Extracellular vesicle bioactivity and potential for clinical development are determined by mesenchymal stromal cell clonal subtype

• MSC clonal subtype determines EV bioactivity, with Y201 EVs showing enhanced biomarker content and functional effects compared to Y202 EVs. • Y201 EVs promote chondrocyte proliferation via an RGD-integrin-FAK-ERK1/2 axis, highlighting a mechanistic pathway for EV-mediated tissue repair. • Both Y201 and Y202 EVs suppress T cell proliferation, but only Y201 EVs demonstrate anti-inflammatory efficacy in vivo, underscoring the importance of EV source selection. • The study emphasizes the need for clonally defined MSC lines to produce consistent and potent EV-based therapies for clinical translation.