Specification of primordial germ cells (PGCs) marks the beginning of the totipotent state. However, without a tractable experimental model, the mechanism of human PGC (hPGC) specification remains unclear. Here, we demonstrate specification of hPGC-like cells (hPGCLCs) from germline competent pluripotent stem cells. The characteristics of hPGCLCs are consistent with the embryonic hPGCs and a germline seminoma that share a CD38 cell-surface marker, which collectively defines likely progression of the early human germline. Remarkably, SOX17 is the key regulator of hPGC-like fate, whereas BLIMP1 represses endodermal and other somatic genes during specification of hPGCLCs. Notable mechanistic differences between mouse and human PGC specification could be attributed to their divergent embryonic development and pluripotent states, which might affect other early cell-fate decisions. We have established a foundation for future studies on resetting of the epigenome in hPGCLCs and hPGCs for totipotency and the transmission of genetic and epigenetic information.

We have developed a robust approach for hPGCLC specification from germ cell competent hESCs/hiPSCs (). We show that SOX17, a critical transcription factor for endoderm lineages, is the earliest marker of hPGCLCs and is in fact the key regulator of hPGCLC fate, which is not the case in mice (). BLIMP1 is downstream of SOX17, and it represses endodermal and other somatic genes during hPGCLC specification. Comparisons among hPGCLCs, embryonic hPGCs, and a seminoma indicate likely progression of the early human germline. These cells also exhibit CD38 cell surface marker, which is shared by cells with germ cell characteristics. We anticipate that genome editing approaches with our robust in vitro model for hPGCLC specification, combined with patient-specific human-induced pluripotent stem cells (hiPSCs), will lead to major advances in human germ cell biology, including on the unique germline-specific epigenetic program with potential consequences for subsequent generations.

Human PGCLCs (hPGCLCs) have been generated at a low frequency by spontaneous differentiation of human ESCs (hESC) in vitro (), but systematic studies to characterize and identify the key regulators of hPGCs remain to be elucidated. Because there are evident differences between the regulation of mouse and human pluripotent ESCs () and during their early postimplantation development (), this might affect the mechanism and the role of the key regulators of hPGCLC specification (). Once the mechanism of hPGCLC specification is established, it could provide insights on the progression of the early human germline with reference to embryonic hPGCs and seminomas that originate from human germ cells in vivo and retain key characteristics of the lineage ().

The ontogeny of cKIT+ human primordial germ cells proves to be a resource for human germ line reprogramming, imprint erasure and in vitro differentiation.

In mice, BMP4 induces expression of BLIMP1 (encoded by Prdm1) and PRDM14 in the postimplantation epiblast at E6.25; together with AP2γ (encoded by Tfap2c), a direct target of BLIMP1, they induce PGC fate (). The tripartite genetic network acts combinatorially to repress somatic genes, induce expression of PGC genes, such as Nanos3, reinduce pluripotency genes, and initiate the epigenetic program (). PGC-like cells (PGCLCs) can also be induced in vitro from naive pluripotent mouse embryonic stem cells (mESCs) after they acquire competence for germ cell fate after ∼48 hr culture in basic fibroblast growth factor (bFGF) and Activin A (). These competent cells acquire PGC-like fate in response to either BMP4 signal or directly to Blimp1, Prdm14, and Tfap2c, which is similar to PGCs in vivo ().

Primordial germ cells (PGCs) are the precursors of sperm and eggs, which generate the totipotent state. The genetic basis of mammalian PGC specification was first established in mice (), which are specified from postimplantation epiblast cells on embryonic day (E)6.25 in response to bone morphogenetic protein 4 (BMP4) (). Subsequently, ∼35 founder PGCs are detected at E7.25. Similar studies on human PGCs (hPGCs) would require E9–E16 embryos, which is not practicable. However, embryonic hPGCs at approximately week 5 to 10 of development, which correspond to mouse PGCs at E10.5–E13.5, can in principle be examined (). These cells retain characteristic of PGCs while they undergo resetting of the epigenome and global DNA demethylation ().

We also asked whether hiPSCs could be used to generate and isolate hPGCLCs using the combination of surface markers CD38 with TNAP ( Figures 2 C and 3 A–3D). Using FX71.1 hiPSCs (see Experimental Procedures ) maintained in 4i medium for >2 weeks that lack CD38 expression, we detected∼31% of TNAP/CD38 double-positive cells after 4 days in response to cytokines ( Figure 7 B). TNAP/CD38 double-positive hPGCLCs showed expression of NANOS3, BLIMP1, TFAP2C, SOX17, STELLA, T, OCT4, NANOG, and PRDM14, but not of SOX2 ( Figure 7 C). Similar results were obtained with another hiPSC line (C1,). Thus, hPGCLC specification could be induced efficiently and directly in hiPSCs that are maintained in the 4i medium, which could be used for disease modeling using patient-derived iPSCs.

Global gene expression analysis indicated overall similarities between hESCs in the conventional medium versus those in “4i” medium (r = 0.923) but with notable differences ( Figure S7 A). Although these cells showed similar expression levels of core pluripotency factors OCT4, NANOG, and SOX2, 4i hESCs had higher expression of mesoderm and gastrulation genes, including T, RUNX1, and PDGFRA ( Figures S7 B and S7C and Table S2 ). Furthermore, OCT4-positive cells in 4i hESCs had varying levels of T protein, possibly due to inhibition of GSK3β (), which is not the case in hESC cultured in conventional condition ( Figure S7 D). These differences might be relevant for the mechanism of competence of ESCs for PGCLC, which merits further investigation.

(C) Heat map showing expression of representative pluripotency and mesodermal genes expression in WIS2 hESCs cultured under 4i or conventional conditions (Conv). RNASeq data from two biological replicates were shown (#1 and #2). Asterisk indicates differential expression with statistical significance (log 2 (fold change) > 2 and adjusted p value < 0.05).

(B) Gene ontology (GO) term enrichment analysis of upregulated genes in 4i WIS2 hESC (upper panel) and conventional WIS2 hESC (lower panel). Top 15 GO biological process terms that were enriched in each condition were shown (DAVID GOTERM_BP_FAT with gene count > = 5 followed by GO Trimming to reduce term redundancy).

(A) Scatter plot showing global gene expression levels (mean log 2 (normalized read counts) of two replicates) between WIS2 hESC cultured under 4i and conventional condition. Each dot represents one gene. Differentially expressed genes (log 2 (fold change) > 2 and adjusted p value < 0.05) were presented as red dots (upregulated in 4i condition) or blue dots (upregulated in conventional condition).

Because gene expression of hESCs in 4i medium resembles that of hESC after preinduction for 2 days in bFGF/TGFβ ( Figures 2 A, 2B, and S2 A), we decided to investigate hPGCLC induction directly in hESCs maintained in 4i medium ( Figure 1 A). Indeed, hPGCLCs could be induced directly from 4i hESCs with apparent enhanced response resulting in ∼46% hPGCLCs ( Figure 7 A ). These hPGCLCs showed a slightly higher intensity of NANOS3/TNAP by FACS, and a greater proportion of them were CD38 positive ( Figure 7 A). Notably, cells maintained for more than 2 weeks in the conventional hESC medium, regardless of whether they were initially maintained in 4i medium, showed a significantly lower numbers of hPGCLCs (∼5%) with a reduced intensity of NANOS3-mCherry/TNAP and CD38 expression ( Figure 7 A). This demonstrates that hESCs in 4i medium are highly competent for the hPGCLC fate. Importantly, the competent state is conferred reversibly because it is gained and lost in 4i and conventional culture conditions, respectively.

(D) Overview of human germline development. hESCs in 4i reversibly attains competence for germ cell fate. Exposure of 4i cells to cytokines containing BMPs results in strong induction of hPGCLCs following expression of SOX17-BLIMP1, which are among the key regulators of germ cell fate. SOX17 and BLIMP1 are detected in in vivo gonadal hPGC and TCam-2 seminoma, indicating a likely progression of early human germ cell lineage. CD38, a cell-surface glycoprotein, is shared by all cells with germ cell characteristics, but not by hESC. Loss of SOX17 or BLIMP1 abrogates hPGCLC specification.

(C) Expression analysis by RT-qPCR on TNAP-positive hiPSCs (iPSC TNAP+), TNAP/CD38 double-negative (TNAP−CD38−) population and TNAP/CD38 double-positive population (TNAP+CD38+) on day 4 after hPGCLC induction. Relative expression levels are shown with normalization to β−ACTIN. Error bars indicate mean ± SD from two independent biological replicates.

(A) FACS analysis of TNAP and NANOS3-mCherry (top) and TNAP and CD38 (bottom) on day 4 embryoids induced from 4i hESCs after preinduction (left), directly without preinduction (middle) or from conventional hESCs (right, Conv hESC).

To determine the competency of the SOX17 null hESCs, we transfected an inducible SOX17 fusion construct with human glucocorticoid receptor ligand-binding domain (GR) into the SOX17 null hESCs. This would allow dexamethasone (Dex) to activate the SOX17-GR and induce translocation of SOX17 fusion protein from the cytoplasm into the nucleus (). After 5 days of induction with cytokines and Dex in the SOX17 null SOX17-GR hESCs, expression of germ cell genes BLIMP1, TFAP2C, OCT4, NANOG, and KIT and the TNAP/CD38-positive population was restored ( Figures 6 E and 6G). This demonstrates that SOX17 null hESCs maintain competency for hPGCLC specification. Strikingly, activation of SOX17 alone in the absence of cytokines was sufficient to induce germ cell genes and TNAP/CD38-positive cells from 4i hESCs ( Figures 6 F and 6G). Taken together, SOX17 is indispensable and sufficient for hPGCLC gene induction from competent hESCs, and it acts upstream of BLIMP1 and other genes to initiate the human germ cell transcriptional network ( Figure 6 H). Interestingly, loss of SOX17 in TCam-2 also causes a repression of germ-cell- and pluripotency-associated genes ( Figure S6 A). This suggests that SOX17 might also be important for the maintenance of the germ cell state because it is also highly expressed in embryonic hPGCs.

To determine whether SOX17 acts cell autonomously, we mixed wild-type NANOS3-mCherry hESCs with the SOX17 null hESCs in 1:1 ratio during induction of hPGCLCs by cytokines. All NANOS3-mCherry positive cells detected by immunofluorescence on day 4 were SOX17 positive ( Figure 6 D), indicating that SOX17 null hESCs did not undergo hPGCLC specification even in the presence of wild-type cells. The overall number of NANOS3-mCherry-positive cells in the embryoid with mixed cells was about half of that in the control consisting of wild-type cells only ( Figure S6 D), suggesting that SOX17 null cells did not affect PGCLC induction from wild-type cells. Thus, SOX17 null cells have intrinsic defect for hPGCLC specification.

