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  1. Author Response

    We thank the reviewers for their comments and helpful suggestions. We are currently preparing a revised version of this manuscript. Notable changes we are making include:

    • adding a diagram to the introduction to show the overall workflow of the study,
    • quantitatively analyzing the fraction of OCT4+ and DDX4+ cells in our immunofluorescence images over time,
    • collecting and analyzing additional bulk RNA-seq data on KGN cells and adult human ovarian tissue,
    • performing estradiol assays on additional lines of hiPSC-derived granulosa-like cells,
    • presenting images from day 70 ovaroids which clearly show follicle formation,
    • changing the colors in the figures to be more accessible to colorblind readers,
    • clarifying which TFs are present in which of our clonal lines.

    These changes will address the weaknesses identified by the reviewers. Along with our revised manuscript, we will also prepare a more comprehensive author response for these reviewer comments.

  2. eLife assessment

    This is an important and significant study that focuses on deriving ovarian granulosa-like cells from hiPSC lines. The strengths of the study include bioinformatics analysis to identify relevant candidate transcription factors that drive the iPSCs into the ovarian granulosa pathway, an attempt to derive ovaroid model by combining human PGC-like cells with the iPSC-derived granulosa-like cells, and a variety of endpoint analysis including hormone measurements. Some limitations of the study include poor quality of images, lack of convincing demonstration that follicle-like structures are indeed derived in vitro, lack of clear rationale for using different cell lines with different endpoints chosen for analysis, and lack of clear methods indicating stepwise which transcription factors were used.

  3. Reviewer #1 (Public Review):

    Smela and colleagues used in silico predictions as well as reports from the literature to identify candidate transcription factors that were likely to promote granulosa-like differentiation of hiPSCs. After careful evaluation and validation using granulosa marker expression and estradiol production as read-outs, the authors identify combinations of NR5A1 with RUNX1 or RUNX2 that are necessary and sufficient to derive granulosa-like cells from hiPSCs. This section of the study is well-controlled and carefully explained, and the authors' conclusions are supported by the data. The authors then use their granulosa-like cells in concert with previously developed human primordial germ cell-like cells (hPGCLCs) to generate human ovaroids. They show that while their TF-induced granulosa-like cells initially and rapidly support the maturation of hPGCLCs into DAZL+ gonadal germ cells, DAXL+ cells are eventually lost to cell death or off-target differentiation. The authors candidly report the need for troubleshooting this aspect of the study, but this is an encouraging and important first step toward a fully human TF-induced organoid model of human ovary development. I am slightly less convinced by the data presented in the ovaroid section of the manuscript, as the immunostaining and gene expression data do not seem to fully align with in vivo conditions, and the authors do not address this discrepancy to my satisfaction in the current version of the text.

    Weaknesses: The manuscript would benefit from a diagram illustrating the experimental approach from the selection of transcription factors to the generation of granulosa-like cells to the assembly of ovaroids. This would increase the accessibility of the data to an audience unfamiliar with iPSC and organoid strategies. In its current form, the data presented does not convince me that follicle-like structures form in the human ovaroid model.

    Strengths: The authors address a critical gap in resources by providing a model for human hiPSC-derived granulosa-like cells. This resource will undoubtedly advance our molecular understanding of human ovary development and allow critical functional studies on the establishment and preservation of human female fertility. The manuscript is very didactic and easy to follow. The conclusions are well supported by the data, and the discussion candidly raises caveats and further directions of the work.

  4. Reviewer #2 (Public Review):

    The study by Smela et al describes the direct differentiation of human "Granulosa-like cells" via the overexpression of a limited number of transcription factors in pluripotent cells. This approach builds on other contemporary work to produce functional support cells of the mammalian gonad.

    The work does succeed to establish cultured cells that retain some characteristics of these ovarian support cells, including the expression of granulosa cell markers, steroid biosynthesis in response to stimulation, and some evidence of acute germline support. The study also marks an important technological development towards the production of in vitro conditions for the production of human gametes from iPS cells. Prior efforts using human germline cells have mostly focussed on xeno-organoid approaches, and so the human-human nature of the present study represents a useful advance. Of particular note, the present study identifies a remarkably fast acquisition of DDX4 and DAZL-positive cells when both germline and support cells are both derived from a human source. This is an intriguing finding, as other groups have reported a substantial delay in acquiring this germline state when cells from mixed species are used. While these findings are of key technological importance towards ongoing efforts to create in vitro gametes, there appear to be some issues of reproducibility, and a lack of deep functional characterisation.

    Several conclusions of the paper need to be described in much greater detail. For example, the regulatory effects of the over-expression of transcription factors are stated in Figures 1 and 2, but how this regulatory logic was assembled is not presented, and the methods by which this logic was experimentally probed are not presented. Second, the abstract highlights two transcription factors that are both necessary and sufficient for granulosa-like cell production. While sufficiency is tested through the overexpression of the transcription factors, necessity is not conventionally assessed through a genetic approach. The upregulation of factors in response to transcription factor overexpression does not seem appropriately described. Third, the transcriptional comparison of granulosa-like cells with cancer cell lines that do not reflect normal granulosa cells should be reconsidered.

    The study contributes an important step towards the production of functional human granulosa cells from pluripotent cells, though the central conclusions would benefit from a more robust interrogation of the cell status achieved.

  5. Reviewer #3 (Public Review):

    The authors sought to develop an efficient protocol for granulosa-like cells by identifying and testing transcription factors identified through secondary analyses of RNA-seq data. The transcription factors were exogenously expressed in human iPSCs and tested for their ability to induce expression of granulosa cell genes, produce estradiol, and form ovaroids with human primordial germ cell-like cells.

    There are weaknesses in some descriptions of experiments and results. Additionally, the follicle formation in the ovaroid experiments was not adequately identified or described. Finally, additional lines of human iPSCs (biological replicates) to demonstrate granulosa cell expression after the final transcription factors were determined, would increase the robustness of the granulosa-like cell differentiation protocol.

    The major strengths of this manuscript include the comparison of granulosa-like cells in vitro and in the ovaroid aggregates to previously published RNA-seq analyses of human fetal ovaries. Additionally, several human, murine, and cell line controls were used where appropriate to compare cell expression.

    Overall, the authors have achieved their aims of identifying transcription factors that induce a granulosa-like phenotype in human pluripotent stem cells. The production of estradiol and the presence of DAZL4+ cells in an aggregate culture that includes human primordial germ cell-like cells confirmed the functionality of the granulosa-like cells (with the caveat that the cell origins within the ovaroid culture need to be confirmed).

    There are several challenges to studying human fetal ovary development and an efficient, robust granulosa-like cell protocol for human pluripotent stem cells, as described here, will lead to major advancements in this field.