Scientia Agricultura Sinica ›› 2012, Vol. 45 ›› Issue (12): 2474-2481.doi: 10.3864/j.issn.0578-1752.2012.12.017

• ANIMAL SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

Bovine Embryonic Stem Cells Culture on Mixed Feeder Layers with Mouse and Bovine Embryonic Fibroblasts in Vitro

 CONG  Shan, WANG  Rui, WEN  Jian-Xun, HAO  Fei, LI  Shuo, LIANG  Hao, LIU  Dong-Jun   

  1. 内蒙古大学哺乳动物生殖生物学教育部重点实验室，呼和浩特 010021
  • Received:2011-10-17 Online:2012-06-15 Published:2011-12-31

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Abstract: 【Objective】The aim of this study was to establish a suitable culture environment for bovine embryo stem cell.【Method】Bovine and mouse fibroblast cells were treated for 2.0-3.0 h with mitomycin C, respectively. Mixed feeder layer was that cells which were mixed according to 1﹕0, 1﹕1, 1﹕2, 2﹕1, 0﹕1 were cultured in the center plate coated by gelatin at 2-2.5×104 per well. Growth states of bovine embryonic stem cells were observed in different feeder layers and undifferentiated phenotypes were detected, including expression of alkaline phosphatase, and presence of OCT-4, SOX2, NANOG and cell marker OCT-4, SSEA-1. Without feeder layer, bovine embryonic stem cell differentiation was observed in vitro.【Result】Comparison of bovine embryonic stem cell growth state on different feeder layers, the clonal morphology on the mixed feeder layer was significantly better than that on the mouse embryonic fibroblast feeder layer or bovine embryonic fibroblast feeder layer. When mouse embryonic fibroblasts and bovine embryonic fibroblasts were mixed at a ratio of 1﹕1 bovine embryonic stem cells accumulated significantly and cloning edge was clear, obvious and full. Expression of alkaline phosphatase, antigen of OCT-4 and SSEA-1 were strongly positive. Specific bands of OCT-4, SOX2 and NANOG mRNA appeared, respectively. Bovine embryonic stem cells on mixed feeder layer was able to form embryoid bodies.【Conclusion】Compared with conventional feeder layers prepared by mouse embryonic fibroblasts or bovine embryonic fibroblasts, the mixed cells feeder layer mixed at a ratio of 1﹕1 may be better suitable for bovine embryonic stem cell culture in vitro and obtain better bovine rembryonic stem cell clonal morphology.

Key words: bovine, ebryonic stem cell, ebryonic fibroblasts, mixed feeder layer

[1]Evans M J, Kaufman M H. Establishment in culture of pluripotential cells from mouse embryos. Nature, 1981, 292(5819): 154-156.

[2]Christoforou N, Oskouei B N, Esteso P, Hill C M, Zimmet J M, Bian W, Bursac N, Leong KW, Hare J M, Gearhart J D. Implantation of mouse embryonic stem cell-derived cardiac progenitor cells preserves function of infarcted murine hearts. PLoS One, 2010, 5(7): e11536.

[3]Zeevi-Levin N, Itskovitz-Eldor J, Binah O. Functional properties of human embryonic stem cell-derived cardiomyocytes. Critical Reviews in Eukaryotic Gene Expression, 2010, 20(1): 51-59.

[4]Thomson J A, Itskovitz-Eldor J, Shapiro S S, Waknitz M A, Swiergiel J J, Marshall V S, Jones J M. Embryonic stem cell lines derived from human blastocysts. Science, 1998, 282(5391): 1145-1147.

[5]Xu C, Inokuma M S, Denham J. Feeder-free growth of undifferentiated human embryonic stem cells. Nature, 2001, 19(10): 971-974.

[6]Miyoshi K, Taguchi Y, Sendai Y, Hoshi H, Sato E. Establishment of a porcine cell line from in vitro-produced blastocysts and transfer of the cells into enucleated oocytes. Biology of Reproduction, 2000, 62(6): 1640-1646.

[7]李碧春, 陈国宏, 吴信生, 徐  淇, 吴圣龙, 蔡中涛. 牛体外受精胚胎衍生干细胞能力影响因素的研究. 畜牧兽医学报, 2006, 37(9): 928-932.

