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    • DE cells were recently generated from mouse embryoid bodies

    2018-10-22

    DE cysteine protease were recently generated from mouse embryoid bodies using a combination of ActA (TGFβ activator), Noggin (BMP antagonist), and lithium chloride (Wnt pathway activator) (Li et al., 2011). Whereas ActA and Wnt activators are commonly used for DE induction, Noggin supplementation is justified by the requirement for low BMP signaling to direct the mesendoderm towards anterior primitive streak derivatives (D\'Amour et al., 2005; Sumi et al., 2008; Wang et al., 2012). In the present study, we implemented this protocol in mESC monolayer cultures using different GSK3β inhibitors to activate the Wnt pathway. We show an efficient DE-derivation from these cultures and the generation of Pdx1+Nkx6.1+ pancreatic progenitors following the strategies that we previously developed with hESCs (Mfopou et al., 2010a; Sui et al., 2012). Furthermore, we present data indicating that the mESC-derived DE cells also give rise to hepatocyte-like cells. Therefore, these findings constitute an optimal and rapid model for further screening growth factor and small molecule combinations in view of the differentiation of endoderm progenies such as the pancreatic beta cells.
    Materials and methods
    Results
    Discussion and conclusion In these studies, we primarily addressed the low efficiency of DE differentiation from mESCs when performed on adherent cultures. It was already known from hESC cultures that exposure to ActA and Wnt3a for DE induction is associated with a high degree of cell death during the first two days, however this phenomenon is more pronounced in mESCs where more than 90% cell death can be observed (Mfopou et al., 2010a; Morrison et al., 2008). This explains the preference given to the embryoid body formation prior to DE induction from mESCs or the use of low ActA concentration (Sulzbacher et al., 2009; Tada et al., 2005; Yasunaga et al., 2005). The drawbacks of these two alternatives are the stochastic differentiation within EBs, which is limitative for achieving high efficiency as on monolayer (D\'Amour et al., 2005; Kubo et al., 2004), and the concomitant mesoderm induction at low ActA concentrations. Conversely, the high degree of cell loss on monolayers precludes further study of endoderm differentiation in vitro. Considering the similarities between several developmental features in mouse and human, the easier manipulation of mESC cultures should constitute an advantage for its use as a rapid screening model to identify new combinations of growth factors and small molecules to be further tested or implemented in hESC differentiation, for instance towards the beta-cells. Because the developmental counterparts of hESCs are mouse EpiSCs but not mESCs, and given their very recent discovery, it will be interesting in the future to evaluate their use as a screening model instead. Nevertheless, it is expected that DE generated from EpiSCs would be quite similar to those obtained from mESCs differentiation. Our findings that FGF2, BMP4 or EGF improved cell yield but in a context of increased T and reduced Sox17 expression suggest the mesendoderm phenotype of differentiated cells. When further exposed to the BMP inhibitor Noggin, they progressed towards Foxa2+Sox17+ DE-like cells, which is concordant with the findings that mesendoderm progenitors induced by activating the Wnt pathway require Noggin treatment in order to differentiate into anterior endoderm with features that are much similar to native E8.25 definitive endoderm (Sumi et al., 2008; Wang et al., 2012). This beneficial effect of Noggin prompted us to examine its combination with ActA and a GSK3β inhibitor (Li et al., 2011) on monolayer cultures of mESCs. Interestingly, this resulted in large epithelial sheaths with morphological and molecular characteristics of DE cells. Therefore, activation of TGFβ and Wnt pathways should be combined with BMP antagonism to efficiently generate DE from mESC adherent cultures. These findings therefore represent a significant progress versus the initial protocol with ActA+Wnt3a. Considering the shorter timeline of mouse development, we assume that this study offers a rapid screening model for improving differentiation towards desired cell types.