Mechanical Regulation of NCSCs Figure 6. The combined effects of mechanical strain and biochemical factors. NCSCs were cultured in three different types of media: DMEM with10% FBS, neural basal medium (NB) and DMEM with 10% FBS and 10 ng/ml of TGF-b1, and subjected to cyclic strain for 1 day (for qPCR in A) or 2 days (for protein expression analysis in B). * p,0.05 in comparison to respective no-strain control by using log- transformed one-sample t-test, n$3. 1 p,0.05 in comparison to ‘‘no- strain’’ sample by using log-transformed one-sample t-test, n$3. " p,0.05 in comparison to ‘‘strain’’ sample by using log-transformed one-sample t-test, n$3. doi:10.1371/journal.pone.0026029.g006 the proliferation and ERK phosphorylation but did not cause Akt activation. However, mechanical strain could also regulate the activity of other mitogen-activated protein kinases such as p38 and JNK, thus modulating cell proliferation. More in-depth studies are needed to dissect the mechanisms of how mechanical strain regulates the proliferation and differentiation of NCSCs. The fact that mechanical strain promotes NCSC differentiation into SMC lineage provides an explanation of how NCSCs differentiate into mature SMC in vivo, and suggests that undifferentiated NCSCs could be directly used for vascular tissue engineering. Previous studies have demonstrated the roles of mechanical stimulation in promoting MSC differentiation into SMC lineage [16,17,18,33]. Several studies have shown that mechanical strain Figure 7. Effect of sequential treatment with TGF-b and mechanical strain. NCSCs were pre-treated with DMEM supplement- ed with10% FBS and 10 ng/ml of TGF-b1 for 2 weeks, and then subjected to cyclic uniaxial strain for 2 days. (A–I) Immunostaining of undifferentiated NCSCs and NCSCs with or without TGF-b1 treatment for 2 weeks. Scale bar = 100 mm. (J) Gene expression was analyzed by qPCR analysis. * p,0.05 in comparison to respective no-strain control by using log-transformed one-sample t-test, n$3. doi:10.1371/journal.pone.0026029.g007 also induces the up-regulation of smooth muscle markers such as CNN1, SMA, and MHC in SMCs [34,35]. Here we showed that mechanical strain significantly induced the expression of CNN1 (within 2 days) and MHC (after 5 days). However, there was no incorporation of mature SMC marker MHC into stress fibers, suggesting that mechanical strain alone was not sufficient to induce the terminal differentiation of SMCs and that other factors, specifically biochemical factors, are required for the terminal differentiation. In contrast, mechanical strain did not induce the markers of several other lineages, including neural, chondrogenic, adipogenic, osteogenic and endothelial lineages, indicating that mechanical strain has specific effect on NCSC differentiation into SMC lineage. Although mechanical strain did not promote the terminal differentiation of NCSCs, it did affect the differentiation potential of NCSCs into neural linage such as Schwann cells, implicating that mechanical strain could drive NCSC differenti- ation into an intermediate stage at which the cells partially loss the differentiation potential into other lineages. PLoS ONE | www.plosone.org 7 October 2011 | Volume 6 | Issue 10 | e26029