Abstract
Maintenance of pluripotency is crucial to the mammalian embryo's ability to generate the extra-embryonic and embryonic tissues that are needed for intrauterine survival and foetal development. The recent establishment of embryonic stem cells from human blastocysts (hESCs) provides an opportunity to identify the factors supporting pluripotency at early stages of human development. Using this in vitro model, we have recently shown that Nodal can block neuronal differentiation, suggesting that TGFbeta family members are involved in cell fate decisions of hESCs, including preservation of their pluripotency. Here, we report that Activin/Nodal signalling through Smad2/3 activation is necessary to maintain the pluripotent status of hESCs. Inhibition of Activin/Nodal signalling by follistatin and by overexpression of Lefty or Cerberus-Short, or by the Activin receptor inhibitor SB431542, precipitates hESC differentiation. Nevertheless, neither Nodal nor Activin is sufficient to sustain long-term hESC growth in a chemically defined medium without serum. Recent studies have shown that FGF2 can also maintain long-term expression of pluripotency markers, and we find that inhibition of the FGF signalling pathway by the tyrosine kinase inhibitor SU5402 causes hESC differentiation. However, this effect of FGF on hESC pluripotency depends on Activin/Nodal signalling, because it is blocked by SB431542. Finally, long-term maintenance of in-vitro pluripotency can be achieved with a combination of Activin or Nodal plus FGF2 in the absence of feeder-cell layers, conditioned medium or Serum Replacer. These findings suggest that the Activin/Nodal pathway maintains pluripotency through mechanism(s) in which FGF acts as a competence factor and therefore provide further evidence of distinct mechanisms for preservation of pluripotency in mouse and human ESCs.
Highlights
Human embryonic stem cells are pluripotent cells derived from the inner cell mass of human blastocysts (Thomson et al, 1998)
We have recently shown that overexpression of Nodal can block neuroectoderm differentiation of Human embryonic stem cells (hESCs) that had been grown in adherent conditions in chemically defined medium (CDM) during a prolonged period of time (Vallier et al, 2004a)
These results suggest that Nodal could be involved in the maintenance of hESC pluripotency
Summary
Human embryonic stem cells (hESCs) are pluripotent cells derived from the inner cell mass of human blastocysts (Thomson et al, 1998) Their embryonic origin confers upon them unique properties. They can proliferate indefinitely in vitro (Amit et al, 2000) while maintaining their pluripotent status, and they can differentiate into a large number of somatic cell types (Odorico et al, 2001). Their potential use to produce clinically effective somatic cells confers exceptional value on hESCs for regenerative medicine. Several studies suggest that FGF and TGF1 could be potential candidates to regulate these mechanisms, the function and the interaction of these pathways are still largely unexplored (Amit et al, 2000; Amit et al, 2004; Beattie et al, 2005; James et al, 2005; Schuldiner et al, 2000; Wang et al, 2005a; Wang et al, 2005b; Xu et al, 2005a; Xu et al, 2005b)
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