Spontaneous differentiation of mouse embryonic stem cells in vitro: Characterization by global gene expression profiles Jeonghoon Heo, Ju-Seog Lee, In-Sun Chu 1 , Yasushi Takahama, Snorri S. Thorgeirsson * Laboratory of Experimental Carcinogenesis, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA Received 28 April 2005 Available online 23 May 2005 Abstract We characterized the temporal gene expression changes during four weeks of spontaneous differentiation of mouse ES cells in a monolayer culture in order to obtain better insight into the differentiation process. The overall gene expression pattern was changed dramatically during the first two weeks of spontaneous differentiation, but stabilized after the second week. Most of the genes regulated within the first two weeks of spontaneous differentiation were genes related to development including mor- phogenesis, cell differentiation, embryonic development, pattern specification, mesoderm development, post-embryonic develop- ment, and blastocyst development. While most of the ectoderm lineage related genes were down-regulated, genes related to the mesoderm or endoderm lineage were up-regulated through the first week and second week, respectively. This study revealed that the development of ectoderm lineage is a recessive process during the spontaneous differentiation of mouse ES cells in monolayer culture. Our time-course characterization might provide a useful time line for directed differentiation of mouse ES cells. Published by Elsevier Inc. Keywords: Embryonic stem cell; Oligonucleotide microarray; Spontaneous differentiation; Monolayer culture; Germ layer lineages Mouse embryonic stem (ES) cells were originally iso- lated from the inner cell masses of developing blasto- cysts [1,2]. ES cells are able to undergo an unlimited self-renewal, and to generate all cell types derived from the three germ layers of the embryo. There has been unprecedented interest in ES cells, mainly because of the potential they offer for cell therapy. Much effort has been applied to understand the mechanisms main- taining the pluripotency as well as those determining lineage commitments that make it possible to manipu- late the differentiation of ES cells into specific cell types. Since the processes of differentiation and lineage com- mitment of embryonic cells in vivo are very complicated, in vitro differentiation of ES cells might provide a sim- pler model system for addressing the questions related to differentiation and lineage commitments. The undifferentiated state of mouse ES cells is main- tained by the presence of leukemia inhibitory factor (LIF) in the culture medium [3,4]. While spontaneous and random differentiation of ES cells can be easily trig- gered by the withdrawal of LIF from the medium [5], the directed differentiation of ES cells into specified cell types was only partially achieved by using cytokine treatments [6,7]. Development of embryoid bodies (EBs) by the aggregation of ES cells in suspension or hanging drops leads to the formation of structures which can generate populations of cells expressing genes 0006-291X/$ - see front matter. Published by Elsevier Inc. doi:10.1016/j.bbrc.2005.04.173 * Corresponding author. Fax: +1 301 496 0734. E-mail address: snorri_s_thorgeirsson@nih.gov (S.S. Thorgeirs- son). 1 Present address: National Genome Information Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-333, Korea. www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 332 (2005) 1061–1069 BBRC