Construction of Vascular Tissues with Macro-Porous Nano-Fibrous Scaffolds and Smooth Muscle Cells Enriched from Differentiated Embryonic Stem Cells Jiang Hu1., Changqing Xie2., Haiyun Ma 1 , Bo Yang 3 , Peter X. Ma1,4,5,6*, Y. Eugene Chen 2 * 1 Department of Biologic and Materials Sciences, University of Michigan, Ann Arbor, Michigan, United States of America, 2 Department of Internal Medicine, Cardiovascular Center, University of Michigan, Ann Arbor, Michigan, United States of America, 3 Department of Cardiac Surgery, Cardiovascular Center, University of Michigan, Ann Arbor, Michigan, United States of America, 4 Department of Biomedical Engineering, University of Michigan, Ann Arbor, Michigan, United States of America, 5 Macromolecular Science and Engineering Center, University of Michigan, Ann Arbor, Michigan, United States of America, 6 Department of Materials Science and Engineering, University of Michigan, Ann Arbor, Michigan, United States of America Abstract Vascular smooth muscle cells (SMCs) have been broadly used for constructing tissue-engineered blood vessels. However, the availability of mature SMCs from donors or patients is very limited. Derivation of SMCs by differentiating embryonic stem cells (ESCs) has been reported, but not widely utilized in vascular tissue engineering due to low induction efficiency and, hence, low SMC purity. To address these problems, SMCs were enriched from retinoic acid induced mouse ESCs with LacZ genetic labeling under the control of SM22a promoter as the positive sorting marker in the present study. The sorted SMCs were characterized and then cultured on three-dimensional macro-porous nano-fibrous scaffolds in vitro or implanted subcutaneously into nude mice after being seeded on the scaffolds. Our data showed that the LacZ staining, which reflected the corresponding SMC marker SM22a expression level, was efficient as a positive selection marker to dramatically enrich SMCs and eliminate other cell types. After the sorted cells were seeded into the three-dimensional nano-fibrous scaffolds, continuous retinoic acid treatment further enhanced the SMC marker gene expression level while inhibited pluripotent maker gene expression level during the in vitro culture. Meanwhile, after being implanted subcutaneously into nude mice, the implanted cells maintained the positive LacZ staining within the constructs and no teratoma formation was observed. In conclusion, our results demonstrated the potential of SMCs derived from ESCs as a promising cell source for therapeutic vascular tissue engineering and disease model applications. Citation: Hu J, Xie C, Ma H, Yang B, Ma PX, et al. (2012) Construction of Vascular Tissues with Macro-Porous Nano-Fibrous Scaffolds and Smooth Muscle Cells Enriched from Differentiated Embryonic Stem Cells. PLoS ONE 7(4): e35580. doi:10.1371/journal.pone.0035580 Editor: Qingzhong Xiao, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, United Kingdom Received January 13, 2012; Accepted March 18, 2012; Published April 24, 2012 Copyright: 2012 Hu 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 work is partially funded by an Inaugural Grant (YEC and PXM) from the Cardiovascular Center at University of Michigan and by the National Institutes of Health (HL092421 and HL068878: YEC; DE015384 and DE017689: PXM). CX is supported by an AHA National Scientific Development Grant (09SDG2260023). YEC is an AHA established investigator (0840025N). PXM is partially supported by the Richard H. Kingery Endowed Collegiate Professorship. 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: mapx@umich.edu (PXM); echenum@umich.edu (YEC) . These authors contributed equally to this work. Introduction Vascular smooth muscle cells (SMCs) constitute the principal layer of all small and large blood vessels in the body. Functional deficiency of vascular SMCs plays a central role in the progression of a large number of major human diseases [1,2]. Many therapeutic treatments to cardiovascular diseases have either enhanced in situ blood vessel formation or implanted in vitro constructed blood vessels. The efforts to construct tissue- engineered blood vessels in vitro using mature vascular SMCs isolated from donor vascular tissues have been extensively reported [3–5]. However, mature vascular SMCs have limited ability to proliferate and the cell source is very limited. Embryonic stem cells (ESCs) have shown promise as a therapeutic cell source because of the capacity of self-renewal and the potential to differentiate into various cell types in the adult body. A growing number of studies have demonstrated the differentiation capabil- ities of ESCs into lineage-specific cells under the appropriate stimuli and the control of a range of signal pathway complex [6]. We and others have utilized a two-dimensional monolayer culture system for cardiovascular differentiation [7–9], which has facilitated the dissection of molecular and cellular mechanisms of cardiovascular development [10,11]. However, one of the major challenges for the application of ESC-derived SMCs to the construction of functional vascular tissues is the heterogeneous cell population derived from pluripotent stem cells, which may lead to teratoma formation or inferior tissue organization. Thus strategies have to be developed to efficiently induce and enrich SMCs before the cells can be used for vascular tissue engineering. Another challenge is to develop a suitable porous scaffold to support vascular tissue development. We recently developed three- dimensional (3D) macro-porous nano-fibrous (NF) scaffolds and found them to preferentially support contractile phenotype of human aorta SMCs under in vitro culture conditions [3], which PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e35580