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Yong-Kook Kang
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Yong-Mahn Han Korea Research Institute of Bioscience and Biotechnology (KRIBB), Department of Biological Sciences and Center for Stem Cell Differentiation, Center for Regenerative Medicine, 52 Eoeun-Dong, Yuseong-Gu, Daejeon 305-806, South Korea

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Complex signaling pathways operate in human embryonic stem cells (hESCs) and are coordinated to maintain self-renewal and stem cell characteristics in them. Protein tyrosine kinases (PTKs) participate in diverse signaling pathways in various types of cells. Because of their functions as key molecules in various cellular processes, PTKs are anticipated to have important roles also in hESCs. In this study, we investigated the roles of PTKs in undifferentiated and differentiated hESCs. To establish comprehensive PTK expression profiles in hESCs, we performed reverse transcriptase PCR using degenerate primers according to the conserved catalytic PTK motifs in both undifferentiated and differentiated hESCs. Here, we identified 42 different kinases in two hESC lines, including 5 non-receptor tyrosine kinases (RTKs), 24 RTKs, and 13 dual and other kinases, and compared the protein kinase expression profiles of hESCs and retinoic acid-treated hESCs. Significantly, up- and downregulated kinases in undifferentiated hESCs were confirmed by real-time PCR and western blotting. MAP3K3, ERBB2, FGFR4, and EPHB2 were predominantly upregulated, while CSF1R, TYRO3, SRC, and GSK3A were consistently downregulated in two hESC lines. Western blot analysis showed that the transcriptional levels of these kinases were consistent with the translational levels. The obstruction of upregulated kinases’ activities using specific inhibitors disturbed the undifferentiated status and induced the differentiation of hESCs. Our results support the dynamic expression of PTKs during hESC maintenance and suggest that specific PTKs that are consistently up- and downregulated play important roles in the maintenance of stemness and the direction of differentiation of hESCs.

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Gabbine Wee Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea,Korea Advanced Institute of Science and Technology (KAIST), Center for Stem Cell Differentiation, Daejeon 305-701, Republic of Korea andKorea Research Institute of Bioscience and Biotechnology (KRIBB), Center for Development and Differentiation, Daejeon 305-806, Republic of Korea

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Jung-Jae Shim Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea,Korea Advanced Institute of Science and Technology (KAIST), Center for Stem Cell Differentiation, Daejeon 305-701, Republic of Korea andKorea Research Institute of Bioscience and Biotechnology (KRIBB), Center for Development and Differentiation, Daejeon 305-806, Republic of Korea

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Deog-Bon Koo Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea,Korea Advanced Institute of Science and Technology (KAIST), Center for Stem Cell Differentiation, Daejeon 305-701, Republic of Korea andKorea Research Institute of Bioscience and Biotechnology (KRIBB), Center for Development and Differentiation, Daejeon 305-806, Republic of Korea

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Jung-Il Chae Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea,Korea Advanced Institute of Science and Technology (KAIST), Center for Stem Cell Differentiation, Daejeon 305-701, Republic of Korea andKorea Research Institute of Bioscience and Biotechnology (KRIBB), Center for Development and Differentiation, Daejeon 305-806, Republic of Korea

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Kyung-Kwang Lee Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea,Korea Advanced Institute of Science and Technology (KAIST), Center for Stem Cell Differentiation, Daejeon 305-701, Republic of Korea andKorea Research Institute of Bioscience and Biotechnology (KRIBB), Center for Development and Differentiation, Daejeon 305-806, Republic of Korea

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Yong-Mahn Han Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea,Korea Advanced Institute of Science and Technology (KAIST), Center for Stem Cell Differentiation, Daejeon 305-701, Republic of Korea andKorea Research Institute of Bioscience and Biotechnology (KRIBB), Center for Development and Differentiation, Daejeon 305-806, Republic of Korea

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Epigenetic reprogramming is a prerequisite process during mammalian development that is aberrant in cloned embryos. However, mechanisms that evolve abnormal epigenetic reprogramming during preimplantation development are unclear. To trace the molecular event of an epigenetic mark such as DNA methylation, bovine fibroblasts were epigeneticallyaltered by treatment with trichostatin A (TSA) and then individually transferred into enucleated bovine oocytes. In the TSA-treated cells, expression levels of histone deacetylases and DNA methyltransferases were reduced, but the expression level of histone acetyltransferases such as Tip60 and histone acetyltransferase 1 (HAT1) did not change compared with normal cells. DNA methylation levels of non-treated (normal) and TSA-treated cells were 64.0 and 48.9% in the satellite I sequence (P < 0.05) respectively, and 71.6 and 61.9% in the α-satellite sequence respectively. DNA methylation levels of nuclear transfer (NT) and TSA-NT blastocysts in the satellite I sequence were 67.2 and 42.2% (P < 0.05) respectively, which was approximately similar to those of normal and TSA-treated cells. In the α-satellite sequence, NT and TSA-NT embryos were substantially demethylated at the blastocyst stage as IVF-derived embryos were demethylated. The in vitro developmental rate (46.6%) of TSA-NT embryos that were individually transferred with TSA-treated cells was higher than that (31.7%) of NT embryos with non-treated cells (P < 0.05). Our findings suggest that the chromatin of a donor cell is unyielding to the reprogramming of DNA methylation during preimplantation development, and that alteration of the epigenetic state of donor cells may improve in vitro developmental competence of cloned embryos.

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