Vascular Engineering Using Embryonic Stem Cells Figure 1. Sorting of SMCs from RA-induced SM22a2/2LacZ ESCs. (A) Morphology change of SM22a2/2LacZ ESCs under the treatment of DMSO (upper panel) or 1025 M RA (lower panel) respectively as indicated time. (B) Comparison of b-gal staining of cells treated with either DMSO (left panel) or 1025 M RA (right panel). The upper panel showed that b-gal staining positive cells accumulated with RA treatment time, while only sporadic b-gal staining positive cells existed in DMSO-treated cell population. The lower panel showed representative magnified images by day 8. Scale bar = 100 mm. (C–D) Using 5-chloromethylfluorescein di-b-D-galactopyranoside (FDG) to react with intracellular glutathione. In LacZ-positive cells derived from RA-induced SM22a2/2LacZ ESCs, the FDG–glutathione adduct was converted to a bright green fluorescent product and LacZ-positive cells were subsequently sorted through a GFP channel (C) and cultured (D). Left panel: bright field; middle panel: GFP channel; right panel: merge. Scale bar = 100 mm. (E) Immunofluorescence staining of SMCs derived from sorted LacZ-positive cells with SMC-specific marker a-SMA antibody. The nuclei were co-stained with DAPI. Scale bar = 100 mm. (F) Gene expression of sorted cells analyzed by quantitative real-time PCR, compared to undifferentiated ESCs (control, Ctrl) or cells before sorting. Before: before sorting; After: after sorting. *p,0.05. doi:10.1371/journal.pone.0035580.g001 Statistical analysis Numerical data were reported as mean 6 S.D. from triplicate cell culture (n = 3). The Student’s t-test was applied to test the significance between the groups. The value of p,0.05 was considered statistically significant. Results Enrichment of SMCs from differentiated SM22a2/2LacZ ESCs Under the treatment of RA, the SM22a2/2LacZ ESCs monolayer underwent dramatic morphological changes. Through- out this process, the cells kept proliferating and gradually grew to confluence. In the meantime, the cells acquired an SMC-like shape starting from day 2 (Fig. 1A). Gradually, the SMC-like cells dominated the culture from day 5 and became confluent with a significant morphological difference compared with the DMSO treated spontaneous differentiation culture, which showed more random cellular shapes. The b-gal staining result indicated that RA treatment induced SM22a2/2LacZ ESCs to differentiate into SMCs in a time-dependent manner (Fig. 1B). The number of b-gal positive cells increased with inducing time. In contrast, only sporadic staining was observed in spontaneously differentiated culture. Moreover, b-gal activity was dramatically enhanced in RA-treated SM22a2/2LacZ ESCs compared to DMSO-treated cells at day 8 (data not shown). The differentiated cells were then sorted. After 6 days of RA induction, the derived SMC population from SM22a2/2LacZ ESCs were sorted by FACS with hypotonic introduction of 5-chloromethylfluorescein di-b-D-galactopyrano- side (FDG), which is designed to react with intracellular glutathione and subsequent hydrolysis by b-galactosidase and generate fluorescent adduct [13]. To eliminate the possibility of contamination of non-specific cell types with low-level transient SM22a gene expression, the sorting gate threshold was set to a value higher than 102 fluorescence intensity. As a result, the sorted cells for further studies accounted for 20–25% of total LacZ PLoS ONE | www.plosone.org 3 April 2012 | Volume 7 | Issue 4 | e35580