Transcriptome Study of Human Embryonic Stem Cells and Knockdown Study of a Pluripotency Marker, LIN28

Abstract

Self-renewal and pluripotency are the key features of human embryonic stem (ES) cells. However, to date, the mechanism of pluripotency has not been well elucidated. In this study, we compared our in-house SAGE libraries constructed from the human ES cell line, HES 3, with other SAGE libraries from human ES cell lines that are available in public databases. Hierarchical Clustering Analysis (HCA) and Transchisq clustering were used to identify distinct expression patterns. In order to validate these patterns, a time-series of embryoid bodies (EBs), representing various differentiation stages were generated, and quantitative real-time PCR were carried out to profile the expression of selected genes that occurred as the EBs differentiated. These results hence provided us with reliable markers to assess both the status of pluripotency and the onset of differentiation taking place in these EBs. We then subsequently carried out a knockdown study on a key pluripotency marker, LIN28, in NCCIT cells, which is a human embryonal carcinoma cell line. A stable NCCIT cell line containing an inducible LIN28sh construct under the regulation of Doxycline was generated. Upon LIN28 knockdown in this NCCIT stable line, a set of genes related to post-transcriptional regulation were identified using microarray analysis. We proposed that LIN28 might act as a master regulator in pluripotency and differentiation by directly controlling genes responsible for pluripotency and/or by modulating other regulators including microRNAs to form a hierarchical post-transcriptional control.