On 2015 Jun 06, A Martinez-Arias commented:

This manuscript claims the discovery of a new kind of Embryonic Stem (ES) cell, so called ‘region specific Epiblast stem cells (rsEpiSC). A close look at the protocols, genetics and cell behaviours indicates that what is reported is likely to be an improved method for the maintenance of EpiSCs and the closely related human ES cells; it would be misleading to describe a more stable population as a new entity.

The main finding reported here is that inhibition of Wnt signalling in cultures of EpiSCs, and also human ES cells, leads to a stable pluripotent population. As briefly indicated below, this has been reported a number of times before, as has the ability of EpiSCs to integrate in postimplantation embryos, the other finding presented here as new. The observation that stabilized human ES cells can undergo a similar integration is, however, novel, though the degree to which this happens and its value will await further experiments.

It is well known that ES and EpiS cells are, for the most part, heterogeneous populations in dynamic equilibria. In the case of ES cells this can be biased towards a stable state, called ‘ground state’, by application of two inhibitors, one for MEK and another for GSK3 [1]. It is also well known that in contrast with ES cells, when EpiSCs and human ES cells are presented with high levels of Wnt signalling they differentiate [2, 3] and over the last few years a number of reports have shown that inhibition of Wnt secretion or Wnt/ß-catenin signalling increases the self renewal of EpiSCs populations [4-6]. Thus a cocktail of Activin, FGF2 together with Wnt/ß-catenin inhibitors leads to efficient derivation of EpiSCs as well as their stable culture. This is confirmed in this report without acknowledging the earlier studies –interestingly some of these studies are referred to but only as discussing Wnt signalling in pluripotency. Having repeated this observation, the authors now notice that removal of Activin from the cocktail –but not inhibition of Activin signalling, which is not tested- improved the stability of the culture; this tweak is novel and mechanistically intriguing, though it is not pursued further. Thus the main message of the work is that EpiSCs grown or derived in the presence of FGF2 and Wnt inhibitors are stable (this is no new kind of ES cell).

With regard to the ability of the cells to integrate in the posterior part of postimplantation embryos, there are also precedents showing that EpiSCs, which indeed are not able to integrate in preimplantation embryos under normal conditions, can and will integrate in postimplantation embryos [7, 8]. The point made here that ‘stable EpiSCs integrate preferentially in posterior regions of the embryo is not surprising as integration is likely to be easier in the region undergoing gastrulation, which is ongoing in the posterior region at the time of the injection. Furthermore, the conclusion that ‘rsEpiSCs’ are related to the posterior proximal epiblast of stage E6.5 is similar to that obtained by Kojima et al. that although it is possible to obtain EpiSCs from a range of stages during gastrulation, EpiSC lines tend to correspond to anterior primitive streak i.e. posterior proximal E6.5 [8]. The results presented here are compatible with the observation that EpiSCs derived from embryos at different stages of gastrulation can be different [8] i.e there might be many rsEpiSCs, which is probably not a helpful notion.

One of the arguments used by the authors to claim the identification of a new type of stem cell is their transcriptional profile. However, EpiSC lines derived from different stages around gastrulation have different properties and transcriptional profiled and much of these differences are likely to be down to signalling [8]. Thus it is not that surprising that a population of EpiSCs severely deprived of Wnt and Activin signalling exhibits a special transcriptional profile and maps, in a PCA plot, away from other cells in different states and notably from EpiSCs grown in Activin and FGF2, a very different cocktail. It is likely that under appropriate experimental conditions, rsEpiSCs will be shown to correspond to one of the EpiSCs derived by Kojima et al. [8], though the changes and adaptations associated with growth in specific signalling environments would make the comparison challenging.

In summary, this report is a protocol to obtain a state which is to EpiSCs what the ground state (2i) is to the ES cells. This is certainly useful but not enough to talk about a new kind of ES cells. Of course, this is an opinion. Nevertheless, the work provides further support to the importance of Wnt signalling in the control of the dynamics of stem cell populations.

