Cartilage defects have a limited ability to self-heal. Stem cell treatment is a promising approach; however, replicative senescence is a challenge to acquiring large-quantity and high-quality stem cells for cartilage regeneration. Synovium-derived stem cells (SDSCs) are a tissue-specific stem cell for cartilage regeneration. Our recent findings suggest that decellularized stem cell matrix (DSCM) can rejuvenate expanded SDSCs in cell proliferation and chondrogenic potential. In this study, we were investigating (1) whether transforming growth factor (TGF)-β1 and TGF-β3 played a similar role in chondrogenic induction of SDSCs after expansion on either DSCM or plastic flasks (plastic), and (2) whether DSCM-expanded SDSCs had an enhanced capacity in repairing partial-thickness cartilage defects in a minipig model. SDSCs were isolated from synovium in two 3-month-old pigs and DSCM was prepared using SDSCs. Passage 2 SDSCs were expanded on either DSCM or plastic for one passage. The expanded cells were evaluated for cell morphology, chondrogenic capacity, and related mechanisms. TGF-β1 and TGF-β3 were compared for their role in chondrogenesis of SDSCs after expansion on either DSCM or plastic. The chondrogenic induction medium without TGF-β served as a control. In 13 minipigs, we intraarticularly injected DSCM- or plastic-expanded SDSCs or saline into knee partial-thickness cartilage defects and assessed their repair using histology and immunohistochemistry. We found DSCM-expanded SDSCs were small, had a fibroblast-like shape, and grew quickly in a three-dimensional format with concomitant up-regulation of phosphocyclin D1 and TGF-β receptor II. Plastic-expanded SDSCs exhibited higher mRNA levels of chondrogenic markers when incubated with TGF-β3, while DSCM-expanded SDSCs displayed comparable chondrogenic potential when treated with either TGF-β isotype. In the minipig model, DSCM-expanded SDSCs were better than plastic-expanded SDSCs in enhancing collagen II and sulfated glycosaminoglycan expression in repair of partial-thickness cartilage defects, but both groups were superior to the saline control group. Our observations suggested that DSCM is a promising cell expansion system that can promote cell proliferation and enhance expanded cell chondrogenic potential in vitro and in vivo. Our approach could lead to a tissue-specific cell expansion system providing large-quantity and high-quality stem cells for the treatment of cartilage defects.