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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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Blastomere-derived hESC lines, SAB-113B and SAB-113D, can be differentiated into derivatives of all three germ layers.In vitro differentiation of SAB-113B (A) and SAB-113D (B) into all 3 germ layers was confirmed by immunocytochemical analysis. αFP – α fetoprotein (endoderm), β3T – βIII-tubulin (ectoderm), SMA – smooth muscle actin (mesoderm). The expression of derivatives of mesodermal – T (Brachyury homolog), WT1 (Wilms tumor 1) and ACTA2 (smooth muscle actin, α2); endodermal – GATA4 (GATA binding protein 4), SOX17 (sex determining region Y- box 17) and AFP (α-fetoprotein); and ectodermal – TUBB3 (tubulin, β3), NES (nestin) and PAX6 (paired box 6) genes were also confirmed by RT-PCR analysis (C). In vivo differentiation was confirmed by histopathological analysis of teratoma tissue obtained 3 months post-initiation under the dorsal flank of immunocompromised NOD/ SCID mice (D). Images of representative areas of H & E stained histological sections of the tumors: derivatives of endoderm – epithelial tissue (Ep) (i); mesoderm – bone tissue fragment (B) (ii) and early cartilage tissue (C) (iii); and ectoderm – neuroectodermal tissue (NE) (iv) and cells with melanin granules (red arrowheads) (iv, inset).
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pone-0026570-g003: Blastomere-derived hESC lines, SAB-113B and SAB-113D, can be differentiated into derivatives of all three germ layers.In vitro differentiation of SAB-113B (A) and SAB-113D (B) into all 3 germ layers was confirmed by immunocytochemical analysis. αFP – α fetoprotein (endoderm), β3T – βIII-tubulin (ectoderm), SMA – smooth muscle actin (mesoderm). The expression of derivatives of mesodermal – T (Brachyury homolog), WT1 (Wilms tumor 1) and ACTA2 (smooth muscle actin, α2); endodermal – GATA4 (GATA binding protein 4), SOX17 (sex determining region Y- box 17) and AFP (α-fetoprotein); and ectodermal – TUBB3 (tubulin, β3), NES (nestin) and PAX6 (paired box 6) genes were also confirmed by RT-PCR analysis (C). In vivo differentiation was confirmed by histopathological analysis of teratoma tissue obtained 3 months post-initiation under the dorsal flank of immunocompromised NOD/ SCID mice (D). Images of representative areas of H & E stained histological sections of the tumors: derivatives of endoderm – epithelial tissue (Ep) (i); mesoderm – bone tissue fragment (B) (ii) and early cartilage tissue (C) (iii); and ectoderm – neuroectodermal tissue (NE) (iv) and cells with melanin granules (red arrowheads) (iv, inset).

Mentions: hESC derived from the ICM of blastocyst stage embryos have the inherent capacity to differentiated into tissues of all three germ layers both in culture and when injected into the animals. To examine whether our newly derived blastomere lines exhibit similar pluripotent capacity, we have subjected SAB-113B and SAB-113D lines to spontaneous in vitro differentiation and teratoma formation in vivo. Differentiation in culture was performed in serum-containing differentiation medium on human matrix-coated plates. After 2–3 weeks of differentiation, cells were examined by RT-PCR and immunocytochemical analysis to determine expression of markers of all 3 germ layers. Our data showed that upon differentiation, similarly to ICM-derived lines, blastomere-derived lines also expressed markers of all three germ layers (Fig. 3A–C). We have previously reported that the differentiated products from blastomere-derived W10 line also showed similar expression of markers of all 3 germ layers.


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)

Blastomere-derived hESC lines, SAB-113B and SAB-113D, can be differentiated into derivatives of all three germ layers.In vitro differentiation of SAB-113B (A) and SAB-113D (B) into all 3 germ layers was confirmed by immunocytochemical analysis. αFP – α fetoprotein (endoderm), β3T – βIII-tubulin (ectoderm), SMA – smooth muscle actin (mesoderm). The expression of derivatives of mesodermal – T (Brachyury homolog), WT1 (Wilms tumor 1) and ACTA2 (smooth muscle actin, α2); endodermal – GATA4 (GATA binding protein 4), SOX17 (sex determining region Y- box 17) and AFP (α-fetoprotein); and ectodermal – TUBB3 (tubulin, β3), NES (nestin) and PAX6 (paired box 6) genes were also confirmed by RT-PCR analysis (C). In vivo differentiation was confirmed by histopathological analysis of teratoma tissue obtained 3 months post-initiation under the dorsal flank of immunocompromised NOD/ SCID mice (D). Images of representative areas of H & E stained histological sections of the tumors: derivatives of endoderm – epithelial tissue (Ep) (i); mesoderm – bone tissue fragment (B) (ii) and early cartilage tissue (C) (iii); and ectoderm – neuroectodermal tissue (NE) (iv) and cells with melanin granules (red arrowheads) (iv, inset).
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Related In: Results  -  Collection

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pone-0026570-g003: Blastomere-derived hESC lines, SAB-113B and SAB-113D, can be differentiated into derivatives of all three germ layers.In vitro differentiation of SAB-113B (A) and SAB-113D (B) into all 3 germ layers was confirmed by immunocytochemical analysis. αFP – α fetoprotein (endoderm), β3T – βIII-tubulin (ectoderm), SMA – smooth muscle actin (mesoderm). The expression of derivatives of mesodermal – T (Brachyury homolog), WT1 (Wilms tumor 1) and ACTA2 (smooth muscle actin, α2); endodermal – GATA4 (GATA binding protein 4), SOX17 (sex determining region Y- box 17) and AFP (α-fetoprotein); and ectodermal – TUBB3 (tubulin, β3), NES (nestin) and PAX6 (paired box 6) genes were also confirmed by RT-PCR analysis (C). In vivo differentiation was confirmed by histopathological analysis of teratoma tissue obtained 3 months post-initiation under the dorsal flank of immunocompromised NOD/ SCID mice (D). Images of representative areas of H & E stained histological sections of the tumors: derivatives of endoderm – epithelial tissue (Ep) (i); mesoderm – bone tissue fragment (B) (ii) and early cartilage tissue (C) (iii); and ectoderm – neuroectodermal tissue (NE) (iv) and cells with melanin granules (red arrowheads) (iv, inset).
Mentions: hESC derived from the ICM of blastocyst stage embryos have the inherent capacity to differentiated into tissues of all three germ layers both in culture and when injected into the animals. To examine whether our newly derived blastomere lines exhibit similar pluripotent capacity, we have subjected SAB-113B and SAB-113D lines to spontaneous in vitro differentiation and teratoma formation in vivo. Differentiation in culture was performed in serum-containing differentiation medium on human matrix-coated plates. After 2–3 weeks of differentiation, cells were examined by RT-PCR and immunocytochemical analysis to determine expression of markers of all 3 germ layers. Our data showed that upon differentiation, similarly to ICM-derived lines, blastomere-derived lines also expressed markers of all three germ layers (Fig. 3A–C). We have previously reported that the differentiated products from blastomere-derived W10 line also showed similar expression of markers of all 3 germ layers.

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