Profiling Of Embryonic Stem Cells Research Articles

Global Proteomic Profiling of Embryonic Stem Cells Using iTRAQ Isobaric Tags with LC-MS/MS Quantification.

In this methods chapter, we describe the use of isobaric tags for relative and absolute quantification (iTRAQ) for the differential expression analysis of global proteins between embryonic stem cell samples. This protocol describes how proteins are collected from cell culture, digested and prepared so that peptides are labeled with these isobaric tags. Labeled digests are pooled, fractionated offline, and quantified using liquid chromatography-mass spectrometry (LC-MS). This offline fractionation allows for a greater separation and thus increased identification/quantification of peptides. This combined method enables large-scale, deep penetration into the proteome of embryonic stem cells. During quantification, the relative intensities of label-derived reporter ions represent the relative amount of peptide in each sample. Using search algorithms that integrate the generated data for the identified and quantified peptides allows the relative quantification of proteins in the samples. The isobaric tags can be used in a 4 or 8 multiplexed manner; however, using an 8-plex experimental setup allows for the simultaneous analysis of biological and technical replicates within the same mass spectrometry run, thus minimizing experimental variation and increasing the confidence in any identified expression differences.

P53: emerging roles in stem cells, development and beyond.

Most human cancers harbor mutations in the gene encoding p53. As a result, research on p53 in the past few decades has focused primarily on its role as a tumor suppressor. One consequence of this focus is that the functions of p53 in development have largely been ignored. However, recent advances, such as the genomic profiling of embryonic stem cells, have uncovered the significance and mechanisms of p53 functions in mammalian cell differentiation and development. As we review here, these recent findings reveal roles that complement the well-established roles for p53 in tumor suppression.

Genome-Wide Prediction of DNA Methylation Using DNA Composition and Sequence Complexity in Human.

DNA methylation plays a significant role in transcriptional regulation by repressing activity. Change of the DNA methylation level is an important factor affecting the expression of target genes and downstream phenotypes. Because current experimental technologies can only assay a small proportion of CpG sites in the human genome, it is urgent to develop reliable computational models for predicting genome-wide DNA methylation. Here, we proposed a novel algorithm that accurately extracted sequence complexity features (seven features) and developed a support-vector-machine-based prediction model with integration of the reported DNA composition features (trinucleotide frequency and GC content, 65 features) by utilizing the methylation profiles of embryonic stem cells in human. The prediction results from 22 human chromosomes with size-varied windows showed that the 600-bp window achieved the best average accuracy of 94.7%. Moreover, comparisons with two existing methods further showed the superiority of our model, and cross-species predictions on mouse data also demonstrated that our model has certain generalization ability. Finally, a statistical test of the experimental data and the predicted data on functional regions annotated by ChromHMM found that six out of 10 regions were consistent, which implies reliable prediction of unassayed CpG sites. Accordingly, we believe that our novel model will be useful and reliable in predicting DNA methylation.

Proteomic profile of embryonic stem cells with low survival motor neuron protein is consistent with developmental dysfunction.

Spinal muscular atrophy is an autosomal recessive motor neuron disease caused by a genetic defect carried by as many as one in 75 people. Unlike most neurological disorders, we know exactly what the genetic basis is of the disorder, but in spite of this, have little understanding of why the low levels of one protein, survival motor neuron protein, results in the specific progressive die back of only one cell type in the body, the motor neuron. Given the fact that all cells in the body of a patient with spinal muscular atrophy share the same low abundance of the protein throughout development, an appropriate approach is to ask how lower levels of survival motor neuron protein affects the proteome of embryonic stem cells prior to development. Convergent biostatistical analyses of a discovery proteomic analysis of these cells provide results that are consistent with the pathomechanistic fate of the developed motor neuron.

Integrated genomics has identified a new AT/RT-like yet INI1-positive brain tumor subtype among primary pediatric embryonal tumors.

BackgroundPediatric embryonal brain tumors (PEBTs), which encompass medulloblastoma (MB), primitive neuroectodermal tumor (PNET) and atypical teratoid/rhabdoid tumor (AT/RT), are the second most prevalent pediatric brain tumor type. AT/RT is highly malignant and is often misdiagnosed as MB or PNET. The distinction of AT/RT from PNET/MB is of clinical significance because the survival rate of patients with AT/RT is substantially lower. The diagnosis of AT/RT relies primarily on morphologic assessment and immunohistochemical (IHC) staining for a few known markers such as the lack of INI1 protein expression. However, in our clinical practice we have observed several AT/RT-like tumors, that fulfilled histopathological and all other biomarker criteria for a diagnosis of AT/RT, yet retained INI1 immunoreactivity. Recent studies have also reported preserved INI1 immunoreactivity among certain diagnosed AT/RTs. It is therefore necessary to re-evaluate INI1(+), AT/RT-like cases.MethodSanger sequencing, array CGH and mRNA microarray analyses were performed on PEBT samples to investigate their genomic landscapes.ResultsPatients with AT/RT and those with INI(+) AT/RT-like tumors showed a similar survival rate, and global array CGH analysis and INI1 gene sequencing showed no differential chromosomal aberration markers between INI1(−) AT/RT and INI(+) AT/RT-like cases. We did not misdiagnose MBs or PNETs as AT/RT-like tumors because transcriptome profiling revealed that not only did AT/RT and INI(+) AT/RT-like cases express distinct mRNA and microRNA profiles, their gene expression patterns were different from those of MBs and PNETs. The most similar transcriptome profile to that of AT/RTs was the profile of embryonic stem cells. However; the transcriptome profile of INI1(+) AT/RT-like tumors was more similar to that of somatic neural stem cells, while the profile of MBs was closer to that of fetal brain tissue. Novel biomarkers were identified that can be used to distinguish INI1(−) AT/RTs, INI1(+) AT/RT-like cases and MBs.ConclusionOur studies revealed a novel INI1(+) ATRT-like subtype among Taiwanese pediatric patients. New diagnostic biomarkers, as well as new therapeutic tactics, can be developed according to the transcriptome data that were unveiled in this work.Electronic supplementary materialThe online version of this article (doi:10.1186/s12920-015-0103-3) contains supplementary material, which is available to authorized users.

