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Human embryonic stem cells derived from embryos at different stages of development share similar transcription profiles.

Giritharan G, Ilic D, Gormley M, Krtolica A - PLoS ONE (2011)

Bottom Line: Blastomere-derived hESC lines exhibited all the standard characteristics of hESC including undifferentiated proliferation, genomic stability, expression of pluripotency markers and the ability to differentiate into the cells of all three germ layers both in vitro and in vivo.Furthermore, this profile was evident in very early passages of the cells and did not appear to be affected by extensive passaging.These results suggest that during derivation process cells which give rise to hESC acquire virtually identical stable phenotype and are not affected by the developmental stage of the starting cell population.

View Article: PubMed Central - PubMed

Affiliation: SLL Sciences, StemLifeLine, Inc., San Carlos, California, United States of America.

ABSTRACT
We have derived hESC from biopsied blastomeres of cleavage stage embryos under virtually the same conditions we used for the derivation of hESC lines from inner cell mass of blastocyst stage embryos. Blastomere-derived hESC lines exhibited all the standard characteristics of hESC including undifferentiated proliferation, genomic stability, expression of pluripotency markers and the ability to differentiate into the cells of all three germ layers both in vitro and in vivo. To examine whether hESC lines derived from two developmental stages of the embryo differ in gene expression, we have subjected three blastomere-derived hESC lines and two ICM-derived hESC lines grown under identical culture conditions to transcriptome analysis using gene expression arrays. Unlike previously reported comparisons of hESC lines which demonstrated, apart from core hESC-associated pluripotency signature, significant variations in gene expression profiles of different lines, our data show that hESC lines derived and grown under well-controlled defined culture conditions adopt nearly identical gene expression profiles. Moreover, blastomere-derived and ICM-derived hESC exhibited very similar transcriptional profiles independent of the developmental stage of the embryo from which they originated. Furthermore, this profile was evident in very early passages of the cells and did not appear to be affected by extensive passaging. These results suggest that during derivation process cells which give rise to hESC acquire virtually identical stable phenotype and are not affected by the developmental stage of the starting cell population.

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Gene expression analysis of cleavage stage blastomere-derived and blastocyst stage ICM-derived hESC lines.Venn diagram of microarray analysis showing differentially expressed genes after pairwise comparison of blastomere-derived (SBD) and whole embryo-derived (WED) lines to reference RNA (A). Clustering tree generated by unsupervised hierarchical clustering analysis of summed median difference in the top 10,000 probe sets relative to reference RNA, and the normalized expression value of top 25 overexpressed and top 25 underexpressed genes (B). Blue and red colors represent lower and higher expression, respectively. Pie charts depict distribution of uniquely expressed genes of blastomere-derived (C) and whole embryo-derived (D) lines compared to reference RNA into known cellular biological processes such as cell cycle, regulation of transcription, cell differentiation, signaling, cell migration, apoptosis, cell adhesion, and cytoskeleton organization.
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pone-0026570-g004: Gene expression analysis of cleavage stage blastomere-derived and blastocyst stage ICM-derived hESC lines.Venn diagram of microarray analysis showing differentially expressed genes after pairwise comparison of blastomere-derived (SBD) and whole embryo-derived (WED) lines to reference RNA (A). Clustering tree generated by unsupervised hierarchical clustering analysis of summed median difference in the top 10,000 probe sets relative to reference RNA, and the normalized expression value of top 25 overexpressed and top 25 underexpressed genes (B). Blue and red colors represent lower and higher expression, respectively. Pie charts depict distribution of uniquely expressed genes of blastomere-derived (C) and whole embryo-derived (D) lines compared to reference RNA into known cellular biological processes such as cell cycle, regulation of transcription, cell differentiation, signaling, cell migration, apoptosis, cell adhesion, and cytoskeleton organization.

Mentions: We performed transcriptome analysis on two whole embryo-derived lines (WED) from blastocyst-stage (SG4 and SG7) and three single blastomere derived lines (SBD) from cleavage stage embryos (SAB-113B, SAB-113D and W10). SAB-113B and SAB-113D lines were derived from two different blastomeres of one embryo and W10 line was derived from a single blastomere of another embryo. The SG4 and SG7 lines were derived from ICM of two different whole embryos. Three replicates of SAB-113B, SAB-113D blastomere lines, and two replicates of W10 blastomere line and SG4 and SG7 whole embryo lines, and human universal reference total RNA from two different lot numbers were included in the experiment. In order to minimize culture induced variations in gene expression, all blastomere and whole embryo lines were grown under identical defined feeder-free serum-free culturing conditions on human matrix-coated surfaces. Cell pellets were collected and RNA samples were generated from cells harvested when the colonies reached 70–80% confluency to ensure similar cell cycle profiles. Human Universal Reference Total RNA was used as control to identify genes which are specifically over- and under- expressed in hESC lines. These commercially available RNA samples were made by pooling total RNA extracts from a collection of different whole tissue sources, thus providing the broadest coverage of expressed genes. The similarities and differences between the single embryo derived (SBD) and whole embryo derived (WED) lines were established by first comparing each to the reference RNA, then performing a 4-way venn of the identified up- and down-regulated genes. Of the 6894 and 6683 significantly different genes in SBD and WED respectively (relative to reference RNA), some 6387 were shared (over 90%). Specifically, 3405 genes were commonly up-regulated and 2982 were commonly down-regulated. None of the genes were oppositely regulated (Fig. 4A). This indicates that less than 10% of the genes may be uniquely expressed in ESC line of specific origin.


