This study demonstrates that culture of hESC in defined media without feeder cells is not a trivial undertaking even for laboratories with significant experience in the field. Apart from the commercial preparations, most of the formulations did not support maintenance of hESC for even the relatively short period of this study. Retesting of the media that failed to support stem cell maintenance by an independent laboratory, using freshly formulated growth factors, indicated that it was unlikely that the outcomes were due to problems specific to the preparations used in the original studies, but rather to a general difficulty in preparing these media. It is notable however that while the independent laboratory had difficulties with media no. 3, their results were substantially better when they used this medium supplied direct by the laboratory of origin, suggesting a critical need for selecting reagent batches and/or subtleties in media preparation.
It is now well established that both FGF and Activin/nodal/TGF-beta signaling are critical for hESC maintenance (Beattie et al. 2005
; James et al. 2005
; Vallier et al. 2005
; Xiao et al. 2006
; Greber et al. 2007
; Xu et al. 2008
). Of the formulations tested, only TeSR, Stem Pro, and no. 3 contain agonists for both pathways: of these, the two commercial media, mTeSR1 and STEM PRO, were the most successful in supporting stem cell growth. These commercial media also contained agonists of other signaling systems, such as GABA receptors and ErbB2, which while less well characterized, are thought to play a role in hESC maintenance.
Considerable effort in development, manufacture, and quality control goes into the formulation and production of commercial media, and it may be that it is difficult to implement equivalent reproducibility in the context of an academic laboratory, particularly when transferring a medium preparation process to many laboratories. This factor might account for the relative success of the commercial preparations observed in our study. On the other hand, there are ample reports of successful long-term propagation of hESC in media supplemented with FGF-2, or other reagents, from a number of different academic laboratories, and the only preparation required of the test laboratories in this study was addition of factors and a few other components to manufactured basal media.
Another explanation for the variability of outcomes observed here compared to published results might lie in the hESC themselves. hESC produce a number of polypeptides that can influence their growth and differentiation. Both FGF and nodal are expressed in hESC cultures (Sperger et al. 2003
; Sato et al. 2003
; Ginis et al. 2004
), as are antagonists of nodal/activin signaling (Brandenberger et al. 2004
), as well as BMPs, which activate SMAD1/5/8 signaling to drive hESC differentiation (Sato et al. 2003
). Differences in production of such factors might reflect subtle differences in methods of passaging (e.g., sizes of cell clumps that are passaged), which are difficult to standardize between laboratories or glean from published details. It is also possible that epigenetic adaptation in vitro leads to modulation of the activity of these pathways in hESC cultures, and that this process varies between different cell lines, or even between the same cell line maintained in different laboratories. In particular genotypic and epigenetic differences between cell lines may give rise to differences in the expression levels of different receptor subtypes such as FGF and TGF-beta receptors and cell attachment modulators such as the Integrin family of receptors. Changes in cell surface receptor expression and cell adhesion modulators between cell lines or indeed at different phases of the hESC culture regrowth may necessitate that different hESC isolates have quite distinct exogenous factor requirements in a given growth media to achieve proper cell adhesion and maintenance of the undifferentiated state. It would be possible to compare the endogenous activity of some of these critical signaling pathways between ES cell lines and sublines and then relate this to dependence upon exogenous factors.
Whatever the role of endogenous autocrine or paracrine-signaling in hESC maintenance, the activation of multiple pathways driving stem cell maintenance and the inhibition of pathways that drive differentiation by combinations of agonists/antagonists with distinct mechanisms of action represent a robust strategy for development of defined culture systems.