Abstract

Therapeutic cloning has broad application prospects in the medical field. However, interspecies cloning efficiency is very low. It is generally believed that G0/G1 stages of cell cycle are more beneficial to cell reprogramming of cloning. The purpose of this study was to evaluate the effects of serum starvation, contact inhibition and 2-methoxyestradiol (2-ME) treatment on cell-cycle synchronization in the G0/G1 stage in human foreskin fibroblasts. Our results show that the proportion of G0/G1 cells from the serum-starved group at 3 and 4 days was significantly higher when compared with 2 days and cells not subjected to serum starvation (97.3% and 97.99% vs. 93.8% and 81.59%, respectively; p < 0.01). No significant difference was observed among cells with 3 and 4 days of starvation. The proportion of contact-inhibited G0/G1 cells significantly increased when compared with cells with 70 to 80% confluence (84.89 vs. 81.59%; p < 0.05) and decreased significantly when compared with cells subjected to serum starvation for 3 and 4 days. In cells treated with 2-ME (0.1 to 10 mM) combined with vibration, the proportion of G0/G1 cells was not significantly different (74.32, 77.75, 78.65, 76.96, 80.39 and 81.4%; p > 0.05). After the recovery of cells that were frozen for 4 to 5 months, the proportion of cells in the G0/G1 phase was significantly lower when compared with normal cells (75.14 vs. 81.59%; p < 0.05). Our results show that serum-starvation for 3 days is the most effective method for synchronizing human foreskin fibroblasts in the G0/G1 phase, which is may more suitable as donor cells for cloning.

Key words: Cell cycle synchronization, 2-methoxyestradiol, serum starvation, culture to confluence, human foreskin fibroblast, induced pluripotent stem cells.

Abbreviation

2-ME, 2-Methoxyestradioin