Purpose <p>Multiple myeloma (MM) is a hematologic malignancy associated with a poor prognosis. MM-derived mesenchymal stromal cells (MM-MSCs) contribute to disease progression by creating a supportive stromal microenvironment for malignant cells. Elucidating transcriptomic alterations in MSCs may therefore facilitate the development of novel therapeutic strategies for treatment-resistant MM.</p> Methods <p>Total RNA was extracted from cultured MSCs isolated from bone marrow aspirates of patients with MM and normal donors (ND-MSCs). Expression of stemness markers (<i>NANOG</i>, <i>OCT4</i>) and senescence-associated genes (<i>P16</i>, <i>P21</i>, <i>IL-6</i>, <i>IL-8</i>) was analyzed using reverse transcription-quantitative polymerase chain reaction. Cellular senescence was assessed by senescence-associated β-galactosidase (SA-β-gal) staining.</p> Results <p>Compared with ND-MSCs, MM-MSCs demonstrated a higher percentage of SA-β-gal-positive cells. Gene expression analysis showed upregulation of <i>P21</i> and <i>IL-6</i> in MM-MSCs, whereas <i>NANOG</i> and <i>OCT4</i> were significantly downregulated. Notably, this downregulation was consistent with analysis of a publicly available RNA sequencing dataset, supporting the validity of our findings.</p> Conclusions <p>Consistent with previous reports of increased senescence, our findings demonstrate significant downregulation of stemness-related genes (<i>NANOG</i>, <i>OCT4</i>) in MSCs from newly diagnosed, untreated patients with MM. This concurrent dysregulation of stemness and increased senescent phenotype may contribute to the pathogenicity of the tumor microenvironment and represents a potential target for therapeutic intervention.</p> Clinical Trial Registration <p>Not applicable. This study did not involve a clinical trial.</p> Graphical Abstract <p></p>