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Heterogeneous lineage marker expression in naive embryonic stem cells is mostly due to spontaneous differentiation.

Nair G, Abranches E, Guedes AM, Henrique D, Raj A - Sci Rep (2015)

Bottom Line: Here, we show the transcriptome of Nanog-negative cells exhibits expression of classes of genes associated with differentiation that are not yet active in cells exposed to differentiation conditions for one day.These results are consistent with the concept that Nanog-negative cells may contain subpopulations of both lineage-primed and differentiated cells.Our results suggest that the observed enrichment of lineage-specific marker gene expression in Nanog-negative cells is associated with spontaneous differentiation of a subset of these cells rather than the more random expression that may be associated with reversible lineage priming.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA.

ABSTRACT
Populations of cultured mouse embryonic stem cells (ESCs) exhibit a subfraction of cells expressing uncharacteristically low levels of pluripotency markers such as Nanog. Yet, the extent to which individual Nanog-negative cells are differentiated, both from ESCs and from each other, remains unclear. Here, we show the transcriptome of Nanog-negative cells exhibits expression of classes of genes associated with differentiation that are not yet active in cells exposed to differentiation conditions for one day. Long non-coding RNAs, however, exhibit more changes in expression in the one-day-differentiated cells than in Nanog-negative cells. These results are consistent with the concept that Nanog-negative cells may contain subpopulations of both lineage-primed and differentiated cells. Single cell analysis showed that Nanog-negative cells display substantial and coherent heterogeneity in lineage marker expression in progressively nested subsets of cells exhibiting low levels of Nanog, then low levels of Oct4, and then a set of lineage markers, which express intensely in a small subset of these more differentiated cells. Our results suggest that the observed enrichment of lineage-specific marker gene expression in Nanog-negative cells is associated with spontaneous differentiation of a subset of these cells rather than the more random expression that may be associated with reversible lineage priming.

No MeSH data available.


Related in: MedlinePlus

Experiment Diagram and Differential Expression of Selected Genes.(A) Simplified diagram of the construction of the Nd Nanog reporter cell line12. In the Nd cell line Venus (VNP) fluorescence is a reporter of Nanog expression. (B) Diagram and Nanog:VNP flow cytometry profiles of the samples from which RNA was extracted for RNA-Seq. The solid grey profile in all panels corresponds to fluorescence cytometry of the parent E14tg2a cell line used as a background control. In the Diff panel, the Stem data is shown with a grey outline for comparison. (C) RNA expression fold changes between the Nanog:VNP(+) and Nanog:VNP(−) (filled circles) samples and between the Stem and Diff (open circles) samples for several genes involved in pluripotency or in early extraembronic and embryonic lineages. Negative points denote genes whose expression is higher in Nanog:VNP(+) or Stem samples, while positive points denote genes whose expression is higher in Nanog:VNP(−) or Diff samples. Dots mark genes that also turn up as hits for significant differential expression at a 10% false discovery rate.
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f1: Experiment Diagram and Differential Expression of Selected Genes.(A) Simplified diagram of the construction of the Nd Nanog reporter cell line12. In the Nd cell line Venus (VNP) fluorescence is a reporter of Nanog expression. (B) Diagram and Nanog:VNP flow cytometry profiles of the samples from which RNA was extracted for RNA-Seq. The solid grey profile in all panels corresponds to fluorescence cytometry of the parent E14tg2a cell line used as a background control. In the Diff panel, the Stem data is shown with a grey outline for comparison. (C) RNA expression fold changes between the Nanog:VNP(+) and Nanog:VNP(−) (filled circles) samples and between the Stem and Diff (open circles) samples for several genes involved in pluripotency or in early extraembronic and embryonic lineages. Negative points denote genes whose expression is higher in Nanog:VNP(+) or Stem samples, while positive points denote genes whose expression is higher in Nanog:VNP(−) or Diff samples. Dots mark genes that also turn up as hits for significant differential expression at a 10% false discovery rate.

Mentions: To accomplish this, we used the Nd reporter ESC line1112, which contains a short-lived fluorescent reporter (Venus-NLS-PEST, VNP) under the control of the Nanog genomic region (Fig. 1A). We sorted cells grown for 48 hours in medium containing FGF/ERK and GSK3 inhibitors (2i) and LIF (ground-state conditions) based on levels of reporter expression into both high (Nanog:VNP(+)) and low (Nanog:VNP(−)) populations (Fig. 1B). We also isolated a subpopulation that expressed VNP at higher levels (Nanog:VNP(++)), but found its population-level transcriptome to be similar to that of Nanog:VNP(+), and so did not consider it further (Supplementary Fig. S1). In parallel, we removed 2i and LIF from the unsorted culture for 24 hours to study early events of culture-wide differentiation (N2B27 media) (Fig. 1B). We isolated RNA from each of these populations and performed RNA-seq to compare expression levels between the various conditions, in particular: i) Nanog:VNP(+) to Nanog:VNP(−) and ii) culture-wide differentiation (denoted “Diff”) to ESCs maintained in 2i + LIF medium (denoted “Stem”). Note that in our experimental design, it is possible that transient effects due to the switch between culture conditions may confound some of our comparisons. However, additional analyses we describe below suggested that the transcriptome of the Nanog:VNP(−) cells we analyzed are similar in character to those grown for longer periods in 2i + LIF conditions.


