Dataset Information

Analyses of the SAGE expression of Human Embryonic Stem Cells

ABSTRACT: A plethora of research has focused on the human embryonic stem cells (hESC) ever since they were first reported mainly due to their distinct features of self renewal and pluripotency. Probing of the hESC transcriptome using global expression profiling tools such as DNA microarray, Serial Analysis of Gene Expression (SAGE), Massively Parallel Signature Sequencing (MPSS) and Expressed Sequence Tag (EST) analysis have contributed significantly in our current understanding of hESCs. In fact, a large number of markers to assess the pluripotent and differentiation status of hESCs have been presented by such studies. However, till date clarity is lacking in identifying a robust set of markers to assess the true state of the hESCs. In this paper, we report the generation of long SAGE libraries for partially differentiated and differentiated HES3 along with a deeper profiling of our previously reported HES3 undifferentiated library. Clustering analysis of these libraries in concert with long and short SAGE libraries available in public databases using Hierarchical Cluster Analysis (HCA) as well as a poison-based approach helped in identification of expression patterns distinct from those reported for the well established pluriptency/differentiation markers. Almost all the previous studies reporting a robust set of markers have focused on a gene by gene comparison in listing out the upregulated and downregulated genes rather than look at the expression patterns in establishing a list of markers. In this analysis, however, we report a new set of markers as well as add confidence to some previously reported markers based on their novel expression patterns as identified by SAGE analysis and also confirm it by real-time PCR analysis. For the real-time PCR confirmation, instead of taking two extreme data sets such as undifferentiated and a late stage embryoid body, we profiled a time series of embryoid body stages so that we could identify those genes which show a dramatic increase or decrease upon differentiation and hence would serve as more reliable markers.

ORGANISM(S): Homo sapiens

PROVIDER: GSE12611 | GEO | 2010/02/17

SECONDARY ACCESSION(S): PRJNA112877

REPOSITORIES: GEO

ACCESS DATA

Similar Datasets

Project description:A plethora of research has focused on the human embryonic stem cells (hESC) ever since they were first reported mainly due to their distinct features of self renewal and pluripotency. Probing of the hESC transcriptome using global expression profiling tools such as DNA microarray, Serial Analysis of Gene Expression (SAGE), Massively Parallel Signature Sequencing (MPSS) and Expressed Sequence Tag (EST) analysis have contributed significantly in our current understanding of hESCs. In fact, a large number of markers to assess the pluripotent and differentiation status of hESCs have been presented by such studies. However, till date clarity is lacking in identifying a robust set of markers to assess the true state of the hESCs. In this paper, we report the generation of long SAGE libraries for partially differentiated and differentiated HES3 along with a deeper profiling of our previously reported HES3 undifferentiated library. Clustering analysis of these libraries in concert with long and short SAGE libraries available in public databases using Hierarchical Cluster Analysis (HCA) as well as a poison-based approach helped in identification of expression patterns distinct from those reported for the well established pluriptency/differentiation markers. Almost all the previous studies reporting a robust set of markers have focused on a gene by gene comparison in listing out the upregulated and downregulated genes rather than look at the expression patterns in establishing a list of markers. In this analysis, however, we report a new set of markers as well as add confidence to some previously reported markers based on their novel expression patterns as identified by SAGE analysis and also confirm it by real-time PCR analysis. For the real-time PCR confirmation, instead of taking two extreme data sets such as undifferentiated and a late stage embryoid body, we profiled a time series of embryoid body stages so that we could identify those genes which show a dramatic increase or decrease upon differentiation and hence would serve as more reliable markers. The hESC lines HES3 from ES Cell International, Singapore (http://www.escellinternational.com) were cultured on mouse embryonic fibroblast feeders (MEFs) as described previously. Selection of cells for library construction was done by micro-dissection following our established protocols. Spontaneous differentiation was induced by prolonged culture without changing the feeder layer. Briefly the HES3 cell line was grown to 18 passages (P18) on MEFs, after which no sub-culturing was performed. The medium was changed daily for 25 days and the differentiated cell population was harvested at the end of the culture period by micro-dissection. Cell differentiation was confirmed by Immuno-cytochemistry for markers to the three germ layers, as well as RT-PCR for the differentiation markers. For construction of the partially differentiated library, colonies which had started differentiation, as suggested by the morphological changes towards the centre and periphery of the colonies were used. Entire HES3 colonies (24P) which had started differentiation were harvested by micro-dissection and used for library construction. MmeI was used as tagging enzyme and libraries were constructed using the LS-SAGE kit from Invitrogen (http://www.invitrogen.com). Cloning of concatemerized ditags and sequencing of tags were done as outlined earlier. The 10 bp SAGE tags were extracted using Microsoft Excel for direct comparisons between libraries.

