Author response: Efficient generation of marmoset primordial germ cell-like cells using induced pluripotent stem cells
Year: 2022
Type: peer-review
Abstract: Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Appendix 1 Data availability References Decision letter Author response Article and author information Metrics 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. Editor's evaluation This nicely done 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. https://doi.org/10.7554/eLife.82263.sa0 Decision letter Reviews on Sciety eLife's review process Introduction The germline, a lineage that ultimately form the gametes, is the fundamental component of the life cycle in metazoan species, ensuring perpetuation and diversification of the genome across generations. In addition, the germline is the foundation of totipotency, since combination of gametes at fertilization gives rise to totipotent zygotes that establish all embryonic and extraembryonic lineages necessary for production of a new organism. The germline first arises during early embryonic development as primordial germ cells (PGCs), which subsequently migrate to the developing gonads and ultimately produce either spermatozoa or oocytes through complex and sex-specific developmental pathways (Saitou and Miyauchi, 2016). Accordingly, aberrancies associated with PGC development can lead to infertility and a variety of genetic and epigenetic disorders in offspring. Therefore, a precise understanding of how PGCs develop bears significant implications not only for reproductive medicine but also toward a better understanding of a breadth of human diseases. Although much has been learned from murine genetic studies regarding the cellular dynamics, signaling, genetic, and epigenetic requirements accompanying PGC specification (Saitou and Miyauchi, 2016; Saitou and Yamaji, 2012), the scarcity of germ cells and complexity of their development and cellular interactions has limited deep understanding of transcriptional regulatory networks and epigenetic bases of germ cell development. The last decade, however, has witnessed remarkable progress toward establishing in vitro gametogenesis (IVG) technologies as an alternative approach to study germ cell development. Remarkably, through the stepwise recapitulation and validation of developmental milestones starting with pluripotent stem cells (embryonic stem cells [ESCs] or induced pluripotent stem cells [iPSCs]), the entirety of mouse germline development has been reconstituted in vitro, culminating in the successful generation of fertilization-competent oocytes and spermatozoa, and healthy offspring (Hikabe et al., 2016; Ishikura et al., 2021). These landmark studies have been followed by successful development of human iPSC-based germline reconstitution methods, in which pre-meiotic oogonia and prospermatogonia-like cells generated through PGC-like cells (PGCLCs) bear remarkable transcriptional similarities to in vivo counterparts (Hwang et al., 2020; Yamashiro et al., 2018; Sasaki et al., 2015). IVG platforms have provided valuable tools to dissect the transcriptional and epigenetic mechanisms underlying germline specification and subsequent gametogenesis. Recent studies using IVG-derived germ cells or primate embryos in vivo have revealed a substantial divergence in the origin of germ cells and transcriptional networks governing germ cell specification between mice and humans (Saitou and Miyauchi, 2016). For example, in mice, core germ cell transcription factors, Prdm14, Blimp1, and Tfap2c, that are deployed by bone morphogenetic protein 4 (BMP4)-induced TBXT, sufficiently establish germ cell fate (Aramaki et al., 2013; Nakaki et al., 2013), whereas SOX17 and TFAP2C, deployed by EOMES and GATA2/3, make up the analogous transcriptional network and fate in humans (Kojima et al., 2017; Kojima et al., 2021). Such divergence between mice and humans necessitates additional layers of caution in direct translation of IVG technologies to human