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Bovine bone marrow mesenchymal stem cells (bBMSC) are potential stem cell source which can be used for multipurpose. However, their application is limited because the in vitro maintenance of these cells is usually accompanied by aging and multipotency losing. Considering transforming growth factor‐β (TGF‐β) pathway inhibitor Repsox is beneficial for cell reprogramming, here we investigated its impacts on the maintenance and differentiation of bBMSC. The bBMSC were enriched and characterized by morphology, immunofluorescent staining, flow cytometry, and multilineage differentiation. The impacts of Repsox on their proliferation, apoptosis, cell cycle, multipotency, and differentiation were examined by Cell Counting Kit‐8 (CCK‐8), real‐time polymerase chain reaction, induced differentiation and specific staining. The results showed that highly purified cluster of diffrentiation 73+ (CD73 +)/CD90 +/CD105 +/CD34 −/CD45 − bBMSC with adipogenic, osteogenic, and chondrogenic differentiation capacities were enriched. Repsox treatments (5 μM, 48 hr) enhanced the messenger RNA mRNA levels of the proliferation gene (telomerase reverse transcriptase [ TERT]; basic fibroblast growth factor [ bFGF]), apoptosis‐related gene ( bax and Bcl2), antiapoptosis ratio ( Bcl2/bax), and pluripotency marker gene ( Oct4, Sox2, and Nanog), instead of changing the cell cycle, in bBMSC. Repsox treatments also enhanced the osteogenic differentiation but attenuated the chondrogenic differentiation of bBMSC, concomitant with decreased Smad2 and increased Smad3/4 expressions in TGF‐β pathway. Collectively, inhibiting TGF‐β/Smad signaling by Repsox regulates the in vitro maintenance and differentiation of bBMSC.

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