Mesenchymal stem cells (MSC) are multipotent cells that can differentiate into various mesodermal lineages. However, their long-term in vitro culture leads to replicative senescence, a process that affects their morphology, surface markers, and differentiation potential. This study shows that MSC undergo continuous changes in gene expression and miRNA profiles during in vitro expansion, starting from the first passage. These changes include reduced cell cycle activity, DNA repair, and expression of genes related to cell cycle and DNA replication. Genes on chromosome 4q21 are overrepresented in differentially regulated transcripts. miRNA expression profiling reveals upregulation of hsa-mir-371, hsa-mir-369-5P, hsa-mir-29c, hsa-mir-499, and hsa-let-7f during in vitro propagation. These findings indicate that replicative senescence of MSC is a continuous process that impacts their phenotype, differentiation potential, and global gene expression patterns. This has important implications for the therapeutic application of MSC preparations. The study also highlights the need for quality control in MSC therapies due to the progressive changes in MSC characteristics during in vitro expansion.Mesenchymal stem cells (MSC) are multipotent cells that can differentiate into various mesodermal lineages. However, their long-term in vitro culture leads to replicative senescence, a process that affects their morphology, surface markers, and differentiation potential. This study shows that MSC undergo continuous changes in gene expression and miRNA profiles during in vitro expansion, starting from the first passage. These changes include reduced cell cycle activity, DNA repair, and expression of genes related to cell cycle and DNA replication. Genes on chromosome 4q21 are overrepresented in differentially regulated transcripts. miRNA expression profiling reveals upregulation of hsa-mir-371, hsa-mir-369-5P, hsa-mir-29c, hsa-mir-499, and hsa-let-7f during in vitro propagation. These findings indicate that replicative senescence of MSC is a continuous process that impacts their phenotype, differentiation potential, and global gene expression patterns. This has important implications for the therapeutic application of MSC preparations. The study also highlights the need for quality control in MSC therapies due to the progressive changes in MSC characteristics during in vitro expansion.