infertility treatment and warrants careful scrutinization and functional validation of IVG-derived gametes in comparison to those developing naturally in vivo. Since ethical and legal constraints make research with human embryos difficult to impossible, IVG studies using model organisms that are phylogenetically close to humans is an important next step. The common marmoset (Callithrix jacchus) is a new-world monkey that shares many biological characteristics with humans, and thus, has been widely used for biomedical research to bridge the gap between rodent models and clinical translation (Kishi et al., 2014). Marmoset embryo development, including implantation and formation of fetal membranes, is well conserved with that in humans, serving as a powerful surrogate model for human post-implantation development (Moore et al., 1985). Moreover, the relatively short reproductive lifespan, small body size, and reasonable cost for breeding compared to other primates render the marmoset a tractable preclinical model for IVG. In particular, use of marmosets permits vigorous validation of intermediary cellular derivatives by comparing them with their in vivo counterparts (Kishi et al., 2014) and may enable future functional validation of resultant IVG-derived gametes by fertilization and embryo transfer. In this study, we provide a highly efficient method to generate, expand, and maintain C. jacchus (cj)PGCLCs from cjiPSCs and demonstrate that these cells are immunophenotypically and transcriptionally similar to pre-migratory stage cjPGCs. Results Immunohistochemical characterization of pre-migratory cjPGCs To validate germ cell generation in vitro, we must first have a precise understanding of the molecular features of cjPGCs in vivo. In particular, molecular characterization of early stage endogenous PGCs is critical to guide the first step of IVG – the induction of PGCLCs that appear to represent pre-migratory PGCs in humans (Sasaki et al., 2015; Sasaki et al., 2016). However, there is a dearth of information describing primate PGCs at stages before gonad colonization, primarily due to their scarcity. Therefore, we collected marmoset embryos from a triplet pregnancy at embryonic day (E)50 for immunofluorescence (IF) and molecular analyses (Carnegie stage [CS]11, 19 somites, corresponding to ~E8.5–9.0 in mice) (Figure 1A, Figure 1—figure supplement 1A). As we and others have previously identified TFAP2C, SOX17, and PDPN as specific markers of pre-migratory/migratory PGCs and PGCLCs in humans and macaque monkeys (Sasaki et al., 2015; Sasaki et al., 2016; Sakai et al., 2020; Li et al., 2017), we traced cjPGCs using these markers. At this stage, TFAP2C+SOX17+PDPN+ cjPGCs were predominantly localized within the ventral portion of the hindgut endoderm and exhibited round nuclei with generally lower DAPI intensity (Figure 1B and C). A few scattered cjPGCs were also seen in the adjacent hindgut mesenchyme outside of the basement membranes, suggestive of the initiation of active migration (Figure 1B). Additional IF analyses revealed that cjPGCs were mostly non-proliferative (i.e., MKI67-) and co-expressed pluripotency-associated markers (e.g., POU5F1, NANOG), but were negative for SOX2 (Figure 1D and E, Figure 1—figure supplement 1B, Figure 1—source data 1A). Notably, cjPGCs did not express later germ cell markers (e.g., DDX4 and DAZL), that are typically observed in testicular germ cells (i.e., prospermatogonia) (Figure 1—figure supplement 1C). IF analysis on cjPGCs showed increased global levels of histone H3 lysine 27 trimethylation (H3K27me3) and reduced global levels of histone H3 lysine 9 dimethylation (H3K9me2), consistent with the germline epigenetic reprogramming that occurs in mice, cynomolgus monkeys, and humans (Sasaki et al., 2016; Tang et al., 2015; Seki et al., 2007; Figure 1F). Figure 1 with 1 supplement see all Download asset Open asset Immunophenotypic characterization of pre-migratory Callithrix jacchus primordial germ cells (cjPGCs) at