Unknown,Transcriptomics,Genomics,Proteomics

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Analyses of the SAGE expression of Human Embryonic Stem Cells

ABSTRACT: A plethora of research has focused on the human embryonic stem cells (hESC) ever since they were first reported mainly due to their distinct features of self renewal and pluripotency. Probing of the hESC transcriptome using global expression profiling tools such as DNA microarray, Serial Analysis of Gene Expression (SAGE), Massively Parallel Signature Sequencing (MPSS) and Expressed Sequence Tag (EST) analysis have contributed significantly in our current understanding of hESCs. In fact, a large number of markers to assess the pluripotent and differentiation status of hESCs have been presented by such studies. However, till date clarity is lacking in identifying a robust set of markers to assess the true state of the hESCs. In this paper, we report the generation of long SAGE libraries for partially differentiated and differentiated HES3 along with a deeper profiling of our previously reported HES3 undifferentiated library. Clustering analysis of these libraries in concert with long and short SAGE libraries available in public databases using Hierarchical Cluster Analysis (HCA) as well as a poison-based approach helped in identification of expression patterns distinct from those reported for the well established pluriptency/differentiation markers. Almost all the previous studies reporting a robust set of markers have focused on a gene by gene comparison in listing out the upregulated and downregulated genes rather than look at the expression patterns in establishing a list of markers. In this analysis, however, we report a new set of markers as well as add confidence to some previously reported markers based on their novel expression patterns as identified by SAGE analysis and also confirm it by real-time PCR analysis. For the real-time PCR confirmation, instead of taking two extreme data sets such as undifferentiated and a late stage embryoid body, we profiled a time series of embryoid body stages so that we could identify those genes which show a dramatic increase or decrease upon differentiation and hence would serve as more reliable markers. The hESC lines HES3 from ES Cell International, Singapore (http://www.escellinternational.com) were cultured on mouse embryonic fibroblast feeders (MEFs) as described previously. Selection of cells for library construction was done by micro-dissection following our established protocols. Spontaneous differentiation was induced by prolonged culture without changing the feeder layer. Briefly the HES3 cell line was grown to 18 passages (P18) on MEFs, after which no sub-culturing was performed. The medium was changed daily for 25 days and the differentiated cell population was harvested at the end of the culture period by micro-dissection. Cell differentiation was confirmed by Immuno-cytochemistry for markers to the three germ layers, as well as RT-PCR for the differentiation markers. For construction of the partially differentiated library, colonies which had started differentiation, as suggested by the morphological changes towards the centre and periphery of the colonies were used. Entire HES3 colonies (24P) which had started differentiation were harvested by micro-dissection and used for library construction. MmeI was used as tagging enzyme and libraries were constructed using the LS-SAGE kit from Invitrogen (http://www.invitrogen.com). Cloning of concatemerized ditags and sequencing of tags were done as outlined earlier. The 10 bp SAGE tags were extracted using Microsoft Excel for direct comparisons between libraries.

ORGANISM(S): Homo sapiens

SUBMITTER: Zhenyang Lai

PROVIDER: E-GEOD-12611 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:A plethora of research has focused on the human embryonic stem cells (hESC) ever since they were first reported mainly due to their distinct features of self renewal and pluripotency. Probing of the hESC transcriptome using global expression profiling tools such as DNA microarray, Serial Analysis of Gene Expression (SAGE), Massively Parallel Signature Sequencing (MPSS) and Expressed Sequence Tag (EST) analysis have contributed significantly in our current understanding of hESCs. In fact, a large number of markers to assess the pluripotent and differentiation status of hESCs have been presented by such studies. However, till date clarity is lacking in identifying a robust set of markers to assess the true state of the hESCs. In this paper, we report the generation of long SAGE libraries for partially differentiated and differentiated HES3 along with a deeper profiling of our previously reported HES3 undifferentiated library. Clustering analysis of these libraries in concert with long and short SAGE libraries available in public databases using Hierarchical Cluster Analysis (HCA) as well as a poison-based approach helped in identification of expression patterns distinct from those reported for the well established pluriptency/differentiation markers. Almost all the previous studies reporting a robust set of markers have focused on a gene by gene comparison in listing out the upregulated and downregulated genes rather than look at the expression patterns in establishing a list of markers. In this analysis, however, we report a new set of markers as well as add confidence to some previously reported markers based on their novel expression patterns as identified by SAGE analysis and also confirm it by real-time PCR analysis. For the real-time PCR confirmation, instead of taking two extreme data sets such as undifferentiated and a late stage embryoid body, we profiled a time series of embryoid body stages so that we could identify those genes which show a dramatic increase or decrease upon differentiation and hence would serve as more reliable markers.

Project description:The self-renewal and differentiation potential of human embryonic stem cells (hESCs) suggests that hESCs could be used for regenerative medicine, especially for restoring neuronal functions in brain diseases. However, the functional properties of neurons derived from hESC are largely unknown. Moreover, since hESCs were derived under diverse conditions, the possibility arises that neurons derived from different hESC lines exhibit distinct properties, but this possibility remains unexplored. To address these issues, we developed a protocol that allows step-wise generation from hESCs of ~70-80% pure human neurons that form spontaneously active synaptic networks in culture. Comparison of neurons derived from the well-characterized HSF1 and HSF6 hESC lines revealed that HSF1- but not HSF6-derived neurons exhibit forebrain properties. Accordingly, HSF1-derived neurons initially form primarily GABAergic synaptic networks, whereas HSF6-derived neurons initially form glutamatergic networks. microRNA profiling revealed significant expression differences between the two hESC lines, suggesting that microRNAs may influence their distinct differentiation properties. These observations indicate that although both HSF1 and HSF6 hESCs differentiate into functional neurons, the two hESC lines exhibit distinct differentiation potentials, suggesting that they are pre-programmed. Information on hESC line-specific differentiation biases is crucial for neural stem cell therapy and establishment of novel disease models using hESCs. Experiment Overall Design: We directly compared the transcriptome of hNPCs derived from HSF-1 and HSF-6 hESC lines under two different neural induction conditions (embryonic body formation in presence of caudalizing factors (retinoic acid and bFGF) and monolayer conversion in presence of BMP signaling inhibitor (Noggin)). Since HSF6 hESC cannot undergo efficient neural differentiation without caudalizing factors, we cannot obtain RNA samples for HSF6 (Noggin). Experiment Overall Design: In summary, consistent with our immunostaining results, HSF1 hNPCs display more forebrain properties as compared to HSF6 hNPCs, indicated by expression of multiple forebrain specific markers. Conversely, HSF6 hNPCs show regional identity towards posterior central nervous system.

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Analyses of the SAGE expression of Human Embryonic Stem Cells

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