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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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Expression of pluripotency genes in cleavage stage blastomere-derived and blastocyst stage ICM-derived hESC.SAB-113B (A) and SAB-113D (B) cells express markers of pluripotency as shown by immunocytochemical analyses (OCT4, NANOG, SSEA-4, TRA 1-60, and TRA 1-81) and alkaline phosphatase activity (AP). Insets of the images show the nuclei of the cells stained with HOECHST 33342. Normalized expression of well-known 14 pluripotency genes (DNMT3B, DPPA2, DPPA4, GABRB3, GDF3, MYBL2, NANOG, PHC1, POU5F1, SALL4, SOX2, TCF7L1, TERT and ZFP42) in all the replicates of blastomere lines (SBD1, SBD2, SBD3, SBD4, SBD5, SBD6, SBD7 and SBD8), and whole embryo lines (WED1, WED2, WED3 and WED4) showed consistent high values compared to reference RNA (ref) (C). Flow cytometric analysis of these lines also showed consistent high expression of OCT4 and NANOG. Representative diagram for SAB-113B is depicted (D). In addition, SAB-113B (E) and SAB-113D (F) lines exhibited a stable male karyotype (46, XY) after 10 passages in culture.
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pone-0026570-g002: Expression of pluripotency genes in cleavage stage blastomere-derived and blastocyst stage ICM-derived hESC.SAB-113B (A) and SAB-113D (B) cells express markers of pluripotency as shown by immunocytochemical analyses (OCT4, NANOG, SSEA-4, TRA 1-60, and TRA 1-81) and alkaline phosphatase activity (AP). Insets of the images show the nuclei of the cells stained with HOECHST 33342. Normalized expression of well-known 14 pluripotency genes (DNMT3B, DPPA2, DPPA4, GABRB3, GDF3, MYBL2, NANOG, PHC1, POU5F1, SALL4, SOX2, TCF7L1, TERT and ZFP42) in all the replicates of blastomere lines (SBD1, SBD2, SBD3, SBD4, SBD5, SBD6, SBD7 and SBD8), and whole embryo lines (WED1, WED2, WED3 and WED4) showed consistent high values compared to reference RNA (ref) (C). Flow cytometric analysis of these lines also showed consistent high expression of OCT4 and NANOG. Representative diagram for SAB-113B is depicted (D). In addition, SAB-113B (E) and SAB-113D (F) lines exhibited a stable male karyotype (46, XY) after 10 passages in culture.

Mentions: Using previously described methods for blastomere biopsy and hESC derivation on human feeders, we have derived two new hESC lines from biopsied blastomeres of cleavage (8-cell) stage embryos (Fig. 1). These newly derived hESC lines, SAB-113B and SAB-113D, expressed known markers of stemness including OCT4, NANOG, SSEA4, TRA-1-60, TRA-1-81 as determined by immunocytochemical analysis and exhibited alkaline phosphatase activity (Fig. 2A, 2B). Similar results have already been obtained for previously derived blastomere line W10 [4] and whole embryo lines SG4 and SG7 (data not shown). Next, we compared normalized expression of 14 established hESC stemness genes (DNMT3B, DPPA2, DPPA4, GABRB3, GDF3, MYBL2, NANOG, PHC1, POU5F1, SALL4, SOX2, TCF7L1, TERT and ZFP42) in newly generated blastomere-derived SAB-113B and SAB-113D lines, previously derived W10 blastomere line, and whole embryo ICM-derived lines, SG4 and SG7, and observed consistent high expression of all stemness genes examined and lack of any major differences in their expression levels between blastomere-derived and whole embryo-derived hESC lines (Fig. 2C). Furthermore, FACS analysis showed that more than 80% of cells in blastomere-derived (Fig. 2D) and whole embryo ICM-derived (data not shown) hESC lines expressed both OCT4 and NANOG. In addition, these lines also showed no significant difference in growth rate, with approximate doubling time of 40 h on feeders and 24 h under feeder-free conditions. Cell and colony morphology was examined semiweekly by inverted microscopy using phase contrast light and Hoffman optics and did not reveal any significant differences between these lines. Newly derived hESC lines exhibited stable normal male karyotype (46, XY) after 10 passages in culture (Fig. 2E, 2F) as examined by G-banding analysis. Blastomere-derived W10 line showed a stable female karyotype (46, XX) as previously reported and both whole embryo SG4 and SG7 lines exhibited a stable normal male karyotype (46, XY; data not shown).


