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Isolation of Oct4-expressing extraembryonic endoderm precursor cell lines.

Debeb BG, Galat V, Epple-Farmer J, Iannaccone S, Woodward WA, Bader M, Iannaccone P, Binas B - PLoS ONE (2009)

Bottom Line: In vitro, the epiblast precursor is most closely represented by the well-characterized embryonic stem (ES) cell lines that maintain the expression of Oct4, but analogous ExEn precursor cell lines are not known and it is unclear if they would express Oct4.Further, they lack markers characteristic of the more differentiated primitive/visceral and parietal ExEn stages, but exclusively differentiate into these stages in vitro and contribute to them in vivo.Our findings (i) suggest strongly that the ExEn precursor is a self-renewable entity, (ii) indicate that active Oct4 gene expression (transcription plus translation) is part of its molecular identity, and (iii) provide an in vitro model of early ExEn differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas, United States of America.

ABSTRACT

Background: The extraembryonic endoderm (ExEn) defines the yolk sac, a set of membranes that provide essential support for mammalian embryos. Recent findings suggest that the committed ExEn precursor is present already in the embryonic Inner Cell Mass (ICM) as a group of cells that intermingles with the closely related epiblast precursor. All ICM cells contain Oct4, a key transcription factor that is first expressed at the morula stage. In vitro, the epiblast precursor is most closely represented by the well-characterized embryonic stem (ES) cell lines that maintain the expression of Oct4, but analogous ExEn precursor cell lines are not known and it is unclear if they would express Oct4.

Methodology/principal findings: Here we report the isolation and characterization of permanently proliferating Oct4-expressing rat cell lines ("XEN-P cell lines"), which closely resemble the ExEn precursor. We isolated the XEN-P cell lines from blastocysts and characterized them by plating and gene expression assays as well as by injection into embryos. Like ES cells, the XEN-P cells express Oct4 and SSEA1 at high levels and their growth is stimulated by leukemia inhibitory factor, but instead of the epiblast determinant Nanog, they express the ExEn determinants Gata6 and Gata4. Further, they lack markers characteristic of the more differentiated primitive/visceral and parietal ExEn stages, but exclusively differentiate into these stages in vitro and contribute to them in vivo.

Conclusions/significance: Our findings (i) suggest strongly that the ExEn precursor is a self-renewable entity, (ii) indicate that active Oct4 gene expression (transcription plus translation) is part of its molecular identity, and (iii) provide an in vitro model of early ExEn differentiation.

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Related in: MedlinePlus

Contributions of rat XEN-P cell lines to postimplantation embryos.Representative fluorescence (A–C) and bright field (A'–C') photographs demonstrating in vivo contributions of microinjected rat cells to (A, A') parietal yolk sac of an 11.5 dpc rat conceptus (inset showing magnification); (B, B') visceral endoderm of an 8.5 dpc rat conceptus; (C, C') visceral endoderm (arrowheads; one patch magnified in inset) of an ∼7 dpc mouse conceptus. Pregnancy timing is distorted by the embryo manipulations and therefore only approximate.
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pone-0007216-g007: Contributions of rat XEN-P cell lines to postimplantation embryos.Representative fluorescence (A–C) and bright field (A'–C') photographs demonstrating in vivo contributions of microinjected rat cells to (A, A') parietal yolk sac of an 11.5 dpc rat conceptus (inset showing magnification); (B, B') visceral endoderm of an 8.5 dpc rat conceptus; (C, C') visceral endoderm (arrowheads; one patch magnified in inset) of an ∼7 dpc mouse conceptus. Pregnancy timing is distorted by the embryo manipulations and therefore only approximate.

Mentions: In order to judge their developmental potential, we labeled the XEN-P cell lines with green fluorescent protein (GFP) by lentiviral transduction. Upon injection into rat and mouse blastocysts and subsequent embryo transfer, the labeled cells proliferated and contributed to the parietal (84%) and visceral (12%) layers of rat and mouse yolk sacs (Fig. 7; Table 2). Thus, the cultured rat cells contributed more than sporadically to the visceral endoderm, although they did more often contribute to the parietal endoderm. This preponderance of parietal endoderm integration contrasts with our finding that a majority of cultured rat cells carried the primitive/visceral marker SSEA3 (Figs. 3F, 4D, 6A) but is in line with the preferential contribution of blastocyst-injected primary primitive endoderm and visceral endoderm cells to the parietal endoderm [26]. Also of note, the percentage of visceral endoderm contribution we observed roughly corresponded to the percentage of XEN-P cells in the cultures. Given that all the cell types of the rat cell lines can be derived from a single cell in vitro (Fig. 6), these results imply that the in vivo integrants were at least indirectly derived from cultured XEN-P cells.


