Influence of oriented nanofibrous PCL scaffolds on quantitative gene expression during neural differentiation of mouse embryonic stem cells Naghmeh Abbasi, 1,2 Seyed Mahmoud Hashemi, 3 Mohammad Salehi, 4 Hoda Jahani, 2 Seyed Javad Mowla, 5 Masoud Soleimani, 2,6 Hossein Hosseinkhani 7 1 Department of Biology, School of Basic Science, Science and Research Branch, Islamic Azad University, Tehran, Iran 2 Department of Stem Cell Biology, Stem Cell Technology Research Center, Tehran, Iran 3 Department of Immunology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran 4 Department of Biotechnology, School of advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran 5 Department of Genetics, Faculty of Biological Science, Tarbiat Modares University, Tehran, Iran 6 Department of Hematology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran 7 Graduate Institute of Biomedical Engineering, National Taiwan University of Science and Technology, Taipei 10607, Taiwan Received 7 June 2015; revised 14 July 2015; accepted 7 August 2015 Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35551 Abstract: Neural differentiation of mouse embryonic stem cells in combination with three-dimensional electrospun nanofibers as an artificial extracellular matrix can be utilized to reconstruct a spinal cord defect. In this study, random and parallel-aligned nanofibrous poly E-caprolactone was fabri- cated using electrospinning. Its hydrophobicity was modified by O 2 plasma treatment to facilitate enhanced cell attach- ment. Embryoid bodies (EBs), which contain all three embry- onic germ layers, were cultured on poly E-caprolactone scaffolds to study the effect of fiber orientation on cell mor- phology and differentiation. Cell morphology and neuron- specific gene and protein expressions were, respectively, evaluated by scanning electron microscopy, real-time poly- merase chain reaction, and immunocytochemistry. Although two types of nanofibrous scaffolds showed neural marker expression at the protein level, cells on randomly oriented scaffolds showed short-range topographical guidance and stretched across multiple directions, whereas cells on the parallel scaffolds exhibited long extension with enhanced neuron outgrowth along the fiber, producing oriented extrac- ellular matrix, leading to direct cell migration and nerve regeneration. Quantitative real-time polymerase chain reac- tion showed that both aligned and random electrospun nano- fibers downregulated the precursor neural marker Nestin compared with that in the control group, a gelatin-coated tis- sue culture plate (T). Analysis also showed higher expression of dorso-ventral neural markers (Isl1/2 and Lim1/2) than motor neuron progenitor markers (Pax6, Nkx6.1, and olig2) in aligned nanofibers than in the T group. Moreover, aligned nanofibers showed higher expression of mature neural spe- cific markers such as b-tub and Map2 than those in the ran- domly oriented scaffolds. Therefore, we conclude that nanofibers with different orientations can support the neural lineage, but aligned nanofibrous scaffolds are superior candi- dates to promote the advancement of neural precursors to achieve maturity during the differentiation process. V C 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 00B:000–000, 2015. Key Words: embryonic stem cell, neural differentiation, gene expression, poly (E-caprolactone), nanofibers, electrospinning How to cite this article: Abbasi N, Hashemi SM, Salehi M, Jahani H, Mowla SJ, Soleimani M, Hosseinkhani H. 2015. Influence of oriented nanofibrous PCL scaffolds on quantitative gene expression during neural differentiation of mouse embryonic stem cells. J Biomed Mater Res Part A 2015:00A:000–000. INTRODUCTION Following disease or trauma, the adult central nervous sys- tem (CNS) is unable to regenerate by itself, because of the physical barrier of cystic cavity in the lesion. 1 Regeneration of lost tissues in the body generally requires three starting materials: cells, extra cellular matrices, and cell growth fac- tors. Scaffolds can replicate the function and structure of natural extracellular matrix (ECM) and provide a temporal support for cells, until seeded cells form a new matrix. 2 The cell response will be similar to the cell behavior in vivo if they are cultured in interconnective, nanostruc- tured, and porous three-dimensional (3D) scaffolds with a large surface area. 3 In 2011, Cunha et al. showed that the highly porous poly (L-lactic acid) (PLLA) nanostructured scaffold, which was fabricated using phase separation with a diameter range of 50–500 nm, acted as a positive cue to support neuron outgrowth while NSCs were seeded onto it in vitro. 4 Correspondence to: H. Hosseinkhani; e-mail: hosseinkhani@mail.ntust.edu.tw or M. Soleimani; e-mail: Soleim_m@modares.ac.ir Contract grant sponsor: National Science Council (NSC) of Taiwan; contract grant number: 104-2221-E-011 -072 Contract grant sponsor: Stem Cell Technology Research Center of Tehran, Iran V C 2015 WILEY PERIODICALS, INC. 1