Transcriptome of embryonic stem cells cultured in enzymatic and mechanic passaging methods [RNA-seq]
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE47946 | GEO | 2015/06/01
SECONDARY ACCESSION(S): PRJNA208433
REPOSITORIES: GEO
Similar Datasets
Project description:hESC have morphologic, genetic and genomic alternatiions when cells cultured in different passaging condition. Here transcriptome of four different hESC lines were compared in two passaging methods.
2015-06-01 | GSE47916 | GEO
Project description:hESC have morphologic, genetic and genomic alternatiions when cells cultured in different passaging condition. Here sub-karyotypic genome change in three different hESC lines were compared in two passaging methods.
2015-06-01 | GSE47914 | GEO
Project description:To realize the potential of human embryonic stem cells (hESCs) in regenerative medicine and drug discovery applications, large numbers of cells that accurately recapitulate cell and tissue function must be robustly produced. Previous studies have suggested that genetic instability and epigenetic changes occur as a consequence of enzymatic passaging. However, the potential impacts of such passaging methods on the metabolism of hESCs have not been described. Using stable isotope tracing and mass spectrometry-based metabolomics, we have explored how different passaging reagents impact hESC metabolism. Enzymatic passaging caused significant decreases in glucose utilization throughout central carbon metabolism along with attenuated de novo lipogenesis. In addition, we developed and validated a method for rapidly quantifying glycan abundance and isotopic labeling in hydrolyzed biomass. Enzymatic passaging reagents significantly altered levels of glycans immediately after digestion but surprisingly glucose contribution to glycans was not affected. These results demonstrate that there is an immediate effect on hESC metabolism after enzymatic passaging in both central carbon metabolism and biosynthesis. HESCs subjected to enzymatic passaging are routinely placed in a state requiring re-synthesis of biomass components, subtly influencing their metabolic needs in a manner that may impact cell performance in regenerative medicine applications.
| S-EPMC4885641 | biostudies-literature
Project description:Ascorbate activates CD30 expression and causes widespread specific demethylation of the epigenome of serum free cultured hESC. The genetic and epigenetic integrity of human Embryonic Stem cells (hESCs) is critical to their future applications in research and medicine. hESC cultured in serum free media can accumulate point mutations, aneuploidy and progressive epigenetic changes over prolonged culture in vitro. We have identified ascorbate as the only molecule in the very widely used Knock-out serum replacement medium that is sufficient to induce expression of CD30, a biomarker for aneuploidy in hESCs. In fact, we show that hESC cultured in the presence of ascorbate for 20 passages not only display demethylation of the CD30 locus, but exhibit widespread and remarkably specific and consistent demethylation of 1,847 genes in both HES2 and HES3 cells. The specific ascorbate induced demethylation changes in the hESC epigenome, of which 86% are shared between the two lines, identify a subset of genes in hESC, including CD30, that are sensitive to serum free culture medium induced epigenetic changes. Experiment. HES2 cells were maintained on 20% Fetal calf serum on mouse embryonic fibroblast feeder layers with mechanical dissection. Using HES2 after 99 mechanical passages (P99), hESC were grown for 17-20 (+17 - +20) passages in the presence of 20% Knock-Out Serum Replacement (Invitrogen) either with (+ASC) or without (-ASC) Ascorbate with enzymatic culturing.
2010-06-10 | GSE16590 | GEO
Project description:Human embryonic stem cell derived retinal pigmented epithelial cells (hESC-RPE) are in clinical trials for the treatment of macular diseases. Currently, these cells take over three months to derive and subsequent months to mature and characterize. After only five to six passages the cells begin to undergo an epithelial-to-mesenchymal transition and are unsuitable for cellular therapies. We describe a novel passaging protocol and show that inhibition of Rho-associated, coiled coil containing protein kinases (ROCK1 and ROCK2) using Y-27632, allows extended passage of hESC-RPE in serum-free culture with maintenance of the RPE phenotype. After 30 population doublings, hESC-RPE at passage 13 maintain normal karyotype, display typical, polarized epithelial morphology, and continue to express RPE-specific genes. Passage 13 hESC-RPE show protein localization patterns similar to passage 2 cells, and display similar levels of growth factor secretion and phagocytosis of photoreceptor outer segments. Microarray analysis from day 2 cells shows several key pathways are altered by ROCK inhibition, including stimulation of the cell cycle and suppression of TGFM-NM-2 and Wnt signaling. These findings describe a means to greatly increase the yield of functional hESC-RPE for use in research and clinical trials. Two-condition experiment, Control vs Y-27632 treated hESC-RPE passage 5. Biological replicates: 4 control, 4 Y-27632. The experiments were technically carried out as dual channel (eg, Cy3 and Cy5-labeled samples hybridized to the same array) but processed as though they are single channel (Cy3 and Cy5 signals are calculated; Cy3/Cy5 ratios are not calculated). Therefore, there are 4 raw data files for total 8 samples.