We addressed the role of SOX17 during hPGCLC specification by generating SOX17 KO NANOS3-mCherry hESC line ( Figure S6 B) and validated absence of SOX17 expression in day 4 embryoids from mutant cells by western blot and immunofluorescence ( Figures 6 A and S6 C). Notably, we did not detect any NANOS3-mCherry or TNAP-positive cells in the embryoids from SOX17 mutant cells ( Figure 6 B). Further, RT-qPCR analysis of day 4 SOX17 null embryoids showed absence of NANOS3, TFAP2C, DND1, UTF1, KLF4, OCT4, NANOG, and, importantly, BLIMP1 ( Figure 6 C). Instead, there was upregulation of mesodermal genes PDGFRA, KDR, and HOXA1 ( Figure 6 C). Although a few TFAP2C-positive cells were detected on day 4, they were BLIMP1 negative and most likely belong to other lineages ( Figure S6 C).

Expression of SOX17 among T-positive cells prior to BLIMP1 apparently marks the onset of hPGCLC specification, which is a key difference between the specification of human and mouse germline fate (see Figure 4 ). Notably, SOX17 and BLIMP1 are also expressed in the authentic in vivo hPGCs and in TCam-2 () ( Figure 2 ). Knockdown of SOX17 in TCam-2, which exhibits key germ cell characteristics () ( Figure 2 ), induced repression of the pluripotency genes NANOG, as well as of the PGC-genes BLIMP1, NANOS3, TFAP2C, STELLA, and KIT ( Figure S6 A). This suggests that SOX17 might be important for regulating the established germline gene expression network.

(C) Immunofluorescence of SOX17, BLIMP1 and TFAP2C (left panel); and TFAP2C and NANOS3-mCherry on wild-type (WT) and SOX17 KO day 4 embryoids. Scale bars = 70 μm

(A) Expression analysis by RT-qPCR of pluripotency and germ cell genes after knock-down of SOX17 in TCam-2. Two independent, miRs against SOX17 (S17 miR #1 and #2) were used for the knockdown with a scramble miR as control. Error bars are mean ± SD. Relative expression levels are shown with normalization to GAPDH. Representative data were shown from two independent biological replicates.

We isolated and characterized the TNAP-positive cells by FACS and confirmed loss of BLIMP1, except for low expression of mutant transcripts ( Figure 5 D). These cells also showed loss of NANOS3, UTF1, and KLF4 and reduced expression of TFAP2C, DND1, OCT4, NANOG, and T ( Figures 5 D and S5 B). In addition, they showed prominent upregulation of mesodermal/primitive streak and HOX genes, as well as endodermal genes, including GATA4, GATA6, FOXA1 HNF1β, and HNF4α ( Figure 5 D). By contrast, endodermal genes were not upregulated in Blimp1 mutant mouse PGCs (). This suggests that BLIMP1 probably suppresses endoderm and other somatic genes, which might otherwise be induced by SOX17 and BMP signaling during hPGCLCs specification ( Figure 6 H). Loss of BLIMP1 and TFAP2C also caused upregulation of HOX genes in TCam-2 (). This suggests that one of the roles of BLIMP1 is to continually suppress the somatic program during human germline development.

(H) Model for establishment of hPGC transcription network by SOX17 and BLIMP1. SOX17 induces germ cell genes and, potentially, endoderm gene. Expression of BLIMP1, downstream of SOX17, suppresses endodermal genes, as well as mesodermal genes. As a result, the SOX17-BLIMP1 axis initiates hPGC program from competent cells upon induction by BMP signaling. The hPGC specification gene network is abrogated in the absence of SOX17 or BLIMP1.

(G) RT-qPCR analysis of day 5 hPGCLC derived from WT and SOX17 KO (S17KO) and SOX17 KO + SOX17-GR (S17KO+S17GR) hESCs with (+) or without (−) dexamethasone (Dex) and in the presence (+) or absence (−) of cytokines. FACS-sorted NANOS3-mCherry/TNAP double-positive cells or whole embryoids (for S17KO) were used. Relative expression levels are shown with normalization to GAPDH. Error bars indicate mean ± SD from two biological replicates.

(E and F) FACS analysis of TNAP and CD38 on day 5 embryoids derived from SOX17 knockout 4i hESCs containing SOX17 fusion construct with human glucocorticoid receptor ligand-binding domain (SOX17 KO+ SOX17 GR). Embryoids were derived in the presence (E) or absence (F) of cytokines with (Dex+) or without (Dex−) addition of dexamethasone.

(D) Immunofluorescence of day 4 embryoids derived from WT, SOX17 knockout (SOX17 KO), and from 1 to 1 mixture of WT and SOX17 KO 4i hESCs. The number of NANOS3-mCherry+ cells with or without SOX17 expression is shown. Quantification was based on seven to nine confocal images from four independent embryoids of each condition. Scale bars, 50 μm.

(C) RT-qPCR analysis of TNAP/NANOS3-mCherry FACS-sorted WT double-negative (TNAP-N3-) or -positive (TNAP+N3+) cells sorted from day 4 embryoids and whole SOX17 KO embryoids (SOX17 KO). Relative expression levels are shown with normalization to β−ACTIN. Error bars indicate mean ± SD from two independent biological replicates.

