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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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Generation of NANOS3-mCherry Reporter Knockin hESC Lines and hPGCLC Differentiation, Related to Figure 1(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).(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.(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.(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.(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.(F) Immunofluorescence of TFAP2C and NANOS3-mCherry, and OCT4 and BLIMP1 on day 4 embryoids. Scale bar = 70 μm.
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figs1: Generation of NANOS3-mCherry Reporter Knockin hESC Lines and hPGCLC Differentiation, Related to Figure 1(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).(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.(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.(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.(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.(F) Immunofluorescence of TFAP2C and NANOS3-mCherry, and OCT4 and BLIMP1 on day 4 embryoids. Scale bar = 70 μm.

Mentions: First, we generated three independent hESC lines (WIS2 and LIS1 male hESC and WIBR3 female hESC line) (Gafni et al., 2013) with a NANOS3-mCherry knockin reporter (Figure S1A available online), a highly conserved PGC-specific gene (Gkountela et al., 2013; Julaton and Reijo Pera, 2011). These hESCs maintained in bFGF and responded to BMP2/BMP4 with ∼0%–5% NANOS3-mCherry positive putative hPGCLCs at day 4 (see Figure 7A). Like hESC, mouse epiblast stem cells (mEpiSC) also respond poorly to specification of PGCLCs (Hayashi and Surani, 2009). In contrast, epiblast-like cells (EpiLCs) derived from naive mESCs have a significant potential for germ cell fate (Hayashi et al., 2011). However, the approach used for mouse ESCs did not confer competence for germline fate on hESCs.


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)

Generation of NANOS3-mCherry Reporter Knockin hESC Lines and hPGCLC Differentiation, Related to Figure 1(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).(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.(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.(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.(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.(F) Immunofluorescence of TFAP2C and NANOS3-mCherry, and OCT4 and BLIMP1 on day 4 embryoids. Scale bar = 70 μm.
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figs1: Generation of NANOS3-mCherry Reporter Knockin hESC Lines and hPGCLC Differentiation, Related to Figure 1(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).(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.(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.(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.(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.(F) Immunofluorescence of TFAP2C and NANOS3-mCherry, and OCT4 and BLIMP1 on day 4 embryoids. Scale bar = 70 μm.
Mentions: First, we generated three independent hESC lines (WIS2 and LIS1 male hESC and WIBR3 female hESC line) (Gafni et al., 2013) with a NANOS3-mCherry knockin reporter (Figure S1A available online), a highly conserved PGC-specific gene (Gkountela et al., 2013; Julaton and Reijo Pera, 2011). These hESCs maintained in bFGF and responded to BMP2/BMP4 with ∼0%–5% NANOS3-mCherry positive putative hPGCLCs at day 4 (see Figure 7A). Like hESC, mouse epiblast stem cells (mEpiSC) also respond poorly to specification of PGCLCs (Hayashi and Surani, 2009). In contrast, epiblast-like cells (EpiLCs) derived from naive mESCs have a significant potential for germ cell fate (Hayashi et al., 2011). However, the approach used for mouse ESCs did not confer competence for germline fate on hESCs.

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