embryonic day (E)50. (A) Bright field images of a cj embryo at E50 (Carnegie stage [CS]11). Scale bar, 1 mm. (B) (Left) Immunofluorescence (IF) images of the hindgut in the cj embryo as in (A) (transverse section), stained as indicated. Laminin outlines the basement membranes of the hindgut endoderm. The white dashed line highlights the hindgut endoderm. Scale bars, 50 μm. (Right) Pie chart showing the number and location of cjPGCs present in representative cross sections. (C) IF of the same cj embryo for TFAP2C (green), SOX17 (red), PDPN (cyan), and DAPI (white). Magnified images of hindgut endoderm are shown at the bottom. Arrows denote nuclei of cjPGCs with lower DAPI intensity than that of surrounding endodermal cells. Scale bar, 50 μm. (D) (Top) IF of the cj embryo stained for MKI67 (green), NANOG (red), and PDPN (cyan), merged with DAPI (white). An arrowhead indicates MKI67+ cjPGC. (Bottom) Pie chart showing the number of MKI67+ cells in PGCs. Scale bars, 50 μm. (E) IF of the cj embryo for pre-migratory PGC markers (POU5F1 [green], SOX17 [red], and NANOG [red]) or gonadal stage PGC markers (DDX4 [red] and DAZL [green]), co-stained for PDPN (cyan). Merged images with DAPI (white) are shown on the right of each panel. Scale bars, 50 μm. (F) IF of the cj embryo for PDPN (cyan), co-stained for H3K27me3 or H3K9me2 (green). Scale bars, 50 μm. Relative fluorescence intensities of H3K27me3 and H3K9me2 in PDPN+ cjPGCs in comparison to those of surrounding somatic cells are shown on the left of each IF panel. Bar, mean. Statistical significance is determined by two-tailed Welch’s t test. Figure 1—source data 1 Negative control images for immunofluorescence studies. https://cdn.elifesciences.org/articles/82263/elife-82263-fig1-data1-v2.pdf Download elife-82263-fig1-data1-v2.pdf Transcriptomes of pre-migratory cjPGCs Having identified cjPGCs residing in the hindgut endoderm by IF studies, we next set out to determine the transcriptome of endogenous cjPGCs. Given the scarcity of cjPGCs and the lack of reliable surface markers to isolate them, we first enriched cjPGCs by dissecting the posterior portions of two marmoset embryos at E50, followed by trimming of the amnion and yolk sac (Figure 1A). These tissues were dissociated into single-cell suspensions and subjected to high-throughput single-cell RNA-sequencing (RNA-seq) using a 10× Genomics platform. In total, 34,458 cells (6 libraries comprising 12,665 and 21,793 cells from embryos A and B, respectively) were captured for downstream analyses (Figure 1—figure supplement 1D–I). These cells contained a median of 2224–4198 genes/cell at a mean sequencing depth of 46–102k reads/well and 27–45% sequence saturation. To determine the cell types, we conducted hierarchical clustering and uniform manifold approximation and projection (UMAP) mapping on the combined single-cell transcriptomes from both embryos. Using known markers and differentially expressed genes (DEGs), we identified a cluster representing cjPGCs (cluster 11 marked by TFAP2C, SOX17, and PDPN) along with other clusters including CLDN5+ endothelium (cluster 3), OSR1+PAX8+ intermediate mesoderm (cluster 4), FOXF1+ lateral plate mesoderm (cluster 5), and SOX2+ neutral tube (cluster 9) (Figure 2A–C). A full listing of all cell types that we identified and their DEGs are shown in Figure 2B and Figure 2—source data 1. Figure 2 Download asset Open asset Single-cell transcriptome analyses of Callithrix jacchus primordial germ cells (cjPGCs) at embryonic day (E)50 (Carnegie stage [CS]11). (A) Uniform manifold approximation and projection (UMAP), showing different cell types in cj embryos at E50. Cell clusters are annotated on the basis of marker genes. A cluster representing cjPGCs is encircled. (B) Heatmap showing differentially expressed genes identified among cell types. DEGs are defined as log2-fold change >0.25, p-value <0.01, and adjusted p-value <0.01. Representative top ranked genes are shown. (C) Key marker genes used for cell type