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SOX17 is a critical specifier of human primordial germ cell fate.

Irie N, Weinberger L, Tang WW, Kobayashi T, Viukov S, Manor YS, Dietmann S, Hanna JH, Surani MA - Cell (2014)

Bottom Line: Here, we demonstrate specification of hPGC-like cells (hPGCLCs) from germline competent pluripotent stem cells.Remarkably, SOX17 is the key regulator of hPGC-like fate, whereas BLIMP1 represses endodermal and other somatic genes during specification of hPGCLCs.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.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Cancer Research UK Gurdon Institute, Tennis Court Road, University of Cambridge, Cambridge CB2 1QN, UK; Department of Physiology, Development and Neuroscience, Downing Street, University of Cambridge, Cambridge CB2 3EG, UK; Wellcome Trust-Medical Research Council Stem Cell Institute, Tennis Court Road, University of Cambridge, Cambridge CB2 3EG, UK.

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hPGCLC Shares Transcriptional Profile with Human Embryonic PGCs and TCam-2 Seminoma(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.(B) PCA of RNA-seq data. Arrowline indicates potential germline progression from 4i hESC to hPGCLC and gonadal hPGC.(C) Heat map of gene expression of key PGC-associated genes (early and late) and of pluripotency, mesoderm, endoderm, and gonadal somatic (Soma) markers.(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.(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.
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fig2: hPGCLC Shares Transcriptional Profile with Human Embryonic PGCs and TCam-2 Seminoma(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.(B) PCA of RNA-seq data. Arrowline indicates potential germline progression from 4i hESC to hPGCLC and gonadal hPGC.(C) Heat map of gene expression of key PGC-associated genes (early and late) and of pluripotency, mesoderm, endoderm, and gonadal somatic (Soma) markers.(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.(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.

Mentions: 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 1C). Remarkably, SOX17 was significantly upregulated, whereas SOX2 was downregulated in the putative hPGCLCs that reflects their expression in embryonic hPGCs and seminomas (de Jong et al., 2008; 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 1D and S1F), as did OCT4 with BLIMP1 (Figure S1F). This suggests that the NANOS3-mCherry-positive cells are very likely nascent germ cells.


SOX17 is a critical specifier of human primordial germ cell fate.

Irie N, Weinberger L, Tang WW, Kobayashi T, Viukov S, Manor YS, Dietmann S, Hanna JH, Surani MA - Cell (2014)

hPGCLC Shares Transcriptional Profile with Human Embryonic PGCs and TCam-2 Seminoma(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.(B) PCA of RNA-seq data. Arrowline indicates potential germline progression from 4i hESC to hPGCLC and gonadal hPGC.(C) Heat map of gene expression of key PGC-associated genes (early and late) and of pluripotency, mesoderm, endoderm, and gonadal somatic (Soma) markers.(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.(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.
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fig2: hPGCLC Shares Transcriptional Profile with Human Embryonic PGCs and TCam-2 Seminoma(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.(B) PCA of RNA-seq data. Arrowline indicates potential germline progression from 4i hESC to hPGCLC and gonadal hPGC.(C) Heat map of gene expression of key PGC-associated genes (early and late) and of pluripotency, mesoderm, endoderm, and gonadal somatic (Soma) markers.(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.(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.
Mentions: 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 1C). Remarkably, SOX17 was significantly upregulated, whereas SOX2 was downregulated in the putative hPGCLCs that reflects their expression in embryonic hPGCs and seminomas (de Jong et al., 2008; 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 1D and S1F), as did OCT4 with BLIMP1 (Figure S1F). This suggests that the NANOS3-mCherry-positive cells are very likely nascent germ cells.

Bottom Line: Here, we demonstrate specification of hPGC-like cells (hPGCLCs) from germline competent pluripotent stem cells.Remarkably, SOX17 is the key regulator of hPGC-like fate, whereas BLIMP1 represses endodermal and other somatic genes during specification of hPGCLCs.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.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Cancer Research UK Gurdon Institute, Tennis Court Road, University of Cambridge, Cambridge CB2 1QN, UK; Department of Physiology, Development and Neuroscience, Downing Street, University of Cambridge, Cambridge CB2 3EG, UK; Wellcome Trust-Medical Research Council Stem Cell Institute, Tennis Court Road, University of Cambridge, Cambridge CB2 3EG, UK.

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Related in: MedlinePlus