FIG. 5.

Pharmacological inhibition of the telomerase activity using a phosphorothioate oligonucleotide telomere mimic does not cause spontaneous hESC differentiation. H9 hESCs were grown in feeder-free conditions and treated with a small-molecule telomerase inhibitor III (TI-III; Calbiochem). (A) Representative immunofluorescence analysis of SSEA4 and SSEA1 levels in hESCs treated with 5 μM of telomerase RNA component (TERC) inhibitor for 3 days in high (20%) and low (2%) O₂. Scale bars=200 μm. (B) Relative telomerase activity levels of whole-cell lysates as measured by the RQ-TRAP assay. Error bars represent SE, n=3. Different letters above the histogram bars indicate significant differences (P<0.05). (C, E) Representative flow cytometry data plots representing fluorescence signal for hESCs treated with 5 μM of TERC inhibitor for 3 days in 2% O₂. SSEA4 (C) and SSEA1 (E) fluorescence in TI-III-treated (blue) and nontreated controls (red) relative to isotype control (black). Viability was determined using 7-AAD staining. Mean fluorescence intensity plots derived from flow cytometry data representing SSEA4 (D) and SSEA1 (F) signal intensities, respectively, in hESCs treated with 3 and 5 μM of TERC inhibitor for 3 days in 2% and 20% O₂. Error bars represent SE, n=3. Letters above histogram bars indicate significant differences (P<0.05). Color images available online at www.liebertpub.com/scd