annotation, shown as violin plots with log normalized expression. Violin plots for PGC marker genes are outlined by red dotted lines. (D) Gene ontology enrichment analysis of genes with significantly higher expression in cjPGCs. Bar color denotes enrichment fold changes over background. (E) Boxplot showing expression of key pluripotency-associated genes; germ cell, mesoderm/endoderm, and ectoderm marker genes; and DNA methylation and histone modification-associated genes. Center line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range. Figure 2—source data 1 Differentially expressed genes (DEGs) among cell clusters in Figure 2B. https://cdn.elifesciences.org/articles/82263/elife-82263-fig2-data1-v2.xlsx Download elife-82263-fig2-data1-v2.xlsx Analysis of DEGs in the cjPGC cluster revealed upregulated expression of potential germ cell specifier/regulator genes (e.g., DND1, KIT, PRDM1, SOX15, SOX17, TFAP2C), pluripotency-associated genes (e.g., DPPA3, KLF4, NANOG, POU5F1, TFCP2L1, UTF1, ZFP42), mesoderm/endoderm-associated genes (e.g., GATA4, TBXT), and other germ cell-related markers (Figure 2B–E). Accordingly, these genes were enriched with those bearing GO terms such as ‘germ cell development’ (Figure 2D). Among pluripotency-associated genes, SOX2 was not expressed by cjPGCs, and PRDM14 was expressed only weakly, a feature conserved with other primates (i.e., humans, cynomolgus monkeys; Figure 2E; Sasaki et al., 2015; Sasaki et al., 2016; Li et al., 2017; Tang et al., 2015). Expression of key proliferation markers was low, in agreement with MKI67 labeling (Figure 1D), suggesting that cjPGCs are largely quiescent (Figure 1—figure supplement 1J). Germ cell markers known to be activated upon arrival at the gonads were not expressed (e.g., DAZL, DDX4, RNF17) (Figure 2E), consistent with the pre-migratory state of these cells (Sasaki et al., 2016; Li et al., 2017). In agreement with globally low H3K9me2 levels (Figure 1F), cjPGCs expressed low levels of enzymes for the deposition of H3K9me2 (e.g., EHMT2, SUV39H1, SUV39H2), and instead, expressed several H3K9 demethylases (e.g., KDM1A/3A/3B/4A) (Figure 2E). Among enzymes involved in the deposition of H3K27me3, cjPGCs expressed EED, EZH2, SUZ12, whereas EZH1 expression was low (Figure 2E). These findings are consistent with PGCLCs/PGCs in humans and cynomolgus monkeys (Sasaki et al., 2015; Sasaki et al., 2016; Tang et al., 2015). Among genes related to DNA demethylation, TET1 was expressed at high levels, whereas TET2 and TET3 were expressed at low levels (Figure 2E). DNMT1 and DNMT3A were expressed at modest levels, whereas UHRF1, DNMT3L, DNMT3B were markedly lower, suggesting that passive demethylation might be operative due to diminished UHRF1 activity required for maintenance DNA methylation, as suggested in other species (Sasaki et al., 2016; Tang et al., 2015; Kagiwada et al., 2013; Guo et al., 2015; Gkountela et al., 2015). Derivation of cjiPSCs through peripheral blood monoculear cell reprogramming Our next goal was to derive cjiPSCs, from which germ cells could potentially be induced. Three cell lines, 20201_6, 20201_7, and 20201_10, were established by reprogramming of peripheral blood mononuclear cells (PBMCs) (Materials and methods). Although hematological chimerism is frequently observed in marmosets (Ross et al., 2007; Benirschke et al., 1962), whole-exome sequencing confirmed that the established cjiPSCs originated from the intended PBMC donor (ID number, 38189) (Figure 3—figure supplement 1A, B). CjiPSCs were initially established using conventional on-feeder (OF) culture conditions (see below), but were subsequently switched to feeder-free (FF) culture conditions (PluriSTEM for basal medium and iMatrix-silk for a substrate) for its ease of maintenance. Under these conditions, FF cjiPSCs could be stably maintained over multiple passages (more than 20 passages) when passaged every 4–6 days in the presence of Y27632, a ROCK inhibitor. FF cjiPSCs bore a high nuclear to cytoplasmic