Vascular Engineering Using Embryonic Stem Cells Figure 2. Representative SEM micrographs of NF scaffold and sorted SMCs seeded on the scaffold. (A–B) Low and high magnification images of the NF scaffold respectively. The cells were seeded and cultured for 24 hours and observed at low (C) or high (D) magnifications. Arrows indicate the cell aggregates inside the pores of scaffolds. doi:10.1371/journal.pone.0035580.g002 positive cell population (Fig. 1C). Sorted cells displayed typical SMC-like morphology with a strong fluorescence signal (Fig. 1D) and strong expression of a-SMA evidenced by immunofluores- cence staining (Fig. 1E). Gene expression analyses confirmed that much higher levels of the SMC specific genes, including a-SMA and SMMHC, were expressed in the purified cell population after FACS and simultaneously the expression level of both pluripotent marker (Oct4) and other cell lineage markers, such as endoderm marker AFP, mesoderm marker GATA2 and ectoderm marker NeuroD, was dramatically reduced (Fig. 1F). Collectively these results demonstrated that the above described method can be used for the enrichment of SMCs from the mixture of differentiated ESCs and thus allow us to generate purified SMCs in large quantities for further vascular tissue engineering applications. In vitro culture of sorted SMCs on 3D NF scaffolds Sorted SMCs were then seeded on 3D NF scaffolds (Fig. 2A&B) and cultured for 2 weeks in the medium containing DMSO or 1025 M RA. After seeding for 24 hours, the cells aggregated inside the pores of the scaffolds (Fig. 2C&D). Gene expression analysis showed enhanced SMC marker gene expressions, including myocardin (MyoCD), a-SMA and SMMHC, for cells cultured on 3D NF scaffolds under continuous RA treatment, compared to DMSO treatment, while the expression of pluoripotent marker Nanog was kept inhibited by RA treatment (Fig. 3A). H–E staining showed that the cells distributed throughout the scaffold after 2 weeks of culture for both groups (Fig. 3B). However, immunoflu- orescence staining showed strong a-SMA positive staining in the constructs with continuous RA treatment. In contrast, weaker a- SMA staining was observed in the constructs treated with DMSO (Fig. 3C). Subcutaneous implantation of cell-scaffold constructs After the sorted SMCs were seeded on the scaffold for 24 hours, the cell-scaffold constructs were subcutaneously implanted into nude mice. After 2 weeks of implantation, the tissue infiltration into the scaffolds was observed on all samples. Positive LacZ staining of implanted cells was observed, while no detectable LacZ staining was observed in the blank scaffold implants (Fig. 4A), which indicated the survival of implanted SMCs inside the scaffold under in vivo conditions and retained the marker gene expression. Meanwhile, the teroatoma development was not observed on the cell-scaffold construct implants (Fig. 4B). Discussion In this study, we presented a strategy to enrich SMCs from the differentiated ESCs and use the purified cells to construct vascular tissues on a macro-porous NF scaffold, which can serve as a potential platform for engineering therapeutic vascular tissues and for establishing vascular disease models. Although a number of in vitro SMC differentiation systems have been established, in which stem cells were induced to differentiate under the treatment of cytokines, growth factors or ECM components [8,9,15–19], there are limited publications showing the application of ESC-derived SMCs in vascular tissue engineering, largely owing to the heterogeneous properties of the cells. Given that ESCs have been considered to be a promising cell source for regenerating various tissues, we explored the capability of ESC-derived SMCs for constructing vascular tissues. SM22a is a 22 kDa protein that physically associates with cytoskeletal apparatus in contractile SMCs. SM22a-deficient (SM22a2/2LacZ) mice has been generated by gene targeting and used to study blood vessel development and gene function during embryogenesis, wherein, certain initiation codon of SM22a were replaced by the LacZ reporter [20,21]. Previous study has shown that this replacement does not alter PLoS ONE | www.plosone.org 4 April 2012 | Volume 7 | Issue 4 | e35580