PONE-D-21-14615

Heading towards a dead end: the role of DND1 in germ line differentiation of human iPSCs

PLOS ONE

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"This work was financially supported by the Deutsche Forschungsgemeinschaft, Clinical Research Unit

500 326—‘Male Germ Cells: from Genes to Function’ (CRU326, SCHO 340/8-1 and SCHL 394/15-1) and by

501 a pilot project funding by the CRU326 (pilot project 6)."

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Additional Editor Comments:

While all three reviewers found this manuscript interesting, several major concerns were raised about figure legends, data in some figures, cell line/lineage markers, and manuscript writing. Please refer to reviewers' comments for details.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #2: Partly

Reviewer #3: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: No

Reviewer #3: I Don't Know

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Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this manuscript, the authors try to show functions of DND1, an evolutionally conserved genes crucial for germ cell development, in human primordial germ cell-like cell (PGCLC) differentiation from iPS cells (iPSC) by its knockout. Loss of DND1 did not result in significant changes of pluripotency and proteomic profiles as well as of global gene expression profiles in the two different iPSCs, while efficiency of PGCLC differentiation from the iPSCs and the expression of NANOS3 and PRDM1, which are important for PGC development, in the induced PGCLCs, were reduced by DND1 deficiency. Roles of DND1 in human PGC development are potentially interesting, but this study does not reach a definitive conclusion, because it is unreliable that only subtle reduction (~1.5 folds) of the two gene expression without additional changes of required gene expression affects PGCLC specification. In addition, data presentation and discussion are often inadequate as described below.

Specific comments:

1. Labeling for Fig.1~Fig.3 may be wrong. This reviewer made comments for the figures according to the figure number in figure legends in the text.

2. Fig.3A is not informative, which should be deleted, because DND1 KO did not influence efficiency of PGCLC differentiation from TS iPSCs, and did not result in consistent effects in CB iPSC in this method (Spin-EB). Instead, the authors should show the effect of DND1 KO by the low adhesion plate methods shown in Fig.3B-D in more detail, including the effects of DND1 KO in different TS DND1-/- cell lines (#2, #3) as well as CB DND1-/- lines. In Fig. 3C, D, statistical evaluation is necessary.

3. Fig. 1A; Sequences of the deleted regions should be shown.

4. Fig. 1B, D; Number of biological replicates should be shown.

5. Reduced expression of NANOS3 and PRDM1 in DND1 KO PGCLCs should be confirmed by RT-qPCR.

6. Supplementary Fig. S1; FACS profiles of the undifferentiated iPSCs as well as of the cells stained by the control antibodies should be shown.

7. Transcriptomes of PGCLCs from parent WT iPSCs in this study and by Irie et al. are substantially different as shown in Supplementary Fig. S3, and the authors discussed that difference of the culture period may cause the difference (line 411~). If they think so, they should repeat transcriptome analysis by using day 9~10 PGCLCs. Although the authors discussed that their cells were indeed PGCLCs due to the expression of a few marker genes without showing it (line 419), it is likely that the cells in this study may not be proper PGCLCs judged by the data presented in this manuscript. More definitive criteria and experimental evidence supporting that their cells are PGCLCs, are necessary.

8. The authors discussed a possibility that loss of DND1 cause trans-differentiation of PGCLCs, but it is too speculative without definitive experimental evidence (line 466~). It is more likely that iPSCs or iMeLCs differentiated from iPSCs may differentiate not to PGCs, but to different types of cells with active proliferation.

Reviewer #2: Mall et al. generated DND1-null human iPSC clones by CRISPR/Cas9 and tested their differentiation potencies. Authors claimed a significant reduction in efficiency of PGCLC production for the DND1-null iPSCs compared to the parental iPSCs whereas the potencies to generate the three germ layers seemed unaffected. Authors also argued that the DND1-null PGCLCs expressed reduced levels of genes associating PGC differentiation (e.g., NANOS3 or PRDM1).

The claimed specific importance of DND1 in PGCLC production from iPSCs is interesting, and the proposed requirement of DND1 in the PGC-like transcriptomal characteristics of PGCLCs seems significant. However, this Reviewer feels that more convincing data are necessary to publish such significant conclusions. Especially, the RNA-seq data shown as Figure 4B need more work to convincingly support the claims of Authors.

[Major Points]

1) Figure 2A. Authors are requested to explain why three datum points of the same sample show significant variations. See CB WT, CB #2, and CB #3 datum points that are located far from the other points.

1) The RNA-seq heatmap data (Figure 4B) show very strong difference between the two columns under “TS WT” – namely, the left column is mostly bright red whereas the right column is mostly black with only a few dark red genes. If the wild type-derived PGCLCs already show quite unstable transcriptomal characteristics, comparison of their transcriptomes with DND1-null PGCLCs would be difficult to interpret. Authors are requested to repeat PGCLC production from TS WT to confirm that the differential expression of the PGC markers shown in Figure 4 is not due to technical inconsistencies.

2) Figure 4B does not include CD38. Because PGCLCs were FACS-enriched using CD38 and TNAP, Authors are requested to show CD38 expression in Figure 4B to make sure that all cells presented in Figure 4B are indeed CD38+ PGCLCs. If CD38 expression is also very low in DND1-knockout cells than wild type cells, the identities of cells presented in this figure would be questionable.

3) Figure 4B shows only representative marker genes and/or differentially expressed genes. Authors are requested to show genes whose mRNA expression is not affected by DND1 knockdown. It is expected that many genes are not affected by DND1 knockdown; however, if this is the case, Authors need to evaluate viabilities of cells to exclude the possibility that the remarkable transcriptional contrasts shown in Figure 4B is not due to non-specific damages.

