Human Feeder Layers for Human Embryonic Stem Cells1

Abstract

Human embryonic stem (hES) cells hold great promise for future use in various research areas, such as human developmental biology and cell-based therapies. Traditionally, these cells have been cultured on mouse embryonic fibroblast (MEF) feeder layers, which permit continuous growth in an undifferentiated stage. To use these unique cells in human therapy, an animal-free culture system must be used, which will prevent exposure to mouse retroviruses. Animal-free culture systems for hES cells enjoy three major advantages in the basic culture conditions: 1). the ability to grow these cells under serum-free conditions, 2). maintenance of the cells in an undifferentiated state on Matrigel matrix with 100% MEF-conditioned medium, and 3). the use of either human embryonic fibroblasts or adult fallopian tube epithelial cells as feeder layers. In the present study, we describe an additional animal-free culture system for hES cells, based on a feeder layer derived from foreskin and a serum-free medium. In this culture condition, hES cells maintain all embryonic stem cell features (i.e., pluripotency, immortality, unlimited undifferentiated proliferation capability, and maintenance of normal karyotypes) after prolonged culture of 70 passages (>250 doublings). The major advantage of foreskin feeders is their ability to be continuously cultured for more than 42 passages, thus enabling proper analysis for foreign agents, genetic modification such as antibiotic resistance, and reduction of the enormous workload involved in the continuous preparation of new feeder lines.

Derivation and long-term culture of human parthenogenetic embryonic stem cells using human foreskin feeders

“…Li et al indicated that homologous somatic cells from monkey could support the growth of monkey ES cells regardless of the tissue from which the feeder cells were derived [33]. In humans, some studies also indicated that cells from the foreskin [13] or adult Fallopian tube epithelium [14] could support the propagation and self-renewal of hES cells. These cells not only support the growth of ES cells, but also enable the formation of ICM outgrowths and the derivation of ES cells with a similar efficiency to mouse feeder cells.…”

“…The normal characteristics of hES cells can be maintained using a feeder-free culture system, and successful derivation has been reported using this system [11,12], however, this system has not yet been applied widely because of its complexity and associated difficulties. Feeder cells from human tissues, including foreskin fibroblasts [13] and adult Fallopian tube epithelial cells [14], have been used to derive and culture hES cells, and all of these types of feeder cell can support the prolonged undifferentiated growth of hES cells.…”

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

“…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).…”

“…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.…”

Evaluation of a laser technique to isolate the inner cell mass of murine blastocysts

“…On the other hand, regardless of the isolation method used, some groups have come to the general conclusion that cells grown on mouse feeder cells would not be appropriate for clinical applications, and this has lead to significant efforts to grow ESC lines on human feeders [17], immortalized cells [18], feeder cell lines that have been derived from the original ESC themselves [19] and the culture on Matrigel TM [20]. We obtained better adhesion rates when we used the MEF instead of the gelatin growth surface using the laser drill (80 versus 27.3 %).…”