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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: 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.

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.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4310934&req=5

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: 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.

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