Efficient generation of marmoset primordial germ cell-like cells using induced pluripotent stem cells
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This paper describes a method for robust differentiation of the common marmoset induced pluripotent stem cells (iPSCs) into primordial germ cell-like cells and subsequently into spermatogonia-like cells when combined with testis somatic cells. The data suggest that marmosets are very similar to humans and macaques. The paper is nicely done but needs further characterization and a better explanation of the methodology.
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Abstract
Reconstitution of germ cell fate from pluripotent stem cells provides an opportunity to understand the molecular underpinnings of germ cell development. Here, we established robust methods for induced pluripotent stem cell (iPSC) culture in the common marmoset ( Callithrix jacchus [cj]), allowing stable propagation in an undifferentiated state. Notably, iPSCs cultured on a feeder layer in the presence of a WNT signaling inhibitor upregulated genes related to ubiquitin-dependent protein catabolic processes and enter a permissive state that enables differentiation into primordial germ cell-like cells (PGCLCs) bearing immunophenotypic and transcriptomic similarities to pre-migratory cjPGCs in vivo. Induction of cjPGCLCs is accompanied by transient upregulation of mesodermal genes, culminating in the establishment of a primate-specific germline transcriptional network. Moreover, cjPGCLCs can be expanded in monolayer while retaining the germline state. Upon co-culture with mouse testicular somatic cells, these cells acquire an early prospermatogonia-like phenotype. Our findings provide a framework for understanding and reconstituting marmoset germ cell development in vitro, thus providing a comparative tool and foundation for a preclinical modeling of human in vitro gametogenesis.
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Author Response
Reviewer #1 (Public Review):
This is an exciting paper describing the development of a robust differentiation of the common marmoset induced pluripotent stem cells (iPSCs) into primordial germ cell-like cells and subsequently into spermatogonia-like cells when combined with testis somatic cells. The work is of high quality, but some experimental details and protocols are missing which are necessary for a new protocol development - for example, reconstitution methods and protocols are missing completely in the manuscript and additional details in various aspects of the differentiation and cell maintenance are missing. Despite this, the work is valuable and would be of interest to the germ cell and in vitro gametogenesis communities. The data suggest that marmosets are very similar to humans and macaques, and indeed …
Author Response
Reviewer #1 (Public Review):
This is an exciting paper describing the development of a robust differentiation of the common marmoset induced pluripotent stem cells (iPSCs) into primordial germ cell-like cells and subsequently into spermatogonia-like cells when combined with testis somatic cells. The work is of high quality, but some experimental details and protocols are missing which are necessary for a new protocol development - for example, reconstitution methods and protocols are missing completely in the manuscript and additional details in various aspects of the differentiation and cell maintenance are missing. Despite this, the work is valuable and would be of interest to the germ cell and in vitro gametogenesis communities. The data suggest that marmosets are very similar to humans and macaques, and indeed previously established protocols for PGCLC induction and likely previously published testis reconstitution methods/differentiation were employed here to generate the spermatogonia-like cells.
We greatly appreciate the positive comments of the reviewer on our manuscript. We have added experimental details of our germ cell differentiation schemes in Materials and Methods.
Reviewer #2 (Public Review):
This paper identifies the need for improved pre-clinical models for the study of human primordial germ cells (PGCs) and suggests the common marmoset (Callithrix jacchus) as a suitable primate model. In vitro gametogenesis offers an alternative method to generate germ cells from pluripotent stem cells for study and potential pre-clinical applications. Therefore, the authors aimed to take the first steps toward developing this technology for the marmoset. Here, iPSCs have been derived from the marmoset and differentiated to PGC like-cells (PGCLCs) in vitro that have similarities in gene expression with PGCs identified from single-cell studies of marmoset embryos, as demonstrated through immunofluorescence and RT-qPCR approaches, as well as RNA-sequencing.
The authors have successfully developed a protocol that produces PGCLCs from marmoset iPSCs. These are shown to express key germline gene markers and are further shown to correlate in gene expression with PGCs from the marmoset. This study uses a 2D culture system for further expansion of the PGCLCs. When cultured with mouse testicular cells in a xenogeneic reconstituted testis culture, evidence is provided that cjPGCLCs have the capacity to develop further, expressing marker genes for later germline differentiation. However, the efficiency of generating these prospermatogonia-like cells in culture is unclear. Nonetheless, with the importance of developing protocols across species for in vitro gametogenesis, this paper takes a key step towards generating a robust preclinical system for the study of germ cells in the marmoset.
We thank the reviewer for the encouraging comment. By IF analyses, we identified 0.89 and 3.3% of DAZL or DDX4 positive cells, respectively (DDX4+TFAP2C+ cells [4/123, 3.3% among all TFAP2C+ cells] and DAZL+TFAP2C+ cells [2/232, 0.86% among all TFAP2C+ cells]). Overall scarcity of cells and lack of fluorescence reporters (DAZL and DDX4 are cytoplasmic proteins necessitating technically challenging intracellular staining procedure to be assessed by flow cytometry), we were not able to provide the flow cytometric plots in this study. This has been described in the revised manuscript (page 11, Results, “Maturation of cjPGCLCs into early prospermatogonia-like state”).
