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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

Single Cell Analysis of Transcriptome Heterogeneity.(A) Maximum projection images from Nd cells stained for Nanog and Oct4 RNA. Scale bar in all panels is 5 um long. The Nanog(−)Oct4(−) cell in the center is flanked by Nanog(+)Oct4(+) cells. (bottom) Summary of RNA FISH results on 1189 single cells. The vast majority of cells are Nanog(+)Oct4(+). 60 cells are Nanog(−), and 44 of these are also Oct4(−). The (+/−) cutoffs were 30 and 80 transcripts for Nanog and Oct4, respectively (see Supplementary Fig. S9). (B) RNA FISH staining for Nanog and Crabp2 (ectoderm marker) showing a Nanog(+)Crabp2(−) and a Nanog(−)Crabp2(+) cell. (bottom) Summary of simultaneous staining for Crabp2, T (Brachyury) and Nanog. The (+/−) cutoffs were 30 and 50 transcripts for Crabp2 and T respectively (Supplementary Fig. S9). (C) RNA FISH staining for Tbx6, T and Oct4, showing a Tbx6(+) T(+) Oct4(−) cell. (bottom) Summary. The (+/−) cutoff for Tbx6 was 10 transcripts (Supplementary Fig. S9).
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f4: Single Cell Analysis of Transcriptome Heterogeneity.(A) Maximum projection images from Nd cells stained for Nanog and Oct4 RNA. Scale bar in all panels is 5 um long. The Nanog(−)Oct4(−) cell in the center is flanked by Nanog(+)Oct4(+) cells. (bottom) Summary of RNA FISH results on 1189 single cells. The vast majority of cells are Nanog(+)Oct4(+). 60 cells are Nanog(−), and 44 of these are also Oct4(−). The (+/−) cutoffs were 30 and 80 transcripts for Nanog and Oct4, respectively (see Supplementary Fig. S9). (B) RNA FISH staining for Nanog and Crabp2 (ectoderm marker) showing a Nanog(+)Crabp2(−) and a Nanog(−)Crabp2(+) cell. (bottom) Summary of simultaneous staining for Crabp2, T (Brachyury) and Nanog. The (+/−) cutoffs were 30 and 50 transcripts for Crabp2 and T respectively (Supplementary Fig. S9). (C) RNA FISH staining for Tbx6, T and Oct4, showing a Tbx6(+) T(+) Oct4(−) cell. (bottom) Summary. The (+/−) cutoff for Tbx6 was 10 transcripts (Supplementary Fig. S9).

Mentions: First, to characterize the cells expressing low levels of Nanog mRNA, we co-hybridized cells with probes targeting Nanog mRNA together with probes targeting the pluripotency marker Oct4. By examining the single cell mRNA distributions for both Nanog and Oct4, we identified two clear subpopulations, which we denote Nanog mRNA (+/−) (cutoff of 30 mRNA molecules per cell) and Oct4 mRNA (+/−) (cutoff of 80 mRNA molecules per cell) (Supplementary Fig. S9). As apparent from the distributions, this classification was not particularly sensitive to the particular threshold chosen because the distinction between the positive and negative populations was clear. Within the overall population, we found that around 5% (N = 1189) of cells had very low levels of Nanog mRNA (less than 30 transcripts per cell). We found that 70% of these Nanog mRNA(−) cells have very low Oct4 mRNA levels (less than 80 transcripts per cell) (Fig. 4A, Supplementary Fig. S9). Since maintaining Oct4 gene expression within a tight concentration interval is considered essential to the ESC state24, we inferred that these cells have likely exited pluripotency. In a separate experiment, we also found that the majority of Nanog mRNA(−) cells have also very low levels of Rex1 transcripts (Supplementary Fig. S10). It is also important to point out that the lack of mRNA does not necessarily imply a complete lack of protein expression. In the case of Oct4, the protein half-life is roughly 12 hours and the mRNA half-life is ~7.5 hours25. Thus, the relatively large fold change difference of mRNA suggests that the cell has not transcribed Oct4 for some time, thus making it likely that the protein level has decreased significantly.