(A) Western blot analysis of SOX17 expression of WT day 4 TNAP/NANOS3-mCherry-positive hPGCLCs (WT, TNAP+N3+), and whole SOX17 knockout day 4 embryoids. TUBULIN was used as loading control.

The zinc finger transcriptional repressor Blimp1/Prdm1 is dispensable for early axis formation but is required for specification of primordial germ cells in the mouse.

BLIMP1 is the first and key regulator of mouse PGC, and loss of function abrogates PGC fate (). However, BLIMP1 expression is apparently downstream of SOX17 in hPGCLCs ( Figures 4 A and 4C). We examined its mechanistic role by generating BLIMP1 knockout (KO) NANOS3-mCherry hESC line ( Figure S5 A). These cells showed loss of BLIMP1 by western blot ( Figure 5 A) and immunofluorescence ( Figure S5 B) on day 4 of hPGCLCs induction. Notably, there was also a loss of NANOS3-mCherry-positive cells, together with a significant reduction of NANOG, OCT4, and TFAP2C expression on day 4 ( Figures 5 C and S5 B), indicating a failure of hPGCLC specification, and all of these cells disappeared by day 8 ( Figure 5 C). However, we detected ∼8% of TNAP-positive cells in day 4 embryoids ( Figure 5 B). This observation is highly reminiscent of the effects of Blimp1 mutation on mouse PGC specification ().

(D) Expression analysis by RT-qPCR for WT TNAP/NANOS3-mCherry double-positive cells (WT; TNAP+N3+) and BLIMP1 KO TNAP single-positive cells (BLIMP1 KO; TNAP+) sorted from day 4 embryoids. Relative expression levels are shown with normalization to β−ACTIN. Error bars indicate mean ± SD from two independent biological replicates.

(C) Immunofluorscence for OCT4 and SOX17 in cryosections of WT and BLIMP1 KO day 4 and 8 embryoids. OCT4-positive cells are highlighted. Scale bar, 50 μm.

(A) Western blot analysis of BLIMP1 and SOX17 in TNAP-positive (TNAP+) cells sorted from wild-type (WT) and BLIMP1 knockout (BLIMP1 KO) day 4 embryoids after hPGCLC induction. TUBULIN was used as loading control.

The zinc finger transcriptional repressor Blimp1/Prdm1 is dispensable for early axis formation but is required for specification of primordial germ cells in the mouse.

The SOX17/BLIMP1 double-positive cells were initially distributed randomly in day 1 embryoids ( Figure 4 A) but were then loosely organized in clusters and often a single cluster in day 2 embryoids. By day 4, generally one and occasionally two tight clusters of hPGCLCs were observed either at the core or periphery of each embryoid ( Figure 4 E). Cumulative observations suggest that SOX17/BLIMP1 might be among the key regulators of hPGCLC specification. Although OCT4 and NANOG were detected between days 1 and 2 in conjunction with NANOS3-mCherry and other PGC-specific genes from days 2–4, PRDM14 was upregulated more gradually in hPGCLCs and was subsequently detected in the cytoplasm of embryonic hPGCs. Following the early expression of SOX17 and BLIMP1 in hPGCLCs, these two transcription factors were also detected in embryonic hPGCs in vivo, as well as in TCam-2 ( Figures 2 E and 2H). These observations suggest that SOX17-BLIMP1 might be among the critical determinant of hPGC specification and maintenance.

PRDM14 is a key regulator of pluripotency in mouse and human ESCs () and is a key regulator of mouse PGC specification (). PRDM14 was generally downregulated in day 1–2 embryoids but was detectable in the nucleus of most BLIMP1+ cells by day 4 ( Figure S4 A). Notably, in a minority of BLIMP1/NANOG-positive hPGCLCs at day 8, PRDM14 was enriched in the cytoplasm ( Figure S4 A), which was the case in most of the gonadal hPGCs ( Figure 2 F). This is in marked contrast to the persistent nuclear PRDM14 expression in mouse PGCs ().

Expression of OCT4 was low but widespread in the day 1 embryoids, including 75% of the BLIMP1+ cells ( Figures S4 B and 4D). Although the overall OCT4 expression declined dramatically in day 2 embryoids, it was strongly expressed in ∼86% of the BLIMP1+ cells. Subsequently, all BLIMP1+ cells became highly OCT4+ by day 4. By contrast, NANOG was expressed in ∼35% of BLIMP1+ cells on day 1, but it was generally absent in other cells in the embryoids ( Figures 4 D and S4 A). Thereafter, NANOG was also rapidly upregulated in the majority of BLIMP1+ cells by day 2–4. The upregulation of key pluripotency genes, such as OCT4 and NANOG, is also reminiscent of their re-expression in mouse PGCs (). Although NANOS3-mCherry expression was weakly detected in 24% of OCT4+ cells at day 2 ( Figure S4 C), it was detected in all OCT4+ cells on day 4, confirming their PGCLC identity.

(A–C) Immunofluoresence of (A) PRDM14 and NANOG; (B) OCT4; and (C) NANOS3-mCherry on cryosections of day 1-8. hPGCLC were counterstained with BLIMP1 or OCT4 as highlighted. Arrowheads indicate enrichment of PRDM14 in cytoplasm. Scale bars = 70 μm.

