Clinical interest in the use of human embryonic stem (hES) cells has greatly increased since the ex vivo culture conditions for hES and embryonic germ cells were established.1-3 However, clinical application of hES cells is hindered by several of the culture parameters that make ex vivo hES cell maintenance possible. The greatest of these obstacles is the requirement of hES cells to be co-cultured with mouse/human-derived “feeder” cells.1,4 The feeder cells deliver an unknown collection of factors that allow hES cells to preserve their “stemness.” Unfortunately, the presence of these feeder cells poses a real risk of transferring animal/human viruses to hES cells. Therefore, to facilitate the development of clinical applications for hES cells, a feeder-free culture system, which includes the critical factors secreted by feeders or other activators of the same signaling pathways, needs to be identified.
Some of the early work on this problem was performed with mouse embryonic stem cells (mES). Several of these studies show that the presence of LIF and the activation of STAT3 via LIF receptor signaling is sufficient for main-tenance of mES cell pluripotency in the absence of feeders.5-7 However, when these conditions were applied to hES cell cultures, the maintenance effect of feeders proved to be LIF and STAT3 independent.8-10
|Figure 1. Model for the involvement of TGF-ß superfamily member signaling in the maintenance of human embryonic stem cell pluripotency. [Note: figure adapted from Besser, D. (2004) J. Biol. Chem. 279:45076.]|
Further work with hES cells lacking feeders implicated the Wnt pathway, as the addition of either Wnt-3a or a GSK-3ß inhibitor allowed successful hES cell expansion.11 There has been one report of cross-talk between the Wnt pathway and the Activin/Nodal pathway,12 and several, more recent studies show that TGF-ß superfamily signaling through Smads is necessary for the maintenance of pluripotency in hES cells.13-15 In these studies, hES cell differentiation into the neuro-ectodermal default pathway is inhibited by Nodal,13 and Activin A promotes the maintenance of hES cells in an undifferentiated state for more than 20 passages in the absence of feeders.15 High expression of Cripto, a co-receptor for Nodal, by undifferentiated hES cells may also indicate the significance of Nodal in maintaining pluri-potency of hES cells.16 Most recently, Xu et al. reported that the BMP antagonist, Noggin, could synergize with FGF basic to sustain undifferentiated proliferation of hES cells under feeder-free culture conditions.17 Taken together, these observations establish that TGF-ß superfamily member-mediated Smad2/3 activation is a key mechanism in the regulation of hES cell pluripotency (Figure 1). The challenge now will lie in understanding how these diverse signaling pathways interact both temporally and spatially to regulate cell fate specification.
Beyond the development of feeder-free culture conditions for hES cells, there are additional hurdles limiting the clinical application of hES cells. These problems will surely receive similar attention over the next several years. Nevertheless, this new group of studies defines several of the signals important for hES cell culture, reduces the need for feeders, and further facilitates the clinical application of hES cells.