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

Reviewers' comments: 
 
Reviewer #1 (Remarks to the Author): 
 
Kuijk et al. quantified and characterized genetic aberrations in human iPSCs and adult stem cells 
during in vitro cell culture. They further compared in vitro and in vivo mutation data, revealing several 
novel and important insights. Detailed analysis of genetic aberration in cultured iPSCs in direct 
comparison with other stem cells is an important step towards the medical application of these stem 
cells. The manuscript is well written, and the topic is also timely. However, the authors need to 
consider the following major remarks. 
 
Major points 
#1. My major concern in this study is that the authors only use one iPS cell line which they 
established. The authors conclude that mutation frequency in iPSCs is about half of those in ASCs 
(without even “biological” replicates for the 1st clonal step). One of the major issues of iPSCs is the 
genetic and epigenetic variations as reviewed by Liang and Zhang in Cell Stem Cells (2013) doi: 
[10.1016/j.stem.2013.07.001]. Although the similarities between their iPSCs and H9 hESCs are 
presented in Supplemental figure 6, the reviewer think it is important to include several iPS cell lines 
(and optionally human ESCs), and to compute solid mutation rate of iPSCs. Awing to the sharp drop of 
sequencing cost in recent years, some more experiments would not be cost prohibitive. 
 
#2. The authors show that in vitro mutation signature is characterized by C to A changes, which are 
linked to oxidative stress conditions. As the authors discuss, lowering the levels of ROS in culture may 
be a way to reduce the number of mutations. It would be ideal to provide experimental data to 
support this for the readers of this journal. 
 
Minor points 
#1. Color code in Figure 1d and 1f. The color code for observed and expected seems only valid for iPS 
but not for SI and Liver. The abbreviation for SI (spelled S.I. in Figure 2) can also be shown in the 
figure legend. 
 
 
 
Reviewer #2 (Remarks to the Author): 
 
In their study “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±1.0 and 8.4±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) Stem cell-specific mutational signatures and differences in transcriptional strand bias were 
observed, indicating differential activity of DNA repair mechanisms between stem cell types in culture. 
 

Also, the authors claim that their approach, in contrast to previous studies, is able to discriminate 
mutations that originate in culture from those that have an in vivo origin or arise as a result of the 
derivation process (see page 5, discussion). 
 
In general, the bioinformatics filtering and analyses appear appropriate, however the paper is difficult 
to read as comprehensive explanations and definitions are missing. 
 
General questions / comments on the methodology: 
 
Suppl Figure 1 is important to understand the study and should become main Fig. 1. However, figure 
and corresponding legend are not comprehensive enough and should be more detailed, or it should at 
least be referred to sections of the manuscript where relevant details are provided. 
 
In particular following aspects that are important to judge the experimental design and results do not 
become clear in the paper: 
 
1) As I understood, genomic DNA from urothel bulk culture of the corresponding donor in case prior to 
reprogramming was used as reference sample in case of iPSCs, correct? How many population 
doublings did happen in the primary culture of urinary epithelium between isolation until 
reprogramming and picking of single cell iPSC clones? This has to be described more clearly. 
2) In case of liver and intestinal stem cell lines, blood of the corresponding donor was used as 
reference sample, correct? It would be better to use an intestinal or liver biopsy as reference sample 
(with the disadvantage that also other cell types than stem cells are included) or alternatively the bulk 
population that was used for the 1st cloning step as reference! 
3) If blood has been used as reference sample, it has to be considered that somatic mutations in the 
sampled white blood cells may be very different than in the cell population utilized for isolation of 
intestinal stem cells and hepatic stem cells. Besides germ line mutations there are somatic mutations 
in individual cell clones that undergo a positive or negative selection pressure dependent on the 
affected cell lineage. Therefore it may be that certain mutations are rare (and undetectable by direct 
WGS) in blood cells, but become enriched in intestinal or livers stem cells. 
4) The authors should analyse and present a list of mutations that were not detectable in the parental 
cells but were detectable in the first clonal culture (representing de novo mutations during 
reprogramming and/or initial culture phase, or rare mutations inherited by the analysed clones from 
their parental cells). 
5) How many population doublings occur for the different cell types between first single cell cloning 
until sequencing of the clonal bulk culture? How man population doublings occur until the second 
cloning step for each cell type (53-146 days)? This should be included in supplementary table 1. 
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. 
7) In the Material and Methods section it is mentioned that “Welch Two Sample t-tests were 
performed to determine whether the mutation rates differ significantly between the stem cell types”. 
To my knowledge, more samples (in particular more than 3 iPSC samples) are required to use a T-test 
for analysis. In the results section, only the ANOVA test is mentioned. Which test has finally been used? 
The authors should comment on the choice and applicability of the applied tests. 
8) Number of three analysed iPSC subclones from only one donor may be not sufficient to be 
representative. At least three additional iPSC subclones from another primary clone should be included. 
Are the four liver and the six intestinal subclones each derived from one primary clone? 
 

