streptomycin (Invitrogen Corp.) and 1% fungizone (Invitrogen Corp.). For the samples subjected to uniaxial strain, the polysulfone bar at one end of the chamber was attached to a motorized cam/ rotor system, while the other end of the frame remained stationary, thereby stretching the membrane [12]. For the no-strain controls, the polysulfone bar still moved back and forth, but the other end of the frame was not held stationary so that the entire membrane moved without being strained but experienced similar fluid disturbance to the stretched membranes in the stretch chamber. NCSCs were subjected to a physiological level of cyclic uniaxial strain at a 5% magnitude and 1 Hz frequency. Immunostaining and microscopy NCSCs were fixed with 4% paraformaldehyde in phosphate- buffered saline (PBS) for 15 min, followed by permeabilization with 0.5% Triton X-100 in PBS for 10 min. For F-actin staining, the specimens were stained with FITC-conjugated phalloidin (A12379, Invitrogen Corp.) for 1 h. For other stainings, the specimens were incubated with respective primary antibodies for 2 h, and with appropriate secondary antibodies for 1 h. Nuclei were stained by DAPI (D1306, Invitrogen Corp.) in blue or propidium iodide (Sigma-Aldrich, Corp.) in red. The following antibodies were used: p75 (AB8874, Abcam Inc., Cambridge, MA, USA), HNK1 (C6680, Sigma-Aldrich Corp.), vimentin (M0725, Dako North America Inc., Carpinteria, CA, USA), nestin (SC- 21247, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), S100b, GFAP, peripherin, TUJ1, CNN1, SMA, ALP, Col-II and MHC (SC-6956, Santa Cruz Biotechnology Inc.). The fluorescently stained samples were imaged by using a Leica (Wetzlar, Germany) True Confocal Scanner SL confocal micros- copy system, including He/Ne laser sources and a Leica DM IRB microscope, to capture multiple Z-section images (0.3- to 0.5-mm- thick sections) for a given specimen. The optical sections were subsequently projected to a single plane to create an overall image of the specimen. All images in a given group were collected with the same hardware and software settings. Other staining samples were examined using a Zeiss fluorescence microscope. The setting of microscopy system such as exposure time was kept constant for each experiment. RNA isolation and quantitative polymerase chain reaction (qPCR) Cells were lysed with 1 ml of Trizol (15596-026, Invitrogen Corp.) per membrane. RNA was extracted by using chloroform and phenol extractions, precipitated by isopropanol, and the resulting RNA pellet was washed with 75% ethanol. RNA pellets were resuspended in 20 ml DEPC-treated H2O and were quantified by RiboGreenH RNA Quantification Reagent and Kit (R11490, Invitrogen Corp.). cDNA was synthesized by using two-step reverse transcription (RT) with the ThermoScript RT-PCR system (11146- 024, Invitrogen Corp.), followed by qPCR using SYBR green reagent and the ABI Prism 7000 Sequence Detection System (Life Tech Corp., Carlsbad, CA, USA). Primers for the genes of interest were all designed by using the ABI Prism Primer Express software v.2.0 (Life Tech Corp.) and are listed in Table S1. The gene expression of each sample was normalized to the level of 18S ribosomal RNA of the same sample: 0.5 mg RNA for cDNA synthesis, and 1 mg cDNA for each qPCR reaction. Data was analyzed by using ABI Prism 7000 SDS software (Life Tech Corp.). Protein Isolation and immunoblotting Cells on each membrane were lysed with 100 ml of lysis buffer containing 25 mM Tris (pH 7.4), 0.5 M NaCl, 1% Triton X-100, Mechanical Regulation of NCSCs 0.1% SDS, 1 mM PMSF, 10 mg/ml leupeptin and 1 mM Na3VO4. Protein lysates were centrifuged to pellet cellular debris, and the supernatant was removed and quantified by DC Protein Assay (Bio-Rad, Hercules, CA). Protein samples (15 mg per well) were run in SDS/PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 3% nonfat milk and incubated with the primary antibodies diluted in TBST (Tris- Buffered Saline Tween-20) buffer containing 25 mM Tris?HCl (pH 7.4), 60 mM NaCl, and 0.05% Tween-20. This was followed by incubation with HRP-conjugated IgG secondary antibodies (Santa Cruz Biotechnology Inc.). To verify the equal loading of proteins in all lanes, nitrocellulose membranes were re-probed with an antibody against actin (SC-1616, Santa Cruz Biotechnol- ogy Inc.), including different actin isoforms. Protein bands were visualized by using the Western Lightning Plus-ECL (NEL 10500, Perkin Elmer Life & Analytical Sciences, Shelton, CT, USA), and signal intensity was analyzed by using ImageJ software (NIH). The following primary antibodies were used: p-ERK1/2 (4370s, Cell Signaling Technology, Inc., Danvers, MA, USA), ERK2 (SC-154, Santa Cruz Biotechnologies, Inc.), Tubulin (SC-5286, Santa Cruz Biotechnologies, Inc.), SMA (1184-1, Epitomics Inc.) and CNN1. Analysis of cell proliferation After 1 day of cyclic strain, NCSCs were pulsed with 10 mM BrdU (RPN201, GE Healthcare, Piscataway, NJ, USA) for 2 hours. Then the cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and incubated for 30 min in 2N HCl at 37uC. Subsequently, the samples were washed and incubated for 30 min in a buffer containing phosphate buffered saline (PBS), 0.05% Tween-20, and 1 mg/mL BSA to minimize background adsorption of antibodies. The samples were incubated overnight at 4uC with primary antibodies against BrdU (BD347580, BD Biosciences, San Jose, CA, USA) and an FITC- conjugated anti-mouse IgG secondary antibody (715-095-150, Jackson ImmunoResearch Inc., West Grove, PA, USA). After washing with PBS, the stained samples were counterstained with DAPI for 5 min, mounted in Fluoromount G (0100-01, Southern Biotech, Birmingham, AL, USA), and observed with a Zeiss fluorescence microscope. The percentage of NCSCs that incor- porated BrdU (i.e., the cells with DNA synthesis) was correlated to the proliferation rate of NCSCs. Statistical analysis Mean and standard deviation (SD) values were calculated for each group of data. Analysis of variance (ANOVA) was performed to detect whether a significant difference existed between groups of different treatments, and a multiple comparison procedure. The Holm t-test was used to identify any differences. Student’s t-test was used to analyze experimental groups with two samples. Results In vitro characterization of NCSCs Our previous study has shown that NCSCs could be derived from human iPSCs, and NCSCs were positive for neural crest markers (p75, HNK1, vimentin and nestin) and transcriptional factors (slug, AP2) [32]. To verify the multipotency of a specific iPSC-derived NCSC line, we determined whether NCSCs were capable of differentiating into a variety of cell types including neural lineages (Schwann cells, peripheral neurons) and mesen- chymal lineages (SMCs, adipocytes and chondrocytes, osteoblasts). As shown in Figure 1, with the treatment of specific differentiation factors, NCSCs differentiated into Schwann cells (Fig. 1A–B), peripheral neurons (Fig. 1C–D), SMC lineage (Fig. 1E–F), PLoS ONE | www.plosone.org 3 October 2011 | Volume 6 | Issue 10 | e26029