The claims of the authors are generally justified by the data provided; however, some conclusions should be clarified. In particular, the authors have failed to show convincingly that cjPGCLCs are a distinct cell type to the iPSCs that generated them. cjiPSCs cultured in feeder conditions (OF) with IWR1 are reported to cluster closely with the derived cjPGCLCs using principal component analysis of RNA-Seq data. This contrasts with the cjiPSCs cultured in feeder-free (FF) conditions which maintain a more undifferentiated/less primed state, and are not capable of differentiating to the germline lineage. Therefore, the OF/IWR1 cjiPSCs could rather be an intermediate cell-state between iPSCs and cjPGCLCs.
Although OF/IWR1 cjiPSCs are closer to cjPGCLCs than cjiPSCs cultured in other conditions, they are pluripotent (as evidenced by trilineage differentiation assay, morphological assessment, and expression of pluripotency markers, Figure 3–figure supplement 2) and do not express most of key germ cell markers (Figure 6–figure supplement 1C). Our newly added scRNA-seq analyses also highlighted the differences between OF/IWR1 and cjPGCLCs and the molecular dynamics associated with the transition.
The reasons behind improved germline competence of iPSCs in the different media conditions are unclear. The authors reject the idea that this is due to the presence of IWR1, since this condition has not affected FF iPSCs. However, the efficiency of differentiation was greatly increased in OF conditions when IWR1 was used, indicating inhibition of WNT does indeed have a positive effect on induction to the germline lineage. This area requires further clarification.
As the reviewer pointed out, inclusion of IWR1 in cultures of OF cjiPSCs upregulates some pluripotency markers (SSEA3, SSEA4) and reduces meso/endodermal differentiation. Thus, the undifferentiated/less primed state under the Wnt inhibition might positively affect germ cell differentiation of OF cjiPSCs. However, FF cjiPSCs are pluripotent and are not germline competent, even in the presence of IWR1, suggesting that there are factors in FF culture conditions that make them incompetent for germline differentiation. Because FF cultures utilize PluriStem™ medium, a proprietary product of MilliporeSigma, we were unable to define the factor that confers such germline incompetence.
Another area requiring clarification is the reporting of RNA sequencing data as representative of a developmental trajectory, without defining which cell lines produced clusters, or defining the stages of this trajectory. The authors refer to the identification of four clusters representative of a developmental trajectory, however, they provide unclear information as to what this refers to. Importantly, detailed transcriptomic comparisons between in vivo-derived PGCs and in vitro PGCLCs are not provided.
Our original analysis revealed which cell lines produced clusters (Figure 6A) and defined the stages of the trajectory (iPSCs feeder free, iPSCs on feeder, PGCLCs, expansion, Figure 6C). The four clusters to which the reviewer refers are gene clusters that are defined by unsupervised clustering analysis of variably expressed genes across the samples (Figure 6D). As it is defined computationally, it is not possible to unequivocally define gene clusters by particular cell types. However, we found that these gene clusters revealed insightful patterns (1, genes higher in cjiPSCs; 2, genes higher in cjPGCLCs; 3, genes higher in expansion culture cjPGCLCs; 4, genes higher in d2 cjPGCLCs). We have added sample information to the Figure 6D to further clarify the meaning of the data and a brief explanation of gene clusters in the figure legend. To define the trajectory in a more unbiased manner, we performed scRNA-seq and have added additional trajectory analyses (Figure 7A-K in the revised manuscript). Moreover, we also added the transcriptomic comparison as the reviewer suggested (Figure 7L, M in the revised manuscript).
Functional validation of iPSC lines generated in the study is not provided besides confirming that the cells express pluripotency markers OCT3/4, SOX2, and NANOG. It is important to confirm tri-lineage differentiation of iPSCs, e.g., through an embryoid body assay. Since FF cjiPSCs were unable to differentiate into cgPGCLCs, it is even more important to confirm cells are genuine iPSCs.
We performed a trilineage differentiation assay and confirmed that they can generate three germ layers.
In summary, although there are issues surrounding clarity, this paper is generally justified in its conclusions. The authors present an optimised protocol for the derivation of PGCLCs from marmoset iPSC-like cells, with defined expansion conditions and evidence of further differentiation to prospermatogonia-like cells.
We thank the reviewer for the encouraging comment.
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eLife assessment
This paper describes a method for robust differentiation of the common marmoset induced pluripotent stem cells (iPSCs) into primordial germ cell-like cells and subsequently into spermatogonia-like cells when combined with testis somatic cells. The data suggest that marmosets are very similar to humans and macaques. The paper is nicely done but needs further characterization and a better explanation of the methodology.
