Directed differentiation of human iPSCs to purposeful ovarian granulosa-like cells through transcription issue overexpression

2023-08-05 19:41:26

Important revisions:

1) Embody a diagram as an instance the experimental workflow of the examine

Now we have made this diagram and included it as Determine 1.

2) Enhance the standard of pictures and colours in Determine 3 and 4. In Determine 3, the Foxl2+ cells don’t appear to be ready comparable to what’s usually noticed in vivo in ovaries.

Now we have modified our graphs to have a colorblind-friendly palette. Moreover, now we have improved the brightness of our immunofluorescence pictures (beforehand we have been solely exhibiting the uncooked brightness values), and exported the figures at a better decision.

Now we have additionally collected further immunofluorescence pictures from day 70 of ovaroid tradition, which clearly present follicle-like buildings (Determine 6).

3) Present a transparent rationale for utilizing totally different endpoints for various cell strains.

Now we have added this to the Dialogue part. Additionally, we collected further estradiol manufacturing and FSH response information, in order that we now have that information for 9 cell strains as an alternative of 4 as earlier than.

4) The ovaroid information requires an OCT4+ and DAZL+ expression over the entire evaluation of the examine.

Now we have included this as Determine 5C.

5) The precise transcription components used within the examine should be clearly described within the Dialogue part.

Now we have added this to the Dialogue part, added a desk exhibiting which TFs are during which strains (Determine 3 – Supply Knowledge 2), and likewise supplied PCR information verifying this (Determine 3—determine complement 1C). Moreover, now we have re-named our cell strains to be extra informative about which TFs they categorical.

Reviewer #1 (Suggestions for the authors):

I received misplaced at first of the manuscript making an attempt to know precisely all of the steps that led to the era of the granulosa-like cells. It will be useful to incorporate a diagram as an instance the experimental workflow of the examine much like a graphical summary.

Now we have included this as Determine 1.

In Determine 3, I’ve a number of suggestions to facilitate the interpretation of the information

– The authors point out a number of occasions that they observe follicle-like formation of their ovaroid mannequin, however within the IF pictures the FOXL2+ and AMHR2 staining is scattered and appears nothing just like the spherical association of squamous (primordial) or cuboidal (rising) cells one would count on to see within the in vivo ovary. As well as, there isn’t a proof in figures 3 or S5 that DAZL+ hPGCLCs change into surrounded by FOXL2+ granulosa-like cells. I recommend changing the pictures with better-quality IF stains, or the authors ought to soften the conclusion that follicle-like buildings do kind of their ovaroid mannequin.

Now we have collected further immunofluorescence pictures from day 70 of ovaroid tradition, which clearly present follicle-like buildings of FOXL2+ AMHR2+ cells (Determine 5).

We agree that the follicles are empty (not containing hPGCLCs), and we point out this within the Outcomes part.

– The pictures in panel A Day 2 and Day 4 seem like on a distinct scale than the remainder, which is complicated.

Now we have swapped these for various pictures which have the identical magnification as the remainder.

– The merged pictures on the fitting aren’t legible, think about eradicating the DAPI channel from these in order that the reader can higher admire the staining overlap or lack thereof.

Now we have accomplished this, and likewise modified the colours to make them extra legible.

– In determine 4C, I discover the combination onto the human cell atlas very useful. Nevertheless, it’s complicated that the UMAP not corresponds to the cluster distribution proven in determine 4A. I’d recommend including a further visible that exhibits the projection of the human cell atlas cell ID onto the unique clusters of the ovaroid scRNAseq.

Now we have accomplished this.

Additionally, how do the authors clarify that RUNX1 expression may be very low within the FOXL2+ granulosa-like cluster (Determine 4B)?

RUNX1 expression shouldn’t be very low within the granulosa cluster. Within the unique model of the violin plot (which is now determine 7B), the default setting of the width scaling issue was too small, which made it appear to be there wasn’t a lot RUNX1. Now we have since corrected this. The distribution of RUNX1 expression is proven in Author response image 1.

Among the information is troublesome to judge within the figures.

– The bar histograms in figures second and e are very compact and troublesome to inform aside. Additionally, the colours aren’t colorblind-friendly. I like to recommend altering the colours to extra accessible coloration combos in these and in determine 4D.

Now we have modified the colours in these figures to a colorblind-friendly palette. Additionally, we separated the ELISA and bulk RNA-seq figures in order that the bar plots will be greater.

