Peer review of: IGF1-mediated human embryonic stem cell self-renewal recapitulates the embryonic niche (2020)


Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
Dear Authors, dear editor, 
 
The authors have addressed the most important comments, and clarified and substantiated their 
claims. The detailed single cell transcriptomic analysis is a valuable source of information for the 
community and clearly demonstrate the power of using a rational approach to define the 
conditions necessary for the in vitro maintenance of the pluripotent states. The website is of great 
interest, and complementary to other websites that are being prepared at the moment. However, 
due to possible problems of maintenance of websites, it would be important to ensure that all the 
data necessary to substantiate the author’s claims are also present in the paper itself, including 
the raw (even if previously published) and the processed data. I would like to propose the 
following minor textual changes. 
. The signalling pathway activity being very dynamical, it would be important to mention, in the 
text and in the figures, the embryonic stages that were investigated in the transcriptomic analysis, 
to the best of the authors knowledge. 
. It is difficult to firmly assess signalling pathway activity based on transcriptomic data. It might 
not be reasonable to discard the possibility that MAPK/ERK plays a role in the Epi. I believe that, in 
order to insure the durability of the author’s claims in the future, some sentences should be 
rewritten. For example: 
1. the statement on lines 166-168 “although FGFR signalling was highlighted as enriched in the EPI 
compared to the PE, the enrichment map category was related to the negative regulation of this 
signaling pathway (Supplementary Table 1)” should be modified as the negative regulation of 
FGFR3 and FGFR4 signalling observed in Epi vs. PE can reflect the classical negative feedback 
loops reflecting a pathway activation. 
2. The statement on line 168 should be tempered. “Consistent with this, we did not find FGF-
related receptors specifically highly enriched in the EPI. Boxplot analysis showed FGFR1, FGFR2 
and FGFR4 were more highly expressed in the PE, while transcripts for all FGF ligands, with the 
exception of FGF18, were not expressed above the required 5 RPKM threshold (Supplementary Fig. 
3; https://shiny.crick.ac.uk/embryo_signalling/). The expression level of FGFR1 (~25 RPKM) are 
lower in Epi as compared to PE but probably sufficient to support MAPK/ERK activity. Note that 
these receptors might however not mediate signalling activity at this precise stage, but rather 
prepare the Epi cells for further developmental transitions. The lack of FGF ligands at this specific 
stage as presented in Sup figure 3 along with the functional inhibition using small molecules (ref 
22-23) are the most convincing arguments supporting the lack of FGF-mediated activity. However, 
other FGF ligands are expressed in stages preceding the blastocyst stage and are thus likely to 
play a role (e.g., FGF8, FGF12, FGF13). Finally, the uterine environment might contain FGFs that 
play a role in the Epi. In the future, a timed analysis from morula to late blastocyst stages would 
be necessary to further strengthen these claims. 
. Please mention the downstream MAPK pathway components that are used to support the claim 
on line 172. 
. I find the “E2” in figure 4e unclear. It could be replaced by “Treatment from E2 to E6.5” 
. Line 531: “Altogether, this suggests that rather than operating via an alternative pathway in the 
human embryo, FGF is unlikely to be required for EPI development”. Here, I would modify to 
“Altogether, this suggests that rather than operating via an alternative pathway in the human 
embryo, FGF is unlikely to be crucial for EPI development”. 
 
 
 
 
 
Reviewer #2: 
Remarks to the Author: 

After revision, the response has addressed everything asked to be addressed, and I can happily 
give my recommendation for publication.