombre total élevé d’allèles du risque (9 à 12) étaient plus jeunes à l’apparition de la FA (âge médian de 58 ans ; intervalle de confiance [IC] à 95 %, 55-60 ans) que ceux ayant un faible nombre total (1 à 4) (âge médian de 63 ans ; IC à 95 %, 61-64 ans) (P = 0,0015). Nous avons observé une différence de 4,38 fois plus grande dans le risque de FA (IC à 95 %, 3,69-5,19) entre les individus ayant des scores dans les quartiles supérieurs et inférieurs du SRG. L’analyse de la fonction d’efficacité du récepteur indiquait une surface sous la courbe de 0,641 (IC à 95 %, 0,628-0,653 ; P < 0,0001).Nous avons validé l’association de 6 locus à la FA dans la population japonaise. Cette étude suggère qu’une combinaison de marqueurs génétiques courants facilite modestement la distinction de la FA. À notre connaissance, il s’agit du premier rapport à démontrer que l’âge de l’apparition de la FA est influencé par les allèles du risque courants.

To avoid artefacts introduced by culturing cells for extended periods of time, it is crucial to use low-passage patient-derived tumour cells. The ability to enrich, isolate and assay sub-populations of cells that behave as cancer stem cells (CSCs) from these primary cell lines is essential before performing characterizations such as gene-expression profiling. We have isolated cells from glioblastomas which show characteristics of CSCs. Although glioblastomas contain only a relatively small amount of putative CSCs, these cells express many genes which seem to be worthy targets for future therapies.

The identification of cancer stem cells in various malignancies led to the hypothesis that these cells have the exclusive ability of self-renewal, contribute to the plasticity of the tumours and may be the cause for ineffective cancer therapies. Several markers of melanoma stem cells have been described in recent studies including CD133, CD166, Nestin and BMI-1. Further studies are necessary to identify, better define and understand the origin and function of cancer stem cells. If confirmed that cancer stem cells play an important role in malignancy, therapeutic strategies may need to be redirected towards these cells to circumvent the failure of conventional therapies.

Cancer stem cells (CSCs) were discovered about 15 years ago in hematopoietic cancers. Subsequently, cancer stem cells were discovered in various solid tumors. Based on parallels with normal stem cells, a developmental process of cancer stem cells follows paths of organized, hierarchical structure of cells with different degrees of maturity. While some investigators have reported particular markers as identification of cancer stem cells, these markers require further research. In this review, we focus on the functional genomics of cancer stem cells. Functional genomics provides useful information on the signaling pathways which are consecutively activated or inactivated amongst those cells. This information is of particular importance for cancer research and clinical treatment in many respects. (1) Understanding of self-renewal mechanisms crucial to tumor growth. (2) Allow the identification of new, more specific marker for CSCs, and (3) pathways that are suitable as future targets for anti-cancer drugs. This is of particular importance, because today's chemotherapy targets the proliferating cancer cells sparing the relatively slow dividing cancer stem cells. The first step on this long road therefore is to analyze genome-wide expression-profiles within the same type of cancer and then between different types of cancer, encircling those target genes and pathways, which are specific to these cells.