– The pictures of the ovaroids lack annotations to information the readers to what’s related, and the staining high quality is suboptimal, making it troublesome to interpret the similarity of those ovariods to in vivo ovaries.

Now we have added annotations to higher present the follicle-like buildings in determine 6.

The determine information within the unique manuscript have been at decrease decision as a result of there was a file measurement restrict on the PDF add. Now we have supplied original-resolution variations of the figures in our resubmission. We word that the unique variations of our immunofluorescence figures have been merely the uncooked picture information not adjusted for brightness/distinction. Now we have since adjusted the brightness of those pictures within the figures (as most different papers do), and this improves their interpretability. Now we have additionally chosen a greater coloration palette. There’s nonetheless some background staining in just a few of the pictures however this could not have an effect on the conclusions of our paper.

Reviewer #2 (Suggestions for the authors):

This examine from Smela et al. describes the manufacturing and purposeful characterisation of granulosa-like cells from induced pluripotent stem cells. The important thing declare is that two transcription components – NR5A1, and both RUNX1 or RUNX2 – are collectively obligatory and enough for the manufacturing of those cells (summary line 9, outcomes web page 7 line 21, dialogue web page 16 line 9). Whereas sufficiency for a specific set of traits is partly supported by purposeful and transcriptional analyses, the declare of necessity shouldn’t be straight examined. If this declare of necessity is to face, then the authors should make clear their use of this time period, or alternatively, describe experiments they’ve undertaken that might justify this declare. Given the in depth information offered in determine 1b, the brink used for this justification must also be described in a lot better element.

Now we have eliminated the declare of necessity, because it’s doable that different TFs outdoors of the set we examined may additionally produce granulosa-like cells.

Impressively, this paper describes the fast improvement of the germline when human-human chimeras are used, and it is a main power of the examine (determine 3). I do nonetheless discover the usage of the time period “ovaroid” to be considerably problematic. An ovary incorporates many greater than two cell sorts, and a extra nuanced time period is strongly suggested, doubtlessly associated to the manufacturing of a follicle-like construction.

Though our organoids don’t comprise all of the cell sorts of the ovary, they do comprise a number of essential ones resembling FOXL2+ granulosa-like cells, NR2F2+ stroma-like cells, and hPGCLCs. The title “ovaroid” is meant to explain a system resembling an ovary, not an precise ovary.

Whereas many appropriate approaches are carried out, the usage of COV434 cells shouldn’t be well-founded. Because the authors word, there may be some uncertainty concerning the id of COV434 cells, and as a most cancers cell kind, are unlikely to replicate embryonic granulosa cells. A extra acceptable management could be the beginning iPS cells (as offered), or the varied sublines developed, from which a extra real looking evaluation of the TROM worth will be drawn. As offered, this transcriptional evaluation can’t be used to justify the id of the TF-induced granulosa-like cells. The authors acknowledge the problems with COV434 cells on web page 9 strains 11-14, which additional justifies an alternate strategy.

Now we have collected further RNA-seq information from KGN cells, that are phenotypically nearer to bona fide granulosa cells when it comes to steroidogenesis and marker gene expression. In our TROM evaluation, the management is the beginning iPSCs (which we discover to have a TROM rating of zero with the entire different samples).

The regulatory logic described in Determine 2c shouldn’t be clearly described, and a way more rigorous description of the information underlying these findings must be included.

Now we have clarified that this chart shows upregulation of genes of curiosity in response to TF overexpression. Now we have additionally included volcano plots to numerically present the regulatory results.

The part that describing the purposeful maturation of germline cells (through activation of DAZL and DDX4) is proscribed in scope, and the manuscript would profit from orthogonal approaches to show the engraftment of those cells into follicle-like buildings, or transcriptional analyses supporting the claims.

Though we now have robust proof for the formation of follicle-like buildings (Determine 6) now we have not noticed any germ cells inside of those follicles. We do point out that these follicles are empty within the Outcomes part. Future research utilizing optimized tradition circumstances to advertise germ cell survival can be obligatory to deal with this. Relating to transcriptional evaluation, now we have now carried out a differential expression and gene ontology enrichment evaluation on DAZL+ vs DAZL- germ cells in our ovaroids, which we describe within the Outcomes part.

Reviewer #3 (Suggestions for the authors):

Further Analyses:

1. Are a number of iPSC strains – organic variables – used to distinguish into granulosa-like cells? It appears this was solely examined on strains from ATCC-BXS0116 and never confirmed in one other biologically totally different line. Testing further strains with the established protocol is critical to extend robustness.