Expression of T is of particular interest, as it signifies competence for germ cell fate in mice, and BMPs can induce it in hESCs (). Notably, expression of T was high in the majority of cells on day 1, except for most of the BLIMP1+ cells ( Figure 4 B). By day 2, however, T was dramatically downregulated in most cells, although now the BLIMP1+ nascent hPGCLC retained low T expression, which persisted until at least day 4 ( Figure 4 B), consistent with the T transcripts detected by RNA-seq ( Figure 2 C). It is possible that BMP signaling may initially enhance expression of T in the embryoids (), and it is from this population that hPGCLCs are specified, which reflects the events during mouse PGC induction ().

On day 1, we first detected SOX17 in a few widely scattered cells throughout the embryoids ( Figures 4 A and 4E ). Among the SOX17-positive (+) cells, 55% were also BLIMP1+, and 22% were TFAP2C+ ( Figures 4 A and 4C). However, all BLIMP1+ cells coexpressed SOX17, suggesting that SOX17 is upregulated before BLIMP1. The proportion of BLIMP1+ and TFAP2C+ cells increased to ∼70% on day 2 and to ∼90% on days 4–8 ( Figures 4 A and 4C). These triple-positive cells likely represent specified hPGCLCs, as they also coexpressed other key hPGC genes. However, ∼10% of single SOX17+ cells failed to undergo hPGCLC specification but persisted in day 4–8 embryoids. These may be aberrant cells or else may belong to other lineages.

(E) Summary model for dynamics of hPGCLC specification in embryoids. SOX17-positive cells are first scattered in day 1 embryoids. They gain expression of BLIMP1, TFAP2C, and NANOG sequentially and form a cluster from day 2 onward until the formation of nascent hPGCLC.

(D) Percentage of BLIMP1-positive (+) cells in day 1–8 embryoids that were TFAP2C+, NANOG+, or OCT4+. Corresponds to data in Figures 4 A, S4 A, and S4B.

(C) Percentage of SOX17-positive (+) cells in day 1–8 embryoids that were also TFAP2C+ or BLIMP1+. Corresponds to data in Figure 4 A.

(A and B) Immunofluorscence analysis for (A) BLIMP1, SOX17, and TFAP2C and (B) BLIMP1 and T in cryosections of day 1–8 embryoids after hPGCLC induction. Bottom row in (B) shows high exposure (digital) image of T, indicating low but specific expression in hPGCLC. SOX17-positive or BLIMP1-positive cells are highlighted. Scale bars, 50 μm.

Having established similarities between hPGCLCs and the authentic hPGCs, we set out to investigate the mechanism of hPGCLC specification. First, for establishing the precise sequence of expression of the key hPGC-related genes at the resolution of single cells, we performed systematic time course analysis by immunofluorescence on day 1–8 embryoids after hPGCLC differentiation.

Taken together, day 4 hPGCLCs, which are the nascent human germ cells, already showed evidence for the initiation of epigenetic changes and DNA demethylation that are comparable to E8 mouse PGCs (). Notably, we also found that PRMT5, an arginine methylatransferase that was ubiquitously but weakly present in the cytoplasm of day 1 and 2 embryoids, showed enhanced expression in the nucleus of day 4–8 hPGCLCs ( Figure S3 E). This is a shared characteristic with ∼E8 mouse PGCs, hPGCs, and TCam-2 seminoma (). The translocation of PRMT5 to the nucleus is important for the suppression of transposable elements at the onset of DNA demethylation ().

The RNA-seq of hPGCLC also revealed gene expression changes that indicate initiation of the epigenetic program with downregulation of UHRF1, DNMT3A, and DNMT3B and upregulation of TET1 and TET2 ( Figure S3 D). Notably, we found a significant increase in 5-hydroxymethylacytosine (5hmC) in hPGCLCs, which is consistent with an increase in the expression of TET1, an enzyme that converts 5-methylcytosine (5mC) to 5hmC ( Figures 3 E–3G), together with a small but significant decline in 5mC ( Figures 3 G and S3 A). This indicates that, as in the mouse PGCs, loss of 5mC might be coupled with the conversion of 5mC to 5hmC (). At the same time, we detected a decline in the expression of de novo DNA methyltransferase 3A (DNMT3A) and UHRF1 in hPGCLCs compared to the neighboring somatic cells in the embryoids ( Figures 3 G, S3 B, and S3C). UHRF1 targets DNMT1 to replication foci to confer maintenance of DNA methylation (). The repression of UHRF1 in proliferating (KI-67-positive) hPGCLCs would allow DNA-replication-coupled loss of 5mC, which is analogous to the observations on the early mouse germline.

(E) Immunofluoresence of PRMT5 on cryosections of embryoids collected at day 1, 2, 4 and 8 post-hPGCLC induction. hPGCLCs were counterstained with OCT4 as highlighted. Scale bar = 70 μm.

(A–C) Immunofluorescence analysis was carried out for (A) 5-methylcytosine (5mC); (B) DNMT3A; (C) UHRF1 on day 4 embryoids. TFAP2C, OCT4 or BLIMP1 were used to counterstain for hPGCLCs. hPGCLCs were highlighted by white dashed lines. White arrowheads showed examples of KI-67-positive PGCLCs while yellow arrows showed the KI-67-negative PGCLCs (C). Scale bars = 50 μm.