Results: 
Page 3: “We found a population doubling time of ~23h for human iPS cells which is approximately 
twice as fast as for human liver and intestinal ASCs, that have population doubling times of ~46h and 
~44h, respectively (figure 1a).”. Was the level of cell death determined and taken into account for this 
calculation? 
 
Page 3: “Base pair substitutions were significantly depleted in genic regions for all three stem cell 
types (figure 1d).” What means „depleted“? Less than statistically expected? What is the basis for this 
calculation? 
 
Page 3: “None of the non-synonymous mutations affected known cancer genes based on the census of 
human cancer genes21 or have previously been described to confer a selective advantage over stem 
cells in culture 6,14. We are therefore confident that the observed mutations provide an unbiased 
insight into mutation accumulation free of selection.” Lack of description as cancer gene does not 
necessarily mean that there is no growth advantage for a specific mutation. Also there are not many 
small scale mutations described so far that provide a selective advantage during culture. Therefore, it 
cannot be concluded that there is an “unbiased insight into mutation accumulation free of selection”. 
In addition, it may well be that a certain mutation does not provide any selection during normal 
culture but during single cell cloning, for instance mutations in apoptosis-related genes. 
 
Discussion section: 
The authors claim that “their approach, in contrast to previous studies, is able to discriminate 
mutations that originate in culture from those that have an in vivo origin or arise as a result of the 
derivation process” (see page 5, discussion). 
 
If the WGS data of the clonal cultures are compared with corresponding nonclonal reference sample 
(bulk of urothelial cells that were parental cells of the analysed iPSCs, and corresponding donor blood 
in case of ASCs) it is of course possible to exclude germline variants and variants that are present in 
the reference sample at relatively high levels (higher than detection limit of the direct WGS, which 
depends on the error rate of the applied Illumina sequencing approach (sequencing-by-synthesis). 
What is the detection level? 10%? Or even higher? 
 
Importantly, direct WGS excludes most mutations that have been acquired over a lifetime after the 
first cell doublings in the early embryo and that did not became enriched in urothel cells or blood cells 
during life. 
 
It has to be considered that the parental cell population, which has been used for reprogramming of 
iPSCs or isolation of ASCs, represents a genetic mosaic. Probably every cell has a unique signature of 
mutations that had been acquired over lifetime. Therefore, both, mutations that are too rare in the 
reference cell population but are present in the specific cell clone, and mutations that occur during the 
first culture period and the first cloning step, will be detectable in the bulk culture after the first 
cloning step. Therefore, it is not always possible to discriminate between mutations that originate in 
culture and those those that have an in vivo origin or arise as a result of the derivation process 
 
 
Materials and Methods section: 
Page 9: Sequencing and Bioinformatics: How are “callable regions” specifically defined? Which minimal 
coverage? 
 
Page 9: “To identify all SNVs of the clonal cultures we used our data analysis pipeline that enables the 
identification of somatic SNVs with a confirmation rate of ~91% 18.” The authors should provide more 

information. Why 91%? 
 
Page 9: “The SNV call set is further filtered on the basis of several quality parameters and dbSNP 
v137.b37 40.” Also here more information is required! 
 
Page 9: “Welch Two Sample t-tests were performed to determine whether the mutation rates differ 
significantly between the stem cell types.” I guess T-test is not appropriate if only 3 or 4 samples are 
available. 
 
Page 9: “We subsequently excluded indels that overlap with a SNV, indels that have a dbSNP ID and 
no COSMIC ID, and indels that are present on a blacklist of 3 unrelated samples (BED file available 
upon request).” What is this black list? 
 
Page 10: CNVs were called using Control-FREEC. What is the minimal coverage for those analyses? 
What fraction of the total genome matched this minimal coverage? 
 
 
Figure & Legends: 
Legends should always be placed below the corresponding figure, otherwise it is confusing. 
 
Legend Figur 1: Should termed “Single Nucleotide Variants” instead of “mutations”; 1e) more different 
colours should be used for “Coding” and “non-synonymous”; it would be helpful for many readers to 
have a definition of (non)synonymous included in the legend f) How is promoter-flanking defined? 
Everything that is not in promoters? Certain distance around promoters? 
 
