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

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
In this manuscript, the authors analyse datasets from single cell transcriptomic of human 
blastocysts to unbiasedly mine the signalling pathways that are active in the Epi compartment. 
Beyond the classical Nodal pathway (that they previously assessed), the authors point at the 
IGF1/IGFR1/Pi3K/AKT pathway. Most intriguingly, this dataset does not pinpoint a role for the 
FGFs, which is largely exploited by current hESC culture protocols. Based on this finding, they 
define new culture conditions (IGF1/Activin/L511) for hESCs that more closely ressemble the in 
vivo situation. This novel culture condition allows for the culture of established lines and for the 
establishment of de novo lines from the blastocyst and via reprogramming. These lines are 
competent for differentiation into hepatocyte-like and cardiomyocyte-like cells. This novel state is 
similar to the primed state of pluripotency both at the transcriptome level and regarding the X 
chromosome inactivation status. The authors then show convincing data showing that the 
PI3K/AKT pathway regulates the pluripotent state both in hESCs and in the Epi of the blastocyst. 
Overall, this study establishes a novel systematic and rational way to investigate the regulation of 
human pluripotency and defines a novel culture condition that recapitulates that state in vitro. It 
also reveals that the cross-talk between the AKT and ERK pathway explains the previous 
misunderstanding and seemingly inappropriate use of FGFs to maintain the human pluripotent 
state in vitro. 
 
Line 131: Please add a reference to the statement ’the interleukin 6 receptor subunit IL6R, which 
can bind insulin-related ligands.’ 
Line 135 and Supplementary figure 1b: 
- Provide the full list of all genes, Log2foldchange, value, padj, basemean, lfcSE, and stat for Epi 
enriched, TE enriched, and PE enriched in a XCel tab, to complement SF1a. 
- Provide the plots for the expression levels of all ligands known to activate the MAPK pathway 
including but not restricted to PDGF, EGF, HB-EGF, FGF ligands in Epi, TE, PE, even if they are 
lowly- or non-expressed. 
- Provide insights into the regulation of downstream proteins relative to GPCR pathways that are 
known to regulate the MAPK pathway (e.g. but not restricted to PKA, PKC). 
Line 136: Beyond the factors secreted by the EPI, the pluripotency might be maintained by niche 
factors produced by the TE and PE. To challenge your finding that IGF1 is an important regulator 
of human pluripotency, the authors should provide the lists and plots of all secreted ligands (a.k.a. 
secretome) and of the corresponding receptors produced by the Epi, TE, and PE. This data would 
be especially relevant to assess the putative role of TGFB, WNT, and STAT pathways. 
Line 136: Because FGFR4 is expressed in the Epi, the authors should use REACTOME or KEGG 
analysis to assess the regulation of classical MAPK/ERK target genes in the Epi as compared to the 
TE and PE. This analysis should be extended to the WNT, TGFB, and STAT pathways that were 
proposed to also regulate human pluripotency. 
Line 156: Integrins and FAK have been associated with MAPK activity (e.g., DOI: 
10.1038/372786a0). The authors should test whether the L511 regulates MAPK activity, for 
example as compared to Matrigel and other laminins previously used for hESC culture. This might 
in part explain the differences in ligands and signalling activity that they observed. Other controls 
including the addition into the culture medium of antibodies blocking or activating integrins might 
be insightful as well. 
Line 180: The authors should add controls to their FACS experiment including hESCs cultured in 
F2/KSR and t2iLGö. 
Line 184: The putative superiority of AI over other culture conditions for clonal derivation should 
be quantified via a CFU assay. 
Line 190: The author should clearly mention in the conclusions that they used multiple lines and 
name them (H1, H9, CH2, Shef6). 
Line 200: The authors should mention the number of cell lines that they derived do novo. Was the 
derivation efficiency similar to FGF-driven derivation? Did the authors see reproducible differences 

in the transcriptome of de novo lines as compared to converted lines? This might point at 
irreversible changes due to a derivation in FGF as compared to IGF1. 
Line 258: Due to the sensitivity of the adjustment of batch effects, it would be important to briefly 
mention the method in the text. 
Line 287: The pair-wise analysis should be also done using Panther and KEGG in order to depict 
the differences in signalling activity. 
Line 293-298: The lower WNT activity in Epi as compared to AI- and TeSR-cultured hESCs is very 
interesting as it contradicts the current paradigm that WNT should be activated in naive hESCs. 
The enrichment for genes associated with IL6 activity in both Epi and AI-hESCs is very interesting 
as well. It is thus tempting to ask the authors to perform longer-term culture of hESCs in AI 
supplemented with WNT inhibitors (e.g., IWP2, XAV939), PD17/PD03 (in relation to line 371 and 
382, and possibly at concentration 0.5 uM for PD03, in light of doi:10.1038/s41592-018-0104-1) 
and in the presence of IL6. 
Line 351: The authors should check whether the elements of the mTOR pathway are differentially 
regulated in the Epi as compared to the TE and PE. 
Line 428: The authors should use their refined WB technique to check the levels of pERK in human 
blastocysts as compared to mouse blastocysts (in which the pathway activity is well established) 
and to hESCs in AI-, KSR-, and t2iLGö-culture conditions. The hESCs samples can be dosed from 1 
to 100 cells for example, in order to give an idea of the respective levels. 
 
