SCIENCE CHINA Life Sciences © Science China Press and Springer-Verlag Berlin Heidelberg 2010 life.scichina.com www.springerlink.com *Corresponding author (email: raw600@gmail.com)  RESEARCH PAPER  January 2010 Vol.53 No.1: 78–86 doi: 10.1007/s11427-010-0010-y Interrogating cell signalling network sensitively monitors cell fate transition during early differentiation of mouse embryonic stem cells YUE ZhiCao 1,2,3* , ZHUANG FengFeng 4 , LIU Yi-Hsin 4 & HO Chih-ming 1,2 1 Department of Mechanical and Aerospace Engineering, University of California, Los Angeles, CA 90095, US; 2 Center for Cell Control, University of California, Los Angeles, CA 90095; US 3 Current address: Institute of Life Sciences, Fuzhou University, Fuzhou 350108, China; 4 Department of Ophthalmology and Doheny Eye Institute, University of Southern California, Los Angeles, CA 90033, US Received September 29, 2009; accepted October 28, 2009 The different cell types in an animal are often considered to be specified by combinations of transcription factors, and defined by marker gene expression. This paradigm is challenged, however, in stem cell research and application. Using a mouse em- bryonic stem cell (mESC) culture system, here we show that the expression level of many key stem cell marker genes/tran- scription factors such as Oct4, Sox2 and Nanog failed to monitor cell status transition during mESC differentiation. On the other hand, the response patterns of cell signalling network to external stimuli, as monitored by the dynamics of protein phosphorylation, changed dramatically. Our results also suggest that an irreversible alternation in cell signalling network pre- cedes the adjustment of transcription factor levels. This is consistent with the notion that signal transduction events regulate cell fate specification. We propose that interrogating cell signalling network can assess the cell property more precisely, and provide a sensitive measurement for the early events in cell fate transition. We wish to bring up attention to the potential prob- lem of cell identification using a few marker genes, and suggest a novel methodology to address this issue. signalling network, cell identification, differentiation, mouse embryonic stem cells Citation: Yue Z C, Zhuang F F, Liu Y H, et al. Interrogating cell signalling network sensitively monitors cell fate transition during early differentiation of mouse embryonic stem cells. Sci China Life Sci, 2010, 53: 78 – 86, doi: 10.1007/s11427-010-0010-y Cell therapy holds great promise in treating many clinically significant diseases. Differentiation from embryonic stem cells (ESC) is among the best choices for deriving large amounts of desired cells [1,2]. A major issue here is quality control, because cells are handled differently in each lab. How similar are the starting/final cells from different groups? This cell identification problem is particularly dif- ficult for in vitro differentiation experiments, because the cells go through a range of developmental processes and change their fate multiple times in a mixture format. Al- though a few marker genes can be used for this purpose, most markers are not specific enough in development. Cell fate specification is a central issue in developmental biology. Tissue interactions and multiple signal transduction pathways were involved [3]. Cells often have a specific lineage history when gradually committed into more differ- entiated identities. On the other hand, cell identity is often considered to be specified by a combination of transcription factors. This concept is reinforced by recent success in cell reprogramming. Through the introduction of a few defined transcription factors, adult fibroblasts and other terminally differentiated cells can be turned into more primitive, ESC-like induced pluripotent stem cells (iPSCs) [4−6].