Comparison of the gene expression profile of undifferentiated human embryonic stem cell lines and differentiating embryoid bodies

BACKGROUND: The identification of molecular pathways of differentiation of embryonic stem cells (hESC) is critical for the development of stem cell based medical therapies. In order to identify biomarkers and potential regulators of the process of differentiation, a high quality microarray containing 16,659 seventy base pair oligonucleotides was used to compare gene expression profiles of undifferentiated hESC lines and differentiating embryoid bodies. RESULTS: Previously identified "stemness" genes in undifferentiated hESC lines showed down modulation in differentiated cells while expression of several genes was induced as cells differentiated. In addition, a subset of 194 genes showed overexpression of greater than ≥ 3 folds in human embryoid bodies (hEB). These included 37 novel and 157 known genes. Gene expression was validated by a variety of techniques including another large scale array, reverse transcription polymerase chain reaction, focused cDNA microarrays, massively parallel signature sequencing (MPSS) analysis and immunocytochemisty. Several novel hEB specific expressed sequence tags (ESTs) were mapped to the human genome database and their expression profile characterized. A hierarchical clustering analysis clearly depicted a distinct difference in gene expression profile among undifferentiated and differentiated hESC and confirmed that microarray analysis could readily distinguish them. CONCLUSION: These results present a detailed characterization of a unique set of genes, which can be used to assess the hESC differentiation

A large-scale proteomic analysis of human embryonic stem cells

<p>Abstract</p> <p>Background</p> <p>Much of our current knowledge of the molecular expression profile of human embryonic stem cells (hESCs) is based on transcriptional approaches. These analyses are only partly predictive of protein expression however, and do not shed light on post-translational regulation, leaving a large gap in our knowledge of the biology of pluripotent stem cells.</p> <p>Results</p> <p>Here we describe the use of two large-scale western blot assays to identify over 600 proteins expressed in undifferentiated hESCs, and highlight over 40 examples of multiple gel mobility variants, which are suspected protein isoforms and/or post-translational modifications. Twenty-two phosphorylation events in cell signaling molecules, as well as potential new markers of undifferentiated hESCs were also identified. We confirmed the expression of a subset of the identified proteins by immunofluorescence and correlated the expression of transcript and protein for key molecules in active signaling pathways in hESCs. These analyses also indicated that hESCs exhibit several features of polarized epithelia, including expression of tight junction proteins.</p> <p>Conclusion</p> <p>Our approach complements proteomic and transcriptional analysis to provide unique information on human pluripotent stem cells, and is a framework for the continued analyses of self-renewal.</p

Self-renewal of human embryonic stem cells requires insulin-like growth factor-1 receptor and ERBB2 receptor signaling

Despite progress in developing defined conditions for human embryonic stem cell (hESC) cultures, little is known about the cell-surface receptors that are activated under conditions supportive of hESC self-renewal. A simultaneous interrogation of 42 receptor tyrosine kinases (RTKs) in hESCs following stimulation with mouse embryonic fibroblast (MEF) conditioned medium (CM) revealed rapid and prominent tyrosine phosphorylation of insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R); less prominent tyrosine phosphorylation of epidermal growth factor receptor (EGFR) family members, including ERBB2 and ERBB3; and trace phosphorylation of fibroblast growth factor receptors. Intense IGF1R and IR phosphorylation occurred in the absence of MEF conditioning (NCM) and was attributable to high concentrations of insulin in the proprietary KnockOut Serum Replacer (KSR). Inhibition of IGF1R using a blocking antibody or lentivirus-delivered shRNA reduced hESC self-renewal and promoted differentiation, while disruption of ERBB2 signaling with the selective inhibitor AG825 severely inhibited hESC proliferation and promoted apoptosis. A simple defined medium containing an IGF1 analog, heregulin-1β (a ligand for ERBB2/ERBB3), fibroblast growth factor-2 (FGF2), and activin A supported long-term growth of multiple hESC lines. These studies identify previously unappreciated RTKs that support hESC proliferation and self-renewal, and provide a rationally designed medium for the growth and maintenance of pluripotent hESCs