title: Putative porcine embryonic stem cell lines derived from aggregated four-celled cloned embryos produced by oocyte bisection cloning.
abstract: We attempted to isolate ES cell lines using inner cell masses from high-quality cloned porcine blastocysts. After being seeded onto feeders, embryos had better (P < 0.05) attachment, outgrowth formation and primary colonization in both 2× and 3× aggregated cloned embryos (62.8, 42.6 and 12.8% vs. 76.2, 55.2 and 26.2%, respectively) compared to the non-aggregated group (41.6, 23.4 and 3.9%). Effects of feeder types (STO vs. MEF) and serum sources (FBS vs. KSR) on extraction of cloned embryo-derived porcine ES cells were examined. More (17.1%) ntES cell lines over Passage 3 were generated in the MEF/KSR group. However, ntES cells cultured in KSR-supplemented medium had a low proliferation rate with defective morphology, and eventually underwent differentiation or apoptosis subsequently. Approximately 26.1, 22.7 and 35.7% of primary colonies were formed after plating embryos in DMEM, DMEM/F12 and α-MEM media, respectively. Survival rates of ntES cells cultured in α-MEM, DMEM and DMEM/F12 were 16.7, 4.3 and 6.8%, respectively (P > 0.05). We further examined the beneficial effect of TSA treatment of 3× aggregated cloned embryos on establishment of ntES cell lines. Primary colony numbers and survival rates of ntES cells beyond passage 3 were higher (P < 0.05) in those derived from TSA-treated 3× blastocysts (36.7 and 26.7%) than from the non-treated aggregated group (23.1 and 11.5%). These cells, remaining undifferentiated over 25 passages, had alkaline phosphatase activity and expressed ES specific markers Oct4, Nanog, Sox2, and Rex01. Moreover, these ntES cells successfully differentiated into embryoid bodies (EBs) that expressed specific genes of all three germ layers after being cultured in LIF-free medium. In conclusion, we have successfully derived putative porcine ntES cells with high efficiency from quality cloned embryos produced by embryo aggregation, and optimized the ES cell culture system suitable for establishing and maintaining ntES cell lines in undifferentiated state.
authors: Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan, ROC; Department of Animal Science, National Chung Hsing University, Taichung, Taiwan, ROC.
authors: Chawalit
authors: C
authors: Siriboon
authors: Department of Animal Science, National Chung Hsing University, Taichung, Taiwan, ROC.
authors: Yu-Hsuan
authors: YH
authors: Lin
authors: Department of Animal Science, National Chung Hsing University, Taichung, Taiwan, ROC.
authors: Michel
authors: M
authors: Kere
authors: Department of Animal Science, National Chung Hsing University, Taichung, Taiwan, ROC.
authors: Chun-Da
authors: CD
authors: Chen
authors: Division of Physiology, Livestock Research Institute, Council of Agriculture, Executive Yuan, Tainan, Taiwan, ROC.
authors: Lih-Ren
authors: LR
authors: Chen
authors: Agriculture Technology Research Institute 1, Ln. 51, Dahu Rd., Xiangshan Dist., Hsinchu City, 300, Taiwan, ROC.
authors: Chien-Hong
authors: CH
authors: Chen
authors: Agriculture Technology Research Institute 1, Ln. 51, Dahu Rd., Xiangshan Dist., Hsinchu City, 300, Taiwan, ROC.
authors: Ching-Fu
authors: CF
authors: Tu
authors: Department of Animal Science and Biotechnology, Tunghai University 181, Sec. 3, Taichung Harbor Road, Taichung, 407, Taiwan, ROC.
authors: Neng-Wen
authors: NW
authors: Lo
authors: Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan, ROC; Department of Animal Science, National Chung Hsing University, Taichung, Taiwan, ROC; Core Laboratory for Stem Cell Research, Medical Research Department, China Medical University Hospital, Taichung, Taiwan, ROC; Agricultural Biotechnology and Biotechnology Centers, National Chung Hsing University, Taichung, Taiwan, ROC; Department of Biomedical Informatics, College of Computer Science, Asia University, Taichung, Taiwan, ROC.
authors: Jyh-Cherng
authors: JC
authors: Ju
mesh_terms: D000818:Animals; D015415:Biomarkers; D001755:Blastocyst / Q000166:cytology; D001757:Blastomeres / Q000166:cytology*; D002454:Cell Differentiation; D002460:Cell Line; D002478:Cells, Cultured; D019976:Cloning, Organism; D003470:Culture Media; D046149:Embryo Culture Techniques; D058732:Embryoid Bodies / Q000166:cytology; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D061252:Feeder Cells; D005260:Female; D009865:Oocytes / Q000166:cytology*; D013552:Swine
chemical_list: D015415:Biomarkers; D003470:Culture Media