In addition to their differentiation potential, self-renewal capability is an important characteristic of stem cells. The limited self-renewal activity of mesenchymal stem cells is the greatest obstacle to the application of stem cell therapy in regenerative medicine. The human TERT gene enhances the self-renewal of MSCs, but the mechanism of self-renewal and the interactions among TERT-gene-related molecules remain unknown. The objectives of this study were to generate immortalized MSCs derived from MSCs isolated from placenta (naive) by human TERT gene transfection with the AMAXA gene delivery system, to compare their characteristics, and to investigate whether increased TERT expression affected the pituitary tumor transforming gene (PTTG1; also known as securin), which is involved in chromosome segregation during mitosis. TERT-immortalized cells (TERT+) with a prolonged life span displayed high PTTG1 expression. TERT+ cells also retained the stemness capacity and multipotency of naive cells and displayed high PTTG1 expression. However, down-regulation of PTTG1 by treatment with short interfering RNA induced cell senescence and decreased telomerase activity. Moreover, TERT bound to PTTG1 formed complexes with chaperones such as Ku70 and heat shock protein 90. Thus, placental MSCs immortalized by TERT gene transfection display differentiation potential and exhibit enhanced self-renewal through a balanced interaction of PTTG1 and chaperones. The interaction between TERT and PTTG1 by association of Ku70 might be important for the enhancement of the limited self-renewal activity of MSCs and for understanding the regulatory mechanisms of self-renewal.