Project description:Recent advances in the study of human embryonic stem cells (hESCs) and induced-pluripotent stem cells (iPS) highlight their importance as both model systems for basic research and avenues for therapeutic applications. To gain better insight into these cells, a clearer understanding of their molecular properties is crucial. In this study, we analyze changes in the expression profile of microRNAs (miRNAs) and mRNAs in nine different, National Institutes of Health (NIH)-approved hESC lines. By examining both undifferentiated hESCs and cells exposed to an undirected differentiation scheme at early stages, we found those miRNAs and mRNAs enriched in the hESC lines, and those miRNAs and mRNAs initially regulated upon commitment. In comparing these profiles with those of an embryonal carcinoma (EC) cell line, NTera-2, we observed distinct patterns of miRNA expression in hESCs. Furthermore, we identify several new hESC-enriched miRNAs that respond rapidly to differentiation cues, in addition to miRNAs from a large cluster of hESC-enriched miRNAs located on chromosome 19, and miRNAs from the miR-302 cluster and the miR-17~92 family of clusters. We show that key miRNAs in the chromosome 19 cluster are highly enriched in hESCs in comparison to NTera-2, and might therefore serve as future diagnostic markers for stem cell capacity. Examination of changes in mRNA expression during early commitment further reveals that many differentiation-regulated genes are possible candidates for regulation by these hESC-enriched miRNAs. A set of 9 different NIH-approved hESC lines were treated with conditions to promote either maintenance of an undifferentiated state (mouse embryonic fibroblast- conditioned media) or undirected differentiation of the hESCs into various cell lineages (DMEM + 20% FBS). RNA was harvested from the cells and subjected to miRNA microarray analysis. An embryonal carcinma cell line, NTera-2, which shows similar growth characteristics to hESCs was used an additional comparative sample. Additionally, human placental RNA samples were used as internal controls for miRNA microarray slide (Agilent Technologies). The miRNA expression data was used to analyze differential miRNA expression in each specific cell line, as well as large-scale comparisons of the undifferentiated and differentiated hESC lines.

Project description:Background: Developmental stage-specific globin expression is a complex phenomenon that involves both trans- and cis-acting elements. While functional analyses ensuing recent genome-wide association studies have highlighted the important roles of trans-factors in regulating hemoglobin expression, these factors can not exert their functions without permissive chromatin domains. By transferring thoroughly profiled beta globin locus of undifferentiated human embryonic stem cells (hESCs) or hESC-derived erythroid cells into an adult erythroid transcriptional environment, we studied the influences of histone modifications on the globin expression decision within a fixed transcriptional environment. Shortly after the locus transfer, embryonic epsilon globin was not expressed regardless of original chromatin states, whereas fetal gamma globin was either expressed or not activated depending on original chromatin configurations, and the originally silent adult beta globin either remained silent or became activated depending on the expression status of gamma globin. These data suggest the interplay between transcriptional environment and the chromatin modifications determine the outcome of globin expression. As the ultimate silencing of gamma globin from hESC-derived erythroid cells in the adult transcriptional environment occurred after months-long cell proliferation, our work also has implications on attempts to generate beta globin expressing erythroid cells from hESCs or induced pluripotent stem cells. hESC line H1 (NIH code WA01, WiCell, Madison, WI) and adeno-associated virus (AAV)-targeted lines, were maintained and differentiated as previously described.12 (Details can be found in the supplemental information.) The expression of stem cell markers including SSEA-3, SSEA-4, TRA-1-60, and TRA-1-61 was detected by flow cytometry. To determine whether AAV-targeted lines retained their hematopoietic differentiation potential, confluent hESCs were harvested off the feeder layers and transferred to ultra-low attachment plates to allow for the formation of embryoid bodies (EBs). Day-14 EBs were dissociated into single cells and the expression of surface markers including CD34, CD71, CD45, CD31, CD41 and glycophorin-A was determined by flow cytometry. To induce erythroid differentiation, day-7 EBs were made into single cell suspension and cultured in erythroid-inducing medium for 14 days. Primers for real time PCR analysis of beta locus globin mRNA expression are provided in the supplemental information.

Dataset Information

Analyses of the SAGE expression of Human Embryonic Stem Cells

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure