Figure 2. Effects of uniaxial mechanical strain on cell morphol- ogy. (A) Micropatterning on the silicon wafer with photoresist. (B) Phase contrast imaging of micropatterned PDMS membranes. NCSCs were cultured on micropattened PDMS membranes and kept as no- strain control (C and E) or subjected to cyclic uniaxial strain (5%, 1 Hz) (D and F) for 1 day. F-actin filaments in NCSCs were stained by phalloidin (C–D). Nuclei were stained by propidium iodide (E–F). Scale bars = 100 mm. doi:10.1371/journal.pone.0026029.g002 of NCSCs into SMC lineage, but did not induce the expression of mature SMC marker MHC at the early stage (within 2 days). To determine whether mechanical strain affects the differenti- ation potential of NCSCs into glial cells, NCSCs were subjected to mechanical strain for 2 days, and cultured in Schwann cell differentiation medium for 1 week. Interestingly, NCSCs under no-strain condition expressed Schwann cell markers S100b and GFAP (Fig. 4D), but pre-stretched NCSCs has little expression of Schwann cell markers. These results suggested that mechanical strain suppressed the differentiation of NCSCs into Schwann cells. Long-term effects of mechanical strain on NCSC differentiation were then investigated. NCSCs were subjected to mechanical strain for 5 days. Immunoblotting analysis showed a 3-fold increase in CNN1 expression, a slight increase in MHC expression, and no significant change in SMA expression (Fig. 5A). Immunostaining experiments further confirmed the changes (Fig. 5B). The slight increase of MHC did not result in the Mechanical Regulation of NCSCs Figure 3. Effects of uniaxial mechanical strain on cell prolifer- ation. NCSCs were cultured on parallel micropatterned surfaces and subjected to cyclic uniaxial strain (5%, 1 Hz) for 1 day. (A) NCSC proliferation. The percentage of BrdU-positive cells (cells in S phase) was calculated. (B) Immunoblotting analysis of p-ERK1/2 and ERK2 after 1 day of cyclic uniaxial strain treatment. * p,0.05 in comparison to respective no-strain control using a two-tailed t-test, n$3. doi:10.1371/journal.pone.0026029.g003 incorporation of MHC into stress fibers, suggesting that the cells had not fully differentiated into SMCs. The combined effects of mechanical strain and biochemical factors on NCSC differentiation We postulated that different biochemical factors might affect the responses of NCSCs to mechanical strain. To test this possibility, NCSCs were cultured in the general culture medium with or without TGF-b, or cultured in neural basal (NB) medium. NCSCs were subjected to uniaxial strain for 1 day. Biochemical factors had significant effects on the expression level of CNN1. As shown in Figure 6A, NB medium suppressed while TGF-b increased the expression of CNN1 under static condition. However, in all of these culture media, mechanical strain induced CNN1 expression to a similar extent (by fold change), suggesting that mechanical strain regulates CNN1 expression in parallel to the signaling pathways regulated by TGF-b and NB medium. The results of gene expression were verified by CNN1 protein expression (Fig. 6B). In all of three types of culture media, there was no induction of SMC mature marker MHC by mechanical strain within 2 days (data not shown). The effect of mechanical strain on NCSC differentiation into SMC lineage at a later stage To test whether mechanical strain regulated NCSC differenti- ation at a later stage, we treated NCSCs with TGF-b for 2 weeks, and then subjected the cells to cyclic uniaxial strain for 2 days. Long-term culture of NCSCs in the general culture medium without the supplement of TGF-b resulted in spontaneous differentiation and the increase of SMA, CNN1 and MHC (Fig. 7 A–F) and the assembly of SMA and CNN1 into stress fibers. This ‘‘default’’ differentiation of NCSCs might be resulted from the low level of various growth factors in FBS, e.g., 500–800 pg/ ml of TGF-b in FBS. TGF-b treatment further induced the expression of SMA, CNN1 and MHC (Fig. 7 G–I). Mechanical strain significantly increased the gene expression of CNN1 (7.5- fold), MHC (2.5-fold) and SMTN (a slight increase) (Fig. 7 J) after 2 days. These results demonstrated that mechanical strain increased the expression of mature SMC marker (MHC) at a later stage of NCSC differentiation. Discussion It has been shown that micropatterning techniques could be used to control cell morphology to better mimic cell shape and PLoS ONE | www.plosone.org 5 October 2011 | Volume 6 | Issue 10 | e26029