Limits...
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.

Show MeSH

Related in: MedlinePlus

Induction and Isolation of hPGCLCs from Competent hiPSCs/hESCs(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).(B) FACS analysis of TNAP and CD38 in 4i hiPSCs (top) and day 4 embryoids derived from 4i hiPSCs after direct induction (bottom).(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.(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.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4310934&req=5

fig7: Induction and Isolation of hPGCLCs from Competent hiPSCs/hESCs(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).(B) FACS analysis of TNAP and CD38 in 4i hiPSCs (top) and day 4 embryoids derived from 4i hiPSCs after direct induction (bottom).(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.(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.

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)

Induction and Isolation of hPGCLCs from Competent hiPSCs/hESCs(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).(B) FACS analysis of TNAP and CD38 in 4i hiPSCs (top) and day 4 embryoids derived from 4i hiPSCs after direct induction (bottom).(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.(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.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4310934&req=5

fig7: Induction and Isolation of hPGCLCs from Competent hiPSCs/hESCs(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).(B) FACS analysis of TNAP and CD38 in 4i hiPSCs (top) and day 4 embryoids derived from 4i hiPSCs after direct induction (bottom).(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.(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.
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.

Show MeSH
Related in: MedlinePlus