Monitoring early differentiation events in human embryonic stem cells by massively parallel signature sequencing and expressed sequence tag scan.
Monitoring early differentiation events in human embryonic stem cells by massively parallel signature sequencing and expressed sequence tag scan.
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DOI：
10.1089/scd.2004.13.694
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发表时间：
2004-12
影响因子：
4
通讯作者：
T. Miura;Yongquan Luo;I. Khrebtukova;R. Brandenberger;Daixing Zhou;R. Thies;T. Vasicek;Holly Y. Young;J. Lebkowski;M. Carpenter;M. Rao
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作者：
T. Miura;Yongquan Luo;I. Khrebtukova;R. Brandenberger;Daixing Zhou;R. Thies;T. Vasicek;Holly Y. Young;J. Lebkowski;M. Carpenter;M. Rao
为了鉴定可能参与人胚胎干细胞（hESC）分化过程的基因，我们使用来自未分化（传代40-50）和分化（第14天）H1， H7和H9系的无饲养培养的RNA样本，通过表达序列标签（EST）计数和大规模平行特征测序（MPSS）来分析基因表达。MPSS和EST扫描分析显示了良好的一致性，并发现了大量基因在培养物从多能状态过渡到分化状态时迅速变化。其中包括已知和未知的胚胎干细胞特异性基因，以及随着细胞分化而改变的大量已知基因。选择上调或下调的基因子集，并通过各种独立的方法确认其差异表达，包括进一步分化后的表达比较，公开可用的数据库，以及逆转录酶(RT)- pcr和免疫细胞化学的直接评估。该分析确定了hESC和胚状体（hEBs）阶段特有的标记物以及可能调节分化的信号通路。生成的数据可用于监测不同实验室用独立方法分离的hESC的状态，并在不同的培养条件下维持。
To identify genes that may be involved in the process of human embryonic stem cell (hESC) differentiation, we profiled gene expression by expressed sequenced tag (EST) enumeration and massively parallel signature sequencing (MPSS) using RNA samples from feeder-free cultures of undifferentiated (passages 40-50) and differentiated (day 14) H1, H7, and H9 lines. MPSS and EST scan analysis showed good concordance and identified a large number of genes that changed rapidly as cultures transition from a pluripotent to a differentiated state. These included known and unknown ES cell-specific genes as well as a large number of known genes that were altered as cells differentiate. A subset of genes that were either up- or down-regulated were selected and their differential expression confirmed by a variety of independent methods, including comparison of expression after further differentiation, publicly available databases, and direct assessments by reverse transcriptase (RT)-PCR and immunocytochemistry. The analysis identified markers unique to the hESC and embryoid bodies (hEBs) stage as well as signaling pathways that likely regulate differentiation. The data generated can be used to monitor the state of hESC isolated by different laboratories using independent methods and maintained under differing culture conditions.