Vascular Engineering Using Embryonic Stem Cells Figure 3. In vitro culture of sorted SMCs on 3D NF scaffolds. (A) Gene expression profiles of sorted SMCs before (Ctrl) and after seeded on NF scaffolds and cultured under the treatment of RA or DMSO for 2 weeks, respectively. (B) Histology of constructs cultured for 2 weeks with H–E staining. Left panel: DMSO treatment; right panel: RA treatment. (C) Immunofluorescence staining of a-SMA for constructs cultured for 2 weeks. Upper panel: DMSO treatment; Lower panel: RA treatment. Red: positive a-SMA expression; Blue: nuclei. Scale bar = 50 mm. *p,0.05. doi:10.1371/journal.pone.0035580.g003 basal homeostatic function mediated by vascular SMCs in the developing mice and SM22a2/2 mice are viable and fertile [20]. Here, we used the ESCs, which have been employed for the generation of SM22a2/2LacZ mice, to generate SMCs in vitro with LacZ staining as the positive selection marker. Naturally, the SM22a promoter is highly active in the heart tube and selectively expressed in a subset of arterial SMCs [20,22–25]. Therefore, there was a possibility that cardiomyocytes might also be isolated using the SM22a promoter as the selecting marker. Our previous study has shown the SMCs, derived from ESCs or induced PLoS ONE | www.plosone.org 5 April 2012 | Volume 7 | Issue 4 | e35580