Peer review of: Unique properties of a subset of human pluripotent stem cells with high capacity for self- (2020)



Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

The authors have now addressed most previous criticism. Fig.2 however needs further work. The 
Germline differentiation potential data of hPSC are described in line 168-191 and show increased PGC-
like cell differentiation capacity in the HHH group. However, these data are very hard to extract from 
the Fig.2. Please add the data described in the text to Fig.2 and/or clarify Fig.2. The authors describe 
high degree of inter-assay variability. How much do the repeats in the same assay vary (intra-assay 
variability)? Furthermore, does HHH population have normal Karyotype? 

Reviewer #2 (Remarks to the Author): 

The authors have done a good job of addressing comments made from the previous review. This is 
potentially important work but I would like to see some further characterization of differentiation 
potential for 'archetypical' cells to show that they are really pluripotent. This is critical. 

Specific comments 
1. Can 'archetypical' cells be converted to naive/EPC/primed cells? This would help define where these
cells are with respect to other PSCs.

2. Can 'archetypical' cells be differentiated to the three germ layers? Are they pluripotent and how
does their potential compare to primed cells?
Is it possible that 'archetypical' cells have a reduced capacity for germ layer formation while having
increased germ cell capacity?

3. The authors probably feel boxed in by existing nomenclature (ground-state, naive etc) but perhaps
this would be a good opportunity to suggesting a different nomenclature. I don't really like
'archetypical' as it isn't informative and is an inappropriate use of the word- maybe "founder PSCs";
"fPSCs" would be better terminology?

Reviewer #3 (Remarks to the Author): 

The revised manuscript addresses in part issues raised for the previous submission. The authors have 
further characterised the sub-population of cells within human pluripotent stem cell cultures that have 
higher self-renewal potential. Although several questions remain open, the ATACseq analyses are a 
useful addition that substantiates the conclusion that this sub-population differs in molecular 
properties from the bulk population and may represent an earlier developmental stage. On balance, 
the observations in this study are appropriate for publication due to the fundamental interest and 
importance in describing accurately the status and identity of human pluripotent stem cell cultures. 
The topicality is illustrated by two recent papers that are published in high profile journals (Nakanishi 
et al., Cell; Cornacchia et al., Cell Stem Cell) but are far from definitive and will be well complemented 
by the Lau et al study. However, some results (PGC differentiation, methylome analysis, cell cycle 
analysis) are inconclusive and could be relegated to supplemental with reduced main text description. 
Indeed the manuscript is in general overlong, which rather detracts from the clarity of the authors’ 
main message. 
Suggestions to improve the manuscript: 

Please be explicit at the beginning of the results about the culture medium. I surmise this is mTeSR 
but it is not stated. The methods mention E8 but do they find the same population heterogeneity in E8. 
This issue must be clearly addressed because E8 has become widely adopted and is purported to 
maintain more homogeneous cultures. 
Following on from the above, the authors should compare their findings with the recent paper by 
Cornacchia et al. reporting that hPSCs in E8 have distinct metabolic features and represent an 
intermediate stage of pluripotency. This is particularly pertinent to the metabolomic analyses. 
Please clarify in the text what is meant by GEN. Is this the bulk population or the population after 
sorting ESR cells? 
The PGC cell surface markers used are also expressed by hPSC. The claim of PGC-like identity requires 
demonstration of PGC specific markers such as NANOS3. In any case I do not see what this 
experiment adds to the paper since it is well established that “archetypal” hPSC can be manipulated 
into PGCLC differentiation and the significance of small quantitative differences is unclear when there 
is such inherent variability. 
The methylome analyses are rather superficial and add nothing beyond the statement that there are 
no major differences between ESR and the rest of the population. The claim that there are low levels 
of methylation is not well-supported and the anecdotal comparison with mouse EpiSC is not justified 
by any analysis. 
The cell cycle data presented are potentially misleading because the authors have compared HH cells 
with LL cells, but not with MM cells as in the self-renewal assays. The authors should either repeat the 
experiment with the proper comparison or remove these data. Furthermore, although argued by some, 
it is absolutely not clear that lineage commitment decisions occur in G1. 
The statement that ESR cells “show a pattern of gene expression that is strongly similar to peri-
implantation epiblast stages in the primate embryo, but clearly distinguished from inner cell mass or 
gastrulation stages” should be toned down and phrased more accurately to reflect the post-
implantation (not peri-implantation) stages of primate epiblast examined. 
Please stated clearly in the text the proportion of HH cells in a typical culture and how variable this is. 
The authors must clarify whether LL cells are primed, as the data suggest but they argue against in 
the response letter.