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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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Properties of rat blastocyst outgrowths.(A) Phase contrast photographs showing stages of WKY rat blastocyst outgrowths kept on mitomycin-treated primary rat embryo fibroblasts (PREF). The outgrowths were initially smooth and compact (left), but converted to XEN morphology (right) ∼10 days after blastocyst plating if not passaged, or a few days later if mechanically disaggregated into smaller clumps. Regardless of when the conversion occurred, it was fast (<24 hours) and went through a stage of intermediate morphology (middle). (B) Loss and re-expression of Oct4 mRNA in WKY rat blastocyst outgrowths. In these experiments, the outgrowths were not passaged and showed compact, smooth morphology before day 10, but XEN morphology thereafter. At the indicated days, the outgrowths were individually harvested for RT-PCR analysis, using rat-specific primers for Oct4 and hypoxanthine phosphoribosyl transferase (Hprt) cDNAs. The Oct4 and Hprt cDNAs were amplified in the same reaction; none of the primers amplified intronless products from genomic DNA (not shown). No amplification was achieved when using mouse-specific primers (not shown). Day 0 = blastocyst; W, water control. (C) Semi-quantitative assessment of Oct4 mRNA level. Rat blastocysts (E4.5, strain WKY), XEN-P line RX1, primary XEN-like blastocyst outgrowths (strain WKY), rat embryo fibroblast line Li 1 (feeder for RX1), and PREF (feeder for primary rat cells) were analyzed for Oct4 and Hprt mRNAs by subjecting 10-fold serial dilutions of the RT reactions to PCR. (D) LIF effect (1,000 u/ml) on the formation of secondary XEN-like cell colonies from primary rat blastocyst outgrowths (WKY). Primary cells were seeded at ∼100–500 cells/well onto feeder line Li 1. 6 independent experiments. Similar results were obtained with rat strain BDIX.
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pone-0007216-g001: Properties of rat blastocyst outgrowths.(A) Phase contrast photographs showing stages of WKY rat blastocyst outgrowths kept on mitomycin-treated primary rat embryo fibroblasts (PREF). The outgrowths were initially smooth and compact (left), but converted to XEN morphology (right) ∼10 days after blastocyst plating if not passaged, or a few days later if mechanically disaggregated into smaller clumps. Regardless of when the conversion occurred, it was fast (<24 hours) and went through a stage of intermediate morphology (middle). (B) Loss and re-expression of Oct4 mRNA in WKY rat blastocyst outgrowths. In these experiments, the outgrowths were not passaged and showed compact, smooth morphology before day 10, but XEN morphology thereafter. At the indicated days, the outgrowths were individually harvested for RT-PCR analysis, using rat-specific primers for Oct4 and hypoxanthine phosphoribosyl transferase (Hprt) cDNAs. The Oct4 and Hprt cDNAs were amplified in the same reaction; none of the primers amplified intronless products from genomic DNA (not shown). No amplification was achieved when using mouse-specific primers (not shown). Day 0 = blastocyst; W, water control. (C) Semi-quantitative assessment of Oct4 mRNA level. Rat blastocysts (E4.5, strain WKY), XEN-P line RX1, primary XEN-like blastocyst outgrowths (strain WKY), rat embryo fibroblast line Li 1 (feeder for RX1), and PREF (feeder for primary rat cells) were analyzed for Oct4 and Hprt mRNAs by subjecting 10-fold serial dilutions of the RT reactions to PCR. (D) LIF effect (1,000 u/ml) on the formation of secondary XEN-like cell colonies from primary rat blastocyst outgrowths (WKY). Primary cells were seeded at ∼100–500 cells/well onto feeder line Li 1. 6 independent experiments. Similar results were obtained with rat strain BDIX.

Mentions: When explanted onto mitotically inactivated primary embryo fibroblasts, rat blastocysts produced smooth, compact outgrowths that initially grew rapidly and could be passaged a few times. After 10–20 days, however, these outgrowths usually converted abruptly into cells with a morphology similar to that of earlier published rodent extraembryonic endoderm cell lines [14], [18] that, in the mouse, were termed XEN cell lines [14] (Fig. 1A). Strikingly, this conversion was associated with the re-expression of Oct4 mRNA that had been lost after day 4 (Fig. 1B, C). At low density, the primary rat blastocyst-derived cells formed colonies with a morphology that was also XEN-like, and this colony formation was stimulated by leukemia inhibitory factor (LIF) (Fig. 1D). Both primary rat embryo fibroblasts and a rat embryo-derived permanent fibroblast cell line (Li1) derived in our laboratory were suitable as feeder cells, but mouse embryo fibroblasts produced extremely variable (batch-dependent) results. When the feeder cells were omitted, colony formation at low density was reduced to an insignificant fraction (on plastic) or undetectable (on gelatin-coated plastic).


