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The mechanical properties of a stem cell culture substrate significantly impact cell adhesion, survival, migration, proliferation, and differentiation in vitro. A major challenge in engineering artificial stem cell substrate is to properly identify the relevant physical features of native stem cell niches, which are likely different for each stem cell type. The behavior of tendon stem cells has potentially significant implications for tendon repair. Here, microfiber scaffolds with various modulus of elasticity are fabricated by near-field electrospinning, and their regulating effects on the in vitro behavior of tendon stem cells (TSCs) are discussed in this study. The number of pseudopodia shows a biphasic relationship with the modulus of scaffold. The proliferation, polarization ratio and alignment degree along the fibers of the TSCs increase with the increase of fiber modulus. TSCs cultured on the scaffold with moderate modulus (1429 MPa) show the upregulation of tendon-specific genes (Col-I, Tnmd, SCX and TNCF). These microfiber scaffolds provide great opportunities to modulate TSCs behavior at the micrometer scales. In conclusion, this study provides an instructive mechanical microenvironment for TSCs behaviors and may lead to the development of desirable engineered artificial stem cell substrate for tendon healing. [Display omitted] • The cross PA56 fiber scaffolds with various modulus were fabricated by NEFS. • The proliferation, number and cell polarization ratio of TSCs increased with the modulus. • The number of pseudopodia of TSCs showed a biphasic response to the modulus. • The arrangement of actin cytoskelenton tended to align toward the fiber with the increase of fiber modulus. • Microfiber scaffolds with moderate modulus (1429 MPa) demonstrated the potential to upregulate the tendon-specific genes. [ABSTRACT FROM AUTHOR]