[Minor Points]

1) Figure numbers are incorrect. Figure 1 is actually Figure 2, Figure 2 is Figure 3, and Figure 3 is Figure 1. A figure with no figure number is Figure 4.

2) Figure S3 is not mentioned in the text. Perhaps Authors want to refer to it in the paragraph starting Line 409.

3) There are many typographical errors. For example,

Line 34: is ac conserved (is conserved?)

Line 47: NANOS2 and the CXCR4-NOT complex (CCR4-NOT?)

Line 56: stop codon that RNA binding– is associated (stop codon in the RNA binding domain?)

Line 67: This rases the question (raises the question?)

Line 79: in early steps of differentiation, The CRISPR/Cas9 (differentiation. The CRISPR/Cas9?)

Line 466: Both proteinsare as well (Both proteins are as well?)

4) Erez Raz is listed as a co-author on the title page, but he is acknowledged in the Acknowledgments section – “We want to thank Eres Raz for useful comments on the manuscript.” (Line 498).

5) The zebrafish gene “dead end” appears in the abstract and introduction (Lines 16 and 38). Authors may want to amend the text to make it clear that this is a name of gene.

6) Lines 421-422: (see Supplemental Fig. 2). Are Authors requesting to see a supplemental materials of a reference (Irie et al.) cited 12 lines above?

Reviewer #3: The work in this manuscript investigates the role of the conserved protein DND1 in pluripotency and germline development in human cord blood and testicular somatic cell derived iPSCs. The requirement for Dnd was examined using CRISPR/Cas9 to disrupt Dnd in two distinct human cell lines. The gene expression patterns of “wild-type” and Dnd deficient cell lines were characterized using RNA seq (to examine transcripts) and mass spec (to analyze proteins). Comparison of the RNA and protein expression profiles and Flow cytometry analysis and cell sorting revealed no major differences in pluripotency factors or ability to differentiate along various lineages in the absence of Dnd. Based on the data provided wild-type and mutant datasets clustered according to the cell type they were derived from. Because mutant aggregates were larger than those of wild-type and fewer PGCLCs were recovered in sorting assays, it is suggested that Dnd1 may be required to maintain PGCLC fate in this context, as it is in other animals. Sequencing of PGCLCs from wild-type and mutants and comparison to previously published datasets suggests that expression of differentiation genes was intact, but that extracellular matrix regulators were lower in mutants whereas negative regulators of epigenetic state were elevated in mutant PCGLCs. The data appear to be properly controlled, are overall clearly presented within each figure, and are consistent with the main conclusion that Dnd1 is not required for pluripotency or differentiation, but instead promotes maintenance of germline fate in this system as has been previously observed in animal models. This was clearly a lot of work, but enthusiasm for the manuscript is diminished because grammatical errors within the text, mismatch between the figures and legends, and in a few cases lack of detail in the legend detract from the clarity of the manuscript. The experiments overall are well described; however, the main conclusions are not consistently clearly stated. This may be in part due to the variability observed from line to line.

Specific comments:

1) The main figures and legends appear to be out of order. Figure legend 1 seems to match Figure 3, legend 2 matches Figure 1, Legend 3 matches Figure 2.

2) Were statistical analyses performed for the data presented in Figure 2 (legend 3)?

3) A more detailed legend is needed for Supplementary Fig. 3. It is not clear which dataset is being compared for Mall data – I this both the bulk sequencing of the aggregates and/or sequencing of the sorted PGCLCs? Also, it is not clear which data are from the wild-type cells and the mutant cells? or is this just wild-type? For the GO analysis it is not clear what was compared – the full dataset or specific clusters from the Irie dataset. Please clarify these points.

4) It is suggested that newly formed PGCLCs transdifferentiate toward other lineages and that trans-differentiation in the culture system is not random and may reflect a shift toward fates dependent on BMP4. Is there any hint from the sequencing data that this is the case?

5) It is stated that “DND1 is expressed in pluripotent stem cells at relatively low levels, so we assume that loss of function was compensated by other factors.” It is unclear why low levels of expression would suggest compensation – please clarify.

6) The mass spectrometry experiments are described in the methods and text and a link to the data was provided, but the data or a summary thereof do not appear to be included in the main or supplemental figures. It is unclear why was the mouse Uniprot database used for the MS analysis.

7) One of the authors, Erez Raz, is acknowledged and his name is misspelled in the Acknowledgements section (which has a typo in the title). It is thoughtful but unusual to acknowledge an author in this section as typically authorship itself acknowledges an individual’s contribution.

8) The author lists don’t match between the main paper and supplemental – Erez Raz is listed as an author on the main paper but not on the supplemental data file.

9) There are typos and grammatical errors throughout the manuscript. For example “extend” should be “extent”, “intercipient” should be “incipient”, “sells” should be “cells”, instead of “prone to” consider “subjected to”, articles are missing in several places, and some sentences are unclear (e.g. lines 84-86 on page 5, 146-147 on page 7, and there is a comma instead of a period on line 79 pg 4, words are missing in several sentences including page 3 line 56 where it appears “disrupts” is missing before “RNA binding”, pg2 line 27 appears to be missing “with”, on page 3 ,line 34 “is ac” should be “is a”, and page 4 line 76 has an extra “that”. This list is not all-inclusive, please proof-read carefully.

10) Table 2: is the reverse primer missing for hU6 or was only one primer used?

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Reviewer #1: No

Reviewer #2: Yes: Toshi Shioda

Reviewer #3: No

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Heading towards a dead end: the role of DND1 in germ line differentiation of human iPSCs

PONE-D-21-14615R1

Dear Dr. Schlatt,

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Kind regards,

Wei Cui, Ph.D.

Academic Editor

PLOS ONE

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