Abstract
BackgroundThe 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.ResultsPreviously 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.ConclusionThese results present a detailed characterization of a unique set of genes, which can be used to assess the hESC differentiation.
Highlights
The identification of molecular pathways of differentiation of embryonic stem cells is critical for the development of stem cell based medical therapies
For microarray studies each sample was compared to human universal reference RNA to maintain uniformity and allow comparisons across samples. cDNA from BG02 and pooled samples of Human embryonic stem cell (hESC) were labeled with Cy5 and huURNA with Cy3, and ~17,000 oligonucleotide arrays were hybridized
We previously reported that eight early differentiation marker genes were expressed in hESC, which were further upregulated in human embryoid bodies (hEB) as determined by expressed sequence tags (ESTs) enumeration [28]
Summary
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. We for example utilized a large-scale oligonucleotide based array to identify a set of 92 genes that are highly upregulated in six hESC lines when compared against human universal reference RNA derived from mature tissues [28]. This set of "stemness genes" along with additional novel genes identified has served to assess the state of undifferentiated cells. Microarray studies of hEB's offer the possibility of such an assay that can be used for routine assessment of the state of ES cell differentiation
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