Commitment of Embryonic Stem Cell Differentiation Occurs in 18hrs in the Absence of LIF, But LIF Itself is not Undifferentiated State of Cells

초록

Embryonic stem (ES) cells maintain the undifferentiated state in presence of leukemia inhibitory factor (LIF), and have an ability to differentiate spontaneously into a variety of cell types by removal of LIF in the culture medium. To determine the commitment point of the differentiation, LIF was treated on mouse ES cells at 2, 4, 6, 8, 10, 12, 18, and 24 hrs after seeding, and their colony morphology was examined on the 5th day of culture. The number of differentiated colonies increased abruptly between 18 and 24 hrs after seeding, suggesting that the irreversible differentiation commitment of ES cells occurs in 18 hrs in the absence of LIF. On the other hand, to analyze the effect of LIF concentration on differentiating ES cells, embryoid bodies (EB) formed by a hanging-drop culture of ES cells, were treated with various concentrations of LIF. EBs did not have any differences in their morphology and proliferation rate among experimental groups. However, interestingly, the more concentration of LIF was treated, the less EBs attached on the bottom of culture dishes. Furthermore, expression of differentiation marker genes was delayed, although it could not maintain undifferentiated state even at the highest concentration of LIF. These data suggest that LIF is essential for maintenance of undifferentiated state of ES cells, but unknown factors, including anchorage of cells, are also crucial.