CD38, an established cell-surface glycoprotein on leukocytes, is a prognostic marker of leukemia (). Surprisingly, we detected CD38 expression in hPGCLCs, gonadal hPGCs, and TCam-2, but not in hESCs or gonadal somatic cells ( Figure 2 C). Indeed, fluorescence-activated cell sorting (FACS) analysis showed that CD38 is present on all the TNAP-positive embryonic hPGCs and on TCam-2 with some heterogeneity ( Figures 3 B and 3C ). Although CD38 is absent on hESCs, ∼50% of the NANOS3-mCherry-positive hPGCLCs were CD38 positive on day 4 ( Figure 3 A), which increased to ∼70% by day 5 ( Figure 3 A). Interestingly, the NANOS3-mCherry/CD38 cells had higher expression of NANOS3, BLIMP1, SOX17, OCT4, and NANOG ( Figure 3 D). By contrast, hESCs and embryonic carcinoma cells exhibit CD30 (also known as TNFRSF8) and SOX2 ( Figures 3 D and 2 G) (). Thus, CD38 and CD30 could potentially be used as additional markers of germ cell tumors in vivo ( Figure 7 D).

(G) Quantification of immunofluorescence intensity of various epigenetic marks/modifiers in hPGCLCs and somatic neighbors in day 1–4 embryoids (see also Figures S3 A–S3C). For UHRF1, only KI-67-positive (proliferating) cells were used for quantification. Numbers below each box denotes number of cells analyzed. Black central line represents the median, boxes and whiskers represent the 25and 75, and 2.5and 97.5percentiles, respectively. Wilcoxon signed-rank test was used to test for statistical significance. #p < 0.05;p < 0.0001.

(E and F) Immunofluorescence analysis for 5hmC (E) and TET1 (F) on day 4 embryoids cryosection. OCT4 or BLIMP1 were used to identify hPGCLCs (highlighted). Scale bars, 50 μm.

(D) Expression analysis by RT-qPCR for FACS-sorted TNAP-positive 4i hESCs (TNAP+ hESC) and CD38 low or high/NANOS3-mCherry day 5 hPGCLCs. Relative expression levels are shown with normalization to β−ACTIN. Error bars indicate mean ± SD from two independent biological replicates.

(A) FACS analysis of NANOS3-mCherry and CD38 on WIS2-NANOS3-mCherry cell line cultured in 4i medium and on day 4 and 5 embryoids following hPGCLC induction. Ratios of CD38 low and high expression in the NANOS3-mCherry-positive cells are indicated.

Taken together, hPGCLCs demonstrate germ cell characteristics that are apparently en route to hPGCs, whereas our objective analysis placed TCam-2 in an intermediate position, which reflects their origin from hPGCs in vivo. Notably, hPGCLCs evidently represent the earliest stages of the human germ cell lineage, indicating that our in vitro model provides an important opportunity to explore the mechanism of hPGC specification, which is otherwise not possible because E9–E14 postimplantation human embryos are excluded from investigations. TCam-2 and other seminomas might, however, also serve as important in vitro models of human germ cell biology ().

Principal component analysis (PCA) further illustrates the relationships between the different cell types. PCA reduces dimensionality of whole-genome expression data by transforming into principal components (PCs), in which the variance within the dataset is maximal. A three-dimensional (3D) PCA plot of the first three PCs showed that the 4i hESC, soma, and hPGC-related cells (hPGCLCs, gonadal hPGCs, and TCam-2) settled at three discrete positions ( Figure 2 B). In particular, hPGCLCs, TCam-2, and gonadal hPGCs aligned together at the lower extreme of PC2, whereas 4i hESCs and preinduced cells formed a distinct cluster with medium PC2 scores and soma at the upper extreme ( Figures 2 B and S2 B). The relative contributions (weights) of key germ cell, pluripotency, and gonadal somatic genes to PC2 and PC3 were plotted as two-dimensional (2D) loading plot alongside a corresponding 2D PCA plot ( Figure S2 B). Indeed, the weights of germ cell, pluripotency, and somatic genes highly overlap with the position of germ-cell-related cell types, hESCs, and soma, respectively. Germ-cell-related genes, such as SOX17, CD38, and NANOS3 loaded heavily for lower extreme of PC2, where hPGCLCs, TCam-2, and gonadal hPGCs were aligned. There was a clear difference in weights of early germ cell genes (commonly expressed in hPGCLCs, TCam-2, and gonadal hPGCs—for example, BLIMP1 and TFAP2C) and late germ cell genes (expressed only in gonadal hPGCs or TCam-2—for example, VASA and DAZL) on PC3, with the latter weighing more heavily toward low PC3 scores ( Figure S2 B). Notably, decreasing scores of PC3 reflected potential progression of germ cell development from hPGCLCs toward gonadal hPGCs, whereas TCam-2 aligned between hPGCLCs and gonadal hPGCs ( Figures 2 B and S2 B).

Given the similarities of hPGCLCs, hPGCs, and TCam-2, a three-way Venn diagram was plotted to investigate their relationships ( Figure 2 D). Out of 972 highly upregulated genes compared to soma ( Table S1 ), the three germline-related cell types shared expression of 161 genes, including pluripotency and germline-specific genes: BLIMP1, TFAP2C, CD38, SOX17, OCT4, and NANOG ( Figure 2 D). Gene ontology (GO biological process) analysis revealed ( Table S1 ) that hPGCLCs from male cell line and male gonadal hPGCs were commonly enriched in “spermatogenesis” genes—for example, NANOS3 and HIST1H1T—whereas meiosis-related SYCP3, MAEL, and PIWIL1 genes were upregulated only in embryonic hPGCs ( Figures 2 C and 2D). Interestingly, TCam-2 and hPGCs revealed expression of a number of late germ cell markers, including Tudor-domain-containing TDRD5, TDRD9, and TDRD12 genes, which have been implicated in PIWI-interacting RNA biogenesis pathway () ( Figure 2 D). As expected, TCam-2 showed characteristics associated with cancer cells, including genes that promote cell proliferation with suppression of apoptosis genes ( Figure 2 D). Altogether, hPGCLCs, TCam-2, and hPGCs share key germ cell characteristics and expressed the core germ cell genes, including CD38, whereas the differentially expressed genes reflected their corresponding stages of development and cell identity.