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)

Properties of rat blastocyst outgrowths.(A) Phase contrast photographs showing stages of WKY rat blastocyst outgrowths kept on mitomycin-treated primary rat embryo fibroblasts (PREF). The outgrowths were initially smooth and compact (left), but converted to XEN morphology (right) ∼10 days after blastocyst plating if not passaged, or a few days later if mechanically disaggregated into smaller clumps. Regardless of when the conversion occurred, it was fast (<24 hours) and went through a stage of intermediate morphology (middle). (B) Loss and re-expression of Oct4 mRNA in WKY rat blastocyst outgrowths. In these experiments, the outgrowths were not passaged and showed compact, smooth morphology before day 10, but XEN morphology thereafter. At the indicated days, the outgrowths were individually harvested for RT-PCR analysis, using rat-specific primers for Oct4 and hypoxanthine phosphoribosyl transferase (Hprt) cDNAs. The Oct4 and Hprt cDNAs were amplified in the same reaction; none of the primers amplified intronless products from genomic DNA (not shown). No amplification was achieved when using mouse-specific primers (not shown). Day 0 = blastocyst; W, water control. (C) Semi-quantitative assessment of Oct4 mRNA level. Rat blastocysts (E4.5, strain WKY), XEN-P line RX1, primary XEN-like blastocyst outgrowths (strain WKY), rat embryo fibroblast line Li 1 (feeder for RX1), and PREF (feeder for primary rat cells) were analyzed for Oct4 and Hprt mRNAs by subjecting 10-fold serial dilutions of the RT reactions to PCR. (D) LIF effect (1,000 u/ml) on the formation of secondary XEN-like cell colonies from primary rat blastocyst outgrowths (WKY). Primary cells were seeded at ∼100–500 cells/well onto feeder line Li 1. 6 independent experiments. Similar results were obtained with rat strain BDIX.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2747266&req=5

pone-0007216-g001: Properties of rat blastocyst outgrowths.(A) Phase contrast photographs showing stages of WKY rat blastocyst outgrowths kept on mitomycin-treated primary rat embryo fibroblasts (PREF). The outgrowths were initially smooth and compact (left), but converted to XEN morphology (right) ∼10 days after blastocyst plating if not passaged, or a few days later if mechanically disaggregated into smaller clumps. Regardless of when the conversion occurred, it was fast (<24 hours) and went through a stage of intermediate morphology (middle). (B) Loss and re-expression of Oct4 mRNA in WKY rat blastocyst outgrowths. In these experiments, the outgrowths were not passaged and showed compact, smooth morphology before day 10, but XEN morphology thereafter. At the indicated days, the outgrowths were individually harvested for RT-PCR analysis, using rat-specific primers for Oct4 and hypoxanthine phosphoribosyl transferase (Hprt) cDNAs. The Oct4 and Hprt cDNAs were amplified in the same reaction; none of the primers amplified intronless products from genomic DNA (not shown). No amplification was achieved when using mouse-specific primers (not shown). Day 0 = blastocyst; W, water control. (C) Semi-quantitative assessment of Oct4 mRNA level. Rat blastocysts (E4.5, strain WKY), XEN-P line RX1, primary XEN-like blastocyst outgrowths (strain WKY), rat embryo fibroblast line Li 1 (feeder for RX1), and PREF (feeder for primary rat cells) were analyzed for Oct4 and Hprt mRNAs by subjecting 10-fold serial dilutions of the RT reactions to PCR. (D) LIF effect (1,000 u/ml) on the formation of secondary XEN-like cell colonies from primary rat blastocyst outgrowths (WKY). Primary cells were seeded at ∼100–500 cells/well onto feeder line Li 1. 6 independent experiments. Similar results were obtained with rat strain BDIX.
Mentions: When explanted onto mitotically inactivated primary embryo fibroblasts, rat blastocysts produced smooth, compact outgrowths that initially grew rapidly and could be passaged a few times. After 10–20 days, however, these outgrowths usually converted abruptly into cells with a morphology similar to that of earlier published rodent extraembryonic endoderm cell lines [14], [18] that, in the mouse, were termed XEN cell lines [14] (Fig. 1A). Strikingly, this conversion was associated with the re-expression of Oct4 mRNA that had been lost after day 4 (Fig. 1B, C). At low density, the primary rat blastocyst-derived cells formed colonies with a morphology that was also XEN-like, and this colony formation was stimulated by leukemia inhibitory factor (LIF) (Fig. 1D). Both primary rat embryo fibroblasts and a rat embryo-derived permanent fibroblast cell line (Li1) derived in our laboratory were suitable as feeder cells, but mouse embryo fibroblasts produced extremely variable (batch-dependent) results. When the feeder cells were omitted, colony formation at low density was reduced to an insignificant fraction (on plastic) or undetectable (on gelatin-coated plastic).

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