We thank Memon and Abdelalim (1) for their comments on our article (2). In short, they point out that the induced pluripotent stem cell (iPSC) lines that we used in our experiments contained mosaic trisomy 1 (47,XY,+1) in the mutated lines with the potential consequence that this trisomy would invalidate the study results. Memon and Abdelalim also point out that the genome-edited lines used as control lines did not contain this trisomy 1 (47,XY,+1), i.e., these gene-edited lines were 46,XY (with additional mosaicism, including deletions of chromosome 18) and hence did not represent appropriate controls. Furthermore, Memon and Abdelalim refer to an article by Hua et al. (3) that was retracted (4) due to misrepresentation of karyotypic results. Memon and Abdelalim (1) conclude that “this signifies the importance of ensuring normal chromosomal profiles for [human pluripotent stem cell] lines used in the experiments.”
We would like to provide the following responses to their comments.
First, the karyotype status of a sample of iPSCs is not a static feature. Instead, the karyotype status is dynamic and may change from earlier to later passages through a genomic process brought forward by strong selection processes, as also pointed out in Assou et al. (5) and referenced by Memon and Abdelalim (1).
We demonstrated the abovementioned trisomy karyotyping results (47,XY,+1) from karyotyping mutant iPSC lines at passage 20, whereas in our published experiments performed in our two laboratories we deployed cells from a larger range of passages, ranging from passage 10 to at least passage 20. In another karyotyping of the deployed iPSCs, which was performed by the same independent Cambridge laboratory at approximately the same time but where the results were not included in the article, we found normal karyotype 46,XY of the mutant iPSC at an earlier passage (passage 10) and a normal karyotype 46,XY at passage 12. We are happy to provide these karyotype results upon request. We shipped this second set of samples to Cambridge, U.K., for karyotyping on 21 November 2021. The karyotypic results for these samples were not reported to us until 22 January 2022. These results were probably delayed due to the pandemic. Since Diabetes granted us a submission deadline that we were not allowed to exceed (19 November 2021) for our revised version of the manuscript after peer review, we were not able to include the results of this second karyotyping in the submission, which explains why we did not include the findings from this second karyotype analysis in the final and published report.
In the context of the normal karyotype 46,XY of the mutated iPSC lines at the earlier passage 10, the normal karyotype 46,XY of the gene-edited lines (which also contained acquired mosaic deletions of chromosome 18) makes perfect sense. In contrast, it would be more difficult to explain how mutant 47,XY,+1 iPSC lines would revert into 46,XY gene-edited lines.
Second, Memon and Abdelalim refer to the retraction of the Hua et al. (3) article where the authors’ retraction was a response to misrepresentation (4), suggesting a similarity with the situation of our article (2). We contest any similarities of the situations of these two articles. The article by Hua et al. presented a normal karyotype in the deployed pluripotent cell lines, whereas a postpublishing karyotype routine quality control test revealed an abnormal karyotype in the same cell lines. The situation of our article is the opposite. We reported an abnormal karyotype (47,XY,+1) in passage 20 of the iPSC lines, as provided in Supplementary Fig. 7 in our article, whereas a second and subsequent karyotyping postsubmission showed a normal karyotype (46,XY) of the same iPSC lines frozen at an earlier passage, passage 10. Additionally, the article by Hua et al. (3) could not confirm the previously reported endonuclease-mediated correction of the mutant GCK G299R allele (4).
Third, Memon and Abdelalim (1) conclude that “this signifies the importance of ensuring normal chromosomal profiles for [human pluripotent stem cell] lines used in the experiments.” In our opinion, their statement is problematic. Are they suggesting that once scientists have demonstrated a normal karyotype status of a pluripotent cell line, one should then assume that the karyotype status of this pluripotent cell line will remain unchanged and normal through all subsequent passages? We believe a more transparent scientific approach is required where authors present the data as authentically as possible and let the readership judge the robustness of the data. This was also the approach we chose. We honestly stated the following facts in the article (please refer to the legend to Supplementary Fig. 7): “The extent to which these genomic alterations potentially perturb the experimental readouts is unclear as most of the chromosomes remained intact. However, the information of chromosomal aberrations must be kept in mind when interpreting the results.”
See accompanying article, p. e1.
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Duality of Interest. No potential conflicts of interest relevant to this article were reported.