A heat map of mRNA expression revealed that hPGCLCs and gonadal hPGCs shared expression of early PGCs (BLIMP1, TFAP2C, DND1, NANOS3, UTF1, ITGB3, and KIT) and pluripotency genes (TNAP, OCT4, NANOG, PRDM14, and LIN28A) but with a notable lack of SOX2 expression ( Figure 2 C). Early mesoderm marker T was detected in hPGCLCs ( Figure 2 C), as in mouse early PGCs (). Interestingly, expression of two endodermal genes, SOX17 and GATA4, was detected in hPGCLCs, embryonic hPGCs, and TCam-2, which are absent in the mouse germline. Notably, we identified CD38 expression in hPGCLCs/hPGCs and TCam-2, but not in hESCs or soma ( Figures 2 C and see also Figures 3 A–3C). Overall, hPGCLCs indeed have germ cell characteristics consistent with hPGCs. Late germ cell markers, however, including DAZL, VASA, and MAEL, were only detected in hPGCs ( Figure 2 C). TCam-2 gene expression was similar to hPGCLCs, albeit with lower expression levels of NANOS3, ITGB3, and T and upregulation of a few somatic genes, e.g., HAND1 and RUNX1. Immunofluorescence analysis validated the expression of BLIMP1, TFAP2C, and OCT4 in hPGCLCs/hPGCs and TCam-2 ( Figures 2 E–2H). Interestingly, PRDM14 showed nuclear localization in the majority of hPGCLCs but was predominantly enriched in the cytoplasm of hPGCs ( Figure 2 F). Importantly, although SOX2 was undetectable, there was significant expression of SOX17 in hPGCLCs, hPGCs, and TCam-2 ( Figures 2 G and 2H).

Unsupervised hierarchical clustering of global gene expression showed that the hPGCLCs clustered with hPGCs and TCam-2, whereas 4i hESCs and preinduced cells (4i hESCs treated with bFGF and TGFβ for 2 days) clustered together in another branch away from gonadal somatic cells (soma) ( Figure 2 A). Consistently, hPGCs were globally more related to hPGCLCs (Pearson correlation coefficient [r] = 0.85) and TCam-2 (r = 0.818) than to 4i hESCs (r = 0.799) and preinduced cells (r = 0.773) ( Figure S2 A).

(B) Two-dimensional principal components analysis of gene expression (PC3 against PC2) of the indicated samples (left panel). A corresponding loadings plot indicates the weight of various genes on PC2 and PC3 (right panel). Gene names are color-coded to illustrate association with pluripotency (black), early germ cell development (green), late germ cell development (red) and gonadal somatic cell development (blue). Arrowline indicates potential germline progression from 4i hESC via hPGCLC to gonadal hPGC.

(A) Pearson correlation heat map of gene expression (log 2 (normalized read counts)) in various samples with unsupervised hierarchical clustering. The color key indicates the correlation coefficient.

We carried out RNA sequencing (RNA-seq) on NANOS3/TNAP double-positive cells from day 4 embryoids and compared them with the gonadal hPGCs from week 7 male human embryos (Carnegie stage 18/19), which are equivalent to mouse ∼E12.5–E13.5 PGCs (). These hPGCs retain key characteristics of earlier hPGCs but, consistent with their more advanced state, expresses later germ cell markers such as VASA and DAZL. We also included TCam-2, a human seminoma that originates from the germline in vivo ().

The NANOS3/TNAP double-positive putative hPGCLCs also expressed key PGC genes, including NANOS3, BLIMP1, TFAP2C, STELLA, TNAP, KIT, OCT4, and NANOG, as well as PRDM14, albeit with reduced levels compared to hESC ( Figure 1 C). Remarkably, SOX17 was significantly upregulated, whereas SOX2 was downregulated in the putative hPGCLCs that reflects their expression in embryonic hPGCs and seminomas (; see Figure 2 ), which is not the case in mouse PGCs. Immunofluorescence confirmed that NANOS3-mCherry expression coincided with OCT4, NANOG, and TFAP2C in day 4 embryoids ( Figures 1 D and S1 F), as did OCT4 with BLIMP1 ( Figure S1 F). This suggests that the NANOS3-mCherry-positive cells are very likely nascent germ cells.

(E–H) Immunofluorescence analysis for (E) BLIMP1, (F) PRDM14, (G) SOX2, and (H) SOX17 on 4i hESCs (top row), day 4 hPGCLC embryoids (second row), human week 7 male gonad (third row), and TCam-2 (bottom row). Samples were counterstained with TFAP2C or OCT4 to identify hPGCLCs in embryoids and hPGCs in embryonic gonad. Arrows indicate cytoplasmic enrichment of PRDM14 (F). Scale bars, 70 μm.

