PMC:2211323 / 19763-25017 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/2211323","sourcedb":"PMC","sourceid":"2211323","source_url":"http://www.ncbi.nlm.nih.gov/pmc/2211323","text":"Comparison of proteomic and transcriptional profiles of hESCs\nWe have previously employed the Illumina Bead Array system for the large-scale profiling of gene expression in hESCs using 24,000 transcript probes [11]. To compare proteomic and transcriptional analyses of hESCs, the levels of \u003e 600 proteins detected using large scale blotting were correlated with the levels of transcripts detected with the Illumina platform (Additional File 3). In general, a close match between the expression level of transcript and protein was observed: transcripts for nearly all the detected proteins were also identified in the Illumina analysis, and most proteins expressed at high levels also exhibited high mRNA levels.\nWe reasoned that a focused comparison of specific signaling pathways using a combination of proteomic and transcriptional data was likely to be much more informative than a global interrogation of hESCs. Several major signal pathways that have been suggested to be involved in self-renewal were examined to test this approach. These included the FGF, TGFβ, GSK3β/Wnt/β-catenin and Jak/Stat pathways [17,29,36-39], as well as the more recently suggested MAPK/ERK and Gap junction pathways [32,40]. Correlating transcriptional and proteomic data provided direct confirmation that these pathways were present and likely functional in hESCs (Table 2). For example, FGF2 protein was expressed highly in hESCs and expression of key members of the TGFβ, Wnt, Jak/Stat and Gap junction pathways, namely Stat1, SMADs, GSK3β, β-catenin and Connexin 43, were detected in both transcriptional and proteomic databases.\nTable 2 Signal pathways that may be active in hESCs Protein expression level: \u003e 10,000: ++++; 5,000–10,000: +++; 1,000–5,000:++; 100–1,000: + mRNA gene expression level: \u003e 5,000:++++; 1,000–5,000: +++; 100–1,000: ++; 30–100: +\n*: not included in the gene expression array This independent confirmation of known networks led us to examine other pathways that showed a similar correlation but have not been identified as key regulators of either self-renewal or differentiation, or suggest unappreciated characteristics of hESCs. Four signaling pathways (IGF, ERBB2, GPCR, and GDNF) and the tight junction complex were highlighted by this analysis (Table 2), and expression of key proteins in these pathways was confirmed. A detailed study demonstrating the importance of the IGF and ERBB2 pathways in hESC self-renewal has been performed and enabled the development of a defined medium for hESC maintenance (TCS and AJR, submitted). Tight junctions are apical cell-cell junctions found in epithelia that establish a barrier to the extracellular environment and a border for apical-basolateral polarity. While hESCs grow in colonies that are highly reminiscent of epithelia, and have been shown to be coupled by gap junctions [40], the formation of tight junction complexes has not been described. hESCs expressed the ZO1 and occludin tight junction proteins along cell borders as expected in polarized epithelia. The distribution of ZO1 expression changed dramatically as hESCs proliferated in culture. When tight junction complexes were disrupted by disaggreagation to single cells, only a subset of cells showed ZO1 staining 4 days after plating (Fig. 5). Continued proliferation to a confluent monolayer on day 7 was accompanied by widespread expression of ZO1, suggesting the formation of a general tight junction barrier. These cultures were undifferentiated and retained uniform expression of Oct4 protein (not shown). ERBB2 and 3 are members of the epidermal growth factor (EGF)-receptor family, which regulate