Journal of Bioengineering & Biomedical Science

The nature of floating cells in human embryonic stem cell culture

Abstract

Chen L*, Jin Q, Gong J, Krishna Dasa SS

A consistent presence of floating cells is a common phenomenon in cultures of human embryonic stem cells (hESCs). However, little attention has been paid to their existence. It is currently believed that unavoidable imperfections in culture conditions lead the cells to undergo senescence and apoptosis resulting in unattached cells floating in the culture medium. Inspired by recent studies on mitotic activities in human embryonic stem cell colonies, we believe the existence of floating cells is not simply the result of unfavorable growth conditions but an intrinsic phenomenon resulted from maintaining the pluripotency of hESCs under the culture conditions. We tested this hypothesis with a set of systematic experiments and discovered: 1) the ratio of floating cells to attached cells was significantly increased with culture time; 2) the number of floating cells could be manipulated. For example, we were able to reduce the number of floating cells by providing the colonies with more horizontal or vertical cultural spaces and maintaining the cells’ pluripotency. The results open a new avenue to increase the stem cell culture efficiencies by rescuing the floating cells. On the other hand, by placing a physical barrier on the top of colonies, the number of floating cells was decreased, at the same time, hESCs also showed signs of differentiation. In addition, when inducing cells to differentiate with retinoic acid, the number of floating cells no longer increased with prolonged culture time. Taken together, these results suggested that continuous cell division across the colonies is responsible for the emergence of floating cells during hESC culture. This is quite different from the bacterial colony growth where the cells in the center of colonies are quiescent. Our results indicated that continuous cell division, even at the cost of floating cells formation, is essential for human embryonic stem cell proliferation.