Mechanical Regulation of NCSCs Figure 1. In vitro differentiation potential of NCSCs. (A–B) Immunostaining for Schwann cell markers S100b (A) and GFAP (B). (C–D) Immunostaining for peripheral neuron markers peripherin (C) and Tuj1 (D). (E–F) Immunostaining for SMC lineage markers CNN1 (E) and SMA (F). (G– H) Adipogenic differentiation shown by phase contrast imaging (G) and Oil red staining (H). (I–J) Osteogenic differentiation shown by Alizarin red staining for calcified matrix (G) and immunostaining of alkaline phosphatase (ALP) (H). (K–L) Chondrogenic differentiation shown by Alcian blue staining for glycosaminoglycans (E) and immunofluorescent staining of Col-II (F). In all immunofluorescence images, nuclei were stained by DAPI in blue. Scale bar = 100 mm. doi:10.1371/journal.pone.0026029.g001 adipogenic cells (Fig. 1G–H), osteoblastic cells (Fig. 1I–J), and chondrogenic cells (Fig. 1K–L). These results demonstrated the multipotency of NCSCs. Parallel microgrooves maintained NCSC alignment during mechanical strain To investigate the effects of mechanical strain on NCSCs, NCSCs were cultured in a general cell culture medium (DMEM with 10% FBS, 1% penicillin-streptomycin and 1% fungizone). NCSCs were subjected to cyclic uniaxial strain within physiological range (5%, 1 Hz). Our previous studies have shown that the direction of cell orientation modulates the effect of mechanical strain on the gene expression in MSCs [12]. To control NCSC orientation, micro- patterned elastic PDMS membranes with parallel microgrooves (10 mm wide, 3 mm deep) were fabricated (Fig. 2A–B), and NCSCs were cultured on these micropattened PDMS membranes. NCSCs followed the topographic guidance and aligned with the micro- grooves (Fig. 2C). Under uniaxial strain in the direction of cell alignment, NCSCs on parallel-oriented microgrooves remained aligned with the microgrooves and the axis of strain (Fig. 2D). The staining intensity and structure of F-actin did not show significant changes between no-strain and strain conditions. In addition, cell nuclei did not have significant change in morphology and remained aligned in the presence of mechanical strain (Fig. 2E–F). Mechanical strain with parallel cell alignment increased NCSC proliferation To investigate the effect of mechanical strain on NCSC proliferation, NCSCs cultured on micropatterned surfaces were subjected to cyclic uniaxial strain for 1 day. Mechanical strain slightly increased the proliferation rate of NCSCs (from 13% to 19%), as indicated by the percentage of NCSCs with BrdU incorporation (Fig. 3A). The activation of ERK1/2 has been shown to play an important role in the proliferation of many cell types. Consistently, mechanical strain increased ERK2 phosphor- ylation (the lower band in the top panel of Fig. 3B) after 1 day, while the level of ERK1 phosphorylation (the faint band above phosphorylated ERK2) was low. In contrast, mechanical strain had no effect on Akt activation in NCSCs (data not shown). Mechanical strain increased the expression of SMC contractile markers and suppressed Schwann cell differentiation To determine the effects of mechanical strain on NCSC differentiation, we examined the gene expression of lineage markers including CNN1, MHC, smoothelin (SMTN), TUJ1, Col-II, peroxisome proliferator-activated receptor c (PPARG), core binding factor a-1 (cbfa1) and vascular endothelial (VE)- cadherin (Fig. 4A). Interestingly, mechanical strain had different effects on SMC contractile markers. Mechanical strain caused a 2.3-fold increase of CNN1 gene expression and a slight increase of SMTN expression, but had no significant effects on the gene expression of MHC. In addition, mechanical strain had no significant effects on neural (TUJ1), chondrogenic (Col-II), adipogenic (PPARG), osteogenic (cbfa1) and endothelial (VE- cadherin expression was not detectable) linage markers. Using immunoblotting analysis, we found a nearly 70% increase in CNN1 protein expression after 2 days (Fig. 4B). In addition, immunostaining showed that the mechanical strain increased the number of cells (from 20% to 80%) with higher level of CNN1 expression (Fig. 4C) and SMTN (data not shown). These results demonstrated that mechanical strain promoted the differentiation PLoS ONE | www.plosone.org 4 October 2011 | Volume 6 | Issue 10 | e26029