 
Conclusion: 
I think that the topic of acquired mutations in iPSCs and ASCs is still a very important topic, which has 
particular relevance for cellular therapies. Clearly, many important questions in this field remained 
unanswered yet. 
 
The study of Kuijk et al. provides novel data, but suffers from methodological limitations and also 
partially by conclusions that cannot be drawn from the data. For publication in Nat Communications, 
definitely major revisions would be required. 
 
 
 
 
Reviewer #3 (Remarks to the Author): 
 
In this manuscript, Kuijk et al. analyze the burden and spectrum of mutations that arise during the in 
vitro culture of human pluripotent and adult stem cells. By applying an elegant study design that 
includes two clonal steps, the authors were able to capture and analyze the mutations that arose 
during a defined period of time (and a defined number of cell divisions) in subclones of three stem cell 
types: iPSCs, liver ASCs and intestinal ASCs. Their main findings are that the in vitro mutation rates 
for ASCs are ~40-fold higher than those reported in these cells in vivo; that functionally-important 
genomic regions are less commonly altered in all three stem cells types (as expected); and that the in 
vitro mutational processes are different from the in vivo mutational processes, and are characterized 
by C to A changes that have been previously linked to oxidative stress. 
 
This is an interesting study that takes an important step towards understanding the mutational 

processes that affect cultured stem cells. Such understanding is necessary to improve stem cell 
culture practices and ultimately enable safer use of these cells in regenerative medicine. Overall, the 
experiments and analyses are well-designed and carefully-performed. However, it is not clear how 
generalizable the findings of this study really are. A major limitation is that a single parental 
population was used to derive a few subclones of each stem cell type, so that eventually comparisons 
were performed across clonal populations that originated from a single iPSC line, a single liver ASC 
line and a single intestinal ASC line (i.e., n=1 per SC type). To what extent these cell lines represent 
their stem cell “type” is not clear, and cannot be addressed without further experiments. It would be 
much more informative to compare clonal cultures derived from independent parental populations 
(taken from different individuals) in order to be able to assess the variability that exists within each 
cell type. 
 
The manuscript could also benefit from some additional elaborations and clarifications, as described 
below: 
 
1. The authors argue that their findings “demonstrate that in vivo mutational processes only play 
minor roles in in vitro mutation accumulation”. This is surprising and interesting, and the manuscript 
clearly provides evidence for in vitro mutational processes that are different from the in vivo ones. But 
if the role of the in vivo mutational processes is only minimal, why does hierarchical clustering based 
on the relative contribution of COSMIC signatures (Fig. 2c) show the in vitro ASCs to cluster together 
with the in vivo liver ASCs and apart from the other in vitro groups? Can this be better clarified? 
 
2. The authors state that “none of the non-synonymous mutations affected known cancer genes based 
on the census of human cancer genes, or have previously been described to confer a selective 
advantage over stem cells in culture. We are therefore confident that the observed mutations provide 
an unbiased insight into mutation accumulation free of selection.” I do not believe that we can be so 
confident that selection is not involved. First, the list of genes involved in cancer that was used for the 
comparison (ref 21) is a census of amplified and overexpressed human cancer genes, and is thus 
partial by definition (it does not cover tumor suppressors, for example). Second, the selection 
pressures in culture are likely to be at least somewhat different from those experienced in vivo, and 
somewhat tissue-type specific; however, the list of genes selected for in stem cell cultures, which was 
used for comparison here, is limited to pluripotent stem cells (refs 6 and 14). Can the authors 
compare their mutated gene list to additional/extended gene lists? This statement should also be 
toned down 
 
3. What specific comparisons were performed in Fig. 1b,c? The legend says ANOVA, but multiple p-
values appear (Fig. 1b) and the p-values are presented above specific bars (Fig. 1b,c). Are these p-
values from contrast analyses? Which contrasts do the shown asterisks represent? This should be 
clarified in the Figures and in the Methods. 
 
4. Given that each stem cell type is cultured under distinct culture conditions, it is impossible to tease 
apart the role of the cell lineage and that of the culture conditions in shaping the mutational 
landscapes of the cultured cells. Can the authors comment on this point? The authors keep referring to 
“stem cell-specific” mutational processes but these could really be “culture conditions-specific” 
mutational processes. Can the authors comment on that? 
 
5. One of the most interesting findings of the study – and certainly the finding with most practical 
implications – is that all cultured stem cells are enriched for C to A changes, which may be linked to 
oxidative stress. However, the study does not provide any direct evidence that oxidative stress indeed 
plays a major role in the mutational processes. An experimental demonstration that reducing ROS 

levels could change the rate and the spectrum of culture-acquired mutations (even if provided only for 
one of the SC types) would significantly increase the impact of this work.