Peer review of: The mutational impact of culturing human pluripotent and adult stem cells (2018)

 

 

Reviewers' comments: 
 
Reviewer #1 (Remarks to the Author): 
 
I thank the authors for thoroughly revising the manuscript. My concerns are fully addressed. 
 
 
Reviewer #2 (Remarks to the Author): 
 
In their revised manuscript “The mutational impact of culturing human pluripotent and adult stem cells” 
Kuijk et al. describe the comparative analysis of human induced pluripotent stem cells (iPS cells) and 
adult stem cells (ASCs) from intestine and liver for accumulation of de novo mutations during in vitro 
culture with in vivo mutations by whole genome sequencing. 
 
The major findings include: 
1) iPS cells, intestinal ASCs and liver ASCs have different mutation rates and accumulated 3.5±0.5, 
7.2±1.1 and 8.3±3.6 base substitutions per population doubling, respectively. 
2) The annual in vitro mutation accumulation rate of ASCs is ~40- fold higher than the in vivo rate of 
~40 base pair substitutions per year. 
3) Irrespective of stem cell type, mutational signatures in vitro are distinct from the in vivo mutational 
signature. This in vitro signature is characterized by C to A changes that have previously been linked 
to oxidative stress conditions. 
4) Reducing oxygen tension in pluripotent stem cell cultures lowered the total number of mutations 
and in particular the number of C to A changes. This highlights the need to further optimize stem cell 
culture conditions in particular to reduce culture-associated oxidative stress. 
 
Comments: 
In their revised version of the manuscript, the authors properly addressed the majority of my 
concerns. Importantly, they removed the lack of clarity leading to a manuscript with coherent 
description of the methodology. 
One major new aspect is the reduced mutation rate under low oxygen conditions. Although this effect 
had already been presumed, to my knowledge no other study provided data to prove that. 
Moreover, additional lines have been analyzed, which further improved the data. 
 
There is only one of my comments that has not been properly addressed: 
 
Reviewer comment: 
6) The bulk population after the extended in vitro culture period (used for the 2nd cloning step) should 
also be sequenced and results should be compared to the sequencing results of the early bulk culture 
after 1st cloning and the bulk culture after 2nd cloning. This will allow to discriminate between de 
novo mutations that occured during normal culture and mutations that occur or become selected due 
to the very stressful conditions during single cell cloning. 
Author's response: 
The mutations that occur during single cell cloning will be shared by all the cells in the bulk population. 
These will therefore also be present in the individual cells that are sequenced after the next clonal step. 
Sequencing of a late bulk culture therefore doesn’t have an added value. We chose a lengthy in vitro 
culture period, to minimize the contribution of the mutations induced by the clonal step. 
 
I don’t agree with this statement of the authors. 
Single cell cloning with its stressful conditions is unlikely to be limited to the first cell division. The 
stressful conditions are caused by lack of required cell-cell contacts and lack of autocrine/paracrine 

factors. It is unlikely that the required conditions that reduce cell stress (at least to a large extent) are 
already achieved after the very first cell division. This implicates that a single cell cloning-related 
increased mutation rate can be expected not only during the very first cell division after cell 
dissociation and single cell seeding, and that single cell cloning-related mutations will not be present 
in all cases in the entire cell population but may also be present in a sub-fraction of cells, only. 
 
Subtracting the mutations present in the early bulk culture after the 1st cloning step from the 
mutations present in the early bulk culture after the 2nd cloning step will yield mutations that 
occurred during further expansion of the early bulk culture following the first cloning step (not caused 
by cloning-related stress) and in addition also mutations that occurred during the 2nd cloning step and 
the following cell expansion (potentially caused by cloning-related stress). 
In contrast, subtracting the mutations present in the early bulk culture following the 1st cloning step 
from the mutations present in the late bulk culture prior to the 2nd cloning step will yield only 
mutations that occurred during further expansion of the early bulk culture after the first cloning step 
(not caused by cloning-related stress). 
Of course, sequencing of bulk cultures always suffer from the limitation that only mutations present in 
a subpopulation of cells above the detection limit of the sequencing approach will be detected. On the 
other hand, a cloning step eliminates a majority of mutations that are present in a fraction of the bulk 
culture but not in the cloned cell itself. 
Therefore, I still think that sequencing of the late bulk culture prior to the 2nd cloning step would 
provide important additional information and should enable to identify expansion-related mutations 
that emerged independent of the cloning-related stress, and to define their proportion among the 
entirety of expansion/cloning-related mutations. 
 
Conclusion: 
 
I think that the topic of acquired mutations in stem cells is still a very important topic, which has 
particular relevance for cellular therapies. Clearly, important questions in this field remained 
unanswered yet. 
 
The revised manuscript of Kuijk et al. has clearly been improved and also provides important novel 
data. If also my last comment would be properly addressed, the manuscript should be accepted by 
Nat Communications. 
 
 
 
Reviewer #3 (Remarks to the Author): 
 
The authors addressed the Reviewers’ comments satisfactorily. and the revised manuscript is 
considerably improved. In particular, the analysis of more pluripotent stem cell lines, and the 
experiments in low oxygen are important additions to the manuscript. 
 
One issue that should be addressed prior to acceptance of the manuscript: 
The revised manuscript puts much emphasis on the calculation of the likelihood that stem cell culture 
would result in oncogenic mutations. The authors seem to have switched the database for calling 
‘oncogenic mutations’ (which seems now to be Tamborero et al. Genome Med 2018). However, the 
Methods section does not provide any information on the criteria that were used to call a mutation 
‘oncogenic’ (there is more than one way to do that). An elaboration is required in order to correctly 
interpret the analysis shown in Fig. 4.