References [1] Ying QL, Wray J, Nichols J, Batlle-Morera L, Doble B, Woodgett J, et al. The ground state of embryonic stem cell self-renewal. Nature 2008;453:519-23. [2] Singh AM, Reynolds D, Cliff T, Ohtsuka S, Mattheyses AL, Sun Y, et al. Signaling network crosstalk in human pluripotent cells: a Smad2/3-regulated switch that controls the balance between self-renewal and differentiation. Cell Stem Cell 2012;10:312-26. [3] Davidson KC, Adams AM, Goodson JM, McDonald CE, Potter JC, Berndt JD, et al. Wnt/beta-catenin signaling promotes differentiation, not self-renewal, of human embryonic stem cells and is repressed by Oct4. Proc Natl Acad Sci U S A 2012;109:4485-90. [4] Kurek D, Neagu A, Tastemel M, Tuysuz N, Lehmann J, van de Werken HJ, et al. Endogenous WNT signals mediate BMP-induced and spontaneous differentiation of epiblast stem cells and human embryonic stem cells. Stem cell reports 2015;4:114-28. [5] Kim H, Wu J, Ye S, Tai CI, Zhou X, Yan H, et al. Modulation of beta-catenin function maintains mouse epiblast stem cell and human embryonic stem cell self-renewal. Nat Commun 2013;4:2403. [6] Sumi T, Oki S, Kitajima K, Meno C. Epiblast ground state is controlled by canonical Wnt/beta-catenin signaling in the postimplantation mouse embryo and epiblast stem cells. PLoS One 2013;8:e63378. [7] Huang Y, Osorno R, Tsakiridis A, Wilson V. In Vivo differentiation potential of epiblast stem cells revealed by chimeric embryo formation. Cell Rep 2012;2:1571-8. [8] Kojima Y, Kaufman-Francis K, Studdert JB, Steiner KA, Power MD, Loebel DA, et al. The transcriptional and functional properties of mouse epiblast stem cells resemble the anterior primitive streak. Cell Stem Cell 2014;14:107-20.

On 2015 Jun 06, A Martinez-Arias commented:

This manuscript claims the discovery of a new kind of Embryonic Stem (ES) cell, so called ‘region specific Epiblast stem cells (rsEpiSC). A close look at the protocols, genetics and cell behaviours indicates that what is reported is likely to be an improved method for the maintenance of EpiSCs and the closely related human ES cells; it would be misleading to describe a more stable population as a new entity.

The main finding reported here is that inhibition of Wnt signalling in cultures of EpiSCs, and also human ES cells, leads to a stable pluripotent population. As briefly indicated below, this has been reported a number of times before, as has the ability of EpiSCs to integrate in postimplantation embryos, the other finding presented here as new. The observation that stabilized human ES cells can undergo a similar integration is, however, novel, though the degree to which this happens and its value will await further experiments.

It is well known that ES and EpiS cells are, for the most part, heterogeneous populations in dynamic equilibria. In the case of ES cells this can be biased towards a stable state, called ‘ground state’, by application of two inhibitors, one for MEK and another for GSK3 [1]. It is also well known that in contrast with ES cells, when EpiSCs and human ES cells are presented with high levels of Wnt signalling they differentiate [2, 3] and over the last few years a number of reports have shown that inhibition of Wnt secretion or Wnt/ß-catenin signalling increases the self renewal of EpiSCs populations [4-6]. Thus a cocktail of Activin, FGF2 together with Wnt/ß-catenin inhibitors leads to efficient derivation of EpiSCs as well as their stable culture. This is confirmed in this report without acknowledging the earlier studies –interestingly some of these studies are referred to but only as discussing Wnt signalling in pluripotency. Having repeated this observation, the authors now notice that removal of Activin from the cocktail –but not inhibition of Activin signalling, which is not tested- improved the stability of the culture; this tweak is novel and mechanistically intriguing, though it is not pursued further. Thus the main message of the work is that EpiSCs grown or derived in the presence of FGF2 and Wnt inhibitors are stable (this is no new kind of ES cell).

