Different responses between diploid and tetraploid H1 embryonic stem cells appeared during long-term culturing in L15F10 medium without leukemia inhibitory factor.

ABSTRACT

To examine the alteration in cellular characteristics of polyploid embryonic stem (ES) cells during long-term culturing without leukemia inhibitory factor (LIF), mouse diploid and tetraploid H-1 (ES) cells (2H1 and 4H1 cells, respectively) were cultured without LIF for approximately 5 months. 2H1 and 4H1 cells were adapted to the medium without LIF by decreasing the concentration for several passages, and they were denoted as 2H1(⁻) and 4H1(⁻) cells, respectively. DNA content of 4H1(⁻) cells decreased gradually in the early stage, increased abruptly in the second stage, and then was maintained for a long time. 4H1(⁻) cells exhibited longer doubling time and equivalent phase fraction compared with those of 2H1(⁻) cells. The G1 phase fractions of 2H1(⁻) and 4H1(⁻) cells were increased compared with that of 2H1 cells. Cellular morphology and pluripotency were maintained in 4H1(⁻) cells but not in 2H1(⁻) cells. 2H1(⁻) cells showed a cell population consisting of several kinds of cells, and they lost alkaline phosphatase activity, suggesting that the cells had differentiated. 4H1(⁻) cells, however, exhibited alkaline phosphatase activity and formed teratocarcinoma in mouse abdomen, suggesting that the cells maintained their pluripotency in the medium without LIF.