2014-06-30 | E-GEOD-56618 | biostudies-arrayexpress
Project description:In the following experiment, three different hESC cell lines (HES2, MEL1 and H9) were grown in the presence of KOSR, KOSR or mTESR containing media respectively. KOSR (Knockout serum replacement medium) is a standard media allowing the growth of hESC without the need for manual passaging - Enzymatic passaging is used instread. mTESR (Ludwig et al., 2007) is a media allowing the growth of hESC on matrigel with enzymatic passaging. At day 7 after passaging, these cells were FACs sorted for the presence of GCTM-2 and CD9 into 4 distinct fractions (p4: GCTM-2-neg, CD9-neg; p5: GCTM-2-low, CD9-low; p6: GCTM-2-medium, CD9-medium and p7: GCTM-2-high, CD9-high). For each cell line-subfraction combination, RNA was harvested and subject to microarray. From each experiment (individual cell line), 4 samples were collected (p4, p5, p6 and p7) and these were subject to microarray. In each case the experiment was performed in triplicate
2009-10-01 | GSE13877 | GEO
Project description:To identify the genes that are regulated during passaging of the parental mouse Dunn osteosarcoma cells with low metastatic potential and Dunn-derived LM8 cells with increased metastatic potential, we performed global gene expression profiling in early and late passage cells.
2015-03-10 | GSE66674 | GEO
Project description:In the following experiment, three different hESC cell lines (HES2, MEL1 and H9) were grown in the presence of KOSR, KOSR or mTESR containing media respectively. KOSR (Knockout serum replacement medium) is a standard media allowing the growth of hESC without the need for manual passaging - Enzymatic passaging is used instread. mTESR (Ludwig et al., 2007) is a media allowing the growth of hESC on matrigel with enzymatic passaging. At day 7 after passaging, these cells were FACs sorted for the presence of GCTM-2 and CD9 into 4 distinct fractions (p4: GCTM-2-neg, CD9-neg; p5: GCTM-2-low, CD9-low; p6: GCTM-2-medium, CD9-medium and p7: GCTM-2-high, CD9-high). For each cell line-subfraction combination, RNA was harvested and subject to microarray. From each experiment (individual cell line), 4 samples were collected (p4, p5, p6 and p7) and these were subject to microarray. In each case the experiment was performed in triplicate Three independent experiments of three consecutive passages of ESC cells were grown and subject to FACs sorting, collection and microarray. A total of 36 samples, (3 experiments of 3 replicates of 4 sorted populations of cells (p4, p5, p6, p7))
2010-04-07 | E-GEOD-13877 | biostudies-arrayexpress
Project description:Although human embryonic stem cells (hESCs) are equipped with highly effective machinery for the maintenance of genome integrity, the frequency of genetic aberrations during long-term in vitro hESC culture has been a serious issue that raises concerns over their safety in future clinical applications. By passaging hESCs over a broad range of timepoints, we found that mitotic aberrations, such as the delay of mitosis, multipolar centrosomes, and chromosome mis-segregation, were increased in the late-passaged hESCs (LP-hESCs) in parallel with polyploidy compared to early-passaged hESCs (EP-hESCs). Through high-resolution genome-wide approaches and by following transcriptome analysis, we found that LP-hESCs with a minimal amplicon in chromosome 20q11.21 highly expressed TPX2 (targeting protein for Xklp2), a key protein for governing spindle assembly and cancer malignancy. Consistent with these findings, the inducible expression of TPX2 in EP-hESCs reproduced aberrant mitotic events, such as the delay of mitotic progression, spindle stability, misaligned chromosomes, and polyploidy. This data suggests that the amplification and increased transcription of the TPX2 gene at 20q11.21 could contribute to an increase in aberrant mitosis due to altered spindle dynamics.
2021-02-26 | GSE167495 | GEO
Project description: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.
2011-10-21 | GSE29625 | GEO