Li B C, Chen G H, Wu X S, Xu Q, Wu S L, Cai Z T. Effect of several factors on developmental potential of bovine embryonic stem cells from IVF embryo. Acta Veterinaria et Zootechnica Sinica, 2006, 37(9): 928-932. (in Chinese)

[8]Saito S, Strechenko N, Niemann H. Bovine embryonic stem cell-like cell lines cultured over several passages. Roux’s Archives of Developmental Biology, 1992, 201(3): 134-141．

[9]Saito S, Sawai K, Ugai H, Moriyasu S, Minamihashi A, Yamamoto Y, Hirayama H, Kageyama S, Pan J, Murata T, Kobayashi Y, Obata Y, Yokoyama K K. Generation of cloned Calves and transgenic chimeric embryos from bovine embryonic stem-like cells．Biochemical and Biophysical Research Communications, 2003, 309: 104-113.

[10]Talbot N C, Powell A M, Rexroad C E. In vitro pluripotency of epiblasts derived from bovine blastocysts. Molecular Reproduction and Development, 1995, 42(1): 35-52.

[11]Sim M, First N L. Production of calves by transfer of nuclei from cultured inner cell mass cells. Proceedings of the National Academy of Sciences of the United States of America 1994, 91(13): 6143-6147.

[12]White K L, Polejaeva I A, Bunch T D, Suh T, Reed W A, Spendlove R, Wilkinson R. Effect of non-serum supplemented media of establishment and maintaince of bovine embryonic stem-like cells. Theriogenology, 1995, 43(1): 350.

[13]Ito T. Konishi M, Takedomi T, Itakura H, Yazawa S. Aoyagi Y. Influence of stage and derivation of bovine embryos on formation of embryonic stem (ES) like colonies. Journal of Reproduction and Development, 1996, 42(5): 129-133.

[14]Stice S L, Strelchenko N S, Keefer C L, Matthews L. Pluripotent bovine embryonic cell lines direct embryonic development following nuclear transfer. Biology of Reproduction, 1996, 54(1): 100-110.

[15]Mitalipova M, Beyhan Z, First N L. pluripotency of bovine embryonic stem cells derived from precompacting embryos. Cloning, 2001, 3(2): 59-67.

[16]Seizo H, Yoshie W, Sadahiro A. Establishment of embryonic stem (ES) cell-like cell lines derived from bovine blastocysts obtained by in vitro culture of oocytes matured and fertilized in vitro. Journal of Reproduction and Development, 1998, 44(3): 297-303.

[17]Muñoz M, Rodríguez A, de Frutos C, Caamaño J N, Díez C, Facal N, Gómez E. Conventional pluripotency markers are unspecific for bovine embryonic-derived cell-lines. Theriogenology, 2008, 69(9): 1159-1164.

[18]李  斌, 彭秋萍, 卢伟英, 徐  雯, 金应霞, 黄元华. 小鼠胚胎及人包皮成纤维细胞按比例制成混合饲养层上的人胚胎干细胞生长状态. 临床医学, 2008, 12(3): 423-428.

Li B, Peng Q P, Lu W Y, Xu W, Jin Y X, Huang Y H. Growth state of human embryonic stem cells on mixed feeder layers with mouse embryonic fibroblasts and human foreskin fibroblasts at different ratios. Journal of Clinical Rehabilitative Tissue Engineering Research, 2008, 12(3): 423-428. (in Chinese)

[19]Reubinoff B E, Pera M F, Fong C Y. Embryonic stem cell lines from human blastocysts somatic differentiation in vitro. Nature Biotechonlogy, 2000, 18(4): 399-404.

[20]Roach M, Wang L, Yang X Z, Tian X C. Bovine embryonic stem cells. Methods in Enzymology, 2006, 418: 21-37.

[21]薛庆善. 体外培养的原理与技术. 北京: 北京科学出版社, 2001: 134-135, 516-517.