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Reviewer #1 (Public Review):
This is an exciting paper describing the development of a robust differentiation of the common marmoset induced pluripotent stem cells (iPSCs) into primordial germ cell-like cells and subsequently into spermatogonia-like cells when combined with testis somatic cells. The work is of high quality, but some experimental details and protocols are missing which are necessary for a new protocol development - for example, reconstitution methods and protocols are missing completely in the manuscript and additional details in various aspects of the differentiation and cell maintenance are missing. Despite this, the work is valuable and would be of interest to the germ cell and in vitro gametogenesis communities. The data suggest that marmosets are very similar to humans and macaques, and indeed previously established …
Reviewer #1 (Public Review):
This is an exciting paper describing the development of a robust differentiation of the common marmoset induced pluripotent stem cells (iPSCs) into primordial germ cell-like cells and subsequently into spermatogonia-like cells when combined with testis somatic cells. The work is of high quality, but some experimental details and protocols are missing which are necessary for a new protocol development - for example, reconstitution methods and protocols are missing completely in the manuscript and additional details in various aspects of the differentiation and cell maintenance are missing. Despite this, the work is valuable and would be of interest to the germ cell and in vitro gametogenesis communities. The data suggest that marmosets are very similar to humans and macaques, and indeed previously established protocols for PGCLC induction and likely previously published testis reconstitution methods/differentiation were employed here to generate the spermatogonia-like cells.
-
Reviewer #2 (Public Review):
This paper identifies the need for improved pre-clinical models for the study of human primordial germ cells (PGCs) and suggests the common marmoset (Callithrix jacchus) as a suitable primate model. In vitro gametogenesis offers an alternative method to generate germ cells from pluripotent stem cells for study and potential pre-clinical applications. Therefore, the authors aimed to take the first steps toward developing this technology for the marmoset. Here, iPSCs have been derived from the marmoset and differentiated to PGC like-cells (PGCLCs) in vitro that have similarities in gene expression with PGCs identified from single-cell studies of marmoset embryos, as demonstrated through immunofluorescence and RT-qPCR approaches, as well as RNA-sequencing.
The authors have successfully developed a protocol that …
Reviewer #2 (Public Review):
This paper identifies the need for improved pre-clinical models for the study of human primordial germ cells (PGCs) and suggests the common marmoset (Callithrix jacchus) as a suitable primate model. In vitro gametogenesis offers an alternative method to generate germ cells from pluripotent stem cells for study and potential pre-clinical applications. Therefore, the authors aimed to take the first steps toward developing this technology for the marmoset. Here, iPSCs have been derived from the marmoset and differentiated to PGC like-cells (PGCLCs) in vitro that have similarities in gene expression with PGCs identified from single-cell studies of marmoset embryos, as demonstrated through immunofluorescence and RT-qPCR approaches, as well as RNA-sequencing.
The authors have successfully developed a protocol that produces PGCLCs from marmoset iPSCs. These are shown to express key germline gene markers and are further shown to correlate in gene expression with PGCs from the marmoset. This study uses a 2D culture system for further expansion of the PGCLCs. When cultured with mouse testicular cells in a xenogeneic reconstituted testis culture, evidence is provided that cjPGCLCs have the capacity to develop further, expressing marker genes for later germline differentiation. However, the efficiency of generating these prospermatogonia-like cells in culture is unclear. Nonetheless, with the importance of developing protocols across species for in vitro gametogenesis, this paper takes a key step towards generating a robust preclinical system for the study of germ cells in the marmoset.
The claims of the authors are generally justified by the data provided; however, some conclusions should be clarified. In particular, the authors have failed to show convincingly that cjPGCLCs are a distinct cell type to the iPSCs that generated them. cjiPSCs cultured in feeder conditions (OF) with IWR1 are reported to cluster closely with the derived cjPGCLCs using principal component analysis of RNA-Seq data. This contrasts with the cjiPSCs cultured in feeder-free (FF) conditions which maintain a more undifferentiated/less primed state, and are not capable of differentiating to the germline lineage. Therefore, the OF/IWR1 cjiPSCs could rather be an intermediate cell-state between iPSCs and cjPGCLCs.
The reasons behind improved germline competence of iPSCs in the different media conditions are unclear. The authors reject the idea that this is due to the presence of IWR1, since this condition has not affected FF iPSCs. However, the efficiency of differentiation was greatly increased in OF conditions when IWR1 was used, indicating inhibition of WNT does indeed have a positive effect on induction to the germline lineage. This area requires further clarification.
Another area requiring clarification is the reporting of RNA sequencing data as representative of a developmental trajectory, without defining which cell lines produced clusters, or defining the stages of this trajectory. The authors refer to the identification of four clusters representative of a developmental trajectory, however, they provide unclear information as to what this refers to. Importantly, detailed transcriptomic comparisons between in vivo-derived PGCs and in vitro PGCLCs are not provided.
Functional validation of iPSC lines generated in the study is not provided besides confirming that the cells express pluripotency markers OCT3/4, SOX2, and NANOG. It is important to confirm tri-lineage differentiation of iPSCs, e.g., through an embryoid body assay. Since FF cjiPSCs were unable to differentiate into cgPGCLCs, it is even more important to confirm cells are genuine iPSCs.
In summary, although there are issues surrounding clarity, this paper is generally justified in its conclusions. The authors present an optimised protocol for the derivation of PGCLCs from marmoset iPSC-like cells, with defined expansion conditions and evidence of further differentiation to prospermatogonia-like cells.
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