Uniaxial Mechanical Strain Modulates the Differentiation of Neural Crest Stem Cells into Smooth Muscle Lineage on Micropatterned Surfaces Xian Li1,2, Julia Chu 2 , Aijun Wang 2 , Yiqian Zhu 2 , Wai Keung Chu3, Li Yang1*, Song Li2* 1 Bioengineering College, Chongqing University, Chongqing, China, 2 Department of Bioengineering, University of California, Berkeley, California, United States of America, 3 Department of Bioengineering, University of California San Diego, La Jolla, California, United States of America Abstract Neural crest stem cells (NCSCs) play an important role in the development and represent a valuable cell source for tissue engineering. However, how mechanical factors in vivo regulate NCSC differentiation is not understood. Here NCSCs were derived from induced pluripotent stem cells and used as a model to determine whether vascular mechanical strain modulates the differentiation of NCSCs into smooth muscle (SM) lineage. NCSCs were cultured on micropatterned membranes to mimic the organization of smooth muscle cells (SMCs), and subjected to cyclic uniaxial strain. Mechanical strain enhanced NCSC proliferation and ERK2 phosphorylation. In addition, mechanical strain induced contractile marker calponin-1 within 2 days and slightly induced SM myosin within 5 days. On the other hand, mechanical strain suppressed the differentiation of NCSCs into Schwann cells. The induction of calponin-1 by mechanical strain was inhibited by neural induction medium but further enhanced by TGF-b. For NCSCs pre-treated with TGF-b, mechanical strain induced the gene expression of both calponin-1 and SM myosin. Our results demonstrated that mechanical strain regulates the differentiation of NCSCs in a manner dependent on biochemical factors and the differentiation stage of NCSCs. Understanding the mechanical regulation of NCSC differentiation will shed light on the development and remodeling of vascular tissues, and how transplanted NCSCs respond to mechanical factors. Citation: Li X, Chu J, Wang A, Zhu Y, Chu WK, et al. (2011) Uniaxial Mechanical Strain Modulates the Differentiation of Neural Crest Stem Cells into Smooth Muscle Lineage on Micropatterned Surfaces. PLoS ONE 6(10): e26029. doi:10.1371/journal.pone.0026029 Editor: Wei-Chun Chin, University of California, Merced, United States of America Received April 22, 2011; Accepted September 15, 2011; Published October 7, 2011 Copyright: 2011 Li 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 supported in part by grants from the National Institutes of Health (EB012240 and HL083900 to S.L.), Innovation and Attracting Talents Program for College and University of China (‘111’ Project) (B06023; to L.Y.), a postdoctoral training grant TG2-01164 from California Institute for Regenerative Medicine (to A.W.), and a Predoctoral Fellowship from the American Heart Association (to Y.Z.). 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: yanglicqu@gmail.com (LY); song_li@berkeley.edu (SL) Introduction Human neural crest stem cells (NCSCs) are multipotent stem cells that can be isolated from pluripotent stem cells, the embryonic tissues and the bulge of hair follicles in adult tissue, with relatively low abundance in adult tissues compared to embryo tissues [1,2,3]. NCSCs have the potential to differentiate into cell types of all three germ layers and generate a wide variety of cell types during vertebrate development, such as vascular smooth muscle cells (SMCs), Schwann cells, peripheral neurons, osteo- blasts, chondrocytes, melanocytes and endocrine cells. In addition, NCSCs can be derived from embryonic stem cells (ESCs and iPSCs) , making them a valuable stem cell source for tissue regeneration and an ideal model system for studying the lineage commitment and therapeutic potential of stem cells. The multi- potency of NCSCs can be characterized by their differentiation into neural and mesenchymal lineages. Although there are studies on the regulation of NCSC differentiation by biochemical cues, little is known on whether and how mechanical factors in the microenvironment regulate NCSC differentiation. Blood vessel wall is constantly subjected to cyclic mechanical strain in the circumferential direction due to the pulsatile nature of the blood flow. It has been reported that mechanical strain plays an important role in the remodeling of blood vessels and tissue- engineered vascular grafts [4,5,6,7,8]. The application of mechan- ical strain to SMCs increases the expression of SMC markers, such as smooth muscle myosin heavy chain (MHC) and caldesmon [4,5,6,7,8,9,10], suggesting that mechanical strain may promote SMC contractile phenotype. Recent studies have shown that cyclic mechanical strain increased SMC marker expression in bone marrow mesenchymal stem cells (MSCs) [11,12,13]. NCSCs are an important source of vascular SMCs during the vascular development [14,15]. NCSCs could also be used to construct tissue engineered vascular grafts. Our recent study showed that NCSCs in vascular grafts could differentiate into SMCs in vivo (Zhu and Li, unpublished observation). However, whether and how hemodynamic factors regulate NCSC differentiation is unknown. We hypothesized that mechanical strain regulates NCSC differentiation into SMC lineage, and we used iPSC- derived NCSCs as a model to investigate this possibility. Among biochemical signaling molecules, transforming growth factor b (TGF-b) plays an important role in SMC differentiation. TGF-b regulates gene expression through both Smad-dependent and Smad-independent pathways in a context-dependent manner [16,17,18]. TGF-b has been shown to up-regulate the expression of SMC markers in SMCs [19,20,21,22], MSCs [23,24,25], ESCs PLoS ONE | www.plosone.org 1 October 2011 | Volume 6 | Issue 10 | e26029