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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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Continued proliferation and preferential accumulation of SSEA1-positive cells in old rat colonies derived from rat XEN-P cells.Two magnifications of a representative 16-days old colony (line RX1) are shown. Bright field (left), immunofluorescence (middle), and nuclear stain (right). Control omitting primary antibody was negative and is not shown.
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pone-0007216-g005: Continued proliferation and preferential accumulation of SSEA1-positive cells in old rat colonies derived from rat XEN-P cells.Two magnifications of a representative 16-days old colony (line RX1) are shown. Bright field (left), immunofluorescence (middle), and nuclear stain (right). Control omitting primary antibody was negative and is not shown.

Mentions: In order to understand the origin of culture heterogeneity and the identity of the self-renewing population, we plated line RX1 at low density and studied the resulting colonies over time. Strikingly, nearly all colonies consisted initially (2–3 days after seeding) entirely or almost entirely of round cells that highly co-expressed Oct4, Gata6, Gata4, and SSEA1 (Fig. 4A–C) while lacking the primitive/visceral endoderm marker SSEA3 as well as the basement membrane components Laminin B and Collagen 4 (Fig. 4D–F) that are characteristically produced by extraembryonic endoderm cells and especially parietal endoderm [25]. In line with the lack of a basement membrane, the young colonies were poorly adherent and easily lost during washing steps. By contrast, in older, larger colonies (4–7 days after seeding), the inner cells became epithelial and firmly adherent, and many round as well as the epithelial cells were negative for SSEA1 and very low in Oct4. Rather, many of the round cells now expressed SSEA3 (Fig. 4D), and the epithelial areas contained abundant, extracellular Laminin B and Collagen 4 (Fig. 4E, F). Notably, however, Oct4/SSEA1-positive cells always persisted in the older colonies, usually at the colony fringes and later also accumulating on top; these same cells tended to show also higher levels of Gata6 (and, to a lesser degree, of Gata4) than the rest of the colony, in line with higher expression in rat XEN-P vs. mouse XEN cell lines (Fig. 2C,E; Fig. 3C, D). With further evolution of the colonies (7–14 days after seeding), the round fringe cells kept proliferating, piled up on top of the colonies (Fig. 5; see also Fig. 4B), and eventually (10–20 days after seeding) converted into bridge-like ductal structures while the inner epithelial parts lost their nuclei and then degenerated completely (results not shown). These data strongly suggest that the round, undifferentiated Oct4/SSEA1-positive cells are the principal self-renewing entity and the precursors of the extraembryonic endoderm cells, i.e. are the XEN-P subpopulation within our XEN-P cell lines. In order to obtain formal evidence that one cell can generate the whole culture heterogeneity, we performed two additional experiments. First, we transfected line RX1 stably with a neomycin resistance marker, a method that ensures single cell origin better than trypsinization. All colonies emerging from G418 selection showed the identical round-epithelial morphology of the parent culture, and three randomly chosen clones showed the same heterogeneous Oct4/Gata6/SSEA1/SSEA3 expression and mixed ExEn lineage marker gene expression as the parent line (Fig. 6A). Second, we sub-cloned line RX1 by single-cell FACS deposition into 96-well plates. Out of 384 wells (in two independent experiments), 129 contained colonies, all of which maintained the heterogeneous morphology of the parent line. 20 colonies were randomly selected for immunostaining, and all contained a significant fraction of Oct4-positive cells (Fig. 6B).


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)

Continued proliferation and preferential accumulation of SSEA1-positive cells in old rat colonies derived from rat XEN-P cells.Two magnifications of a representative 16-days old colony (line RX1) are shown. Bright field (left), immunofluorescence (middle), and nuclear stain (right). Control omitting primary antibody was negative and is not shown.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2747266&req=5

pone-0007216-g005: Continued proliferation and preferential accumulation of SSEA1-positive cells in old rat colonies derived from rat XEN-P cells.Two magnifications of a representative 16-days old colony (line RX1) are shown. Bright field (left), immunofluorescence (middle), and nuclear stain (right). Control omitting primary antibody was negative and is not shown.
Mentions: In order to understand the origin of culture heterogeneity and the identity of the self-renewing population, we plated line RX1 at low density and studied the resulting colonies over time. Strikingly, nearly all colonies consisted initially (2–3 days after seeding) entirely or almost entirely of round cells that highly co-expressed Oct4, Gata6, Gata4, and SSEA1 (Fig. 4A–C) while lacking the primitive/visceral endoderm marker SSEA3 as well as the basement membrane components Laminin B and Collagen 4 (Fig. 4D–F) that are characteristically produced by extraembryonic endoderm cells and especially parietal endoderm [25]. In line with the lack of a basement membrane, the young colonies were poorly adherent and easily lost during washing steps. By contrast, in older, larger colonies (4–7 days after seeding), the inner cells became epithelial and firmly adherent, and many round as well as the epithelial cells were negative for SSEA1 and very low in Oct4. Rather, many of the round cells now expressed SSEA3 (Fig. 4D), and the epithelial areas contained abundant, extracellular Laminin B and Collagen 4 (Fig. 4E, F). Notably, however, Oct4/SSEA1-positive cells always persisted in the older colonies, usually at the colony fringes and later also accumulating on top; these same cells tended to show also higher levels of Gata6 (and, to a lesser degree, of Gata4) than the rest of the colony, in line with higher expression in rat XEN-P vs. mouse XEN cell lines (Fig. 2C,E; Fig. 3C, D). With further evolution of the colonies (7–14 days after seeding), the round fringe cells kept proliferating, piled up on top of the colonies (Fig. 5; see also Fig. 4B), and eventually (10–20 days after seeding) converted into bridge-like ductal structures while the inner epithelial parts lost their nuclei and then degenerated completely (results not shown). These data strongly suggest that the round, undifferentiated Oct4/SSEA1-positive cells are the principal self-renewing entity and the precursors of the extraembryonic endoderm cells, i.e. are the XEN-P subpopulation within our XEN-P cell lines. In order to obtain formal evidence that one cell can generate the whole culture heterogeneity, we performed two additional experiments. First, we transfected line RX1 stably with a neomycin resistance marker, a method that ensures single cell origin better than trypsinization. All colonies emerging from G418 selection showed the identical round-epithelial morphology of the parent culture, and three randomly chosen clones showed the same heterogeneous Oct4/Gata6/SSEA1/SSEA3 expression and mixed ExEn lineage marker gene expression as the parent line (Fig. 6A). Second, we sub-cloned line RX1 by single-cell FACS deposition into 96-well plates. Out of 384 wells (in two independent experiments), 129 contained colonies, all of which maintained the heterogeneous morphology of the parent line. 20 colonies were randomly selected for immunostaining, and all contained a significant fraction of Oct4-positive cells (Fig. 6B).

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