Unknown,Transcriptomics,Genomics,Proteomics

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Comparative expression profiles of human embryonic stem cells among different feeders and conditioned media

ABSTRACT: Background: The hES-T3 cell line with normal female karyotype was used to differentiate into autogeneic fibroblast-like cells (T3HDF) as feeder to support the undifferentiated growth of hES-T3 cells (T3/HDF) for 14 passages. A feeder-free culture on Matrigel in hES medium conditioned by these autogeneic feeder cells (T3HDF) was established to maintain the undifferentiated growth of hES-T3 cells (T3/CMHDF) for 8 passages. Results: The gene expression profiles of mRNAs, microRNAs and proteins between the undifferentiated T3/HDF and T3/CMHDF cells were shown to be very similar, and their expression profiles were also found to be similar to those of T3/MEF and T3/CMMEF cells grown on MEF feeder and feeder-free Matrigel in MEF-conditioned medium, respectively. The abundantly expressed genes of T3/HDF, T3/CMHDF, T3/MEF and T3/CMMEF cells were found to play prominent roles in signaling pathways and GO process networks. The miR-302/367 cluster and miR-371/372/373 cluster were extremely abundantly expressed in undifferentiated T3/HDF and T3/CMHDF as well as T3/MEF andT3/CMMEF cells. The undifferentiated state of T3/HDF and T3/CMHDF cells was evidenced by the very high expression levels of ¡§stemness¡¨ genes and low expression levels of differentiation markers of ectoderm, mesoderm and endoderm in addition to the strong staining of OCT4 and NANOG. Conclusion: The T3HDF feeder and T3HDF-conditioned medium were able to support the undifferentiated growth of hES cells, and they would be useful for drug development and toxicity testing in addition to the reduced risks of xenogeneic pathogens when used for medical applications such as cell therapies. Keywords: genetic modification In this investigation, both miRNA and mRNA expression profiles from human embryonic stem cells (hES-T3) grown on MEF feeder (T3_MEF), feeder-free Matrigel in MEF-conditioned medium (T3_CMMEF), T3HDF (hES-T3 differentiated fibroblast-like cells) feeder and feeder-free Matrigel in T3HDF-conditioned medium were quantitatively determined. Several target genes of T3BA and T3CM cells-specific miRNAs were identified. ***This submission represents the mRNA expression component of the study only***

ORGANISM(S): Homo sapiens

SUBMITTER: Sung-Liang Yu

PROVIDER: E-GEOD-19902 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Human embryonic stem (hES) cells have the capacities to propagate for extended periods and to differentiate into cell types from all three germ layers both in vitro and in vivo. These characteristics of self-renewal and pluripotency enable hES cells having the potential to provide an unlimited supply of different cell types for tissue replacement, drug screening, and functional genomics studies. The hES-T3 cells with normal female karyotype cultured on either mouse embryonic fibroblasts (MEF) in hES medium (containing 4 ng/ml bFGF) (T3MF) or feeder-free Matrigel in MEF-conditioned medium (supplemented with additional 4 ng/ml bFGF) (T3CM) were found to express very similar profiles of mRNAs and microRNAs, indicating that the unlimited self-renewal and pluripotency of hES cells can be maintained by continuing culture on these two conditions. However, the expression profiles, especially microRNAs, of the hES-T3 cells cultured on Matrigel in hES medium supplemented with 4 ng/ml bFGF and 5 ng/ml activin A (T3BA) were found to be different from those of T3MF and T3CM cells. In T3BA cells, four hES cell-specific microRNAs miR-372, miR-302d, miR-367 and miR-200c, as well as three other microRNAs miR-199a, miR-19a and miR-217, were found to be up-regulated, whereas five miRNAs miR-19b, miR-221, miR-222, let-7b and let-7c were down-regulated by activin A. Thirteen abundantly differentially expressed mRNAs, including NR4A2, ERBB4, CXCR4, PCDH9, TMEFF2, CD24 and COX6A1 genes, targeted by seven over-expressed miRNAs were identified by inverse expression levels of these seven microRNAs to their target mRNAs in T3BA and T3CM cells. The NR4A2, ERBB4 and CXCR4 target genes were further found to be regulated by EGF and/or TNF. The 50 abundantly differentially expressed genes targeted by five under-expressed miRNAs were also identified. The abundantly expressed mRNAs in T3BA and T3CM cells were also analyzed for the network and signaling pathways, and roles of activin A in cell proliferation and differentiation were found. These findings will help elucidate the complex signaling network which maintains the self-renewal and pluripotency of hES cells. In this investigation, both miRNA and mRNA expression profiles from human embryonic stem cells grown on MEF feeder (T3MF), feeder-free Matrigel in MEF-conditioned medium (T3CM) and in hES medium (containing 4 ng/ml bFGF) supplemented with 5 ng/ml activin A (T3BA) were quantitatively determined. Several target genes of T3BA and T3CM cells-specific miRNAs were identified. ***This submission represents the mRNA expression component of the study only***