epithelial proliferation via EGF-family ligands. ERBB2 and 3 transcripts are expressed by hESCs [8], are known to function as a heterodimer [41], and transmit a strong proliferative signal for hESCs by Heregulin 1β (an EGF-family ligand) (TCS and AJR, submitted). Immunofluorescence revealed general cell surface expression of ERBB2 on hESCs. Conversely, ERBB3 was highly localized to a concentrated area, and observed in cells that also expressed ZO1. Epithelial cells are known to localize ERBB receptors to the basolateral side of tight junctions, which serves to functionally separate receptors from ligands [42,43]. This is a basic epithelial wound healing mechanism, whereby disruption of the tight junction barrier by injury immediately exposes receptors to extracelluar ligands [43]. These staining patterns are also suggestive of basolateral sorting of ERBB3 in hESCs. The pathways and complexes identified by these analyses lay a framework for future functional analyses of signaling networks in hESCs.\nFigure 5 Tight junction proteins and ERBB2/3 expression in hESCs. BG01 hESCs were disaggregated to single cells using accutase [52] and cultured in defined conditions. (A) ZO1 expression four and (B) seven days after plating, indicating progressive tight junction formation. (C) Occludin expression 5 days after plating. (D) General cell surface expression of ERBB2, in the same field of view as (A). (E) Localized expression of ERBB3, in the same field of view as (B). (F) Higher magnification of ERBB3 localization in ZO1 expressing BG01 cells, 5 days after plating. Nuclei were stained with DAPI.\n\nD","divisions":[{"label":"Title","span":{"begin":0,"end":61}},{"label":"Table caption","span":{"begin":1618,"end":1892}},{"label":"Figure caption","span":{"begin":4651,"end":5253}}],"tracks":[{"project":"CellFinder","denotations":[{"id":"T338","span":{"begin":4723,"end":4728},"obj":"CellType"},{"id":"T339","span":{"begin":4718,"end":4722},"obj":"CellLine"},{"id":"T340","span":{"begin":5221,"end":5227},"obj":"CellComponent"},{"id":"T341","span":{"begin":4770,"end":4778},"obj":"GeneProtein"},{"id":"T342","span":{"begin":5172,"end":5175},"obj":"GeneProtein"},{"id":"T343","span":{"begin":5150,"end":5155},"obj":"GeneProtein"},{"id":"T344","span":{"begin":4931,"end":4939},"obj":"GeneProtein"},{"id":"T345","span":{"begin":4824,"end":4827},"obj":"GeneProtein"},{"id":"T346","span":{"begin":5187,"end":5191},"obj":"CellLine"},{"id":"T347","span":{"begin":5187,"end":5197},"obj":"CellType"},{"id":"T348","span":{"begin":4689,"end":4696},"obj":"GeneProtein"},{"id":"T349","span":{"begin":5081,"end":5086},"obj":"GeneProtein"},{"id":"T350","span":{"begin":5012,"end":5017},"obj":"GeneProtein"},{"id":"T351","span":{"begin":4711,"end":4716},"obj":"CellType"},{"id":"T352","span":{"begin":4985,"end":4997},"obj":"CellComponent"},{"id":"T353","span":{"begin":4661,"end":4675},"obj":"CellComponent"}],"attributes":[{"subj":"T338","pred":"source","obj":"CellFinder"},{"subj":"T339","pred":"source","obj":"CellFinder"},{"subj":"T340","pred":"source","obj":"CellFinder"},{"subj":"T341","pred":"source","obj":"CellFinder"},{"subj":"T342","pred":"source","obj":"CellFinder"},{"subj":"T343","pred":"source","obj":"CellFinder"},{"subj":"T344","pred":"source","obj":"CellFinder"},{"subj":"T345","pred":"source","obj":"CellFinder"},{"subj":"T346","pred":"source","obj":"CellFinder"},{"subj":"T347","pred":"source","obj":"CellFinder"},{"subj":"T348","pred":"source","obj":"CellFinder"},{"subj":"T349","pred":"source","obj":"CellFinder"},{"subj":"T350","pred":"source","obj":"CellFinder"},{"subj":"T351","pred":"source","obj":"CellFinder"},{"subj":"T352","pred":"source","obj":"CellFinder"},{"subj":"T353","pred":"source","obj":"CellFinder"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"CellFinder","color":"#ec93e0","default":true}]}]}}