may therefore serve as a suitable scaffold for constructing vascular tissues. In this study, SMCs derived from the differentiated mouse SM22a2/2LacZ ESCs were enriched by positive cell sorting, and the 3D NF scaffolds were shown to support the growth of sorted cells both in vitro and in vivo. Materials and Methods SMC differentiation of SM22a2/2LacZ ESCs SM22a2/2LacZ ESCs were kindly provided by Dr. Li Li from Wayne State University, who published the SM22a-KO mice data previously [12]. The ESCs were expanded and induced to differentiate into SMCs in vitro when treated with 1025 M all trans-retinoid acid (RA) following the protocol described in our previous publication [8]. The vehicle dimethyl sulfoxide (DMSO) treatment served as a spontaneous differentiation control. b-gal staining A b-gal staining kit (Invitrogen, Carlsbad, CA) was used for b- gal staining. The derived cells after treatment with either the DMSO or RA for different time periods were fixed with 2% formaldehyde and incubated with an X-gal containing solution, according to the manufacturer’s instructions. For b-gal staining of the implants, sections (5 mm thick) were generated using a cryostat (CM 1850, Leica Microsystems) and thaw-mounted onto glass slides and stored at 220uC until staining. The cell nuclei were counterstained with fast red (Vector Lab, Burlingame, CA). Implant samples were also stained with hematoxylin and eosin (H&E). Fluorescence activated cell sorting (FACS) Cell suspension preparation and FACS were performed following published methods [13]. Briefly, differentiated cells cultured on Petri dishes were digested with trypsin and then centrifuged to remove the supernatant. Cells were re-suspended in staining medium to a density of ,107 cells/mL and loaded with 5- chloromethylfluorescein di-b-D-galactopyranoside (FDG) by add- ing 100 mL 2 mM pre-warmed FDG working solution. Cells were mixed rapidly and incubated in a 37uC water bath for 1 minute. Then the FDG loading was stopped by adding 1.8 mL ice-cold staining buffer. The cells were kept on ice prior to FACS. The background autofluorescence compensation was set with unstained cells. The cells with fluorescence labeling (LacZ positive) were analyzed and sorted using the FACSCaliburTM system (BD Biosciences, San Jose, CA) following the user’s guide. Immunofluorescence staining Sorted cells were replated on 0.1% gelatin-coated chamber vessels (BD Biosciences) and incubated in the culture medium overnight. Cells were then fixed, permeabilized and incubated overnight with smooth muscle-a-actin (a-SMA) antibody at 1:1000 dilutions (Millipore, Temecula, CA). After rinsing, samples were incubated with Alexa Fluor-488 conjugated secondary antibody (Molecular Probe, Eugene, OR) for 1 hour. The cell nuclei were co-stained with 49,6-diamidino-2-phenylindole (DAPI) (Molecular Probe). Samples were observed under an Olympus BX51 microscope. Fabrication of 3D macro-porous NF scaffolds 3D macro-porous NF scaffolds were fabricated following our previous publication [3]. Briefly, 10wt/v% poly(L-lactide) (PLLA) (Boehringer Ingelheim, Ingelheim, Germany) in tetrahydrofuran solution was cast into an assembled sugar template from bound sugar spheres with a diameter of 125–250 mm under a mild Vascular Engineering Using Embryonic Stem Cells vacuum. After phase separation at 220uC overnight, the polymer- sugar composite was immersed into cyclohexane to exchange tetrahydrofuran for 2 days. The resulting polymer-sugar compos- ites were freeze-dried and the sugar spheres were leached out in distilled water. The obtained macro-porous NF scaffolds were freeze-dried again and cut into circular disks with dimensions of 3.6 mm in diameter and 1 mm in thickness. The scaffolds were sterilized with ethylene oxide before use. Cell culture on 3D macro-porous NF scaffolds The sterile 3D NF scaffolds were pre-wetted in 70% ethanol for 30 minutes, washed three times with PBS for 30 minutes each, and twice in cell culture medium for 2 hours each. 2.56105 cells were seeded on each scaffold. The cell-seeded scaffolds were then transferred to 6-well plates on an orbital shaker [14]. The medium was changed twice a week. Scanning electron microscopy (SEM) observation Samples were rinsed in PBS, fixed in 2.5% glutaraldehyde and 2% paraformaldehyde overnight, and post-fixed in 1% osmium tetroxide for 1 hour. Samples were dehydrated in increasing concentrations of ethanol and hexamethyldisilizane. The samples were then sputter-coated with gold and observed under a scanning electron microscope (Philips XL30 FEG). Gene expression analysis with quantitative real-time PCR Total cellular RNA and cDNA were prepared with an RNeasy mini kit (Qiagen, Valencia, CA) and superscript III first-strand synthesis method (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions respectively. PCR amplification was performed with primers selective for individual genes with SYBR Green supermix kit (Bio-rad, Hercules, CA). PCR primers and reaction conditions were described previously [11] and in Table S1. All gene expression level of individual RNA sample was normalized against 18S RNA as an internal standard. Histological observation The cell-scaffold constructs were washed in PBS, fixed with 3.7% formaldehyde, dehydrated in ethanol, and embedded in paraffin. Sections (5 mm thick) were stained with H&E method. For immunofluorescence analysis of SMC specific markers, the slides were de-paraffinized, blocked with serum, incubated with primary antibodies to a-SMA (1:50 dilution, Millipore) and then incubated with Alexa Fluor-592 conjugated secondary antibodies (Invitrogen, Carlsbad, CA). Samples were observed under an Olympus BX51 microscope. Subcutaneous implantation After the sorted SMCs were seeded and cultured on scaffolds for 24 hours, the cell-scaffold constructs were implanted into subcutaneous pockets on nude mice. Briefly, 6–8 weeks old male nude mice (Charles River Laboratories, Wilmington, MA) were anesthetized with isofluorane. Two dorsal midsagittal incisions were made on the disinfected back. One subcutaneous pocket was created on each side of each incision using blunt dissection and one scaffold was implanted into each pocket. Four samples were implanted randomly for each group (n = 4). The mice were euthanized and the implants were harvested at 2 weeks for b-gal staining. The animal procedures followed the approved protocol by the University of Michigan Committee on Use and Care of Laboratory Animals. PLoS ONE | www.plosone.org 2 April 2012 | Volume 7 | Issue 4 | e35580