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Gene expression profiles during early differentiation of mouse embryonic stem cells.

Mansergh FC, Daly CS, Hurley AL, Wride MA, Hunter SM, Evans MJ - BMC Dev. Biol. (2009)

Bottom Line: A high degree of inter-replicate variability was noted when confirming array results.We found that individual EBs selected from the same dish were highly variable in gene expression profile.Tissue culture conditions that give the widest range of gene expression and maximise EB growth involve the use of 20% serum and starting cell numbers of 1000 per EB. 23 genes of importance to early development have been identified; more than half of these are also identified using similar studies, thus validating our results.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biosciences, Cardiff University, Cardiff, Wales, UK. mansergf@tcd.ie

ABSTRACT

Background: Understanding the mechanisms controlling stem cell differentiation is the key to future advances in tissue and organ regeneration. Embryonic stem (ES) cell differentiation can be triggered by embryoid body (EB) formation, which involves ES cell aggregation in suspension. EB growth in the absence of leukaemia inhibitory factor (LIF) leads EBs to mimic early embryonic development, giving rise to markers representative of endoderm, mesoderm and ectoderm. Here, we have used microarrays to investigate differences in gene expression between 3 undifferentiated ES cell lines, and also between undifferentiated ES cells and Day 1-4 EBs.

Results: An initial array study identified 4 gene expression changes between 3 undifferentiated ES cell lines. Tissue culture conditions for ES differentiation were then optimized to give the maximum range of gene expression and growth. -Undifferentiated ES cells and EBs cultured with and without LIF at each day for 4 days were subjected to microarray analysis. -Differential expression of 23 genes was identified. 13 of these were also differentially regulated in a separate array comparison between undifferentiated ES cells and compartments of very early embryos. A high degree of inter-replicate variability was noted when confirming array results. Using a panel of marker genes, RNA amplification and RT-PCR, we examined expression pattern variation between individual -D4-Lif EBs. We found that individual EBs selected from the same dish were highly variable in gene expression profile.

Conclusion: ES cell lines derived from different mouse strains and carrying different genetic modifications are almost invariant in gene expression profile under conditions used to maintain pluripotency. Tissue culture conditions that give the widest range of gene expression and maximise EB growth involve the use of 20% serum and starting cell numbers of 1000 per EB. 23 genes of importance to early development have been identified; more than half of these are also identified using similar studies, thus validating our results. EBs cultured in the same dish vary widely in terms of their gene expression (and hence, undoubtedly, in their future differentiation potential). This may explain some of the inherent variability in differentiation protocols that use EBs.

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Confirmed genes, up- or down-regulated during differentiation without LIF. Ann temp = PCR annealing temperature, Cycles = number of PCR cycles used. Expression change marks the direction of expression changes as shown by RT-PCR. Consistency 1000C, 750C refers to the number of replicates out of three that show the same change (always at least 2), this information is given as space constraints prevent showing all.
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Figure 8: Confirmed genes, up- or down-regulated during differentiation without LIF. Ann temp = PCR annealing temperature, Cycles = number of PCR cycles used. Expression change marks the direction of expression changes as shown by RT-PCR. Consistency 1000C, 750C refers to the number of replicates out of three that show the same change (always at least 2), this information is given as space constraints prevent showing all.

Mentions: Given problems with reproducibility that have been noted with stem cell arrays [20-22], RT-PCRs were carried out on 3 sets each of 1000 cell and 750 cell EB samples; differentially regulated genes showed a reproducible expression pattern change in at least 4/6 samples. 18 genes were differentially regulated in -LIF samples; a further 8 were differentially regulated in + LIF samples. 3 genes appeared in both datasets (Tubb5 and 2 uncharacterised genes; BG063737 and BG069482, see Figures 8 and 9 and Figures 1 and 2). None of these 23 genes appeared in the list of 3 that were differentially regulated between different undifferentiated ES cell lines (GSE8625). A further 3 genes did not show the exact expression patterns predicted by the array, but were dramatically downregulated on induction of differentiation and are therefore also presented in Figure 8. This gave a total of 26 genes we deemed differentially regulated during ES cell differentiation. Q-PCR analysis of two confirmed genes, Hspa8 and BG063737, gave very similar results to semi-quantitative PCR (Additional File 2).


