One of the key characteristics of human embryonic stem cells (hESC) is their ability to proliferate for an indefinite period of time. Previous studies have shown that a unique network of transcription factors are involved in hESC self renewal. Since hESC lines have the potential to differentiate into cells of all three germ layers, cells derived from hESC may be useful for the treatment of a variety of inherited or acquired diseases. The molecular signal required to differentiate hESC into a particular cell type has not been defined. It is expected that global gene expression profiling of hESC may provide an insight into the critical genes involved in maintaining pluripotency of hESC and genes that are modulated when hESCs differentiate. Several groups have utilized a variety of high throughput techniques and performed gene expression profiling of undifferentiated hESCs and mouse ES cells (mESC) to identify a set of genes uniquely expressed in ES cells but not in mature cells and defined them as "stemness" genes. These molecular techniques include DNA microarray, EST-enumeration, MPSS profiling, and SAGE. Irrespective of the molecular technique used, highly expressed genes showed similar expression pattern in several ES cell lines supporting their importance. A set of approximately 100 genes were identified, which are highly expressed in ES cells and considered to be involved in maintaining pluripotency and self renewal of ES cells. Various studies have also reported on the gene expression profiling of differentiated embryoid bodies (EB) derived from hESCs and mESCs. When hESCs are differentiated, "stemness" genes are down-regulated and a set of genes are up-regulated. Together with down-modulation of "stemness" genes and up-regulation of new genes may provide a new insight into the molecular pathways of hESC differentiation and study of these genes may be useful in the characterization of differentiated cells.
"Since a considerable number of the predicted genes by promoter analysis were not upregulated based on the microarray data , we assumed that they might be repressed during hESC growth or could be due to species differences or culture condition ( Bhattacharya et al . , 2009 ) . Therefore the second PI network was constructed using the all 328 predicted genes by promoter analysis ( Supplementary material 4 ) . Three hundred and two out of 328 genes could be predicted in the network . Interestingly , 126 of the predicted downregulated genes were present in the network ( Supplementary material 5 ) . Fig . 2 r"
[Show abstract][Hide abstract] ABSTRACT: Self-proliferation and differentiation into distinct cell types have been made stem cell as a promising target for regenerative medicine. Several key genes can regulate self-renewal and pluripotency of embryonic stem cells (hESCs). They work together and build a transcriptional hierarchy. Coexpression and coregulation of genes control by common regulatory elements on the promoter regions. Consequently, distinct organization and combination of transcription factor binding sites (TFBSs modules) on promoter regions, in view of order and distance, leads to a common specific expression pattern within a set of genes. To gain insights into transcriptional regulation of hESCs, we selected promoter regions of eleven common expressed hESC genes including SOX2, LIN28, STAT3, NANOG, LEFTB, TDGF1, POU5F1, FOXD3, TERF1, REX1 and GDF3 to predict activating regulatory modules on promoters and discover key corresponding transcription factors. Then, promoter regions in human genome were explored for modules and 328 genes containing the same modules were detected. Using microarray data, we verified that 102 of 328 genes commonly upregulate in hESCs. Also, using output data of DNA-Protein interaction assays, we found that 42 of all predicted genes are targets of SOX2, NANOG and POU5F1 . Additionally, a protein interaction network of hESC genes was constructed based on biological processes and interestingly, 126 downregulated genes along with upregulated ones identified by promoter analysis were predicted in the network. Based on the results, we suggest that the identified genes, coregulating with common hESC genes, represent a novel approach for gene discovery based on whole genome promoter analysis irrespective of gene expression. Altogether, promoter profiling can be used to expand hESC transcriptional regulatory circuitry by analysis of shared functional sequences between genes. This approach provides a clear image on underlying regulatory mechanism of gene expression profile and offers a novel approach in designing gene networks of stem cell.