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)

Single Cell Analysis of Transcriptome Heterogeneity.(A) Maximum projection images from Nd cells stained for Nanog and Oct4 RNA. Scale bar in all panels is 5 um long. The Nanog(−)Oct4(−) cell in the center is flanked by Nanog(+)Oct4(+) cells. (bottom) Summary of RNA FISH results on 1189 single cells. The vast majority of cells are Nanog(+)Oct4(+). 60 cells are Nanog(−), and 44 of these are also Oct4(−). The (+/−) cutoffs were 30 and 80 transcripts for Nanog and Oct4, respectively (see Supplementary Fig. S9). (B) RNA FISH staining for Nanog and Crabp2 (ectoderm marker) showing a Nanog(+)Crabp2(−) and a Nanog(−)Crabp2(+) cell. (bottom) Summary of simultaneous staining for Crabp2, T (Brachyury) and Nanog. The (+/−) cutoffs were 30 and 50 transcripts for Crabp2 and T respectively (Supplementary Fig. S9). (C) RNA FISH staining for Tbx6, T and Oct4, showing a Tbx6(+) T(+) Oct4(−) cell. (bottom) Summary. The (+/−) cutoff for Tbx6 was 10 transcripts (Supplementary Fig. S9).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Single Cell Analysis of Transcriptome Heterogeneity.(A) Maximum projection images from Nd cells stained for Nanog and Oct4 RNA. Scale bar in all panels is 5 um long. The Nanog(−)Oct4(−) cell in the center is flanked by Nanog(+)Oct4(+) cells. (bottom) Summary of RNA FISH results on 1189 single cells. The vast majority of cells are Nanog(+)Oct4(+). 60 cells are Nanog(−), and 44 of these are also Oct4(−). The (+/−) cutoffs were 30 and 80 transcripts for Nanog and Oct4, respectively (see Supplementary Fig. S9). (B) RNA FISH staining for Nanog and Crabp2 (ectoderm marker) showing a Nanog(+)Crabp2(−) and a Nanog(−)Crabp2(+) cell. (bottom) Summary of simultaneous staining for Crabp2, T (Brachyury) and Nanog. The (+/−) cutoffs were 30 and 50 transcripts for Crabp2 and T respectively (Supplementary Fig. S9). (C) RNA FISH staining for Tbx6, T and Oct4, showing a Tbx6(+) T(+) Oct4(−) cell. (bottom) Summary. The (+/−) cutoff for Tbx6 was 10 transcripts (Supplementary Fig. S9).
Mentions: First, to characterize the cells expressing low levels of Nanog mRNA, we co-hybridized cells with probes targeting Nanog mRNA together with probes targeting the pluripotency marker Oct4. By examining the single cell mRNA distributions for both Nanog and Oct4, we identified two clear subpopulations, which we denote Nanog mRNA (+/−) (cutoff of 30 mRNA molecules per cell) and Oct4 mRNA (+/−) (cutoff of 80 mRNA molecules per cell) (Supplementary Fig. S9). As apparent from the distributions, this classification was not particularly sensitive to the particular threshold chosen because the distinction between the positive and negative populations was clear. Within the overall population, we found that around 5% (N = 1189) of cells had very low levels of Nanog mRNA (less than 30 transcripts per cell). We found that 70% of these Nanog mRNA(−) cells have very low Oct4 mRNA levels (less than 80 transcripts per cell) (Fig. 4A, Supplementary Fig. S9). Since maintaining Oct4 gene expression within a tight concentration interval is considered essential to the ESC state24, we inferred that these cells have likely exited pluripotency. In a separate experiment, we also found that the majority of Nanog mRNA(−) cells have also very low levels of Rex1 transcripts (Supplementary Fig. S10). It is also important to point out that the lack of mRNA does not necessarily imply a complete lack of protein expression. In the case of Oct4, the protein half-life is roughly 12 hours and the mRNA half-life is ~7.5 hours25. Thus, the relatively large fold change difference of mRNA suggests that the cell has not transcribed Oct4 for some time, thus making it likely that the protein level has decreased significantly.

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