Heterogeneous lineage marker expression in naive embryonic stem cells is mostly due to spontaneous differentiation.

Nair G, Abranches E, Guedes AM, Henrique D, Raj A - Sci Rep (2015)

Experiment Diagram and Differential Expression of Selected Genes.(A) Simplified diagram of the construction of the Nd Nanog reporter cell line12. In the Nd cell line Venus (VNP) fluorescence is a reporter of Nanog expression. (B) Diagram and Nanog:VNP flow cytometry profiles of the samples from which RNA was extracted for RNA-Seq. The solid grey profile in all panels corresponds to fluorescence cytometry of the parent E14tg2a cell line used as a background control. In the Diff panel, the Stem data is shown with a grey outline for comparison. (C) RNA expression fold changes between the Nanog:VNP(+) and Nanog:VNP(−) (filled circles) samples and between the Stem and Diff (open circles) samples for several genes involved in pluripotency or in early extraembronic and embryonic lineages. Negative points denote genes whose expression is higher in Nanog:VNP(+) or Stem samples, while positive points denote genes whose expression is higher in Nanog:VNP(−) or Diff samples. Dots mark genes that also turn up as hits for significant differential expression at a 10% false discovery rate.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4544010&req=5

f1: Experiment Diagram and Differential Expression of Selected Genes.(A) Simplified diagram of the construction of the Nd Nanog reporter cell line12. In the Nd cell line Venus (VNP) fluorescence is a reporter of Nanog expression. (B) Diagram and Nanog:VNP flow cytometry profiles of the samples from which RNA was extracted for RNA-Seq. The solid grey profile in all panels corresponds to fluorescence cytometry of the parent E14tg2a cell line used as a background control. In the Diff panel, the Stem data is shown with a grey outline for comparison. (C) RNA expression fold changes between the Nanog:VNP(+) and Nanog:VNP(−) (filled circles) samples and between the Stem and Diff (open circles) samples for several genes involved in pluripotency or in early extraembronic and embryonic lineages. Negative points denote genes whose expression is higher in Nanog:VNP(+) or Stem samples, while positive points denote genes whose expression is higher in Nanog:VNP(−) or Diff samples. Dots mark genes that also turn up as hits for significant differential expression at a 10% false discovery rate.
Mentions: To accomplish this, we used the Nd reporter ESC line1112, which contains a short-lived fluorescent reporter (Venus-NLS-PEST, VNP) under the control of the Nanog genomic region (Fig. 1A). We sorted cells grown for 48 hours in medium containing FGF/ERK and GSK3 inhibitors (2i) and LIF (ground-state conditions) based on levels of reporter expression into both high (Nanog:VNP(+)) and low (Nanog:VNP(−)) populations (Fig. 1B). We also isolated a subpopulation that expressed VNP at higher levels (Nanog:VNP(++)), but found its population-level transcriptome to be similar to that of Nanog:VNP(+), and so did not consider it further (Supplementary Fig. S1). In parallel, we removed 2i and LIF from the unsorted culture for 24 hours to study early events of culture-wide differentiation (N2B27 media) (Fig. 1B). We isolated RNA from each of these populations and performed RNA-seq to compare expression levels between the various conditions, in particular: i) Nanog:VNP(+) to Nanog:VNP(−) and ii) culture-wide differentiation (denoted “Diff”) to ESCs maintained in 2i + LIF medium (denoted “Stem”). Note that in our experimental design, it is possible that transient effects due to the switch between culture conditions may confound some of our comparisons. However, additional analyses we describe below suggested that the transcriptome of the Nanog:VNP(−) cells we analyzed are similar in character to those grown for longer periods in 2i + LIF conditions.

Bottom Line: Here, we show the transcriptome of Nanog-negative cells exhibits expression of classes of genes associated with differentiation that are not yet active in cells exposed to differentiation conditions for one day.These results are consistent with the concept that Nanog-negative cells may contain subpopulations of both lineage-primed and differentiated cells.Our results suggest that the observed enrichment of lineage-specific marker gene expression in Nanog-negative cells is associated with spontaneous differentiation of a subset of these cells rather than the more random expression that may be associated with reversible lineage priming.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA.

ABSTRACT
Populations of cultured mouse embryonic stem cells (ESCs) exhibit a subfraction of cells expressing uncharacteristically low levels of pluripotency markers such as Nanog. Yet, the extent to which individual Nanog-negative cells are differentiated, both from ESCs and from each other, remains unclear. Here, we show the transcriptome of Nanog-negative cells exhibits expression of classes of genes associated with differentiation that are not yet active in cells exposed to differentiation conditions for one day. Long non-coding RNAs, however, exhibit more changes in expression in the one-day-differentiated cells than in Nanog-negative cells. These results are consistent with the concept that Nanog-negative cells may contain subpopulations of both lineage-primed and differentiated cells. Single cell analysis showed that Nanog-negative cells display substantial and coherent heterogeneity in lineage marker expression in progressively nested subsets of cells exhibiting low levels of Nanog, then low levels of Oct4, and then a set of lineage markers, which express intensely in a small subset of these more differentiated cells. Our results suggest that the observed enrichment of lineage-specific marker gene expression in Nanog-negative cells is associated with spontaneous differentiation of a subset of these cells rather than the more random expression that may be associated with reversible lineage priming.

No MeSH data available.


Related in: MedlinePlus