(D) Venn diagram illustrates common and differentially expressed genes. Significantly upregulated genes in hPGCLC, gonadal hPGC, and TCam-2 (with log2 (fold change) > 3 and adjusted p value < 0.05 versus gonadal Soma, respectively) were compared. Representative genes that were exclusive to each category are indicated. Text boxes indicate gene ontology biological processes (BP) terms that were significantly enriched as indicated by p values. Asterisk denotes custom categories absent from BP annotation.

(C) Heat map of gene expression of key PGC-associated genes (early and late) and of pluripotency, mesoderm, endoderm, and gonadal somatic (Soma) markers.

(A) Unsupervised hierarchical clustering (UHC) of gene expression in 4i hESC, preinduced cells (Pre-induced), day 4 hPGCLCs (hPGCLC), gonadal hPGC, TCam-2, and gonadal somatic cell (Soma). RNA-seq was performed on two biological replicates (#1 and #2) for each cell type.

Next, we tested hESC-NANOS3-mCherry cells that were maintained in four-inhibitor-containing medium with LIF, bFGF, and TGFβ (adopted and modified from NHSM conditions; see Experimental Procedures ), henceforth called “4i” medium, which endows the cells with a distinct pluripotent state (). These hESCs were then cultured for 2 days in bFGF, TGFβ, and 1% KSR medium, and thereafter, 2,000–4,000 cells were cultured in low-attachment well in the presence of BMP2 or BMP4, LIF, stem cell factor (SCF), epidermal growth factor (EGF), and Rho-kinase (ROCK) inhibitor to induce hPGCLCs () ( Figure 1 A). These cells aggregated to form embryoid bodies (henceforth called embyoids) and responded within 3 days with significant expression of NANOS3-mCherry and tissue-nonspecific alkaline phosphatase (TNAP), a PGC and pluripotency marker in humans and mice ( Figure 1 B). The intensity of the NANOS3-mCherry reporter increased progressively until day 4–5, resulting in ∼27% of NANOS3/TNAP double-positive putative hPGCLCs ( Figures 1 B and S1 B). Similar to mice, hPGCLCs do not proliferate significantly after 5 days under these conditions (). The response was highly reproducible in three independent male and female NANOS3-mCherry hESC lines. Both BMP2 and/or BMP4 (with LIF, SCF, and EGF) were effective in inducing hPGCLC ( Figure S1 C) in a dose-dependent manner in the range of 50–500 ng/ml ( Figures S1 D and S1E).

(C) Expression analysis by RT-qPCR of TNAP-positive 4i hESCs (hESC TNAP+), TNAP/NANOS3-mCherry-positive hPGCLCs (TNAP+N3+), and the remaining cells (TNAP-N3-) of day 4 embryoids (D4 embryoid). Relative expression levels are shown with normalization to β−ACTIN. Error bars indicate mean ± SD from three independent biological replicates.

(B) Development of day 1–7 embryoids derived from WIS2-NANOS3-mCherry hESCs. Top row: images of embryoids. Bottom row: FACS analysis of the dissociated embryoids with anti-TNAP-Alexa Fluor 647 and NANOS3-mCherry to detect hPGCLCs.

First, we generated three independent hESC lines (WIS2 and LIS1 male hESC and WIBR3 female hESC line) () with a NANOS3-mCherry knockin reporter ( Figure S1 A available online), a highly conserved PGC-specific gene (). These hESCs maintained in bFGF and responded to BMP2/BMP4 with ∼0%–5% NANOS3-mCherry positive putative hPGCLCs at day 4 (see Figure 7 A). Like hESC, mouse epiblast stem cells (mEpiSC) also respond poorly to specification of PGCLCs (). In contrast, epiblast-like cells (EpiLCs) derived from naive mESCs have a significant potential for germ cell fate (). However, the approach used for mouse ESCs did not confer competence for germline fate on hESCs.

(E) FACS analysis of NANOS3-mCherry and TNAP positive population from WIS2-NANOS3-mCherry cell line with PGCLC induction with BMP2 alone from day 1 to day 4.

(D) FACS analysis for NANOS3-mCherry and TNAP positive population using WIS2-NANOS3-mCherry cell line after 3 days of hPGCLC induction with BMP2 (50, 250 and 500 ng/ml) or without BMP2 (0) in the presence of ROCK inhibitor.

(C) FACS analysis for NANOS3-mCherry and TNAP positive population using WIS2-NANOS3-mCherry cell line after 4 days of hPGCLC induction by BMP2 (500 ng/ml), BMP4 (500 ng/ml) or BMP2 (250 ng/ml) and BMP4 (250 ng/ml) together with human LIF, SCF, EGF and ROCK inhibitor. Numbers show the percentage of the TNAP/NANOS3 double positive population in the boxes.

(B) Number of the NANOS3-mCherry/TNAP positive cells per embryoid during PGCLC induction with BMP2, human LIF, SCF and EGF (BMP2+L/S/E) or BMP2 alone.

(A) Targeting strategy of generation of NANOS3-mCherry knock-in reporter hESC lines. P2A-mCherry sequence in frame with the last exon of the human NANOS3 gene was inserted. We have generated plasmids encoding TALEN molecules specific to the region covering NANOS3 stop codon. Scissors indicate TALEN cutting site. Southern blot was performed with 5′ external probe (5′ probe) and the BglII restriction enzyme sites. Correct targeting and loop-out of resistance cassette was conducted in three independent human ESC lines (WIS2, LIS1 and WIBR3).

The ontogeny of cKIT+ human primordial germ cells proves to be a resource for human germ line reprogramming, imprint erasure and in vitro differentiation.

Discussion

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