PMC:2211323 / 1410-6953 JSONTXT

{"project":"CellFinder","denotations":[{"id":"T1","span":{"begin":5268,"end":5273},"obj":"CellType"},{"id":"T2","span":{"begin":5333,"end":5338},"obj":"CellType"},{"id":"T3","span":{"begin":5537,"end":5542},"obj":"CellType"},{"id":"T4","span":{"begin":517,"end":530},"obj":"Anatomy"},{"id":"T5","span":{"begin":500,"end":512},"obj":"Anatomy"},{"id":"T6","span":{"begin":486,"end":498},"obj":"Anatomy"},{"id":"T7","span":{"begin":360,"end":373},"obj":"Anatomy"},{"id":"T8","span":{"begin":271,"end":281},"obj":"CellType"},{"id":"T9","span":{"begin":17,"end":26},"obj":"Anatomy"},{"id":"T10","span":{"begin":354,"end":359},"obj":"Species"},{"id":"T11","span":{"begin":1099,"end":1104},"obj":"CellType"},{"id":"T12","span":{"begin":538,"end":543},"obj":"CellType"},{"id":"T13","span":{"begin":623,"end":628},"obj":"Species"},{"id":"T14","span":{"begin":1463,"end":1471},"obj":"CellType"},{"id":"T15","span":{"begin":1463,"end":1479},"obj":"Anatomy"},{"id":"T16","span":{"begin":1521,"end":1536},"obj":"Anatomy"},{"id":"T17","span":{"begin":1547,"end":1565},"obj":"CellType"},{"id":"T18","span":{"begin":1736,"end":1740},"obj":"CellType"},{"id":"T19","span":{"begin":254,"end":266},"obj":"CellType"},{"id":"T20","span":{"begin":1785,"end":1790},"obj":"CellType"},{"id":"T21","span":{"begin":11,"end":37},"obj":"CellType"},{"id":"T22","span":{"begin":1454,"end":1459},"obj":"CellType"},{"id":"T23","span":{"begin":11,"end":16},"obj":"Species"},{"id":"T24","span":{"begin":231,"end":242},"obj":"Anatomy"},{"id":"T25","span":{"begin":4615,"end":4620},"obj":"CellType"},{"id":"T26","span":{"begin":4754,"end":4759},"obj":"CellType"},{"id":"T27","span":{"begin":17,"end":36},"obj":"CellType"},{"id":"T28","span":{"begin":109,"end":119},"obj":"Anatomy"},{"id":"T29","span":{"begin":39,"end":44},"obj":"CellType"},{"id":"T30","span":{"begin":86,"end":101},"obj":"Anatomy"}],"text":"Background\nHuman embryonic stem cells (hESCs) are pluripotent cells isolated from the inner cell mass of the blastocyst [1]. They can be maintained for prolonged periods in culture and differentiate to representatives of the three germ layers as well as trophoblasts and germ cells. This differentiation potential may be used to model certain aspects of human embryogenesis, including the development and differentiation of pluripotent and other stem cell types during the processes of gastrulation, neurogenesis and organogenesis. Thus, hESCs provide a unique and powerful system to study otherwise intractable aspects of human development. Furthermore, these approaches have the potential to provide differentiated cell types for cell replacement therapies of degenerative disorders such as Parkinson's disease and Type I diabetes [2,3]. Before these cell therapy applications are developed, an understanding of the molecular and cellular mechanisms that drive self-renewal and differentiation is required. Fundamental to this understanding is the elucidation of the transcriptome and proteome of hESCs, using approaches that lay a framework for functional analyses of the unique properties of these cells.\nLarge-scale gene expression analyses such as microarray, massive parallel signature sequencing (MPSS), expressed sequenced tag (EST) enumeration, and serial analysis of gene expression (SAGE) have been used to compare multiple hESC lines [4-7]; hESCs to germ cell tumors [8]; or to differentiated derivatives in embryoid bodies [9-11] or neural populations [12]. These approaches have highlighted an expanded set of transcripts that mark the pluripotent state [4,13,14], cross-species commonalities in the molecular profile of ESCs [6,12,15], prominent receptors expressed by hESCs [8] and pathways that may play a role in the regulation of pluripotency [16,17]. Nevertheless, cataloguing the cellular transcriptome is only predictive of protein expression and typically does not shed light on post-transcriptional regulation. For example, while tens of thousands of transcripts can be followed simultaneously with SAGE, microarrays and MPSS, these methods do not routinely detect differences in transcript splice variants, or polyadenylation status. These differences may have profound effects on translation, as well as the isoform and function of the protein produced. Finally, numerous post-translational modifications are known to regulate protein function, including enzymatic cleavage, covalent coupling to other molecules, glycosylation, phosphorylation and ubiquitination. These issues all highlight potential shortfalls in our understanding of the hESC proteome.\nSeveral practical approaches for proteomic analyses are currently available, the most established of which is the 2-dimensional (2D) separation of proteins by polyacrylamide gel electrophoresis (PAGE). HPLC-tandem mass spectrometry (HPLC-MS/MS) based technology is rapidly evolving and has recently been used to detect protein expression in multiple cell types. An alternate approach is the recent large-scale adaptation of standard western blotting [18]. In this procedure, a large well is used to separate the sample by PAGE and lanes are created on the membrane containing immobilized protein with the use of a manifold. Compatible combinations of primary antibodies are predetermined, with the criterion of being able to identify proteins that do not co-migrate. Different combinations of primary antibodies are added to each well, with appropriate dilutions of each primary antibody so that expressed proteins are detected in a single condition. The scalability of the system depends on defining suitable combinations of primary antibodies, with up to 1000 antibodies in 200 lanes being used in the largest screens thus far. Detection software is used to identify proteins based on their expected and observed gel mobility. Unlike 2D PAGE and HPLC-MS/MS, large-scale western blotting only identifies proteins for which antibodies are already available. While this is not an appropriate screen for identifying uncharacterized proteins, it greatly simplifies the verification and functional analyses of proteins that are detected. In addition, this approach is highly flexible, and if desired can be focused to particular sets of proteins or protein function, such as cell signaling molecules. Importantly, the foundation of this approach is the large amount of data on individual antibodies, which are already available and characterized in the literature.\nMore recently, two research groups have conducted proteomic analyses of hESCs using MS [19-22]. In the present study, we used two large-scale western blot systems to examine the expression of \u003e 1000 proteins in hESCs and detected \u003e 600 proteins that were grouped into 18 functional classes. In addition, we identified 42 examples of multiple bands for a single protein, likely to be protein isoforms and/or post-translational modifications, and 22 phosphorylation events in cell signaling molecules. We correlated the expression of members of key active pathways in our transcriptional and proteomic databases and confirmed the validity of this approach. Using these approaches we identified new markers for undifferentiated hESCs and highlighted unrecognized epithelial characteristics of hESCs. Our data confirm the importance of proteomic analyses in complementing transcriptional profiling and provide a framework for continued analyses of the molecular and cellular biology of pluirpotent hESCs."}