Establishment and Maintenance of Human Embryonic Stem Cell Lines on Human Feeder Cells Derived from Uterine Endometrium under Serum-Free Condition1

Abstract

Human embryonic stem (hES) cells are usually established and maintained on mouse embryonic fibroblast (MEFs) feeder layers. However, it is desirable to develop human feeder cells because animal feeder cells are associated with risks such as viral infection and/or pathogen transmission. In this study, we attempted to establish new hES cell lines using human uterine endometrial cells (hUECs) to prevent the risks associated with animal feeder cells and for their eventual application in cell-replacement therapy. Inner cell masses (ICMs) of cultured blastocysts were isolated by immunosurgery and then cultured on mitotically inactivated hUEC feeder layers. Cultured ICMs formed colonies by continuous proliferation and were allowed to proliferate continuously for 40, 50, and 55 passages. The established hES cell lines (Miz-hES-14, -15, and -9, respectively) exhibited typical hES cells characteristics, including continuous growth, expression of specific markers, normal karyotypes, and differentiation capacity. The hUEC feeders have the advantage that they can be used for many passages, whereas MEF feeder cells can only be used as feeder cells for a limited number of passages. The hUECs are available to establish and maintain hES cells, and the high expression of embryotrophic factors and extracellular matrices by hUECs may be important to the efficient growth of hES cells. Clinical applications require the establishment and expansion of hES cells under stable xeno-free culture systems.

Efficient culture system for human embryonic stem cells using autologous human embryonic stem cell-derived feeder cells

“…The hESC lines cultured on Diff Miz-hES6 feeder cells were relatively circular in shape (Figure 2), which is similar to their morphology when cultured on MEF feeder cells (data not shown). In contrast, the literature suggests hESCs cultured on other human feeder cells become rather irregular in shape (Richards et al, 2002;Amit et al, 2003a;Cheng et al, 2003;Hovatta et al, 2003;Lee et al, 2005). We could culture the three hESC lines on Diff Miz-hES6 cells for over 50 passages without any detecatable impairment of their self-renewal capacity (data not shown).…”

“…Thus, Diff Miz-hES6 feeder cells support the proliferation of hESCs as efficiently as MEFs. In contrast, the literature suggests hESCs cultured on other human feeder cells require a 5-7 day passaging interval (data not shown) (Richards et al, 2002;Amit et al, 2003a;Cheng et al, 2003;Hovatta et al, 2003;Lee et al, 2005). The hESC lines cultured on Diff Miz-hES6 feeder cells were relatively circular in shape (Figure 2), which is similar to their morphology when cultured on MEF feeder cells (data not shown).…”

“…To avoid the possibility of feeder cell contamination during the preparation of the undifferentiated hESCs and EBs for the RT-PCR analyses described above, we performed RT-PCR using primers specific for a fibroblast marker (prolyl 4-hydroxylase ); fibroblast marker expression was never detected (data not shown). We have found previously that hESCs cultured on MEFs have a circular shape, whereas hESCs cultured on human feeder cells are relatively irregular in shape (Richards et al, 2002;Amit et al, 2003a;Cheng et al, 2003;Hovatta et al, 2003;Lee et al, 2005). The reason for this difference is unclear.…”

“…However, the use of human biopsies from fetal tissues raises ethical issues. The use of donated adult and neonatal tissues would be less ethically problematic but still would not be feasible for the mass production of hESCs; moreover, the use of different donors introduces an unwelcome variability into the culture system (Amit et al, 2003a;Hovatta et al, 2003;Lee et al, 2005). Furthermore, the risk of contamination with pathogens responsible for newly emerging human diseases, which cannot be screened for yet, remains.…”

“…Recently, we and other groups demonstrated that it is possible to culture hESCs on feeder cells that originate from human sources (Richards et al, 2002;Amit et al, 2003a;Cheng et al, 2003;Hovatta Efficient culture system for human embryonic stem cells using autologous human embryonic stem cell-derived feeder cells Lee et al, 2005). In most cases, however, these human feeder cell lines are derived from samples obtained from patients or discarded human fetuses.…”