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Pig epiblast stem cells depend on activin/nodal signaling for pluripotency and self-renewal - PubMed

Pig epiblast stem cells depend on activin/nodal signaling for pluripotency and self-renewal

Ramiro Alberio et al. Stem Cells Dev. 2010 Oct.

Abstract

Activin/Nodal signaling is required for maintaining pluripotency and self-renewal of mouse epiblast stem cells and human embryonic stem cells (hESC). In this study, we investigated whether this signaling mechanism is also operative in cultured epiblasts derived from Days 10.5-12 pig embryos. Pig epiblast stem cell lines (pEpiSC) were established on mouse feeder layers and medium supplemented with basic fibroblast growth factor (bFGF). pEpiSC express the core pluripotency factors OCT4 (or POU5F1), NANOG, SOX2, and NODAL, but they do not express REX1 or alkaline phosphatase activity. Blocking leukemia inhibitory factor (LIF)/JAK/STAT3 pathway by adding the specific JAK I inhibitor 420099 and an anti-LIF antibody over 3 passages did not affect pluripotency of pEpiSC. In contrast, cells grown with the Alk-5 inhibitor SB431542, which blocks Activin/Nodal pathway, differentiated readily toward the neural lineage. pEpiSC are pluripotent, as established by their differentiation potential to ectoderm, mesoderm, and endoderm. These cells can be induced to differentiate toward trophectoderm and to germ cell precursors in response to bone morphogenetic protein 4 (BMP-4). In conclusion, our study demonstrates that pig epiblasts express the core pluripotency genes and that the capacity for maintaining self-renewal in pEpiSC depends on Activin/Nodal signaling. This study provides further evidence that maintenance of pluripotency via Activin/Nodal signal is conserved in mammals.

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Figures

FIG. 1
FIG. 1

Isolation and culture of pig epiblasts. A) The epiblast of pig embryos appears as a clear area surrounded by darker trophoblast cells. Inset: whole embryo at the ovoid stage. (B) A dissected epiblast prior to culture and (C) 12 h after plating. (D) Higher magnification of C shows prominent nucleoli and high nuclear/cytoplasmic ratio. (E) Stem cell colony at P2 (arrows) surrounded by differentiated cells. (F) A stem cell colony at P6 grows as a flat and compact colony. Scale bar: 10 μm, inset: 50 μm. E: epiblast, TE: trophoblast.

FIG. 2
FIG. 2

Characterization of pig epiblast stem cell lines (pEpiSC). (A) Alkaline phosphatase activity detected in isolated pig epiblasts and in mESC, used as positive controls. (B) Pig epiblasts are SSEA-1-positive and only weakly positive to SSEA-4. (C) OCT4 and NANOG immunostaining in pig epiblasts. (D) Gene expression of isolated epiblasts and pEpiSC at P5 and P22. (E) A representative metaphase spread showing a normal karyotype. Color images are available online at

www.liebertonline.com/scd

.

FIG. 3
FIG. 3

Signaling pathways required for pig epiblast stem cell lines (pEpiSC) pluripotency. (A) Gene expression of EpiSC treated with JAK I inhibitor. −RT: no reverse transcriptase control. (B) Plating efficiency of EpiSC grown with JAK I inhibitor and (C) with anti-LIF antibody for 3 passages. Asterisk indicates significant differences compared with control group (P < 0.05). Error bars: SEM (n = 3). (D) EpiSC grown with SB431542 stained for tubulin (TuJ). Nuclei were counterstained with 4′-6-diamidino-2-phenylindole (DAPI). Scale bar = 10 μm.

FIG. 4
FIG. 4

Differentiation potential of pig epiblast stem cell lines (pEpiSC). (A) Cells from 3 somatic germ layers visualized under phase contrast. (B) Gene expression analysis of cells from (A). RT± indicates with or without reverse transcriptase. (C) pEpiSC differentiated to the neural lineage visualized under phase contrast and after immunostaining for tubulin (TuJ) and MAP2. Color images available online at

www.liebertonline.com/scd

.

FIG. 5.
FIG. 5.

Differentiation to trophectoderm and germ cell lineage in response to BMP-4. (A) Diagram depicting the differentiation strategy through EB formation. (B) Gene expression analysis of trophoblast and (C) germ cell genes in BMP-4-induced pig epiblast stem cell lines (pEpiSC). TE: trophectoderm tissue. —RT: representative no reverse transcriptase group used as control.

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References

    1. Smith AG. Heath JK. Donaldson DD. Wong GG. Moreau J. Stahl M. Rogers D. Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature. 1988;336:688–690. - PubMed
    1. Ying QL. Nichols J. Chambers I. Smith A. BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell. 2003;115:281–292. - PubMed
    1. Vallier L. Alexander M. Pedersen RA. Activin/Nodal and FGF pathways cooperate to maintain pluripotency of human embryonic stem cells. J Cell Sci. 2005;118(Pt 19):4495–4509. - PubMed
    1. Daheron L. Opitz SL. Zaehres H. Lensch MW. Lensch WM. Andrews PW. Itskovitz-Eldor J. Daley GQ. L IF/STAT3 signaling fails to maintain self-renewal of human embryonic stem cells. Stem Cells. 2004;22:770–778. - PubMed
    1. Brons IG. Smithers LE. Trotter MW. Rugg-Gunn P. Sun B. Chuva de Sousa Lopes SM. Howlett SK. Clarkson A. Ahrlund-Richter L. Pedersen RA. Vallier L. Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature. 2007;448:191–195. - PubMed

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