Vascular Engineering Using Embryonic Stem Cells Figure 4. Histology of constructs implanted subcutaneously for 2 weeks. (A) b-gal staining. (Left) blank scaffold implants; (Right) cell- scaffold construct implants. Blue: positive LacZ staining; Red: nuclei. (B) H&E staining of cell-scaffold construct implants. Scale bar = 200 mm. doi:10.1371/journal.pone.0035580.g004 pluoripotent stem cells (iPSCs) by high dose (1025 M) RA induction, had very low level of cardiomyocyte specific marker expression [8], therefore excluding the contamination of cardiomyocytes in our induction-sorting protocol. The results demonstrated that SM22a expression and the corresponding LacZ positive staining within the cell population sorted from differentiated ESCs exhibited a high degree of SMC specificity, as evidenced by the high level expression of SMC specific markers and diminished expression of other cell lineage markers. A concern associated with the use of ESCs for therapies is the retention of undifferentiated pluripotent cells. Studies indicated that undifferentiated ESCs remained in embryoid bodies even after 28 days of differentiation and could be eradicated only by prolonged negative selection [18]. Undifferentiated ESCs are highly proliferative and have the potential to differentiate into unwanted cell lineages including teratoma formation, raising a significant concern over their use for therapeutic applications. The risk can be reduced if the ESCs are differentiated prior to transplantation [18,26]. Here, we showed the absence of teratoma formation in our studies, likely benefited from the high efficiency of RA induced differentiation protocol and the positive sorting strategy. However, we observed that after being seeded and cultured on 3D NF scaffolds, the sorted cells could re-express the pluripotent genes (i.e., Nanog) when RA was withdrawn early from the culture medium, indicating that the sorted cells were not terminally differentiated and longer RA treatment may be necessary to drive the cells to a mature differentiated phenotype. Growth factors have also been shown to greatly affect the cell differentiation [27,28]. The incorporation of a controlled-release capacity for RA or/and growth factors into the porous NF scaffolds [29] may further enhance functional vascular tissue regeneration. Supporting Information Table S1 (DOC) Primers Used for RT-PCR. Author Contributions Conceived and designed the experiments: JH CX YEC PXM. Performed the experiments: JH CX HM. Analyzed the data: JH CX BY. Wrote the paper: JH CX YEC PXM. PLoS ONE | www.plosone.org 6 April 2012 | Volume 7 | Issue 4 | e35580