Iijima Saori, Tanimoto Yoko, Mizuno Seiya, Daitoku Yoko, Kunita Satoshi, Sugiyama Fumihiro, Yagami Ken-ichi
Laboratory Animal Resource Center, University of Tsukuba , Tsukuba, Ibaraki, Japan.
Cell Reprogram. 2010 Dec;12(6):679-88. doi: 10.1089/cell.2010.0018. Epub 2010 Oct 26.
As the phenotype of a given single-gene mutation in mice is modulated by the genetic background of the inbred strain, embryonic stem (ES) cells derived from various inbred mouse strains are required to produce gene-targeted mice without the need for backcrossing and for detailed analysis of gene function in vivo. Here, we performed a comparative investigation of the effects of three culture conditions, LIF + KSR/ES medium described previously, High LIF + KSR/ES medium and iSTEM + LIF medium containing three inhibitors of glycogen synthase kinase 3, mitogen-activated protein kinase kinase, and fibroblast growth factor receptor signaling (3i), on the establishment of germline-competent ES cells derived from strains BALB/c and NZB mice. The results indicated that LIF + KSR/ES medium was permissive for the derivation of ES cells from NZB mice, which contribute to the somatic lineage in vivo, but not to the germline lineage. In contrast, ES cells that contribute to the makeup of chimeric mice were not propagated from blastocysts of BALB/c mice. Both germline and somatic competency were improved by increased LIF concentration in cultures of BALB/c ES cells, although we failed to establish germline-competent NZB ES cells using the same concentration of LIF. Unexpectedly, iSTEM + LIF medium containing 3i showed a negative effect on the derivation of NZB ES cells with normal chromosome numbers, but not on the maintenance of previously established ES cells. Our findings suggest that the stability of pluripotency in the inner cell mass isolated from blastocyst embryos may differ according to the genetic background of inbred mouse strains, and that although the concentration of LIF is a determinant for authentic pluripotency, including germline and somatic competency in BALB/c ES cells, additional factor(s) are required for commitment to germline lineage independent of somatic lineage in NZB ES cells.
由于小鼠中给定单基因突变的表型会受到近交系遗传背景的调节，因此需要来自各种近交小鼠品系的胚胎干细胞（ES细胞）来产生基因靶向小鼠，而无需回交，并用于体内基因功能的详细分析。在这里，我们对三种培养条件的效果进行了比较研究，这三种培养条件分别是：先前描述的LIF + KSR/ES培养基、高LIF + KSR/ES培养基以及含有糖原合酶激酶3、丝裂原活化蛋白激酶激酶和成纤维细胞生长因子受体信号传导三种抑制剂（3i）的iSTEM + LIF培养基，研究它们对源自BALB/c和NZB小鼠品系的具有种系能力的ES细胞建立的影响。结果表明，LIF + KSR/ES培养基允许从NZB小鼠中获得ES细胞，这些细胞在体内对体细胞谱系有贡献，但对种系谱系没有贡献。相反，对嵌合小鼠组成有贡献的ES细胞并非从BALB/c小鼠的囊胚中增殖而来。在BALB/c ES细胞培养物中增加LIF浓度可同时提高种系和体细胞能力，尽管我们未能使用相同浓度的LIF建立具有种系能力的NZB ES细胞。出乎意料的是，含有3i的iSTEM + LIF培养基对具有正常染色体数的NZB ES细胞的获得有负面影响，但对先前建立的ES细胞的维持没有影响。我们的研究结果表明，从囊胚胚胎中分离出的内细胞团中多能性的稳定性可能因近交小鼠品系的遗传背景而异，并且尽管LIF浓度是真正多能性的决定因素，包括BALB/c ES细胞中的种系和体细胞能力，但在NZB ES细胞中，除了体细胞谱系外，还需要其他因素来促使细胞向种系谱系分化。
