<p>Isolating mesenchymal stromal cell (MSC) subpopulations with improved quality based on specific surface markers, such as CD146, is one approach to enhance their therapeutic potential. However, there is limited information on the cytokine-boosted immunomodulatory mechanisms between CD146-expressing and non-expressing MSCs across various inflammatory conditions. This study seeks to address this gap. MSCs from the human periodontal ligament (hPDL-MSCs) were treated with interleukin-(IL)-1β, interferon-(IFN)-γ, or tumor necrosis factor-(TNF)-α, investigating the expression of IDO-1, PD-L1, PTGS-2, and TSG-6 between CD146⁺ and CD146⁻ hPDL-MSCs. Additionally, the expression of the immunomediators was compared between CD146-depleted and -enriched populations, generated by magnetic-bead-sorting. CD146⁺ hPDL-MSCs exhibited a significantly higher proportion of IDO-1⁺ cells, enhanced expression levels in the presence of IL-1β or TNF-α, and higher IL-1β, IFN-γ, or TNF-α-induced PD-L1 protein expression. Conversely, the proportion of TSG-6⁺ hPDL-MSCs was significantly lower in CD146⁺ cells under basal conditions. In CD146-enriched hPDL-MSCs, the immunomediator expression was minimally elevated, with significant differences under specific conditions: a higher PD-L1 protein expression under basal conditions and in the presence of IFN-γ or TNF-α, as well as higher or lower PGE₂ levels with TNF-α or IL-1β, respectively. These results suggest that the immunomediator expression in hPDL-MSCs alters with different CD146 surface expression. CD146⁺ hPDL-MSCs do not always show higher levels of immunomediator expression. Further research is necessary to isolate hPDL-MSC subpopulations with optimal potential for therapeutic applications.</p>