Isolation of Oct4-expressing extraembryonic endoderm precursor cell lines.

Debeb BG, Galat V, Epple-Farmer J, Iannaccone S, Woodward WA, Bader M, Iannaccone P, Binas B - PLoS ONE (2009)

Contributions of rat XEN-P cell lines to postimplantation embryos.Representative fluorescence (A–C) and bright field (A'–C') photographs demonstrating in vivo contributions of microinjected rat cells to (A, A') parietal yolk sac of an 11.5 dpc rat conceptus (inset showing magnification); (B, B') visceral endoderm of an 8.5 dpc rat conceptus; (C, C') visceral endoderm (arrowheads; one patch magnified in inset) of an ∼7 dpc mouse conceptus. Pregnancy timing is distorted by the embryo manipulations and therefore only approximate.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2747266&req=5

pone-0007216-g007: Contributions of rat XEN-P cell lines to postimplantation embryos.Representative fluorescence (A–C) and bright field (A'–C') photographs demonstrating in vivo contributions of microinjected rat cells to (A, A') parietal yolk sac of an 11.5 dpc rat conceptus (inset showing magnification); (B, B') visceral endoderm of an 8.5 dpc rat conceptus; (C, C') visceral endoderm (arrowheads; one patch magnified in inset) of an ∼7 dpc mouse conceptus. Pregnancy timing is distorted by the embryo manipulations and therefore only approximate.
Mentions: In order to judge their developmental potential, we labeled the XEN-P cell lines with green fluorescent protein (GFP) by lentiviral transduction. Upon injection into rat and mouse blastocysts and subsequent embryo transfer, the labeled cells proliferated and contributed to the parietal (84%) and visceral (12%) layers of rat and mouse yolk sacs (Fig. 7; Table 2). Thus, the cultured rat cells contributed more than sporadically to the visceral endoderm, although they did more often contribute to the parietal endoderm. This preponderance of parietal endoderm integration contrasts with our finding that a majority of cultured rat cells carried the primitive/visceral marker SSEA3 (Figs. 3F, 4D, 6A) but is in line with the preferential contribution of blastocyst-injected primary primitive endoderm and visceral endoderm cells to the parietal endoderm [26]. Also of note, the percentage of visceral endoderm contribution we observed roughly corresponded to the percentage of XEN-P cells in the cultures. Given that all the cell types of the rat cell lines can be derived from a single cell in vitro (Fig. 6), these results imply that the in vivo integrants were at least indirectly derived from cultured XEN-P cells.

Bottom Line: In vitro, the epiblast precursor is most closely represented by the well-characterized embryonic stem (ES) cell lines that maintain the expression of Oct4, but analogous ExEn precursor cell lines are not known and it is unclear if they would express Oct4.Further, they lack markers characteristic of the more differentiated primitive/visceral and parietal ExEn stages, but exclusively differentiate into these stages in vitro and contribute to them in vivo.Our findings (i) suggest strongly that the ExEn precursor is a self-renewable entity, (ii) indicate that active Oct4 gene expression (transcription plus translation) is part of its molecular identity, and (iii) provide an in vitro model of early ExEn differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas, United States of America.

ABSTRACT

Background: The extraembryonic endoderm (ExEn) defines the yolk sac, a set of membranes that provide essential support for mammalian embryos. Recent findings suggest that the committed ExEn precursor is present already in the embryonic Inner Cell Mass (ICM) as a group of cells that intermingles with the closely related epiblast precursor. All ICM cells contain Oct4, a key transcription factor that is first expressed at the morula stage. In vitro, the epiblast precursor is most closely represented by the well-characterized embryonic stem (ES) cell lines that maintain the expression of Oct4, but analogous ExEn precursor cell lines are not known and it is unclear if they would express Oct4.

Methodology/principal findings: Here we report the isolation and characterization of permanently proliferating Oct4-expressing rat cell lines ("XEN-P cell lines"), which closely resemble the ExEn precursor. We isolated the XEN-P cell lines from blastocysts and characterized them by plating and gene expression assays as well as by injection into embryos. Like ES cells, the XEN-P cells express Oct4 and SSEA1 at high levels and their growth is stimulated by leukemia inhibitory factor, but instead of the epiblast determinant Nanog, they express the ExEn determinants Gata6 and Gata4. Further, they lack markers characteristic of the more differentiated primitive/visceral and parietal ExEn stages, but exclusively differentiate into these stages in vitro and contribute to them in vivo.

Conclusions/significance: Our findings (i) suggest strongly that the ExEn precursor is a self-renewable entity, (ii) indicate that active Oct4 gene expression (transcription plus translation) is part of its molecular identity, and (iii) provide an in vitro model of early ExEn differentiation.

Show MeSH
Related in: MedlinePlus