Iraj Bahrebar - Department of Biochemistry, Kharazmi (Tarbiat Moallem), Tehran, Iran These authors contributed equally to this work.
Abdulshakour Mohammadnia - National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran These authors contributed equally to this work.
Moein Yaqubi - National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran These authors contributed equally to this work.
Hossein Fallahi - Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran -Department of Biology, School of Science, Razi University, Kermanshah, Iran

Aggregations of human embryonic stem cells (hESCs), generate embryoid body (EB) as preliminary stage for differentiation into three germ layers. Regulators of BMP signaling during EB formation are mainly unknown. We constructgene regulatory network based on expression profile and molecular interactions to investigate most important regulators ofBMP signaling pathway during EB differentiation from hESCs. Methods:Microarray data for EB formation from hESCs were downloaded from GEO. Normalization and detection of differentially expressed genes performed in Flexarray software. Potentially transcription factor binding sites for differentially expressed genes obtained from ChEA database. BioGRID was used for finding experimentally validate protein-protein interactions for differentially expressed transcription factors. Expression and molecular interactions integrate to construct gene regulatorynetwork in Cytoscape software. Network ontology using ClueGO reveal most affected processes during EB formation fromhESCs. Result: Comparison of hESCs with EB indicates 1382 differentially expressed genes, include 695 up regulated and 687 down regulated genes. Gene regulatory network of this transition accommodate 1087 differentially expressed genes and 2745edges. Our constructed network contains 17 differentially expressed genes (based on network ontology), including: CAV1, CER1, CYR61, FBN1, FOXD1, GPC3, GREM1, HES1, HIPK2, HTRA1, SFRP1, SMAD7, SOX11, TFAP2B, TWSG1, WNT5A, ZNF423 that play a role in regulation of BMP signaling during EB formation. BMP signaling pathwaydifferentially expressed genes during EB formation, mostly regulated by MITF, STAT3, SMAD3, MYC, and ATF3 (8, 8, 7, 6, and 4 targets respectively). MITF, SMAD3, and ATF3 transcription factors up regulated whereas STAT3 and MYC down regulated

BMP signaling pathway, Embryoid body, Embryonic stem cells, Regulatory network, Transcription factors