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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

Genome Wide Analysis of Heterogeneity and Differentiation.(A) Distribution of fold changes for all genes in our study between Nanog:VNP(+) and Nanog:VNP(−) subpopulations (left) and between Stem and Diff conditions (right). Black regions indicate genes that are significant hits at a 10% false discovery rate. (B) Joint distribution of fold changes for genes between this work and literature data. Left 2 panels include only genes that are hits in Nanog:VNP(+)/Nanog:VNP(−) and compare Nanog:VNP(+)/Nanog:VNP(−) fold change to reported RNA-Seq fold changes for Rex1+/Rex1- populations grown i serum + LIF8. Right panel: Includes only genes that are hits in Stem/Diff and compares to reported microarray fold changes between ESCs and day1 EpiLCs6. (C) The set of GO categories selected by our “greedy” method for genes that are hits in either Nanog:VNP(+)/Nanog:VNP(−) or Stem/Diff, using a minimal GO size of 750 genes, in the order that they were selected (most relevant first). Leftmost panel: Fraction of genes in GO category that are a hit, with the vertical line showing the (background) fraction of all genes that are hits. Right 4 panels: Within each panel, each hit gene is assigned to the topmost category that it is annotated for. (D) For each transcription factor, number of genes called as bound in literature20 that are also hits of the specified type, divided by the number of bound genes. Black vertical lines indicate the fraction of hits in our gene universe.
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f2: Genome Wide Analysis of Heterogeneity and Differentiation.(A) Distribution of fold changes for all genes in our study between Nanog:VNP(+) and Nanog:VNP(−) subpopulations (left) and between Stem and Diff conditions (right). Black regions indicate genes that are significant hits at a 10% false discovery rate. (B) Joint distribution of fold changes for genes between this work and literature data. Left 2 panels include only genes that are hits in Nanog:VNP(+)/Nanog:VNP(−) and compare Nanog:VNP(+)/Nanog:VNP(−) fold change to reported RNA-Seq fold changes for Rex1+/Rex1- populations grown i serum + LIF8. Right panel: Includes only genes that are hits in Stem/Diff and compares to reported microarray fold changes between ESCs and day1 EpiLCs6. (C) The set of GO categories selected by our “greedy” method for genes that are hits in either Nanog:VNP(+)/Nanog:VNP(−) or Stem/Diff, using a minimal GO size of 750 genes, in the order that they were selected (most relevant first). Leftmost panel: Fraction of genes in GO category that are a hit, with the vertical line showing the (background) fraction of all genes that are hits. Right 4 panels: Within each panel, each hit gene is assigned to the topmost category that it is annotated for. (D) For each transcription factor, number of genes called as bound in literature20 that are also hits of the specified type, divided by the number of bound genes. Black vertical lines indicate the fraction of hits in our gene universe.

Mentions: Owing to the depth of our sequencing and high degree of fidelity between replicates (Supplementary Fig. S5), we were able to characterize differences in the transcriptomes at a high level of precision. Nearly half the genes with fold changes of at least 40% in Nanog:VNP(+) vs Nanog:VNP(−) were called as differential expression hits at a false discovery rate (FDR) of 10% (Fig. 2A). Of these 3293 hits, 1993 are more highly expressed in Nanog:VNP(−) than Nanog:VNP(+) and 1300 are more highly expressed in Nanog:VNP(+). In our differentiation comparison, we calculated 4233 gene hits differentially expressed between the 2i + LIF population (Stem) and after 24 h without 2i and LIF (Diff) (Fig. 2A). Due to even higher replicate reproducibility, these hits include genes with RNA fold changes as small as 20%. In this case, 2235 gene hits were down-regulated and 1998 up-regulated after a day of differentiation. Our Nanog:VNP(+/−) results correlate closely with RNA-seq-based expression differences between Rex1(+) and (−) cells8, and our Stem/Diff results are in concordance with earlier findings which compare ESCs and day 1 EpiLCs in differentiation conditions6 (Fig. 2B). Interestingly, we found that the expression changes we observed in Nanog:VNP(−) cells did not correlate with those previously observed after Nanog depletion (Supplementary Fig. S6)17, suggesting that the altered transcriptome of Nanog:VNP(−) cells does not reflect functional effects of Nanog levels per se.


