Generation of Induced Pluripotent Stem Cell-Derived iTenocytes via Combined Scleraxis Overexpression and 2D Uniaxial Tension

This JoVE protocol describes a combined biochemical and mechanical strategy to generate induced pluripotent stem cell-derived tenocytes (iTenocytes) as a scalable cell source for tendon repair research. Human iPSCs are differentiated into induced mesenchymal stromal cells (iMSCs) using embryoid body formation and growth-factor exposure, then characterized by flow cytometry for classic MSC markers (CD44,CD90,CD105). iMSCs are subsequently transduced with a lentiviral construct to stably overexpress scleraxis (SCX), generating iMSCSCX+ cells with GFP-based tracking and titer optimization to balance transduction efficiency versus cytotoxicity. For mechanical maturation, iMSCSCX+ are seeded onto fibronectin-coated deformable silicone culture plates and subjected to cyclic uniaxial stretch under physiologically relevant conditions (reported as 4% sinusoidal strain at 0.5 Hz for 2 h/day for at least 3 days, up to 7 days). Mechanical loading induces cell re-organization and promotes tenogenic maturation, with demonstrated upregulation of tendon-related genes (e.g., SCX,THBS4,COL1a1,BGN,MKX,TPPP3) and increased collagen deposition versus static controls.