Project description:Analysis of genes that were differentially expressed in mutant VUB03_DM1 as compared to controls VUB01 and SA01 undifferentiated hES cells Embryonic stem (ES) cell lines provide, theoretically, unlimited access to any needed amount of any specific cell phenotype of an organism, due to their unique capacities at indefinite self-renewal and pluripotency (Smith 2001; Trounson 2006). These properties allow using the progeny of ES cell lines to model human pathologies (Martinat, Shendelman et al. 2004; Lerou and Daley 2005; Ben-Nun and Benvenisty 2006). In particular, human ES cell lines derived from embryos characterized as gene-carriers following pre-implantation genetic diagnosis (PGD) for any one of major monogenic diseases (Pickering, Minger et al. 2005; Mateizel, De Temmerman et al. 2006) may be considered as perfect cellular replicas of those diseases, as they exhibit the exact genotypes associated to them. Here, we confirm this hypothesis by demonstrating that the cell progeny of an ES cell line derived from an embryo with myotonic dystrophy type 1 (DM1) displayed the morphological stigma associated to the expression of the mutant gene âso-called intranuclear foci- as well as abnormal alternate splicing of the insulin receptor, a characteristic feature of DM1. Further differential transcriptomic analysis of the DM1 gene-carrying cells with phenotypically similar populations from native ES cell lines revealed abnormal expression of 89 genes, among which 48 were down-regulated and 39 over-expressed. This study demonstrates that DM1, though a disease with relatively late clinical onset, is associated with expression of genetic defects early on during development. It underlines the value of PGD-derived ES cell lines as a tool to decipher molecular mechanisms of genetic diseases. Ben-Nun, I. F. and N. Benvenisty (2006). Human embryonic stem cells as a cellular model for human disorders. Mol Cell Endocrinol 252(1-2): 154-9. Lerou, P. H. and G. Q. Daley (2005). Therapeutic potential of embryonic stem cells. Blood Rev 19(6): 321-31. Martinat, C., S. Shendelman, et al. (2004). Sensitivity to oxidative stress in DJ-1-deficient dopamine neurons: an ES- derived cell model of primary Parkinsonism. PLoS Biol 2(11): e327. Mateizel, I., N. De Temmerman, et al. (2006). Derivation of human embryonic stem cell lines from embryos obtained after IVF and after PGD for monogenic disorders. Hum Reprod 21(2): 503-11. Pickering, S. J., S. L. Minger, et al. (2005). Generation of a human embryonic stem cell line encoding the cystic fibrosis mutation deltaF508, using preimplantation genetic diagnosis. Reprod Biomed Online 10(3): 390-7. Smith, A. G. (2001). Embryo-derived stem cells: of mice and men. Annu Rev Cell Dev Biol 17: 435-62. Trounson, A. (2006). The production and directed differentiation of human embryonic stem cells. Endocr Rev 27(2): 208-19. Keywords: disease state analysis Two controls cell lines (VUB01 and SA01) and one mutant cell line (VUB03_DM1), with three biological replicats for each

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Comparative expression profiles of human embryonic stem cells among different feeders and conditioned media

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