Messenger RNA and microRNA profiling during early mouse EB formation Rashmi Tripathi a , Harpreet Kaur Saini b,⇑ , Roland Rad a , Cei Abreu-Goodger b , Stijn van Dongen b , Anton J. Enright b a Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, United Kingdom b EMBL – European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, United Kingdom article info Article history: Received 25 December 2009 Received in revised form 21 February 2011 Accepted 14 March 2011 Available online 1 April 2011 Keywords: Embryonic stem cells EBs Messenger RNA MicroRNA Pluripotency Germ layer Nervous system development Let-7 Lin28 MicroRNA targets Gene ontology abstract Embryonic stem (ES) cells can be induced to differentiate into embryoid bodies (EBs) in a synchronised manner when plated at a fixed density in hanging drops. This differentiation procedure mimics post- implantation development in mouse embryos and also serves as the starting point of protocols used in differentiation of stem cells into various lineages. Currently, little is known about the potential influence of microRNAs (miRNAs) on mRNA expression patterns during EB formation. We have measured mRNA and miRNA expression in developing EBs plated in hanging drops until day 3, when discrete structural changes occur involving their differentiation into three germ layers. We observe significant alterations in mRNA and miRNA expression profiles during this early developmental time frame, in particular of genes involved in germ layer formation, stem cell pluripotency and nervous system development. Com- putational target prediction using Pictar, TargetScan and miRBase Targets reveals an enrichment of bind- ing sites corresponding to differentially and highly expressed miRNAs in stem cell pluripotency genes and a neuroectodermal marker, Nes. We also find that members of let-7 family are significantly down-regu- lated at day 3 and the corresponding up-regulated genes are enriched in let-7 seed sequences. These results depict how miRNA expression changes may affect the expression of mRNAs involved in EB forma- tion on a genome-wide scale. Understanding the regulatory effects of miRNAs during EB formation may enable more efficient derivation of different cell types in culture. Ó 2011 Elsevier B.V. All rights reserved. 1. Results and discussion Embryonic stem (ES) cells were first established in culture from the inner cell mass of mouse blastocysts (Evans and Kaufman, 1981; Martin, 1981). They can also differentiate into three-dimen- sional structures called EBs (EBs), which mimic many aspects of mammalian embryos. To date most microarray based expression studies of differentiating EBs have focused primarily on mRNAs (Hailesellasse Sene et al., 2007; Leahy et al., 1999; Mansergh et al., 2009). There has been only one study that has looked at both mRNA and miRNA expression changes in undifferentiated and dif- ferentiated human ES cells (Lakshmipathy et al., 2007). MiRNAs have been shown to be important modulators of ES cell growth and differentiation (Marson et al., 2008; Wilson et al., 2009). These are a single stranded 21–23 nt family of RNAs first discovered in Caenorhabditis elegans (C. elegans) as short RNAs regulating devel- opmental timing (Lee et al., 1993; Olsen and Ambros, 1999). They repress gene expression by targeting cognate messenger RNAs (mRNAs) for degradation or translational repression (Bartel, 2004). This activity is based on miRNAs binding to complementary sites in the 3 0 un-translated regions (UTRs) of their target tran- scripts. The so called ‘seed-region’ of the miRNAs (2–8 nucleotides (nt)) appears to be a key specificity determinant in this interaction (Lewis et al., 2005). Analysing mRNA and miRNA expression pro- files simultaneously in an ES cell based developmental model could help unravel potential regulatory effects of miRNAs on mRNA expression during this process. In order to avoid significant heterogeneity in EB size, differenti- ation status and gene expression (Kurosawa, 2007; Lewis et al., 2005) as often observed in suspension cultures (Mogi et al., 2009), we plated ES cells at a fixed cell density of 1000 cells per drop using the hanging drop method (Guan et al., 1999; Kurosawa, 2007; Mogi et al., 2009). The three dimensional environment pro- vided to stem cells allowed them to differentiate synchronously into clusters of cells which then spontaneously differentiated into an outer endoderm layer and an inner cell mass (Fig. 1). RNA was isolated from undifferentiated stem cells at day 0 and days 1, 2 and 3 of EB formation and hybridised on Illumina beadarrays. Illumina expression profiling was performed in triplicate using the R statis- tical package, with overall correlations between replicates ob- served between 0.98 and 0.99. 1567-133X/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.gep.2011.03.004 ⇑ Corresponding author. Tel.: +44 1223 492676; fax: +44 1223 494468. E-mail address: hsaini@ebi.ac.uk (H.K. Saini). Gene Expression Patterns 11 (2011) 334–344 Contents lists available at ScienceDirect Gene Expression Patterns journal homepage: www.elsevier.com/locate/gep