Reduction of TERC by siRNA. (A) To determine whether reduction in TERC could mimic the characteristics observed in TBD-BMSCs, transient reduction of TERC utilizing siRNA was used using a commercially available negative control (siNC-RNA) and siTERC-RNA (siTERC) constructs. Although there was a significant increase in TERC mRNA levels in transfection control (siNC-treated) BMSCs compared with N-BMSCs, TERC was significantly reduced by siTERC-RNA compared with both N-BMSCs and siNC-BMSCs after 72 hrs (n = 6 different N-BMSC cultures, and n = 3 different cultures each for siNC and siTERT treatment). (B) Seventy-two hours after transfection, the cells were trypsinized and plated at 50 000 cells/well of a 6-well plate (nonclonal density), allowed to grow for 7 days, and the number of cells was counted. TERC reduction substantially lowered the proliferation of siTERC-BMSCs compared with siNC-BMSCs (n = 3 different siNC-BMSC and siTERC-BMSC cultures). (C) Likewise, when transfected cells were trypsinized and plated at 500 cells/100 cm² dish (clonal density), the secondary CFE determined after 14 days was also reduced in the siTERC-BMSCs compared with siNC-BMSCs, in spite of the transient nature of the TERC reduction. (D) Seventy-two hours after transfection, siTERC-BMSCs also displayed significantly more SA-β-galactosidase compared with siNC-BMSCs (n = 2 experiments performed in triplicate from 2 different siNC-BMSC and siTERC-BMSC cultures). A representative experiment is shown. The senescent cells, with blue-green staining, were counted (1 field/chamber, 3-4 chambers/group) and the average cell number of positive cells is indicated (*P ≤ .05).