• Urine-derived stem cells (USCs) provide a non-invasive, inexhaustible source of MSCs for EV production, overcoming limitations of invasive tissue extraction.
• USC isolation and expansion are robust across different times of day and donors, with no significant differences in clone number, doubling time, or viability.
• USCs exhibit typical MSC surface markers (CD73, CD90, CD105) and can be efficiently transfected using PEI/DNA transposon technology, enabling genetic engineering for enhanced EV properties.
• USCs maintain viability in serum-free conditions for 72 hours and produce EVs, making them a promising platform for clinical-grade native or engineered extracellular vesicle therapies.