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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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Specification of hPGCLCs from Human Embryonic Stem Cells(A) Schematic protocol for hPGCLCs specification from hESCs.(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.(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.(D) Immunofluorescence of a day 4 embryoid showing coexpression of NANOS3-mCherry, NANOG, and OCT4 in hPGCLCs. Scale bar, 66 μm.
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fig1: Specification of hPGCLCs from Human Embryonic Stem Cells(A) Schematic protocol for hPGCLCs specification from hESCs.(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.(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.(D) Immunofluorescence of a day 4 embryoid showing coexpression of NANOS3-mCherry, NANOG, and OCT4 in hPGCLCs. Scale bar, 66 μm.

Mentions: 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 (Gafni et al., 2013). 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 (Hayashi et al., 2011; Watanabe et al., 2007) (Figure 1A). 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 1B). The intensity of the NANOS3-mCherry reporter increased progressively until day 4–5, resulting in ∼27% of NANOS3/TNAP double-positive putative hPGCLCs (Figures 1B and S1B). Similar to mice, hPGCLCs do not proliferate significantly after 5 days under these conditions (Hayashi et al., 2011). 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 S1C) in a dose-dependent manner in the range of 50–500 ng/ml (Figures S1D and S1E).


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)

Specification of hPGCLCs from Human Embryonic Stem Cells(A) Schematic protocol for hPGCLCs specification from hESCs.(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.(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.(D) Immunofluorescence of a day 4 embryoid showing coexpression of NANOS3-mCherry, NANOG, and OCT4 in hPGCLCs. Scale bar, 66 μm.
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Related In: Results  -  Collection

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fig1: Specification of hPGCLCs from Human Embryonic Stem Cells(A) Schematic protocol for hPGCLCs specification from hESCs.(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.(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.(D) Immunofluorescence of a day 4 embryoid showing coexpression of NANOS3-mCherry, NANOG, and OCT4 in hPGCLCs. Scale bar, 66 μm.
Mentions: 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 (Gafni et al., 2013). 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 (Hayashi et al., 2011; Watanabe et al., 2007) (Figure 1A). 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 1B). The intensity of the NANOS3-mCherry reporter increased progressively until day 4–5, resulting in ∼27% of NANOS3/TNAP double-positive putative hPGCLCs (Figures 1B and S1B). Similar to mice, hPGCLCs do not proliferate significantly after 5 days under these conditions (Hayashi et al., 2011). 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 S1C) in a dose-dependent manner in the range of 50–500 ng/ml (Figures S1D and S1E).

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