Gene expression profiles during early differentiation of mouse embryonic stem cells.

Mansergh FC, Daly CS, Hurley AL, Wride MA, Hunter SM, Evans MJ - BMC Dev. Biol. (2009)

Confirmed genes, up- or down-regulated during differentiation without LIF. Ann temp = PCR annealing temperature, Cycles = number of PCR cycles used. Expression change marks the direction of expression changes as shown by RT-PCR. Consistency 1000C, 750C refers to the number of replicates out of three that show the same change (always at least 2), this information is given as space constraints prevent showing all.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2656490&req=5

Figure 8: Confirmed genes, up- or down-regulated during differentiation without LIF. Ann temp = PCR annealing temperature, Cycles = number of PCR cycles used. Expression change marks the direction of expression changes as shown by RT-PCR. Consistency 1000C, 750C refers to the number of replicates out of three that show the same change (always at least 2), this information is given as space constraints prevent showing all.
Mentions: Given problems with reproducibility that have been noted with stem cell arrays [20-22], RT-PCRs were carried out on 3 sets each of 1000 cell and 750 cell EB samples; differentially regulated genes showed a reproducible expression pattern change in at least 4/6 samples. 18 genes were differentially regulated in -LIF samples; a further 8 were differentially regulated in + LIF samples. 3 genes appeared in both datasets (Tubb5 and 2 uncharacterised genes; BG063737 and BG069482, see Figures 8 and 9 and Figures 1 and 2). None of these 23 genes appeared in the list of 3 that were differentially regulated between different undifferentiated ES cell lines (GSE8625). A further 3 genes did not show the exact expression patterns predicted by the array, but were dramatically downregulated on induction of differentiation and are therefore also presented in Figure 8. This gave a total of 26 genes we deemed differentially regulated during ES cell differentiation. Q-PCR analysis of two confirmed genes, Hspa8 and BG063737, gave very similar results to semi-quantitative PCR (Additional File 2).

Bottom Line: A high degree of inter-replicate variability was noted when confirming array results.We found that individual EBs selected from the same dish were highly variable in gene expression profile.Tissue culture conditions that give the widest range of gene expression and maximise EB growth involve the use of 20% serum and starting cell numbers of 1000 per EB. 23 genes of importance to early development have been identified; more than half of these are also identified using similar studies, thus validating our results.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biosciences, Cardiff University, Cardiff, Wales, UK. mansergf@tcd.ie

ABSTRACT

Background: Understanding the mechanisms controlling stem cell differentiation is the key to future advances in tissue and organ regeneration. Embryonic stem (ES) cell differentiation can be triggered by embryoid body (EB) formation, which involves ES cell aggregation in suspension. EB growth in the absence of leukaemia inhibitory factor (LIF) leads EBs to mimic early embryonic development, giving rise to markers representative of endoderm, mesoderm and ectoderm. Here, we have used microarrays to investigate differences in gene expression between 3 undifferentiated ES cell lines, and also between undifferentiated ES cells and Day 1-4 EBs.

Results: An initial array study identified 4 gene expression changes between 3 undifferentiated ES cell lines. Tissue culture conditions for ES differentiation were then optimized to give the maximum range of gene expression and growth. -Undifferentiated ES cells and EBs cultured with and without LIF at each day for 4 days were subjected to microarray analysis. -Differential expression of 23 genes was identified. 13 of these were also differentially regulated in a separate array comparison between undifferentiated ES cells and compartments of very early embryos. A high degree of inter-replicate variability was noted when confirming array results. Using a panel of marker genes, RNA amplification and RT-PCR, we examined expression pattern variation between individual -D4-Lif EBs. We found that individual EBs selected from the same dish were highly variable in gene expression profile.

Conclusion: ES cell lines derived from different mouse strains and carrying different genetic modifications are almost invariant in gene expression profile under conditions used to maintain pluripotency. Tissue culture conditions that give the widest range of gene expression and maximise EB growth involve the use of 20% serum and starting cell numbers of 1000 per EB. 23 genes of importance to early development have been identified; more than half of these are also identified using similar studies, thus validating our results. EBs cultured in the same dish vary widely in terms of their gene expression (and hence, undoubtedly, in their future differentiation potential). This may explain some of the inherent variability in differentiation protocols that use EBs.

Show MeSH
Related in: MedlinePlus