With regard to the ability of the cells to integrate in the posterior part of postimplantation embryos, there are also precedents showing that EpiSCs, which indeed are not able to integrate in preimplantation embryos under normal conditions, can and will integrate in postimplantation embryos [7, 8]. The point made here that ‘stable EpiSCs integrate preferentially in posterior regions of the embryo is not surprising as integration is likely to be easier in the region undergoing gastrulation, which is ongoing in the posterior region at the time of the injection. Furthermore, the conclusion that ‘rsEpiSCs’ are related to the posterior proximal epiblast of stage E6.5 is similar to that obtained by Kojima et al. that although it is possible to obtain EpiSCs from a range of stages during gastrulation, EpiSC lines tend to correspond to anterior primitive streak i.e. posterior proximal E6.5 [8]. The results presented here are compatible with the observation that EpiSCs derived from embryos at different stages of gastrulation can be different [8] i.e there might be many rsEpiSCs, which is probably not a helpful notion.

One of the arguments used by the authors to claim the identification of a new type of stem cell is their transcriptional profile. However, EpiSC lines derived from different stages around gastrulation have different properties and transcriptional profiled and much of these differences are likely to be down to signalling [8]. Thus it is not that surprising that a population of EpiSCs severely deprived of Wnt and Activin signalling exhibits a special transcriptional profile and maps, in a PCA plot, away from other cells in different states and notably from EpiSCs grown in Activin and FGF2, a very different cocktail. It is likely that under appropriate experimental conditions, rsEpiSCs will be shown to correspond to one of the EpiSCs derived by Kojima et al. [8], though the changes and adaptations associated with growth in specific signalling environments would make the comparison challenging.

In summary, this report is a protocol to obtain a state which is to EpiSCs what the ground state (2i) is to the ES cells. This is certainly useful but not enough to talk about a new kind of ES cells. Of course, this is an opinion. Nevertheless, the work provides further support to the importance of Wnt signalling in the control of the dynamics of stem cell populations.

References [1] Ying QL, Wray J, Nichols J, Batlle-Morera L, Doble B, Woodgett J, et al. The ground state of embryonic stem cell self-renewal. Nature 2008;453:519-23. [2] Singh AM, Reynolds D, Cliff T, Ohtsuka S, Mattheyses AL, Sun Y, et al. Signaling network crosstalk in human pluripotent cells: a Smad2/3-regulated switch that controls the balance between self-renewal and differentiation. Cell Stem Cell 2012;10:312-26. [3] Davidson KC, Adams AM, Goodson JM, McDonald CE, Potter JC, Berndt JD, et al. Wnt/beta-catenin signaling promotes differentiation, not self-renewal, of human embryonic stem cells and is repressed by Oct4. Proc Natl Acad Sci U S A 2012;109:4485-90. [4] Kurek D, Neagu A, Tastemel M, Tuysuz N, Lehmann J, van de Werken HJ, et al. Endogenous WNT signals mediate BMP-induced and spontaneous differentiation of epiblast stem cells and human embryonic stem cells. Stem cell reports 2015;4:114-28. [5] Kim H, Wu J, Ye S, Tai CI, Zhou X, Yan H, et al. Modulation of beta-catenin function maintains mouse epiblast stem cell and human embryonic stem cell self-renewal. Nat Commun 2013;4:2403. [6] Sumi T, Oki S, Kitajima K, Meno C. Epiblast ground state is controlled by canonical Wnt/beta-catenin signaling in the postimplantation mouse embryo and epiblast stem cells. PLoS One 2013;8:e63378. [7] Huang Y, Osorno R, Tsakiridis A, Wilson V. In Vivo differentiation potential of epiblast stem cells revealed by chimeric embryo formation. Cell Rep 2012;2:1571-8. [8] Kojima Y, Kaufman-Francis K, Studdert JB, Steiner KA, Power MD, Loebel DA, et al. The transcriptional and functional properties of mouse epiblast stem cells resemble the anterior primitive streak. Cell Stem Cell 2014;14:107-20.