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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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Derivation of hESC from biopsied blastomere of the cleavage stage embryo.Micrographs showing the stepwise procedure of embryo biopsy using inverted microscope-attached micromanipulator (A–D) and the appearance of initial outgrowth and hESC colony during the derivation procedure (E–L). All pictures were taken using microscope mounted digital camera and Hoffman optics under 200X total magnification.
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pone-0026570-g001: Derivation of hESC from biopsied blastomere of the cleavage stage embryo.Micrographs showing the stepwise procedure of embryo biopsy using inverted microscope-attached micromanipulator (A–D) and the appearance of initial outgrowth and hESC colony during the derivation procedure (E–L). All pictures were taken using microscope mounted digital camera and Hoffman optics under 200X total magnification.

Mentions: Three human embryonic stem cell (hESC) lines were derived from biopsied blastomeres of 2 surplus frozen human cleavage stage embryos donated by two consenting couples that provided written consent according to the protocol approved by WIRB. These lines (W10, SAB-113B, and SAB-113D) were derived using established derivation procedure in our laboratory (illustrated in Fig. 1). Briefly, the embryos were thawed and incubated in Quinn's cleavage medium for minimum 3 h at standard culture conditions. Blastomeres were removed from each embryo using established biopsy procedure [11]. Each biopsied blastomere was cultured in a 20 µl drop of Quinn's cleavage medium in an incubator at 37 °C. After 24 h incubation, the blastomeres were transferred onto irradiated human foreskin fibroblast (HFF) feeders in 50 µl drops of Quinn's cleavage medium supplemented with human laminin (10 µg/ml; day 0). Three days later cultures were assessed for blastomere attachment to feeders. Starting on day 3, medium in drops containing attached blastomeres has been refreshed every day by replacing 1/3 of volume with Quinn's blastocyst medium supplemented with human laminin (10 µg/ml), recombinant human leukemia inhibitory factor (LIF; 10 ng/ml), and recombinant human basic fibroblast growth factor (FGF; 25 ng/ml; R&D Systems, Inc.). From day 5, Quinn's blastocyst medium was replaced with standard hESC medium (80% KO-DMEM, 20% KSR, FGF-25 ng/ml, and LIF-10 ng/ml), and replaced in drops daily. Embryonic stem cell-certified fetal calf serum (10%) was added to the medium for a week. On day 11, initial hESC colonies were dissected and left in the same drop. On day 14, colonies were dissected and transferred into 4-well dish with new HFF feeders. From the next day onwards the cells were cultured in standard hESC medium containing FGF (25 ng/ml) and LIF (10 ng/ml).


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)

Derivation of hESC from biopsied blastomere of the cleavage stage embryo.Micrographs showing the stepwise procedure of embryo biopsy using inverted microscope-attached micromanipulator (A–D) and the appearance of initial outgrowth and hESC colony during the derivation procedure (E–L). All pictures were taken using microscope mounted digital camera and Hoffman optics under 200X total magnification.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026570-g001: Derivation of hESC from biopsied blastomere of the cleavage stage embryo.Micrographs showing the stepwise procedure of embryo biopsy using inverted microscope-attached micromanipulator (A–D) and the appearance of initial outgrowth and hESC colony during the derivation procedure (E–L). All pictures were taken using microscope mounted digital camera and Hoffman optics under 200X total magnification.
Mentions: Three human embryonic stem cell (hESC) lines were derived from biopsied blastomeres of 2 surplus frozen human cleavage stage embryos donated by two consenting couples that provided written consent according to the protocol approved by WIRB. These lines (W10, SAB-113B, and SAB-113D) were derived using established derivation procedure in our laboratory (illustrated in Fig. 1). Briefly, the embryos were thawed and incubated in Quinn's cleavage medium for minimum 3 h at standard culture conditions. Blastomeres were removed from each embryo using established biopsy procedure [11]. Each biopsied blastomere was cultured in a 20 µl drop of Quinn's cleavage medium in an incubator at 37 °C. After 24 h incubation, the blastomeres were transferred onto irradiated human foreskin fibroblast (HFF) feeders in 50 µl drops of Quinn's cleavage medium supplemented with human laminin (10 µg/ml; day 0). Three days later cultures were assessed for blastomere attachment to feeders. Starting on day 3, medium in drops containing attached blastomeres has been refreshed every day by replacing 1/3 of volume with Quinn's blastocyst medium supplemented with human laminin (10 µg/ml), recombinant human leukemia inhibitory factor (LIF; 10 ng/ml), and recombinant human basic fibroblast growth factor (FGF; 25 ng/ml; R&D Systems, Inc.). From day 5, Quinn's blastocyst medium was replaced with standard hESC medium (80% KO-DMEM, 20% KSR, FGF-25 ng/ml, and LIF-10 ng/ml), and replaced in drops daily. Embryonic stem cell-certified fetal calf serum (10%) was added to the medium for a week. On day 11, initial hESC colonies were dissected and left in the same drop. On day 14, colonies were dissected and transferred into 4-well dish with new HFF feeders. From the next day onwards the cells were cultured in standard hESC medium containing FGF (25 ng/ml) and LIF (10 ng/ml).

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