"2 ul of 1∶10 dilution of cDNA product was used in a 25 ul qRT-PCR reaction. QRT-PCR was carried out on an Applied Biosystems StepOne Plus Real Time PCR machine, with StepOne software, using Applied Biosystems 2xSYBR green PCR mix+ROX and 60 degree Celsius anneal, Target gene transcript levels were compared to actin B control (actin B primers, Eurogentec), and subsequently to either fibroblasts harvested 5 days after infection with the reprogramming vectors (for transgene expression) or to undifferentiated HUES2, as appropriate . "
[Show abstract][Hide abstract] ABSTRACT: Human macrophages are specialised hosts for HIV-1, dengue virus, Leishmania and Mycobacterium tuberculosis. Yet macrophage research is hampered by lack of appropriate cell models for modelling infection by these human pathogens, because available myeloid cell lines are, by definition, not terminally differentiated like tissue macrophages. We describe here a method for deriving monocytes and macrophages from human Pluripotent Stem Cells which improves on previously published protocols in that it uses entirely defined, feeder- and serum-free culture conditions and produces very consistent, pure, high yields across both human Embryonic Stem Cell (hESC) and multiple human induced Pluripotent Stem Cell (hiPSC) lines over time periods of up to one year. Cumulatively, up to ∼3×10(7) monocytes can be harvested per 6-well plate. The monocytes produced are most closely similar to the major blood monocyte (CD14(+), CD16(low), CD163(+)). Differentiation with M-CSF produces macrophages that are highly phagocytic, HIV-1-infectable, and upon activation produce a pro-inflammatory cytokine profile similar to blood monocyte-derived macrophages. Macrophages are notoriously hard to genetically manipulate, as they recognise foreign nucleic acids; the lentivector system described here overcomes this, as pluripotent stem cells can be relatively simply genetically manipulated for efficient transgene expression in the differentiated cells, surmounting issues of transgene silencing. Overall, the method we describe here is an efficient, effective, scalable system for the reproducible production and genetic modification of human macrophages, facilitating the interrogation of human macrophage biology.
PLoS ONE 08/2013; 8(8):e71098. DOI:10.1371/journal.pone.0071098 · 3.23 Impact Factor
"Of these, 78% were up-regulated and 22% down-regulated. Up-regulated genes in hESCs versus ICMs and blastomeres included the most significant markers of pluripotency and cellular immortality characterisation, namely the transcriptional core NANOG, POU5F1 (OCT4), and SOX2, and telomerase related TERT1 and TERF1, as well as Activin/Nodal signaling markers such as TGFB1, LEFTY1 and LEFTY2, Wnt signaling such as WNT5A and WNT6, adhesion molecules such as THY1, ribosomal genes involved in cell proliferation (RPL6, RPL14), and the transcriptional repressor TP53
, , , , . The DNA methyltransferases, DNMT3A and DNMT3B, which play key roles in regulating gene expression and chromatin structure ,  were up-regulated in hESCs, as shown in previous studies, and confirmed by these results , , , while the enzyme catalysing their activity, DNMT3L, was up-regulated in the IVVPS. "
[Show abstract][Hide abstract] ABSTRACT: The genetic mechanisms governing human pre-implantation embryo development and the in vitro counterparts, human embryonic stem cells (hESCs), still remain incomplete. Previous global genome studies demonstrated that totipotent blastomeres from day-3 human embryos and pluripotent inner cell masses (ICMs) from blastocysts, display unique and differing transcriptomes. Nevertheless, comparative gene expression analysis has revealed that no significant differences exist between hESCs derived from blastomeres versus those obtained from ICMs, suggesting that pluripotent hESCs involve a new developmental progression. To understand early human stages evolution, we developed an undifferentiation network signature (UNS) and applied it to a differential gene expression profile between single blastomeres from day-3 embryos, ICMs and hESCs. This allowed us to establish a unique signature composed of highly interconnected genes characteristic of totipotency (61 genes), in vivo pluripotency (20 genes), and in vitro pluripotency (107 genes), and which are also proprietary according to functional analysis. This systems biology approach has led to an improved understanding of the molecular and signaling processes governing human pre-implantation embryo development, as well as enabling us to comprehend how hESCs might adapt to in vitro culture conditions.
PLoS ONE 04/2013; 8(4):e62135. DOI:10.1371/journal.pone.0062135 · 3.23 Impact Factor
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