Human embryonic stem cells derived from embryos at different stages of development share similar transcription profiles.

Giritharan G, Ilic D, Gormley M, Krtolica A - PLoS ONE (2011)

Gene expression analysis of cleavage stage blastomere-derived and blastocyst stage ICM-derived hESC lines.Venn diagram of microarray analysis showing differentially expressed genes after pairwise comparison of blastomere-derived (SBD) and whole embryo-derived (WED) lines to reference RNA (A). Clustering tree generated by unsupervised hierarchical clustering analysis of summed median difference in the top 10,000 probe sets relative to reference RNA, and the normalized expression value of top 25 overexpressed and top 25 underexpressed genes (B). Blue and red colors represent lower and higher expression, respectively. Pie charts depict distribution of uniquely expressed genes of blastomere-derived (C) and whole embryo-derived (D) lines compared to reference RNA into known cellular biological processes such as cell cycle, regulation of transcription, cell differentiation, signaling, cell migration, apoptosis, cell adhesion, and cytoskeleton organization.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198782&req=5

pone-0026570-g004: Gene expression analysis of cleavage stage blastomere-derived and blastocyst stage ICM-derived hESC lines.Venn diagram of microarray analysis showing differentially expressed genes after pairwise comparison of blastomere-derived (SBD) and whole embryo-derived (WED) lines to reference RNA (A). Clustering tree generated by unsupervised hierarchical clustering analysis of summed median difference in the top 10,000 probe sets relative to reference RNA, and the normalized expression value of top 25 overexpressed and top 25 underexpressed genes (B). Blue and red colors represent lower and higher expression, respectively. Pie charts depict distribution of uniquely expressed genes of blastomere-derived (C) and whole embryo-derived (D) lines compared to reference RNA into known cellular biological processes such as cell cycle, regulation of transcription, cell differentiation, signaling, cell migration, apoptosis, cell adhesion, and cytoskeleton organization.
Mentions: We performed transcriptome analysis on two whole embryo-derived lines (WED) from blastocyst-stage (SG4 and SG7) and three single blastomere derived lines (SBD) from cleavage stage embryos (SAB-113B, SAB-113D and W10). SAB-113B and SAB-113D lines were derived from two different blastomeres of one embryo and W10 line was derived from a single blastomere of another embryo. The SG4 and SG7 lines were derived from ICM of two different whole embryos. Three replicates of SAB-113B, SAB-113D blastomere lines, and two replicates of W10 blastomere line and SG4 and SG7 whole embryo lines, and human universal reference total RNA from two different lot numbers were included in the experiment. In order to minimize culture induced variations in gene expression, all blastomere and whole embryo lines were grown under identical defined feeder-free serum-free culturing conditions on human matrix-coated surfaces. Cell pellets were collected and RNA samples were generated from cells harvested when the colonies reached 70–80% confluency to ensure similar cell cycle profiles. Human Universal Reference Total RNA was used as control to identify genes which are specifically over- and under- expressed in hESC lines. These commercially available RNA samples were made by pooling total RNA extracts from a collection of different whole tissue sources, thus providing the broadest coverage of expressed genes. The similarities and differences between the single embryo derived (SBD) and whole embryo derived (WED) lines were established by first comparing each to the reference RNA, then performing a 4-way venn of the identified up- and down-regulated genes. Of the 6894 and 6683 significantly different genes in SBD and WED respectively (relative to reference RNA), some 6387 were shared (over 90%). Specifically, 3405 genes were commonly up-regulated and 2982 were commonly down-regulated. None of the genes were oppositely regulated (Fig. 4A). This indicates that less than 10% of the genes may be uniquely expressed in ESC line of specific origin.

Bottom Line: Blastomere-derived hESC lines exhibited all the standard characteristics of hESC including undifferentiated proliferation, genomic stability, expression of pluripotency markers and the ability to differentiate into the cells of all three germ layers both in vitro and in vivo.Furthermore, this profile was evident in very early passages of the cells and did not appear to be affected by extensive passaging.These results suggest that during derivation process cells which give rise to hESC acquire virtually identical stable phenotype and are not affected by the developmental stage of the starting cell population.

View Article: PubMed Central - PubMed

Affiliation: SLL Sciences, StemLifeLine, Inc., San Carlos, California, United States of America.

ABSTRACT
We have derived hESC from biopsied blastomeres of cleavage stage embryos under virtually the same conditions we used for the derivation of hESC lines from inner cell mass of blastocyst stage embryos. Blastomere-derived hESC lines exhibited all the standard characteristics of hESC including undifferentiated proliferation, genomic stability, expression of pluripotency markers and the ability to differentiate into the cells of all three germ layers both in vitro and in vivo. To examine whether hESC lines derived from two developmental stages of the embryo differ in gene expression, we have subjected three blastomere-derived hESC lines and two ICM-derived hESC lines grown under identical culture conditions to transcriptome analysis using gene expression arrays. Unlike previously reported comparisons of hESC lines which demonstrated, apart from core hESC-associated pluripotency signature, significant variations in gene expression profiles of different lines, our data show that hESC lines derived and grown under well-controlled defined culture conditions adopt nearly identical gene expression profiles. Moreover, blastomere-derived and ICM-derived hESC exhibited very similar transcriptional profiles independent of the developmental stage of the embryo from which they originated. Furthermore, this profile was evident in very early passages of the cells and did not appear to be affected by extensive passaging. These results suggest that during derivation process cells which give rise to hESC acquire virtually identical stable phenotype and are not affected by the developmental stage of the starting cell population.

Show MeSH
Related in: MedlinePlus