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.

Human amniotic epithelial cells maintain mouse spermatogonial stem cells in an undifferentiated state due to high leukemia inhibitor factor (LIF) expression

“…Mouse embryonic fibroblasts (MEF) cells were isolated from 13-day-old C57BL/6 mouse embryos. Cells were mitotically inactivated using mitomycin C (Sigma, St Louis, MO) as described previously (Akle et al 1981;Lai et al 2009). …”

“…Human amniotic epithelial cells (hAECs) are temporary and specialized fetal cells derived from the placenta that maintained the pluripotency of early epiblast cells (Koizumi et al 2000;Miki et al 2005;Lai et al 2009). hAECs express many growth factors [epidermal growth factor (EGF), bFGF, transforming growth factor α (TGF-α), TGF-β, and bone morphogenic protein 4 (BMP-4),] as well as stem cell markers (Nanog, Oct-4 and Nestin) (Miki et al 2005;Liu et al 2008;Lai et al 2009).…”

“…hAECs express many growth factors [epidermal growth factor (EGF), bFGF, transforming growth factor α (TGF-α), TGF-β, and bone morphogenic protein 4 (BMP-4),] as well as stem cell markers (Nanog, Oct-4 and Nestin) (Miki et al 2005;Liu et al 2008;Lai et al 2009). If the human placental amnion, routinely discarded as medical waste, could be used as an abundant source of feeder cells, it would simplify mouse ESC culture procedures (Lai et al 2009). Although we have discovered that hAECs can be effectively used as feeder cells, very little is known about how they inhibit mouse SSC differentiation (Lai et al 2009).…”

“…If the human placental amnion, routinely discarded as medical waste, could be used as an abundant source of feeder cells, it would simplify mouse ESC culture procedures (Lai et al 2009). Although we have discovered that hAECs can be effectively used as feeder cells, very little is known about how they inhibit mouse SSC differentiation (Lai et al 2009). In this study, we examine how LIF expression participates in hAEC-induced maintenance of mSSCs in an undifferentiated state.…”

Feeder Cell Sources and Feeder-Free Methods for Human iPS Cell Culture

“…Zhang et al [48] reported that human amniotic MSCs can be used as feeder cells for effective growth of human ESCs. Human amniotic epithelial cells (AECs) can be isolated from the surface membrane of fresh placentas, and they express many growth factors including EGF, bFGF, TGF-β, and BMP-4 [49,59,60] in addition to stem cell markers such as Oct-4 and Nanog [50]. Additionally, Liu et al [51] recently showed that microRNA-145-mediated regulation of Sox2 expression in human AECs maintains the self-renewal and pluripotency of human iPSCs.…”