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Distinct Developmental Ground States of Epiblast Stem Cell Lines Determine Different Pluripotency Features

From the October Edition of Stem Cells
By Stuart P. Atkinson

Epiblast stem cells (EpiSCs) are derived from postimplantation mouse embryos from embryonic day (E) 5.5 to E7.5 (Brons et al and Tesar et al) and appear to share more characteristics with human embryonic stem cells (hESCs) than with mouse ESCs (mESCs) (Greber et al). EpiSCs are considered to be pluripotent, as they form teratomas upon injection into immunocompromised mice, but do not efficiently colonize host embryos when injected into blastocysts. Hans R. Schöler from the Max Planck Institute for Molecular Biomedicine, Münster, Germany has suggested that a better understanding of EpiSCs may allow a greater understanding of hESCs, and a paper in the October edition of Stem Cells from his group (Bernemann et al) describes the systematic characterisation of a panel of independent EpiSC lines derived in several different laboratories to determine whether there are any functional differences. What they have discovered is that although some basic characteristics are conserved, mesodermal gene expression patterns often associated with the EpiSC state are not consistent and are associated with differentiation potential and the ability of the cells to revert to the mESC-like state.

Of the mouse EpiSCs studied, five lines were derived on E5.5 (OG2.1, OG2.2, E3, E5 and T9), one line (C1a1) was derived on E6.5, and all but one cell line (E3) were female. All were shown to have characteristics of EpiSCs, while being distinct from mESCs, and grew in a way more closely resembling hESCs, but expressed Oct4, Sox2 and Nanog in a similar manner to mESCs. Fgf5, a recognised epiblast marker, was higher in the EpiSCs, while the mESC marker Rex1 was reduced. EpiSCs also exhibited higher activity of the epiblast-specific proximal enhancer of Oct4 in all EpiSC lines and lower activity of the inner cell mass (ICM)-specific distal enhancer compared with mESCs. Global gene expression analysis revealed that all EpiSC lines clustered together and differed from mESCs, while upon injection into SCID mice, all previously uncharacterized EpiSC lines (OG2.1, OG2.2, and E5) gave rise to teratomas that contained tissues of all three germ layers, confirming their pluripotent nature.

Some reports have described the expression of mesendodermal marker genes as a characteristic of EpiSCs (Guo et al, Han et al and Chenoweth et al), such as the primitive streak and mesoderm- associated Brachyury/T gene. Strikingly, immunohistochemical analysis showed that Brachyury was detected in some, but not all, EpiSC lines. C1a1 had high expression in most colonies, OG2.1 and OG2.2 also showed high expression but with fewer positive cells per colony, T9 cell line had very weak expression and E3 and E5 did not exhibit any Brachyury expression at all. mRNA analysis confirmed this, with C1a1, OG2.1 and OG2 exhibiting high levels while T9 showed only slightly higher expression compared with lines E3 and E5. This pattern of expression was also observed for the primitive streak and mesendoderm markers Mixl1, Gsc, Sox17, and Eomes and was also shown to be a stable feature during study across multiple passages. This overall suggests that mesendodermal markers should not be considered a general hallmark of the EpiSC state.

The functional consequence of the mesendodermal gene expression signature in the ground state across different EpiSCs were then studied by analysing their differentiation propensities. Initiation of mesodermal differentiation (FCS stimulation) demonstrated that EpiSCs lines exhibit the potential to differentiate into the mesodermal lineage, regardless of whether the EpiSC lines exhibited a mesendodermal marker gene expression signature in the undifferentiated state. Initiation of neural differentiation (blocking FGF and SMAD signaling), however, demonstrated that those cells expressing the lowest level of mesodermal markers showed the highest induction of neural markers (Sox1 and Pax6), although all cells were able to undergo some level of neuronal differentiation as measured by expression of b-III-Tubulin. As well as having an effect on neuroectodermal differentiation, the mesendodermal gene expression signature was shown to have a detrimental effect on the potential of EpiSCs to revert to an mESC-like cell state using stringent mESC culture conditions (MEK inhibitor PD0325901, GSK3b inhibitor CHIR99021, and LIF). Again, cells with lower levels of mesodermal markers were favoured, with E3, E9 and T9 showing a greater reversion potential that OG2.1, OG2.2 and C1a1. The negative effect of the mesendodermal gene expression signature was further shown by the reduction in the reversion potential of EpiSCs upon the upregulation of Brachyury expression, with E5 showing the most pronounced reduction (75% decrease). However, the reciprocal experiment, RNAi mediated reduction in Brachyury levels, failed to show any significant effects, or could not reduce the mesendodermal marker gene expression to a sufficient level for any effect to be observed.

It can be concluded that while EpiSC lines share certain characteristics regardless of their time of derivation, some features, such as mesendodermal gene expression and reversion efficiency, do differ, which the authors suggest stem from line-specific distinct developmental ground states and analysis such as these could be important for the study of mammalian post-implantation development and in the directed differentiation of EpiSCs.



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