br Results br Discussion In the field of

Results
Discussion In the field of stem cell research, the demand for standardized media is rising, allowing for better reproducibility of results and thereby deepening our knowledge of the pathways regulating their maintenance and differentiation. Here, we set out to determine the minimum requirements for TSCs. Our results indicate that TSCs can be grown without FBS in media supplemented with insulin, transferrin, and low levels of the cytokines FGF4 and TGF-ß1 without losing their stem cell characteristics, self-renewal, and differentiation capability. Further, global gene expression and methylation analyses revealed a high degree of similarity of cells cultured in TX media when compared with standard TSC culture. We believe that TX medium now offers a tool for further studies on molecular pathways that govern cell-fate decisions, because of its well-defined extrinsic stimuli. One example for the importance of defined culture systems for the analysis of stem cell behavior was the bone-morphogenetic-protein-4-driven induction of trophoblast from mouse embryonic stem cells, which is inhibited by serum or leukemia inhibitory factor, usually present in standard ESC culture (Hayashi et al., 2010). A study from 2010 comparing different defined media formulations for hESCs used the criteria attachment, death, differentiated morphology, cell growth, and maintenance of stem cell surface marker expression on cells grown for ten passages in eight different media (International Stem Cell Initiative Consortium et al., 2010). According to this standard, our media formulation passed the test for all of the above-mentioned criteria. We could demonstrate that TX media in combination with Matrigel coating could support attachment, cell growth, and maintenance of stem-cell-marker expression without morphological appearance of differentiation in long-term culture. Nevertheless, we were not able to culture TSCs on a more defined substrate than Matrigel. Although we used a growth-factor-reduced Matrigel formulation, possible influences of Matrigel-derived factors cannot be excluded. Despite this limitation, it is striking that TSCs can cope with the absence of FBS, even after derivation in serum-containing medium and protease inhibitors to standard culture conditions for more than 30 passages (i.e., TS3.5 cells). For some TSC lines, growth rates in TX media were lower when compared to TS media. This is most likely due to a combined effect of decreased proliferation rates and the increase of cell death. A possible explanation might be that, in TX medium, inhibition of the proapoptotic factor BIM by FGF4 alone is not sufficient and that there are other factors present in serum which additionally inhibit this factor (Yang et al., 2006). In this study, our first goal was to validate the analyzed medium and to fully demonstrate the ability to maintain stem cell characteristics in defined medium. We did not try to exclude further factors from the original E8 formulation by Chen et al. (2011), albeit ascorbic acid is one of the supplements that may well be dispensable for TSC culture. Unlike hESCs, it is not required by mouse ESCs (mESCs), probably because of the inability of humans in contrast to mice to synthesize this vitamin (Furue et al., 2008). Our medium formulation can be considered a starting point for the defined culture of murine TSCs. We speculate that this medium might enable the derivation of human TSC lines, because the establishment of defined media for murine and human ESCs facilitated the derivation of ESC lines from additional species, such as rats (Nichols and Smith, 2009). Additionally, Furue et al. (2005, 2008) first established a defined medium for mESCs, which was then adjusted for the use of hESC culture. Interestingly, mESCs grown in serum-containing medium display greater morphological heterogeneity when compared with so-called naive mESCs cultured in 2i, a defined medium that inhibits mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase and glycogen synthase kinase 3 kinases (Marks et al., 2012). Because we also observed a similar effect on the morphology of the three different TSC lines when cultured in TX media, it seems that lack of serum rather than inhibition of specific signaling pathways is responsible for the more-homogenous phenotype of different stem cell lines in defined media. This is also reflected by the fact that the TS-EGFP line cultured in TS medium clusters most distinct from the other two analyzed TSC lines in the microarray expression analysis, whereas after culture in TX media, the sample clusters closer to the other samples.