Suwinska Aneta, Tarkowski Andrzej K, Ciemerych Maria A
Department of Embryology, Institute of Zoology, University of Warsaw, Warsaw, Poland.
Int J Dev Biol. 2010;54(1):113-24. doi: 10.1387/ijdb.082704as.
The objective of this study was to investigate the capability of bank vole (Myodes glareolus) embryonic cells to sustain their pluripotent character during in vitro culture, and to determine the optimal conditions for derivation of embryonic stem (ES) cells. We compared the presence of specific pluripotency (Oct4, Ssea1) and differentiation markers (Gata4 - primitive endoderm marker; Cdx2 - trophectoderm marker) in blastocysts and inner cell mass (ICM) outgrowths obtained from blastocysts of bank vole, and two mouse hybrids F1(C57Bl/6xCBA/H) and F1(C57Bl/6x129/Sv), which differ in the permissiveness of giving rise to ES cells. We found that, in contrast to mouse, the expression of pluripotency markers in the cells of bank vole ICM outgrowths is progressively downregulated and rapidly lost by the 4th day of culture. This correlates with the appearance of cells expressing Gata4 and Cdx2, indicating differentiation towards primitive endoderm and derivatives of trophectoderm, respectively. We have also shown that heterologous cytokine leukaemia inhibitory factor (LIF) in conjunction with either homologous or heterologous feeder layer is unable to delay differentiation and preserve pluripotency of bank vole embryonic cells. Thus, the conditions optimised for mouse do not support the maintenance of bank vole embryonic cells in the undifferentiated state and do not allow for the isolation of the ES cells. Instead, combination of fibroblast growth factor 2 and activin A allows retention of Oct4 expression in bank vole blastocyst outgrowths during 4-day culture, indicating that signaling pathways operating in human, rather than mouse ES cells, might be involved in the process of self-renewal of bank vole embryonic cells.
本研究的目的是调查田鼠（Myodes glareolus）胚胎细胞在体外培养期间维持其多能性特征的能力，并确定获得胚胎干细胞（ES细胞）的最佳条件。我们比较了从田鼠囊胚以及两种小鼠杂种F1（C57Bl/6xCBA/H）和F1（C57Bl/6x129/Sv）的囊胚中获得的囊胚和内细胞团（ICM）生长物中特定多能性（Oct4、Ssea1）和分化标志物（Gata4 - 原始内胚层标志物；Cdx2 - 滋养外胚层标志物）的存在情况，这两种小鼠杂种在产生ES细胞的允许性方面存在差异。我们发现，与小鼠不同，田鼠ICM生长物细胞中多能性标志物的表达在培养的第4天逐渐下调并迅速丧失。这与表达Gata4和Cdx2的细胞的出现相关，分别表明向原始内胚层和滋养外胚层衍生物的分化。我们还表明，异源细胞因子白血病抑制因子（LIF）与同源或异源饲养层结合不能延迟田鼠胚胎细胞的分化并维持其多能性。因此，为小鼠优化的条件不支持将田鼠胚胎细胞维持在未分化状态，也不允许分离ES细胞。相反，成纤维细胞生长因子2和激活素A的组合可使田鼠囊胚生长物在4天培养期间保留Oct4表达，这表明在人类而非小鼠ES细胞中起作用的信号通路可能参与了田鼠胚胎细胞的自我更新过程。