ratio, were tightly packed in colonies with sharp borders and exhibited flat morphology, each of which are characteristic features of primate primed-state pluripotent cells (Figure 3—figure supplement 1C). These cells were mycoplasma-free, exhibited normal 46, XY karyotypes, and uniformly expressed key pluripotency-associated genes (Figure 3—figure supplement 1D–G). Notably, similar to FF culture, conventional OF cultures allowed long-term propagation of cjiPSCs. However, OF cjiPSCs tended to differentiate at the center or periphery of colonies 4–5 days after passaging (Figure 3—figure supplement 2A, B). Moreover, OF cjiPSCs required clump passaging because single-cell passaging decreased colony formation after two passages (Figure 3—figure supplement 2C). Accordingly, OF cjiPSCs exhibited modest upregulation of mesodermal (e.g., T, EOMES, MIXL1) and endodermal genes (e.g., FOXA2, SOX17) compared to those maintained under FF conditions (Figure 3—figure supplement 2D). Previous studies showed that inhibition of WNT signaling stabilizes primate iPSC/ESC cultures (Sakai et al., 2020; Kim et al., 2013; Wu et al., 2015). Consequently, we compared our conventional OF culture to cultures containing a WNT signaling inhibitor (IWR1). Notably, OF cjiPSCs cultured with PluriSTEM containing IWR1 (OF/IWR1) maintained an undifferentiated morphology and pluripotency-associated gene expression (Figure 3—figure supplement 2E, F, Figure 1—source data 1A). Under this condition, mesoderm/endoderm genes were suppressed compared with conventional OF culture. Moreover, OF/IWR1 culture conditions allowed efficient single-cell passaging (Figure 3—figure supplement 2C). While we also found that addition of IWR1 to OF culture conditions previously utilized to grow cynomolgus monkey ESCs (AITS+IF20: advancedRPMI1640 and Neurobasal [1:1] supplemented with IWR1 and suppressed the were not as as when PluriSTEM was used as a basal medium (Figure 3—figure supplement with the of inhibition in cjiPSCs two and increased expression of pluripotency-associated surface and (Figure 3—figure supplement Although cjiPSCs cultured under conditions in the expression of genes, all cjiPSCs expressed key pluripotency-associated markers (Figure 3—figure supplement Figure 3—figure supplement and a differentiation confirmed their potential to differentiate into all germ layers (Figure 3—figure supplement our findings that we have identified an culture in both FF and OF conditions that stable propagation of cjiPSCs in an undifferentiated state and with a normal thus serving as a foundation for differentiation toward the of PGCLCs from cjiPSCs Our next goal was to derive cjPGCLCs from cjiPSCs the established in humans and cynomolgus monkeys (Sasaki et al., 2015; Sakai et al., For we first FF cjiPSCs with a induction (i.e., stem cell in medium supplemented with or and supplemented with basal Under these conditions, cjiPSCs with a markedly and did not cjPGCLCs (Figure 3—figure supplement suggesting that may not have germline we next our to OF cjiPSCs WNT inhibition in humans (Sasaki et al., 2015). Remarkably, upon culture with a induction in or cells as a starting at of the of such cells generally after (Figure 3—figure supplement of these at revealed small clusters of PDPN+ cells uniformly cjPGC markers SOX17, PRDM1, NANOG, and which was confirmed by (Figure 3—figure supplement E, that the relatively low induction of cjPGCLCs might be due to their to differentiate under OF Therefore, we next utilized OF/IWR1 cjiPSCs for cjPGCLCs Upon induction in culture, cjiPSCs and uniform compared to those induced from OF cjiPSCs (Figure and B). Moreover, under this condition, the induction of cjPGCLCs was significantly with cells at and of induction (Figure Figure 3—figure supplement Although across the median of PDPN+ cells was at and but (Figure IF of of at revealed clusters of PDPN+ cells uniformly key early germ cell markers (e.g., SOX17, TFAP2C, PRDM1, POU5F1, (Figure Notably, this that PDPN can as a