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)

Genome Wide Analysis of Heterogeneity and Differentiation.(A) Distribution of fold changes for all genes in our study between Nanog:VNP(+) and Nanog:VNP(−) subpopulations (left) and between Stem and Diff conditions (right). Black regions indicate genes that are significant hits at a 10% false discovery rate. (B) Joint distribution of fold changes for genes between this work and literature data. Left 2 panels include only genes that are hits in Nanog:VNP(+)/Nanog:VNP(−) and compare Nanog:VNP(+)/Nanog:VNP(−) fold change to reported RNA-Seq fold changes for Rex1+/Rex1- populations grown i serum + LIF8. Right panel: Includes only genes that are hits in Stem/Diff and compares to reported microarray fold changes between ESCs and day1 EpiLCs6. (C) The set of GO categories selected by our “greedy” method for genes that are hits in either Nanog:VNP(+)/Nanog:VNP(−) or Stem/Diff, using a minimal GO size of 750 genes, in the order that they were selected (most relevant first). Leftmost panel: Fraction of genes in GO category that are a hit, with the vertical line showing the (background) fraction of all genes that are hits. Right 4 panels: Within each panel, each hit gene is assigned to the topmost category that it is annotated for. (D) For each transcription factor, number of genes called as bound in literature20 that are also hits of the specified type, divided by the number of bound genes. Black vertical lines indicate the fraction of hits in our gene universe.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4544010&req=5

f2: Genome Wide Analysis of Heterogeneity and Differentiation.(A) Distribution of fold changes for all genes in our study between Nanog:VNP(+) and Nanog:VNP(−) subpopulations (left) and between Stem and Diff conditions (right). Black regions indicate genes that are significant hits at a 10% false discovery rate. (B) Joint distribution of fold changes for genes between this work and literature data. Left 2 panels include only genes that are hits in Nanog:VNP(+)/Nanog:VNP(−) and compare Nanog:VNP(+)/Nanog:VNP(−) fold change to reported RNA-Seq fold changes for Rex1+/Rex1- populations grown i serum + LIF8. Right panel: Includes only genes that are hits in Stem/Diff and compares to reported microarray fold changes between ESCs and day1 EpiLCs6. (C) The set of GO categories selected by our “greedy” method for genes that are hits in either Nanog:VNP(+)/Nanog:VNP(−) or Stem/Diff, using a minimal GO size of 750 genes, in the order that they were selected (most relevant first). Leftmost panel: Fraction of genes in GO category that are a hit, with the vertical line showing the (background) fraction of all genes that are hits. Right 4 panels: Within each panel, each hit gene is assigned to the topmost category that it is annotated for. (D) For each transcription factor, number of genes called as bound in literature20 that are also hits of the specified type, divided by the number of bound genes. Black vertical lines indicate the fraction of hits in our gene universe.
Mentions: Owing to the depth of our sequencing and high degree of fidelity between replicates (Supplementary Fig. S5), we were able to characterize differences in the transcriptomes at a high level of precision. Nearly half the genes with fold changes of at least 40% in Nanog:VNP(+) vs Nanog:VNP(−) were called as differential expression hits at a false discovery rate (FDR) of 10% (Fig. 2A). Of these 3293 hits, 1993 are more highly expressed in Nanog:VNP(−) than Nanog:VNP(+) and 1300 are more highly expressed in Nanog:VNP(+). In our differentiation comparison, we calculated 4233 gene hits differentially expressed between the 2i + LIF population (Stem) and after 24 h without 2i and LIF (Diff) (Fig. 2A). Due to even higher replicate reproducibility, these hits include genes with RNA fold changes as small as 20%. In this case, 2235 gene hits were down-regulated and 1998 up-regulated after a day of differentiation. Our Nanog:VNP(+/−) results correlate closely with RNA-seq-based expression differences between Rex1(+) and (−) cells8, and our Stem/Diff results are in concordance with earlier findings which compare ESCs and day 1 EpiLCs in differentiation conditions6 (Fig. 2B). Interestingly, we found that the expression changes we observed in Nanog:VNP(−) cells did not correlate with those previously observed after Nanog depletion (Supplementary Fig. S6)17, suggesting that the altered transcriptome of Nanog:VNP(−) cells does not reflect functional effects of Nanog levels per se.

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