ES cells were initially established and maintained by coculture with mouse embryonic feeder cells (Evans and Kaufman 1981
; Martin 1981). Subsequent studies identified leukemia inhibitory factor (LIF) as one of the feeder-cell-derived molecules that plays a pivotal role in the maintenance of these cells (Smith et al. 1988; Williams et al. 1988; Stewart et al. 1992). In the presence of appropriate batches of fetal calf serum (FCS), recombinant LIF can replace the feeder cell function and support the growth of undifferentiated ES cells (Smith et al. 1988; Williams et al. 1988). Recently, Ying et al. (2003a) have uncovered a role for BMP4 in ES cell growth and demonstrated that in the presence of LIF, it can replace the requirement for serum. With these new developments, it is now possible to grow ES cells with defined factors in the absence of serum or feeder cells. Molecular analyses have revealed that LIF functions through the gp130 activation of STAT3 (Niwa et al. 1998; Matsuda et al. 1999), whereas the effect of BMP4 on undifferentiated ES cells is mediated by Smad activation and the subsequent induction of the helix–loop–helix Id factors. In addition to STAT3 and Id, two other transcription factors, Oct3/4 (Niwa et al. 2000) and nanog (Chambers et al. 2003; Mitsui et al. 2003), have been shown to play pivotal roles in maintaining the undifferentiated state of ES cells. The role of these transcription factors in ES cell renewal has been recently reviewed (Chambers and Smith 2004) and will not be discussed further here.
The regulation of hES cell growth is less well understood and differs from that of the mouse in that LIF and STAT3 appear to play no role in their self-renewal (Thomson et al. 1998; Reubinoff et al. 2000; Daheron et al. 2004). With current protocols, hES cells can be maintained on feeder cells in serum-free medium supplemented with bFGF (Amit et al. 2000). hES cells can also be grown in the absence of feeder cells, if cultured on matrigel- or laminin-coated plates in medium supplemented with mouse embryonic fibroblast conditioned medium (MEF CM) (Xu et al. 2001). While not as well defined as the conditions for the growth of mouse cells, this protocol does provide for relatively easy maintenance of hES cell populations. Cells grown in these conditions for >100 population doublings retained normal karyotypes and stem cell characteristics, including their in vitro and in vivo pluripotent differentiation potential. Recently, Sato et al. (2004) demonstrated that activation of the canonical Wnt pathway could replace the requirement of MEF CM in the maintenance of undifferentiated hES cells for short periods of time (5–7 d). Whether or not Wnt signaling has an effect on hES cell self-renewal over longer periods through multiple passages remains to be determined. hES cells do express both Oct4 (Ginis et al. 2004) and nanog (Daheron et al. 2004; Richards et al. 2004; Sato et al. 2004), suggesting that this aspect of their regulation may be similar to that observed in mouse ES cells. Future studies will no doubt define specific molecules for the maintenance of hES cells and uncover the molecular mechanisms that regulate their self-renewal.
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