highly specific surface marker of cjPGCLCs that for of cjPGCLCs for downstream In of PDPN+ cjPGCLCs also expressed pluripotency-associated genes (i.e., POU5F1, NANOG), PGC marker genes (i.e., SOX17, TFAP2C, PRDM1, and and SOX2 and germ cell marker (i.e., DDX4, (Figure features similar to pre-migratory PGCs 1D and 2E; Sasaki et al., 2016). these that our in vitro enables highly efficient and generation of Figure with see all Download asset Open asset of Callithrix jacchus primordial germ cell-like cells from cj induced pluripotent stem cells (A) for (B) images and cell plots for the of cjiPSCs induced to differentiate into The of cells are shown. Scale bars, μm. (C) Boxplot of the induction of cells number of during induction in Center line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range. (D) Immunofluorescence (IF) images of after days of stained as indicated. Scale bars, 50 μm. (E) Gene expression of cjiPSCs and cjPGCLCs at days and as by For each gene the were using the of the two genes and as and for two cjPGCLCs culture Induction of cjPGCLCs from cjiPSCs is and limited in As of PGCLCs that the cellular and molecular characteristics of PGCs our to PGCLCs in a that can be for downstream molecular and functional To we a culture method previously utilized to human (Figure et al., we cultured PDPN+ cjPGCLCs on a layer in medium containing fetal and cjPGCLCs which increased in and by culture day (Figure These cells expressed markers of early but did not germ cell markers (i.e., DDX4, DAZL), suggesting that the cellular state of cjPGCLCs (Figure Figure 1—source data 1A). Moreover, these cells could be passaged every days by and cell of PDPN+ cells and exhibited for at days (Figure and Although marker expression was largely during days of culture, showed modest similar to under culture (Figure which is a surface marker expressed on also exhibited modest upregulation along the (Figure These findings the of culture of cjPGCLCs analogous to Figure 4 Download asset Open asset culture of Callithrix jacchus primordial germ cell-like cells (A) for culture of (B) images of and colonies of The white dashed colonies of Scale bars, μm. (C) cell analysis of cultures of The of cells is shown. (D) Immunofluorescence (IF) images of culture day cjPGCLCs for DAPI TFAP2C and SOX17 (red), and the merged Scale bars, 50 μm. (E) Gene expression of cjPGCLCs and as by For each gene the from the of the two genes and as were and for two not (F) analyses of and The and mean fluorescence intensity of cells are shown. of cells during culture A of PGCLCs were used as a starting cell analyses of the expression of the genes during culture. are by a not of cjPGCLCs into early prospermatogonia-like state of the functional features of PGCLCs is their to develop into germ cells (Hwang et al., 2020; Yamashiro et al., 2018; et al., et al., Therefore, we next utilized a reconstituted testis culture that to into to determine cjPGCLCs could differentiate (Hwang et al., of cjPGCLCs for days by culture, we an culture by PDPN+ cjPGCLCs with mouse fetal testicular somatic cells of endogenous germ cells (Figure 2 days of culture, reconstituted which were subsequently maintained by cultures (Figure and B). At day of culture, we observed reconstituted testicular by cells in cultures (Figure Figure 1—source data 1A). Notably, there were a number of which primarily localized peripheral to cell nuclei (Figure In addition, maintained day of culture revealed proliferation of germ cells, which cells toward the center of the testicular (Figure Remarkably, we found a few scattered cells cells among all and cells among all suggesting into early (Figure et al., these data that cjPGCLCs can be in the testicular and are of and Figure Download asset Open asset of Callithrix jacchus primordial germ cell-like cells into a prospermatogonia-like state. (A) for culture. reconstituted mouse gonadal somatic
Source: eLife
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $2,500
OpenAlex ID: W4321266413