Recombinant rabbit leukemia inhibitory factor and rabbit embryonic fibroblasts support the derivation and maintenance of rabbit embryonic stem cells.
Recombinant rabbit leukemia inhibitory factor and rabbit embryonic fibroblasts support the derivation and maintenance of rabbit embryonic stem cells.
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DOI：
10.1089/cell.2012.0001
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发表时间：
2012-08
影响因子：
1.6
通讯作者：
F. Xue;Yinghong Ma;Y. E. Chen;Jifeng Zhang;T. Lin;Chien-Hong Chen;Wei-Wen Lin;M. Roach;J. Ju;Lan Yang;F. Du;Jie Xu
中科院分区：
文献类型：
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作者：
F. Xue;Yinghong Ma;Y. E. Chen;Jifeng Zhang;T. Lin;Chien-Hong Chen;Wei-Wen Lin;M. Roach;J. Ju;Lan Yang;F. Du;Jie Xu
家兔是一种经典的实验动物。利用兔进行生物医学研究的一个主要限制是缺乏具有生殖系能力的兔胚胎干细胞（rbESCs）。我们推测，同源饲养细胞和重组兔白血病抑制因子（rbLIF）的使用可能会提高生殖系的能力rbES细胞的机会。在本研究中，我们建立了兔胚胎成纤维细胞（REF）饲养层和合成重组rbLIF。我们总共获得了7个推定的rbESC系，其中2个系（M5和M23）来自培养条件I，使用小鼠胚胎成纤维细胞（MEF）作为饲养层，补充人LIF（hLIF）（MEF+hLIF）。另外5个细胞系（R4、R9、R15、R21和R31）来源于条件II，使用REF作为补充有rbLIF的饲养细胞（REF+rbLIF）。在这两种条件下观察到相似的衍生效率（8.7% vs. 10.2%）。在2×3析因设计的单独实验中，我们检查了饲养细胞（MEF vs. REF）和LIF（mLIF、hLIF vs. rbLIF）对rbESC培养的影响。条件I和II均支持令人满意的rbESC培养，与饲养细胞与LIF的其他组合相比，具有相似或更好的群体倍增时间和集落形成效率。在两种条件下衍生和维持的兔ESC显示出典型的ESC特征，包括ESC多能性标志物表达（AP、Oct 4、Sox 2、Nanog和SSEA 4）和基因表达（Oct 4、Sox 2、Nanog、c-Myc、Klf 4和Dppa 5），以及在体外分化成三个初级胚层的能力。本研究首次尝试用同源饲养细胞和重组rbLIF建立rbESC细胞系，并使rbESC细胞得到正常分化和维持。这些细胞系是独特的资源，可以促进生殖系感受态rbESCs的衍生。
The rabbit is a classical experimental animal species. A major limitation in using rabbits for biomedical research is the lack of germ-line-competent rabbit embryonic stem cells (rbESCs). We hypothesized that the use of homologous feeder cells and recombinant rabbit leukemia inhibitory factor (rbLIF) might improve the chance in deriving germ-line-competent rbES cells. In the present study, we established rabbit embryonic fibroblast (REF) feeder layers and synthesized recombinant rbLIF. We derived a total of seven putative rbESC lines, of which two lines (M5 and M23) were from culture Condition I using mouse embryonic fibroblasts (MEFs) as feeders supplemented with human LIF (hLIF) (MEF+hLIF). Another five lines (R4, R9, R15, R21, and R31) were derived from Condition II using REFs as feeder cells supplemented with rbLIF (REF+rbLIF). Similar derivation efficiency was observed between these two conditions (8.7% vs. 10.2%). In a separate experiment with 2×3 factorial design, we examined the effects of feeder cells (MEF vs. REF) and LIFs (mLIF, hLIF vs. rbLIF) on rbESC culture. Both Conditions I and II supported satisfactory rbESC culture, with similar or better population doubling time and colony-forming efficiency than other combinations of feeder cells with LIFs. Rabbit ESCs derived and maintained on both conditions displayed typical ESC characteristics, including ESC pluripotency marker expression (AP, Oct4, Sox2, Nanog, and SSEA4) and gene expression (Oct4, Sox2, Nanog, c-Myc, Klf4, and Dppa5), and the capacity to differentiate into three primary germ layers in vitro. The present work is the first attempt to establish rbESC lines using homologous feeder cells and recombinant rbLIF, by which the rbESCs were derived and maintained normally. These cell lines are unique resources and may facilitate the derivation of germ-line-competent rbESCs.