Comprehensive Gene Expression Analysis of Human Embryonic Stem Cells during Differentiation into Neural Cells Ali Fathi1, Maryam Hatami2, Vahid Hajihosseini1,3, Faranak Fattahi1,3, Sahar Kiani2, Hossein Baharvand1,4*, Ghasem Hosseini Salekdeh1,5* 1 Department of Molecular Systems Biology, Cell Science Research Centre, Royan Institute for Stem Cell Biology and Technology, Avicenna Research Institute (ACECR), Tehran, Iran, 2 Department of Stem Cells and Developmental Biology, Cell Science Research Centre, Royan Institute for Stem Cell Biology and Technology, Avicenna Research Institute (ACECR), Tehran, Iran, 3 Department of Biotechnology, College of Science, University of Tehran, Tehran, Iran, 4 Department of Developmental Biology, University of Science and Culture, Avicenna Research Institute (ACECR), Tehran, Iran, 5 Department of Systems Biology, Agricultural Biotechnology Research Institute of Iran, Karaj, Iran Abstract Global gene expression analysis of human embryonic stem cells (hESCs) that differentiate into neural cells would help to further define the molecular mechanisms involved in neurogenesis in humans. We performed a comprehensive transcripteome analysis of hESC differentiation at three different stages: early neural differentiation, neural ectoderm, and differentiated neurons. We identified and validated time-dependent gene expression patterns and showed that the gene expression patterns reflect early ESC differentiation. Sets of genes are induced in primary ectodermal lineages and then in differentiated neurons, constituting consecutive waves of known and novel genes. Pathway analysis revealed dynamic expression patterns of members of several signaling pathways, including NOTCH, mTOR and Toll like receptors (TLR), during neural differentiation. An interaction network analysis revealed that the TGFb family of genes, including LEFTY1, ID1 and ID2, are possible key players in the proliferation and maintenance of neural ectoderm. Collectively, these results enhance our understanding of the molecular dynamics underlying neural commitment and differentiation. Citation: Fathi A, Hatami M, Hajihosseini V, Fattahi F, Kiani S, et al. (2011) Comprehensive Gene Expression Analysis of Human Embryonic Stem Cells during Differentiation into Neural Cells. PLoS ONE 6(7): e22856. doi:10.1371/journal.pone.0022856 Editor: Joseph Charles Glorioso, University of Pittsburgh School of Medicine, United States of America Received April 16, 2011; Accepted June 29, 2011; Published July 28, 2011 Copyright: 2011 Fathi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was funded by a grant provided from the Royan Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Salekdeh@Royaninstitute.org (GHS); Baharvand@RoyanInstitute.org (HB) Introduction Embryonic stem cells (ESCs) are a promising tool for the study of neural development and cell lineage specification. The current lack of knowledge about cues for mammalian neuronal commit- ment and differentiation is at least partly due to a paucity of available model systems that permit direct observation of developmental processes. Studies of the molecular mechanisms underlying the neural differentiation of human ESCs (hESCs) can help to unravel the complex gene pathways that are involved in neural cell commitment and differentiation processes. High throughput studies of gene expression have been applied to neural stem cells (NSCs) derived from the subventricular zone (SVZ) [1], NSCs derived from mouse ESCs and the fetal brain [2], dopaminergic neurons from mouse ESCs [3], heterogeneous neural cells from mouse ESCs [4] and neural progenitors (NPs) differentiated from mouse ESCs [5] and forebrain periventricular zone NPs compared to neuroectoderm from mESCs [6]. Cytoskeletal genes, cell membrane receptor genes, and transcrip- tion factor genes are differentially expressed in most cell types. Recently, Wu et al. (2010) analyzed the transcriptome of hESCs during differentiation into the neural lineage at the early initiation, neural progenitor, and early glial-like stages [7] and found an extraordinary degree of stage-specific transcription and splicing. The diversity was highest in undifferentiated hESCs and decreased upon differentiation [7]. Previously, we described the differentiation of hESCs into NPs and neural cells with typical cellular, molecular and ultrastructural markers using a defined adherent culture protocol [8,9]. Here we analyzed the transcriptome of hESCs during differentiation into neural cells [8] using a whole genome microarray chip. We identified differentially expressed genes that may be linked to neural fate specification, proliferation, and differentiation. Our data extend the gene expression network for neural differentiation and reveal novel aspects of transcriptional control pathways underlying the multistep process of commitment and differentia- tion of hESCs into neural cells. Materials and Methods Human ESC culture and sample preparation The hESC line Royan H6 [10] was passaged and cultured under feeder-free culture conditions on Matrigel in hESC medium containing DMEM/F12 medium supplemented with 20% knock- out serum replacement, 2 mM L-glutamine, 1% nonessential amino acids, 100 units/ml penicillin and 100 mg/ml streptomycin, insulin-transferrin-selenite, (All from Invitrogen), 0.1 mM b- mercaptoethanol and 100 ng/ml basic-fibroblast growth factor PLoS ONE | www.plosone.org 1 July 2011 | Volume 6 | Issue 7 | e22856