The treatment of LIF plus BIO dramatically changed the morphology of serum- and feeder-free B6 ES cells and significantly increased the expressions of self-renewal and pluripotency regulators. The aggregation or microinjection of serum- and feeder-free B6 ES cells cultured in LIF plus BIO to each eight-cell-stage diploid embryo stably provided germline-competent B6 ES-derived mice. Previously, it was reported that laser-assisted microinjection of ES cells into an eight-cell-stage diploid embryo efficiently provides founder generation mice that are available for immediate phenotypic analyses (Poueymirou et al., 2007
). Certainly, the eight-cell-stage embryo method is useful for obtaining ES-derived mice compared to the conventional blastocyst injection method. However, judging from the data in , the birth rate of ES-derived mice seems to be highly dependent on the developmental potential of ES cells regardless of embryo manipulation. Accordingly, we hereby propose that serum- and feeder-free ES cells cultured with LIF plus GSK3 inhibitor are valuable for generating germline-competent ES-derived mice.
It is accepted that B6 ES cells have low potency to colonize germ cells in their founder mice. In ES cell culture, Nanog expression is thought to be an important factor for the maintenance of germline competency. Compared with LIF single-treated B6 ES cells, LIF plus BIO-treated B6 ES cells showed homogeneous expression pattern of Nanog (). By aggregating them with each eight-cell-stage diploid embryo, they stably differentiated into germ cells in their founder mice, which exhibited full germline transmission (). It has been reported that Nanog is dispensable for expression of somatic pluripotency but is specifically required for formation of germ cells (Chambers et al., 2007
). Moreover, by the selection of Nanog expression, the germline-competent induced pluripotent stem (iPS) cells have been successfully established from embryonic fibroblasts (Okita et al., 2007
). From these findings, we speculate that homogeneous expression of Nanog is a landmark in ES cells and/or iPS cells that have high potential to differentiate particularly into germ cells.
ES cells have been reported to be maintained by three inhibitors (3i) for FGF receptor and GSK3 (Ying et al., 2008
). For GSK3 inhibitors, BIO and CHIR99021 were used in our study and that of Ying and colleagues, respectively. After each treatment of GSK3 inhibitor, the expression levels of Wnt/β-catenin target genes such as Brachyury (T) and Cdx1 were similarly upregulated. However, BIO treatment significantly increased the expression levels of Tbx3, Esrrb, and Tcl1 in B6-derived ES cells, but CHIR99021 did not have the same upregulation effect on these genes in 129-derived ES cells. Moreover, In addition to GSK3 inhibitor, we have used LIF in KSR-based medium, while Ying's group used two inhibitors for the FGF receptor instead of LIF in N2B27-based medium. In our studies, the LIF plus BIO combination culture enhanced cell growth and developmental potential of serum- and feeder-free B6 ES cells as shown in and . Thus, different culture conditions derived from different combinations of chemical compounds, culture media, and ES cell strains may explain these different findings. From the standpoint of stable generation of founder mice, it is interesting for us to examine which conditions bring out the best developmental potential in each ES cell line, although GSK3 inhibition is crucial for ES cell culture.
Finally, diploid embryo aggregation with the serumand feeder-free B6 ES cells cultured in LIF plus GSK3 inhibitor is a rapid, cost-effective, and easy-to-learn method for generating germline-competent B6 ES-derived founder mice. Furthermore, GSK3 inhibition together with LIF/STAT3 signaling makes it possible to enhance the developmental potential of various ES cells without specific skills and experience, because the serum lot check and the preparation for feeder layers are skipped. Generally, in the conventional method, 129 or F1 hybrid ES cells cultured in the presence of serum and feeder layers have been used for the production of germline-competent chimeras. Partial chimeric mice generated from these ES cells require multiple rounds of breeding to produce homozygous mutations within a given mouse. The desired mice are produced with a mixed genetic background at low frequencies in the final breeding step. However, the use of B6 ES cell line instead of 129 or F1 hybrid ES cell line eliminates the need for backcross breeding. Founder mice entirely derived from female XO, heterozygous, or homozygous mutant B6 ES cells were directly available for intercross breeding and phenotypic analysis in vivo. In addition, the access to the public C57BL/6J mouse sequence and the RPCI23 C57BL/6J BAC genomic library is a powerful tool that facilitates the production of gene-targeting vectors for the manipulation of genes in C57/BL6 ES cell lines. Therefore, from the point of view of saving time and labor, the established serum- and feeder-free B6 ES cells will be helpful and useful for large-scale functional genomic analyses with genetically engineered mice on B6 background.