We examined this on each F3 (ATCC-BXS0116) and F66 (an hiPSC line derived in-house from NIA Getting old Cell Repository fibroblast line AG07141). A full listing of strains is given in Determine 3 – Supply Knowledge 2.

2. There aren’t any pictures of ovaroids handled with androstenedione or FSH for the E2 and P4 experiments.

We tried to do whole-mount staining on these, however this was unsuccessful and we weren’t in a position to get any good pictures. We do have brightfield pictures of the ovaroids, however these aren’t very informative (the samples are too thick to see a lot element), and there don’t appear to be any main variations in look between the circumstances.

3. The ovaroid information was descriptive and requires an OCT4+ and DAZL+ cell over complete evaluation additional time to assist the descriptive claims that these cell sorts underwent “fast improvement” and “declined over time” and to change into examine the outcomes from the hPSCs towards the murine fetal cells. Whereas the authors do carry out an evaluation of Dazl-expressing cells in Determine 4D, this isn’t what’s referred to within the paragraphs of outcomes above and there aren’t any comparisons with murine ovaroids right here.

Now we have carried out this evaluation and it’s now offered in Determine 5C.

4. Which cells within the ovaroid come from the granulosa-like cell strains versus those who have been differentiated into PGCLCs? Will probably be essential to differentiate these populations in “follicle-like” cultures and to verify that each are nonetheless current.

We don’t at present have sufficient info to definitively assign the origins of the cells within the ovaroids. Nevertheless, we think about it unlikely that DAZL+ cells may originate from our granulosa-like cells, since DAZL expression is extremely particular to gonadal PGCs (see Refs. 15–17). Additionally we don’t see DAZL expression in bulk RNA-seq information of our FOXL2+ cells (see Supply Knowledge for determine 3A).

Editorial or information description options:

1. “The outline of experimentation inside the Outcomes part have to be extra clear.”

a. The information represented in Determine 2C and SFiles 2 have to be offered in a kind that demonstrates the relative fold change of expression for every doxy-inducible transcription issue strains in a principal determine.

Now we have added volcano plots to Determine 3 which depict the fold-changes for genes of curiosity, together with our transcription components. Full information are supplied within the supply information for that determine.

b. The outline of iPSC strains which have a number of TF have to be clearly acknowledged. And the outcomes have to be clearly described for every line examined. For instance, on web page 11 “The fourth of our granulosa strains produced excessive ranges of estradiol in all circumstances, and this line lacked the RUNX1 expression vector that was current within the others.” Which 4 strains have been used and during which order the authors are referring to them to infer the “fourth line” was not made clear. Which TFs have been within the strains that have been picked for “phenotype” on web page 12? First described on web page 13 – which strains have been used because the granulosa-like strains?

We modified our naming of the clones to obviously describe which TFs are current, and likewise made a listing of all of the clones in Determine 3 – Supply Knowledge 2.

c. Determine 2 – 2 organic replicates for 4 clones? What does G1, G2, G3, G4 imply in Determine 2E? Are these totally different monoclonal granulosa-like cells from these proven in 2D? If that’s the case, why have been they chosen?

Now we have examined further clones (now all 9 of our prime clones, as an alternative of simply 4). We additionally modified our naming of the clones to be extra descriptive.

2. It isn’t apparent which iPSC strains have been used to generate the hPGCLCs or GLCs within the ovaroid cultures. This must be acknowledged within the outcomes and strategies.

Now we have added this.

3. Web page 13 strains 12-14 examine the expression of SOX17, OCT4, DAZL, and TFAP2C. Nevertheless, the day in tradition chosen for these IF analyses have been totally different. This sentence must be reworded to extra precisely state that OCT4 and DAZL expression was nonetheless seen on day 32 and SOX17 expression on day 8 in human…

We reworded this following your suggestion.

4. Are OCT4- and DAZL+ oogonia seen on day 32? The road on web page 13, line 14 states that “at later timepoints (day 16), DaZL+…” That is complicated as a result of there may be a picture for DAZL and OCT4 antibodies at day 32 in Determine 3.

We clarified that DAZL+/OCT4- and DAZL+/OCT4+ cells are each current.

5. The germ cell populations in Determine 4D aren’t talked about within the textual content.

The “germ cells” are the cells annotated as germ cells by our fetal ovary atlas integration. Now we have indicated this within the textual content.

6. The dialogue ought to clearly summarize/restate the transcription components used and steps to effectively generate the granulosa-like cells from hiPSCs.

Now we have added this.