Fig. 2. Differentiation properties of mTERT-overexpressing ES cells. (A) Differentiation of ES cells was induced with 0.3 µM retinoic acid for 6 days. Cell morphology of non-induced and induced cells was monitored with phase-contrast microscopy. (B) Expression of mTERT in differentiated ES cells was determined by RT-PCR in vector-transfected and mTERT-expressing ES cells. (C) Protein level of mTERT in differentiated ES cells was determined by western-blot analysis using 100 µg nuclear extract from vector-transfected and mTERT-expressing ES cells. Topoisomerase-I levels were also determined, which represent loading in each lane. (D) Vector-transfected or mTERT-expressing ES cells were induced to differentiate with 0.3 µM retinoic acid for 2 days, 4 days and 6 days. The loss of surface expression of a stem-cell-specific antigen (ECMA-7) was monitored over time by flow cytometry. (E) Expression of stem-cell and differentiation markers was determined by RT-PCR. ES cells were induced to differentiate with retinoic acid for 6 days. Cells were harvested at different days of differentiation and RNA was extracted for RT-PCR analysis.