Use of Human Amnion Epithelial Cells as a Feeder Layer to Support Undifferentiated Growth of Mouse Embryonic Stem Cells

Abstract

Mouse and human embryonic stem (ES) cells are usually maintained in an undifferentiated state by coculture with mouse embryonic fibroblasts (MEFs) as feeder cells. In the case of mouse ES cells, addition of leukemia inhibitory factor (LIF) to culture media is also necessary to prevent cell differentiation. Here, we report the use of primary human amnion epithelial cells (hAECs) as feeder cells to culture mouse ES cells using our newly developed protocols. We found that mouse ES cells grown on hAECs express ES cell markers including FGF, Oct-4, Nanog, Sox-2, Rex, and TERT. Interestingly, the expression levels of these genes are three- to five fold higher on hAECs than on MEFs by quantitative real-time PCR. The quicker growing ES cells on hAECs showed a normal 19XY karyotype on passages 25, and ruled out the transformation of ES cells. Using flow cytometry analysis, we show that ES cells grown on hAECs have the same cell cycle distribution pattern as those on MEFs. Further, mouse ES cells cultured on hAECs for at least 20 passages retain the capability of teratoma formation in mice. Finally, we reveal that hAECs express highly LIF that allows for ES growth without the need of addition of commercially obtained LIF. Taken together, our data suggest that hAECs are suitable for mouse ES cell culture and may prove to be a useful alternative to MEFs for human ES cell culture.