[25] and adult NCSC lines [26]. It’s also shown that there is TGF- b-mediated signaling pathways can crosstalk with mechanical force-induced signaling and gene expression in MSCs and endothelial cells [27,28,29]. However, whether TGF-b and mechanical strain cooperate to induce SMC differentiation in NCSCs is not clear. In this study we investigated the effects of mechanical strain on human NCSCs, particularly with regard to the differentiation of NCSCs into smooth muscle lineage. Our results showed that mechanical strain induced contractile markers calponin-1 (CNN1) and MHC, and suppressed the differentiation of NCSCs into Schwann cells. The induction of CNN1 by mechanical strain was inhibited by neural induction medium but further enhanced by TGF-b. For NCSCs pre-treated with TGF-b, mechanical strain induced the expression of both calponin-1 and MHC. Our results demonstrated that mechanical strain regulates the differentiation of NCSCs in a manner dependent on biochemical factors and the differentiation stage of NCSCs. These findings are novel and are important for the understanding of vascular development and how mechanical factors regulate NCSC differentiation in vivo. Materials and Methods Cell culture and NCSC derivation An iPSC line derived from human bone marrow MSCs (MSC- iPS1) [30] was used to derive NCSCs. To derive NCSCs, iPSCs were detached by collagenase IV (1 mg/ml) and dispase (0.5 mg/ ml), and grown as embryo body (EB)-like floating cell aggregates in ESC maintenance medium without basic fibroblast growth factor (bFGF) for 5 days. The cell aggregates were allowed to adhere to the CellStart (A10142-01, Invitrogen Corp., Carlsbad, CA, USA)- coated dishes in StemProH NSC serum-free neural induction medium (A10509-01, SFM, Invitrogen Corp.). After 7 more days, the colonies with rosette structures were mechanically harvested, cultured in suspension in SFM for 1 week, replanted onto the CellStart-coated dishes, and cultured for 3 more days. Cells were dissociated into single cells by TryplE Select (A1217702, Invitrogen Corp.), cultured in SFM as a monolayer, and further purified by cloning, subculture and/or flow-activated cell sorting (FACS) to obtain homogeneous populations that were positive for neural crest markers. The NCSCs derived from iPSCs were maintained in SFM for expansion without differentiation. NCSC differentiation To demonstrate the multipotency of NCSCs, we carried out the protocol of NCSC differentiation into neural lineages (Schwann cells, peripheral neurons) and mesenchymal lineages (SMCs, adipocytes, osteoblasts and chondrocytes) [2]. For NCSC differentiation into Schwann cells, NCSCs were cultured for 2 weeks in N2 medium supplemented with 10 ng/ml ciliary neurotrophic factor (CNTF; RD257-NT, R&D Systems Inc., Minneapolis, MN, USA), 10 ng/ml bFGF (PHG0024, Invitrogen Corp.), 1 mM dibutyryl-cAMP (dbcAMP; D0260, Sigma-Aldrich Corp., St. Louis, MO, USA) and 20 ng/ml neuregulin1b (377-HB, R&D Systems Inc.), and the cells were double-immunostained for Schwann cell markers S100b (S1542, Sigma-Aldrich Corp.) and Glial fibrillary acidic protein (GFAP; AB5804, Millipore Corp., Billerica, MA, USA). For NCSC differentiation into peripheral neurons, NCSCs were cultured for 2 weeks in N2 medium supplemented with 20 ng/ml brain-derived neurotrophic factor (BDNF; 248-BD, R&D Systems Inc.), 10 ng/ml nerve growth factor (NGF; 256-GF, R&D Systems Inc.), 10 ng/ml glial cell line-derived neurotrophic factor (GDNF; 212-GD, R&D Systems Inc.) and 1 mM dbcAMP, and the cells Mechanical Regulation of NCSCs were double-immunostained for peripheral neuron markers peripherin (AB1530, Millipore Corp.) and class-III b-tubulin (TUJ1; MMS-435P, Covance Inc., Princeton, NJ, USA). For NCSC differentiation into SMC lineage, NCSCs were treated with 10 ng/ml TGF-b1 (100-21, Pepro Tech Inc., Rocky Hill, NJ, USA) in Minimum Essential Medium a Medium (aMEM medium; Invitrogen Corp.) containing 10% Fetal Bovine Serum (FBS; Invitrogen Corp.) for 2 weeks, and double-immunostained with smooth muscle markers CNN1 (1806-1, Epitomics Inc., Burlingame, CA, USA) and smooth muscle a-actin (SMA; A5228, Sigma-Aldrich Corp.). For adipogenic differentiation, confluent NCSCs were treated with 10 mg/ml insulin (12643, Sigma-Aldrich Corp.), 1 mM dexamethasone (D4902, Sigma-Aldrich Corp.) and 0.5 mM isobutylxanthine (I7018, Sigma-Aldrich Corp.) in aMEM medium containing 10% FBS for 3 weeks. The formation of lipid and fat was examined by phase contrast microscopy and Oil red staining. For osteogenic differentiation, NCSCs were seeded at a low density (103cells/cm2) and grown in aMEM medium containing 10% FBS for 4 weeks in the presence of 10 mM b-glycerol phosphate (Sigma-Aldrich Corp.), 0.1 mM dexamethasone and 200 mM ascorbic acid (Sigma-Aldrich Corp.). Then cells was fixed in 4% paraformaldehyde, and stained with Alizarin red for calcified matrix or immunostained for alkaline phosphatase (ALP; B4-78, DSHB, Iowa city, IA, USA). For chondrogenic differentiation, NCSC pellets were cultured in suspension in aMEM medium containing 10% FBS for 4 weeks in the presence of 10 ng/ml TGF-b3 (243-B3, R&D Systems Inc.), 0.1 mM dexamethasone and 200 mM ascorbic acid. The cell pellets were cryosectioned, and stained for glycosaminoglycans (GAGs) by using Alcian blue staining or immunostained for Collagen type II (Col-II; MAB8887, Millipore Corp.). Microfabrication and soft lithography Microfabrication techniques were used to create micropatterned elastic membranes with parallel microgrooves (10-mm wide, 3-mm deep, 10-mm spacing between adjacent grooves) [12]. Briefly, photoresist (OIR 897-10I; Arch Chemicals, Norwalk, CT, USA) was spin-coated onto a silicon wafer, and a patterned photomask was used to expose the photoresist to UV light. After washing away the unpolymerized photoresist, polydimethylsiloxane (PDMS) was pre- pared according to the manufacturer’s protocol (Sylgard 184; Dow Corning, Midland, MI, USA), spin-coated onto the patterned silicon wafers to the desired thickness (,250 mm), degassed under vacuum, and cured at 70uC for 1 hour. The resulting micropatterned membranes were removed from the template, cut to appropriate dimensions for the assembly into custom-built stretch chambers, and thoroughly washed and sonicated before use. The surface topography of micropatterned PDMS membranes was examined by phase contrast microscopy. Mechanical testing showed that PDMS membranes were elastic under cyclic uniaxial strain (,30% strain, 1 Hz). Applying cyclic uniaxial strain to NCSCs For mechanical strain experiments, micropatterned membranes were treated with O2 plasma in a Plasma-Prep II plasma etcher (Structure Probe Inc., West Chester, PA, USA) for 1 min, and coated with a 2% gelatin solution. Membranes were assembled into custom-built uniaxial stretch chambers with microgrooves oriented parallel to the axis of strain [12,31]. Previously we have shown that aligned microgrooves can maintain cell orientation in the direction of mechanical strain [12]. NCSCs were seeded at 80% confluency on the micropatterned membranes in Dulbecco’s Modified Eagle’s Medium (DMEM; Invitrogen Corp.) supplemented with 10% FBS, 1% penicillin- PLoS ONE | www.plosone.org 2 October 2011 | Volume 6 | Issue 10 | e26029