Xue Q S. Principles and Techniques of in Vitro Culture. Beijing: Beijing Science Press, 2001: 134-135, 516-517. (in Chinese)

[22]Bettiol E, Sartiani L, Chicha L, Krause K H, Cerbai E, Jaconi M E. Fetal bovine serum enables cardiac differentiation of human embryonic stem cells. International Society of Differentiation, 2007, 75(8): 669-681.

[23]Bryja V, Bonilla S, Arenas E. Derivation of mouse embryonic stem cells. Nature Publishing Group, 2006, 1(4): 2082-2087.

[24]Li M, Li Y H, Hou Y, Sun X F, Sun Q, Wang W H. Isolation and culture of pluripotent cells from in vitro produced porcine embryos. Zygote, 2004, 12(1): 43-48.

[25]Brackett B G, Oliphant G. Capacitation of rabbit spermatozoa in vitro. Biology of Reproduction, 1975, 12(2): 260-274.

[26]Tervit H R, Wittingham D G, Rowson L E. Successful culture in vitro of sheep and cattle ova. Journal of Reproduction and Fertility, 1972, 30: 493-497.

[27]郭雨霁, 高英茂, 邴鲁军, 李盛芳. 小鼠胚胎干细胞构建嵌合体的方法. 解剖学报, 2004, 35(3): 324-327.

Guo Y J, Gao Y M, Bing L J, Li S F. The experimental study of chimer as construction from embryonic stem cell. Acta Anatomica Sinica, 2004, 35(3): 324-327. (in Chinese)

[28]崔  雯, 熊  浩, 王兴龙. 饲养层和生长因子对山羊类胚胎干细胞的影响. 中国畜牧杂志, 2011, 47(1): 29-31.

Cui W, Xiong H, Wang X L. Influence of feeder layer and growth factors on goat ESC-lile cells culture. Reproduction and Physiology, 2011, 47(1): 29-31. (in Chinese)

[29]Piedrahita J A, Anderson G B, BonDurant R H. Influence of feeder layer type on the efficiency of isolation of porcine embryo-derived cell lines. Therogenology, 1990, 34(5): 865-877.

[30]李  松, 窦忠英, 华进联, 李相运, 宋文刚, 曲  迅. 影响牛胚胎干细胞分离克隆因素的研究. 生物技术通报, 2002(3): 35-39.

Li S, Dou Z Y, Hua J L, Li X Y, Song W G, Qu X. Influences on isolation and cloning of embryonic stem cells from bovine primordial germ cells. Biotechnology Information, 2002(3): 35-39. (in Chinese)

[31]钱  坤, 陈  红, 张明苏, 朱桂金. 人成纤维细胞对人胚胎干细胞生长的支持作用. 华中科技大学学报: 医学版, 2006, 35(4): 462-464.

Qan K, Chen H, Zhang M S, Zhu G J. Human fibroblast cell supporting undifferentiated human embryonic stem cells proliferation in vitro. Journal of Huazhong University of Ccience and Technology: Health Sciences, 2006, 35(4): 462-464. (in Chinese)

[32]靳木子, 马玉珍, 仓  明, 王  瑞, 任  宇, 刘东军. 以两种不同成纤维细胞为饲养层培养牛胚胎干细胞的比较. 畜牧与兽医, 2010, 42(6): 57-61.

Jin M Z, Ma Y Z, Cang M, Wang R, Ren Y, Liu D J. Comparison of bovine embryonic stem cells on two different fibroblasts as feeder layer. Animal Husbandry and Veterinary Medicine, 2010, 42(6): 57-61. (in Chinese)

[33]Homson J A, Kalishman J, Golos T G. Pluripotent cell lines derived from common marmoset (Callithrix jacchus) blastocysts. Biology of Reproduction, 1996, 55(2): 254-259.

[34]张守宝, 柏学进, 朱金香, 王文明, 刘  玮, 董亚娟. 不同饲养层对牛胚胎干细胞培养的影响. 畜牧与兽医, 2010, 42(5): 44-47.

Zhang S B, Bai X J, Zhu J X, Wang W M, Liu W, Dong Y J. The effect of bovine embryonic stem cells culture on different feeder layers. Animal Husbandry and Veterinary Medicine, 2010, 42(5): 44-47. (in Chinese)
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