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)

Expression of pluripotency genes in cleavage stage blastomere-derived and blastocyst stage ICM-derived hESC.SAB-113B (A) and SAB-113D (B) cells express markers of pluripotency as shown by immunocytochemical analyses (OCT4, NANOG, SSEA-4, TRA 1-60, and TRA 1-81) and alkaline phosphatase activity (AP). Insets of the images show the nuclei of the cells stained with HOECHST 33342. Normalized expression of well-known 14 pluripotency genes (DNMT3B, DPPA2, DPPA4, GABRB3, GDF3, MYBL2, NANOG, PHC1, POU5F1, SALL4, SOX2, TCF7L1, TERT and ZFP42) in all the replicates of blastomere lines (SBD1, SBD2, SBD3, SBD4, SBD5, SBD6, SBD7 and SBD8), and whole embryo lines (WED1, WED2, WED3 and WED4) showed consistent high values compared to reference RNA (ref) (C). Flow cytometric analysis of these lines also showed consistent high expression of OCT4 and NANOG. Representative diagram for SAB-113B is depicted (D). In addition, SAB-113B (E) and SAB-113D (F) lines exhibited a stable male karyotype (46, XY) after 10 passages in culture.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198782&req=5

pone-0026570-g002: Expression of pluripotency genes in cleavage stage blastomere-derived and blastocyst stage ICM-derived hESC.SAB-113B (A) and SAB-113D (B) cells express markers of pluripotency as shown by immunocytochemical analyses (OCT4, NANOG, SSEA-4, TRA 1-60, and TRA 1-81) and alkaline phosphatase activity (AP). Insets of the images show the nuclei of the cells stained with HOECHST 33342. Normalized expression of well-known 14 pluripotency genes (DNMT3B, DPPA2, DPPA4, GABRB3, GDF3, MYBL2, NANOG, PHC1, POU5F1, SALL4, SOX2, TCF7L1, TERT and ZFP42) in all the replicates of blastomere lines (SBD1, SBD2, SBD3, SBD4, SBD5, SBD6, SBD7 and SBD8), and whole embryo lines (WED1, WED2, WED3 and WED4) showed consistent high values compared to reference RNA (ref) (C). Flow cytometric analysis of these lines also showed consistent high expression of OCT4 and NANOG. Representative diagram for SAB-113B is depicted (D). In addition, SAB-113B (E) and SAB-113D (F) lines exhibited a stable male karyotype (46, XY) after 10 passages in culture.
Mentions: Using previously described methods for blastomere biopsy and hESC derivation on human feeders, we have derived two new hESC lines from biopsied blastomeres of cleavage (8-cell) stage embryos (Fig. 1). These newly derived hESC lines, SAB-113B and SAB-113D, expressed known markers of stemness including OCT4, NANOG, SSEA4, TRA-1-60, TRA-1-81 as determined by immunocytochemical analysis and exhibited alkaline phosphatase activity (Fig. 2A, 2B). Similar results have already been obtained for previously derived blastomere line W10 [4] and whole embryo lines SG4 and SG7 (data not shown). Next, we compared normalized expression of 14 established hESC stemness genes (DNMT3B, DPPA2, DPPA4, GABRB3, GDF3, MYBL2, NANOG, PHC1, POU5F1, SALL4, SOX2, TCF7L1, TERT and ZFP42) in newly generated blastomere-derived SAB-113B and SAB-113D lines, previously derived W10 blastomere line, and whole embryo ICM-derived lines, SG4 and SG7, and observed consistent high expression of all stemness genes examined and lack of any major differences in their expression levels between blastomere-derived and whole embryo-derived hESC lines (Fig. 2C). Furthermore, FACS analysis showed that more than 80% of cells in blastomere-derived (Fig. 2D) and whole embryo ICM-derived (data not shown) hESC lines expressed both OCT4 and NANOG. In addition, these lines also showed no significant difference in growth rate, with approximate doubling time of 40 h on feeders and 24 h under feeder-free conditions. Cell and colony morphology was examined semiweekly by inverted microscopy using phase contrast light and Hoffman optics and did not reveal any significant differences between these lines. Newly derived hESC lines exhibited stable normal male karyotype (46, XY) after 10 passages in culture (Fig. 2E, 2F) as examined by G-banding analysis. Blastomere-derived W10 line showed a stable female karyotype (46, XX) as previously reported and both whole embryo SG4 and SG7 lines exhibited a stable normal male karyotype (46, XY; data not shown).

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