The similarity between human embryonic stem cell-derived epithelial cells and ameloblast-lineage cells

This study aimed to compare epithelial cells derived from human embryonic stem cells (hESCs) to human ameloblast-lineage cells (ALCs), as a way to determine their potential use as a cell source for ameloblast regeneration. Induced by various concentrations of bone morphogenetic protein 4 (BMP4), retinoic acid (RA) and lithium chloride (LiCl) for 7 days, hESCs adopted cobble-stone epithelial phenotype (hESC-derived epithelial cells (ES-ECs)) and expressed cytokeratin 14. Compared with ALCs and oral epithelial cells (OE), ES-ECs expressed amelogenesis-associated genes similar to ALCs. ES-ECs were compared with human fetal skin epithelium, human fetal oral buccal mucosal epithelial cells and human ALCs for their expression pattern of cytokeratins as well. ALCs had relatively high expression levels of cytokeratin 76, which was also found to be upregulated in ES-ECs. Based on the present study, with the similarity of gene expression with ALCs, ES-ECs are a promising potential cell source for regeneration, which are not available in erupted human teeth for regeneration of enamel.

Developmental Enhancement of Adenylate Kinase-AMPK Metabolic Signaling Axis Supports Stem Cell Cardiac Differentiation

BackgroundEnergetic and metabolic circuits that orchestrate cell differentiation are largely unknown. Adenylate kinase (AK) and associated AMP-activated protein kinase (AMPK) constitute a major metabolic signaling axis, yet the role of this system in guiding differentiation and lineage specification remains undefined.Methods and ResultsCardiac stem cell differentiation is the earliest event in organogenesis, and a suitable model of developmental bioenergetics. Molecular profiling of embryonic stem cells during cardiogenesis revealed here a distinct expression pattern of adenylate kinase and AMPK genes that encode the AK-AMP-AMPK metabolic surveillance axis. Cardiac differentiation upregulated cytosolic AK1 isoform, doubled AMP-generating adenylate kinase activity, and increased AMP/ATP ratio. At cell cycle initiation, AK1 translocated into the nucleus and associated with centromeres during energy-consuming metaphase. Concomitantly, the cardiac AMP-signal receptor AMPKα2 was upregulated and redistributed to the nuclear compartment as signaling-competent phosphorylated p-AMPKα(Thr172). The cardiogenic growth factor TGF-β promoted AK1 expression, while knockdown of AK1, AK2 and AK5 activities with siRNA or suppression by hyperglycemia disrupted cardiogenesis compromising mitochondrial and myofibrillar network formation and contractile performance. Induction of creatine kinase, the alternate phosphotransfer pathway, compensated for adenylate kinase-dependent energetic deficits.ConclusionsDevelopmental deployment and upregulation of the adenylate kinase/AMPK tandem provides a nucleocytosolic energetic and metabolic signaling vector integral to execution of stem cell cardiac differentiation. Targeted redistribution of the adenylate kinase-AMPK circuit associated with cell cycle and asymmetric cell division uncovers a regulator for cardiogenesis and heart tissue regeneration.

Differential expression profiling between atypical teratoid/rhabdoid and medulloblastoma tumor in vitro and in vivo using microarray analysis

Atypical teratoid/rhabdoid tumor (AT/RT) and medulloblastoma (MB) are the most malignant primary brain tumors in early childhood. AT/RT is frequently misdiagnosed as primitive neuroectodermal tumor/medulloblastoma. The biological features and clinical outcomes of AT/RT and MB are extremely different. In this study, we used microarray as a platform to distinguish these two tumors with the definitive diagnostic marker as well as the profiling of expression genes. In order to clarify the pathogenesis and find the biological markers for AT/RT, we established a derivative AT/RT primary cell culture. The differential profiling between AT/RT and MB were analyzed by using microarray method. With the use of the microarray method, we demonstrated that 15 genes were significantly changed (at least 5-fold in upregulation and 1/5-fold in downregulation) between AT/RT and MB in tissues and cell lines. The quantitative reverse transcription-polymerase chain reaction analyses further confirmed that mRNA expression levels of SERPINI1 and osteopontin were highly expressed in AT/RT cells and tissues than those in MB. Importantly, our microarray result suggested that AT/RT presents the stemness-like pattern and expression profiling of embryonic stem cells as well as high mRNA expressions of Oct-4, Nanog, Sox-2, and c-Myc. Our study demonstrated the differential gene expression profiling between AT/RT and MB. Based on the microarray findings, AT/RTs present embryonic stem-like gene recapitulation and further provide novel insights into their underlying biology.

Gene expression profiling of embryonic stem cells leads to greater understanding of pluripotency and early developmental events.

Embryonic stem cells are characterized by their ability to propagate indefinitely in culture, maintaining a normal karyotype and their undifferentiated state. They have the potential of differentiating into any specialized cell type in the body. An understanding of the transcriptional profile related to pluripotency and early development is necessary to better tap their developmental potential and also maintain their undifferentiated phenotype. Currently, several techniques are in use to ascertain the gene expression profile of embryonic stem cells. This review summarizes the information generated using microarray and other approaches on the gene expression analyses of stem cells in both mouse and human cell lines. We also discuss specific approaches useful in future studies aimed at further deciphering the pluripotent nature of human embryonic stem cells.