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Neural crest stem cells (NCSCs) are multipotent and play an important role during the development and tissue regeneration.However, the anisotropic effects of mechanical factors on NCSCs are not known.To investigate the anisotropic mechanosensing by NCSCs, NCSCs derived from induced pluripotent stem cells were cultured on micropatterned membranes, and subjected to cyclic uniaxial strain in the direction parallel or perpendicular to the microgrooves.Cell and nuclear shape were both regulated by micropatterning and mechanical strain.Among the unpattemed, parallel-patterned and perpendicular-patterned groups, mechanical strain caused an increase in histone deacetylase (HDAC) activity in the parallel-patterned group, accompanied by the increase of cell proliferation.In addition, mechanical strain increased the expression of contractile marker calponin-1 but not other differentiation markers in the unpattemed and parallel-patterned groups.These results demonstrated that NCSCs responded differently to the anisotropic mechanical environment.We then determined whether mechanical strain modulates the differentiation of NCSCs into smooth muscle (SM) lineage in the parallel-patterned group.Mechanical strain induced contractile marker calponin-1 within 2 days and slightly induced SM myosin within 5 days, while mechanical strain suppressed the differentiation of NCSCs into Schwann cells.The induction of calponin-1 by mechanical strain was inhibited by neural induction medium but further enhanced by TGF-β.For NCSCs pre-treated with TGF-β, mechanical strain induced the gene expression of both calponin-1 and SM myosin.Our results demonstrated that mechanical strain regulates the differentiation of NCSCs in a manner dependent on biochemical factors and the differentiation stage of NCSCs.Understanding the mechanical regulation of NCSCs will reveal the role of mechanical factors in NCSC differentiation during development, and provide a basis for using NCSCs for tissue engineering.