As a summary, in order to make this paper a landmark in hESC culture by unbiasedly assessing 
signalling pathways regulating human pluripotency, I would suggest that the authors: 
- perform additional computational analysis of the single cell sequencing data to: 
(i) provide the full list of all genes, Log2foldchange, value, padj, basemean, lfcSE, and stat for Epi 
enriched, TE enriched, and PE enriched, 
(ii) provide the plots for the expression levels of all ligands activating the MAPK pathway including 
but not restricted to PDGF, EGF, HB-EGF, FGF ligands in Epi, TE, PE, 
(iii) provide insights into the downstream proteins of GPCR pathways that are also known to 
regulate the MAPK pathway (e.g., PKA, PKC) 
(iv) provide the list and plots of all secreted ligands and of the corresponding receptors produced 
by the Epi, TE, and PE, especially for the TGFB, WNT, and STAT pathways 
(v) provide panther and KEGG analysis of the AI- as compared to F2/KSR- and t2iLGö-cultured 
hESCs 
- perform an additional experiment to weight the induction of MAPK activity via integrins 
- add controls to the FACS experiment (Fig 1c) to quantify and compare the levels of pluripotency-
related genes 
- test the clonal efficiency of AI- as compared to F2/KSR- and t2iLGö-cultured hESCs 
- attempt the long-term culture and characterise AI-hESCs in the presence of WNT inhibitors 
(IWP2, XAV939), ERK inhibitors (PD17, PD03) and IL6 [modified AI culture] 
- verify the levels of pERK in human blastocysts as compared to mouse blastocysts and to multiple 
doses of hESCs in AI-, modified AI-, F2/KSR- and t2iLGö-cultured hESCs. 
 
 
 
 
Reviewer #2: 
Remarks to the Author: 
This manuscript shows that FGF2 is dispensable in the culture of human embryonic stem cells 
(hESCs) when replaced with IGF1 and insulin. This conclusion is based on a screen from prior 
single cell RNA seq data of a day 6 embryo and based on their work culturing hESCs in IGF1-
containing media. I think their results are interesting and will be valuable to the field. My main 
concern, however, is that this result is not very novel. They show that cells in AI medium have a 
highly similar transcriptomics profile to hESCs conventionally grown in mTesr (with the exception 
of morphology). At the authors’ own admission, “IGF1 and FGF2 may activate overlapping 
downstream mechanisms for the maintenance of hESCs”. Cells cultured in these two media also 

respond similarly to differentiation cues. Based on that, it is unlikely that the altered culture 
conditions will change our current hESC media practices and the value of the paper is in linking the 
IGF pathway to pluripotency. 
 
However, this point is not novel either. The authors missed previous references that have already 
shown that PI3K/AKT signaling is active in hESCs, that it is necessary for pluripotency 
maintenance and that this signaling is activated by insulin/IGF (McLean et al. 2007; Singh et al. 
2012). Furthermore, FGF-free media were also developed and used to maintain pluripotency. It 
included heregulin in addition to IGF and Activin (Singh et al. 2012). Cross-talk between PI3K and 
MAPK pathways in hESCs has also been previously demonstrated at the level of pAKT and pERK 
(Singh et al. 2012). The authors should discuss and cite this prior work. Also given the findings of 
these papers, I think all instances of the word "first" should be removed from the current paper. 
 
Using the human embryo to optimize the culture conditions for hESCs is a novel approach with a 
strong rationale. However, the fact that AI culture conditions maintain pluripotency is not 
surprising given the above-mentioned work. The main new insight that the authors add is to 
demonstrate that the FGF receptor can be inhibited without consequences to pluripotency, thus 
providing clear evidence that FGF signaling is dispensable for hESC maintenance and that the main 
function of FGF in conventional media is to activate PI3K signaling. 
 
Additionally, the authors should consider comparing transciptome data for AI conditions to other 
media conditions in which cells are also grown on laminin-511, so as to highlight any differences 
between using FGF and IGF rather than differences resulting from the extra-cellular matrix. This 
would strengthen the paper and the impact of its findings. If this comparison was already made 
then the authors should discuss it in more detail (e.g. AI vs mTeSR1 on Laminin-511). 
 
It is crucial to discuss these points and revise the paper accordingly before further consideration. 
In all, while I think the work is solid, my concern is that not very much about the work (culture 
conditions or the mechanism itself) is very novel and it is up to the editor to decide whether it is 
suitable for Nature Communications. 
 
 
 
Missed references: 
McLean, Amanda B, Kevin A D'Amour, Karen L Jones, Malini Krishnamoorthy, Michael J Kulik, 
David M Reynolds, Alan M Sheppard, et al. 2007. “Activin a Efficiently Specifies Definitive 
Endoderm From Human Embryonic Stem Cells Only When Phosphatidylinositol 3-Kinase Signaling 
Is Suppressed.” 25 (1). John Wiley & Sons, Ltd.: 29–38. doi:10.1634/stemcells.2006-0219. 
 
Singh, Amar M, David Reynolds, Timothy Cliff, Satoshi Ohtsuka, Alexa L Mattheyses, Yuhua Sun, 
Laura Menendez, Michael Kulik, and Stephen Dalton. 2012. “Signaling Network Crosstalk in Human 
Pluripotent Cells: a Smad2/3-Regulated Switch That Controls the Balance Between Self-Renewal 
and Differentiation..” Cell Stem Cell 10 (3): 312–26. doi:10.1016/j.stem.2012.01.014. 

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