Comparisons between Transcriptional Regulation and RNA Expression in Human Embryonic Stem Cell Lines

Advanced Technology Center, Microarray Facility, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Stem Cells and Development (Impact Factor: 3.73). 07/2006; 15(3):315-23. DOI: 10.1089/scd.2006.15.315
Source: PubMed


Recent studies have focused on transcriptional regulation and gene expression profiling of human embryonic stem cells (hESCs). However, little information is available regarding the relationship between RNA expression and transcriptional regulation, which is critical in the complete understanding of pluripotency and differentiation of hESCs. In the current study, we determined RNA expression of three different hESC lines compared to Human universal reference RNA expression (HuU-RNA) using a full genome expression microarray, and compared our results to target genes previously identified using ChIP-on-chip analysis. The objective was to identify genes common between the two methods, and generate a more reliable list of embryonic signature genes. Even though hESCs were obtained from different sources and maintained under different conditions, a considerable number of genes could be identified as common between RNA expression and transcriptional regulation analyses. As an example, results from ChIP-on-chip studies show that OCT4, SOX2, and NANOG co-occupy SOX2, OCT4, TDGF1, GJA1, SET, and DPPA4 genes. The results are consistent with RNA expression analyses that demonstrate these genes as differently expressed in our hESC lines, further substantiating their role across cell types and confirming their importance as embryonic signatures. In addition, we report the differential expression of growth arrest-specific (GAS) family of genes in hESC. GAS2L1 and GAS3 members of this family appear to be transcriptionally regulated by OCT4, SOX2, or NANOG, whereas GAS5 and GAS6 are not; all of the genes are differentially expressed, as determined by microarray and validated via quantitative (Q)- PCR. Collectively, these data provide insight into the relationship between gene expression and transcriptional regulation, resulting in a reliable list of genes associated with hESCs.

8 Reads
  • Source
    • "One hundred thirty seven GEO datasets appeared using these terms. We examined each dataset and screened for (a) samples processed using the Affymetrix U133 microarray platform (Santa Clara CA; [12-14] and (b) human/breast cancer samples that were not genetically manipulated or previously treated with drugs. Eight datasets were selected but 3 were excluded because they either contained too few breast samples or in the case of one, the dataset contained only TNBC samples. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Background Triple negative breast cancer (TNBC) and often basal-like cancers are defined as negative for estrogen receptor, progesterone receptor and Her2 gene expression. Over the past few years an incredible amount of data has been generated defining the molecular characteristics of both cancers. The aim of these studies is to better understand the cancers and identify genes and molecular pathways that might be useful as targeted therapies. In an attempt to contribute to the understanding of basal-like/TNBC, we examined the Gene Expression Omnibus (GEO) public datasets in search of genes that might define basal-like/TNBC. The Il32 gene was identified as a candidate. Findings Analysis of several GEO datasets showed differential expression of IL32 in patient samples previously designated as basal and/or TNBC compared to normal and luminal breast samples. As validation of the GEO results, RNA and protein expression levels were examined using MCF7 and MDA MB231 cell lines and tissue microarrays (TMAs). IL32 gene expression levels were higher in MDA MB231 compared to MCF7. Analysis of TMAs showed 42% of TNBC tissues and 25% of the non-TNBC were positive for IL32, while non-malignant patient samples and all but one hyperplastic tissue sample demonstrated lower levels of IL32 protein expression. Conclusion Data obtained from several publically available GEO datasets showed overexpression of IL32 gene in basal-like/TNBC samples compared to normal and luminal samples. In support of these data, analysis of TMA clinical samples demonstrated a particular pattern of IL32 differential expression. Considered together, these data suggest IL32 is a candidate suitable for further study.
    BMC Research Notes 08/2014; 7(1):501. DOI:10.1186/1756-0500-7-501
  • Source
    • "The stemness gene signatures composed of 190 genes [1], [2], [25], [32]–[35], [41]–[48] were used to compare the blastomeres from the 5-, 6- and 8- cell stage embryos (Table S2). All the genes were present in the array, and no significant differences were found between either the blastomeres from the same stage or different stages, except RRAS2 and FZD5 which were overrepresented in the 5-cell embryo blastomeres compared to those from the 6- and 8- cell embryos respectively (Figure 2B1). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Blastomere fate and embryonic genome activation (EGA) during human embryonic development are unsolved areas of high scientific and clinical interest. Forty-nine blastomeres from 5- to 8-cell human embryos have been investigated following an efficient single-cell cDNA amplification protocol to provide a template for high-density microarray analysis. The previously described markers, characteristic of Inner Cell Mass (ICM) (n = 120), stemness (n = 190) and Trophectoderm (TE) (n = 45), were analyzed, and a housekeeping pattern of 46 genes was established. All the human blastomeres from the 5- to 8-cell stage embryo displayed a common gene expression pattern corresponding to ICM markers (e.g., DDX3, FOXD3, LEFTY1, MYC, NANOG, POU5F1), stemness (e.g., POU5F1, DNMT3B, GABRB3, SOX2, ZFP42, TERT), and TE markers (e.g., GATA6, EOMES, CDX2, LHCGR). The EGA profile was also investigated between the 5-6- and 8-cell stage embryos, and compared to the blastocyst stage. Known genes (n = 92) such as depleted maternal transcripts (e.g., CCNA1, CCNB1, DPPA2) and embryo-specific activation (e.g., POU5F1, CDH1, DPPA4), as well as novel genes, were confirmed. In summary, the global single-cell cDNA amplification microarray analysis of the 5- to 8-cell stage human embryos reveals that blastomere fate is not committed to ICM or TE. Finally, new EGA features in human embryogenesis are presented.
    PLoS ONE 10/2010; 5(10):e13615. DOI:10.1371/journal.pone.0013615 · 3.23 Impact Factor
  • Source
    • "Gene expression profiling may thus be useful for assessing cultured hESCs since it has been used to identify genes that are uniquely expressed by hESCs [29]. Player et al have found that 1715 genes were differentially expressed between hESCs and differentiated embryonic cells [29]. This finding is likely to be useful in determining if cells in culture have maintained their embryonic stem cell characteristics. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Cellular therapies are becoming increasingly important in treating cancer, hematologic malignancies, autoimmune disorders, and damaged tissue. These therapies are becoming more effective and are being used more frequently, but they are also becoming more complex. As a result, quality testing is becoming an increasingly important part of cellular therapy. Cellular therapies should be tested at several points during their production. The starting material, intermediate products and the final product are usually analyzed. Products are evaluated at critical steps in the manufacturing process and at the end of production prior to the release of the product for clinical use. In addition, the donor of the starting biologic material is usually evaluated. The testing of cellular therapies for stability, consistency, comparability and potency is especially challenging. We and others have found that global gene and microRNA expression analysis is useful for comparability testing and will likely be useful for potency, stability and consistency testing. Several examples of the use of gene expression analysis for assessing cellular therapies are presented.
    The Korean journal of hematology 03/2010; 45(1):14-22. DOI:10.5045/kjh.2010.45.1.14
Show more