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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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Bioinformatic analysis of confirmed genes (part 1). From top left, columns are as follows: Experiment: this column indicates whether the gene confirmed is from the GSE8625 (undifferentiated ES cell lines) or GSE8766 (EB differentiation) gene lists. Acc no: GenBank accession number. Gene ID: the NIA clone ID, which is referred to in the NCBI nucleotide database as Gene ID. Identity refers to the official NCBI gene name to which the gene relates (where this is known). UniGene: refers to the UniGene cluster to which the EST has been assigned (where known). Chromosome indicates the mouse chromosome to which the EST maps. Differential regulation gives the nature of the expression change and the fold change derived from array analysis. Bioinformatics indicates possible functions related to differentiation obtained from searches of the NCBI database and the literature. Differentially regulated:other experiments describes if the EST/gene in question has been identified as differentially regulated in our own study of ES cells versus compartments of early embryos (GSE8881), and/or similar published work. Abbreviations associated with GSE8881: IMT11: undifferentiated IMT11 ES cells. ICM88 and ICM105 = blastocyst inner cell mass, 88 hours post coitum, 105 hours post coitum. DICM136, DICM180 = delayed blastocyst inner cell mass, 136 hours post coitum, 180 hours post coitum. EE5.5, EE6.5 = embryonic ectoderm, 5.5 and 6.5 days post coitum. For methods and more detailed descriptions of this experiment, see GEO, GSE8881, [24].
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Figure 1: Bioinformatic analysis of confirmed genes (part 1). From top left, columns are as follows: Experiment: this column indicates whether the gene confirmed is from the GSE8625 (undifferentiated ES cell lines) or GSE8766 (EB differentiation) gene lists. Acc no: GenBank accession number. Gene ID: the NIA clone ID, which is referred to in the NCBI nucleotide database as Gene ID. Identity refers to the official NCBI gene name to which the gene relates (where this is known). UniGene: refers to the UniGene cluster to which the EST has been assigned (where known). Chromosome indicates the mouse chromosome to which the EST maps. Differential regulation gives the nature of the expression change and the fold change derived from array analysis. Bioinformatics indicates possible functions related to differentiation obtained from searches of the NCBI database and the literature. Differentially regulated:other experiments describes if the EST/gene in question has been identified as differentially regulated in our own study of ES cells versus compartments of early embryos (GSE8881), and/or similar published work. Abbreviations associated with GSE8881: IMT11: undifferentiated IMT11 ES cells. ICM88 and ICM105 = blastocyst inner cell mass, 88 hours post coitum, 105 hours post coitum. DICM136, DICM180 = delayed blastocyst inner cell mass, 136 hours post coitum, 180 hours post coitum. EE5.5, EE6.5 = embryonic ectoderm, 5.5 and 6.5 days post coitum. For methods and more detailed descriptions of this experiment, see GEO, GSE8881, [24].

Mentions: Samples were compared with pooled controls; sets of 4 experimental replicates were also compared with normalised samples from each of the other experimental samples. A "master list" of genes from all analyses was generated (see Figures 1 and 2 (all confirmed genes) and Additional File 1 (all unconfirmed genes)).


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)

Bioinformatic analysis of confirmed genes (part 1). From top left, columns are as follows: Experiment: this column indicates whether the gene confirmed is from the GSE8625 (undifferentiated ES cell lines) or GSE8766 (EB differentiation) gene lists. Acc no: GenBank accession number. Gene ID: the NIA clone ID, which is referred to in the NCBI nucleotide database as Gene ID. Identity refers to the official NCBI gene name to which the gene relates (where this is known). UniGene: refers to the UniGene cluster to which the EST has been assigned (where known). Chromosome indicates the mouse chromosome to which the EST maps. Differential regulation gives the nature of the expression change and the fold change derived from array analysis. Bioinformatics indicates possible functions related to differentiation obtained from searches of the NCBI database and the literature. Differentially regulated:other experiments describes if the EST/gene in question has been identified as differentially regulated in our own study of ES cells versus compartments of early embryos (GSE8881), and/or similar published work. Abbreviations associated with GSE8881: IMT11: undifferentiated IMT11 ES cells. ICM88 and ICM105 = blastocyst inner cell mass, 88 hours post coitum, 105 hours post coitum. DICM136, DICM180 = delayed blastocyst inner cell mass, 136 hours post coitum, 180 hours post coitum. EE5.5, EE6.5 = embryonic ectoderm, 5.5 and 6.5 days post coitum. For methods and more detailed descriptions of this experiment, see GEO, GSE8881, [24].
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Bioinformatic analysis of confirmed genes (part 1). From top left, columns are as follows: Experiment: this column indicates whether the gene confirmed is from the GSE8625 (undifferentiated ES cell lines) or GSE8766 (EB differentiation) gene lists. Acc no: GenBank accession number. Gene ID: the NIA clone ID, which is referred to in the NCBI nucleotide database as Gene ID. Identity refers to the official NCBI gene name to which the gene relates (where this is known). UniGene: refers to the UniGene cluster to which the EST has been assigned (where known). Chromosome indicates the mouse chromosome to which the EST maps. Differential regulation gives the nature of the expression change and the fold change derived from array analysis. Bioinformatics indicates possible functions related to differentiation obtained from searches of the NCBI database and the literature. Differentially regulated:other experiments describes if the EST/gene in question has been identified as differentially regulated in our own study of ES cells versus compartments of early embryos (GSE8881), and/or similar published work. Abbreviations associated with GSE8881: IMT11: undifferentiated IMT11 ES cells. ICM88 and ICM105 = blastocyst inner cell mass, 88 hours post coitum, 105 hours post coitum. DICM136, DICM180 = delayed blastocyst inner cell mass, 136 hours post coitum, 180 hours post coitum. EE5.5, EE6.5 = embryonic ectoderm, 5.5 and 6.5 days post coitum. For methods and more detailed descriptions of this experiment, see GEO, GSE8881, [24].
Mentions: Samples were compared with pooled controls; sets of 4 experimental replicates were also compared with normalised samples from each of the other experimental samples. A "master list" of genes from all analyses was generated (see Figures 1 and 2 (all confirmed genes) and Additional File 1 (all unconfirmed genes)).

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