Nanotopographical control of human embryonic stem cell differentiation into definitive endoderm Mohammad Hossein Ghanian, 1,2 Zahra Farzaneh, 1 Jalal Barzin, 2 Mojgan Zandi, 2 Mohammad Kazemi-Ashtiani, 1,2 Mehdi Alikhani, 1 Morteza Ehsani, 2 Hossein Baharvand 1,3 1 Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran 2 Biomaterials Department, Iran Polymer and Petrochemical Institute, Tehran, Iran 3 Department of Developmental Biology, University of Science and Culture, ACECR, Tehran, Iran Received 17 February 2015; revised 29 March 2015; accepted 20 April 2015 Published online 27 May 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35483 Abstract: Derivation of definitive endoderm (DE) from human embryonic stem cells (hESCs) can address the needs of regener- ative medicine for endoderm-derived organs such as the pan- creas and liver. Fibrous substrates which topographically recapitulate native extracellular matrix have been known to pro- mote the stem cell differentiation. However, the optimal fiber diameter remains to be determined for the desired differentia- tion. Here, we have developed a simple method to precisely fab- ricate electrospun poly(e-caprolactone) fibers with four distinct average diameters at nano- and microscale levels (200, 500, 800, and 1300 nm). Human ESCs were cultured as clumps or single cells and induced into DE differentiation to determine the opti- mal topography leading to the promoted differentiation com- pared with planar culture plates. Gene expression analysis of the DE-induced cells showed significant upregulation of DE- specific genes exclusively on the 200-nm fibers. By Western blot analysis, significant expression of DE-specific proteins was found when hESCs were cultured on the 200 nm substrate as single cells rather than clumps, probably due to more efficient cell– matrix interaction realized by morphological observations of the cell colonies. The results indicated that nanofibrillar substrates, only at ultrathin fiber diameters, provided a better environment for DE differentiation of hESC, which holds great promise in pro- spective tissue engineering applications. V C 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 103A: 3539–3553, 2015. Key Words: human embryonic stem cell, topography, electro- spinning, definitive endoderm, differentiation How to cite this article: Ghanian MH, Farzaneh Z, Barzin J, Zandi M, Kazemi-Ashtiani M, Alikhani M, Ehsani M, Baharvand H. 2015. Nanotopographical control of human embryonic stem cell differentiation into definitive endoderm. J Biomed Mater Res Part A 2015:103A:3539–3553. INTRODUCTION Human embryonic stem cells (hESCs) are capable of differ- entiating into all three primary germ layers, the ectoderm, mesoderm, and endoderm. Therefore, they offer potential for cell-based regenerative therapies. In vitro endodermal differentiation of hESCs is of great interest for curing dis- eases related to endoderm-derived organs, in particular the liver and pancreas. 1,2 As hESC differentiation to clinically applicable cells such as hepatocytes and pancreatic b-cells is complex and stepwise in nature, it is logical to dissect the entire process into separate stages for stepwise optimiza- tion of differentiation conditions. Accordingly, efficient dif- ferentiation of hESCs into definitive endoderm (DE), an early stage of the endoderm development that precedes hepatic or pancreatic specification, is considered a prerequi- site for successful generation of the mature cells. 3 In vitro generation of DE from hESCs cultured on two-dimensional (2D) culture plates has not been efficient enough largely due to oversimplification of the native extracellular microenvironment. 4 In addition to soluble bio- chemical factors, biophysical cues including mechanical forces, matrix elasticity, and topographical patterns are also essential parts of the cell microenvironment that regulate embryonic morphogenesis. 5 Recently, emerging evidence indicates that hESC differentiation can be strongly influ- enced by the underlying topography provided by engineered substrates with micro/nanoscale features such as gratings, pillars, or pits. 6–10 These regular surface features are mostly fabricated by complex and expensive micro/nanofabrication techniques and appear to inadequately mimic the fibrous topography of the native extracellular matrix (ECM). In con- trast, micro- and nanofibrous substrates are ECM-mimicking structures which can be cost-effectively fabricated by elec- trospinning. These substrates have been extensively explored as versatile substrates for stem cell culture and differentiation. 11 Additional Supporting Information may be found in the online version of this article. Correspondence to: H. Baharvand; e-mail: baharvand@royaninstitute.org or J. Barzin; e-mail: J.barzin@ippi.ac.ir Contract grant sponsors: The study was supported by grants from Royan Institute for Stem Cell Biology and Technology, Iran Polymer and Petrochemical Institute, and Iranian Council for Stem Cell Research and Technology. V C 2015 WILEY PERIODICALS, INC. 3539