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Production of chimeric animals is often a necessity for the generation of genetically modified animals and has gained popularity in recent years in regenerative medicine for the reconstruction of xenogeneic organs. Aggregation and injection methods are generally used to produce chimeric mice. In the aggregation method, the chimeras are produced by co-culturing embryos and stem cells, and keeping them physically adhered, although it may not be an assured method for producing chimeric embryos. In the injection method, the chimeras are produced by injecting stem cells into the zona pellucida using microcapillaries; however, this technique requires a high degree of skill. This study aimed to establish a novel method for producing chimeric embryos via water-in-oil droplets that differs from conventional methods. In this study, embryonic stem cells and embryos were successfully isolated in the droplets, and the emergence of chimeric embryos was confirmed by co-culture for 6 h. Using this method, the control and operability of stem cell numbers could be regulated, and reproducibility and quantification were improved during the production of chimeric embryos. In addition to the conventional methods for producing chimeric embryos, the novel method described here could be employed for the efficient production of chimeric animals.

Cultured cells are generally observed through the bottom of dishes or flasks using an inverted microscope. Two-dimensional and horizontal observation is insufficient for histological analysis of several cell lines, such as embryonic stem cells or cancer cells, because they form three-dimensional colonies. In the present study, we aimed to establish a more informative method for analysis of such stereoscopic cultured cells. We cultured mouse embryonic stem cells using a temperature-sensitive culture dish, embedded these cells in paraffin, and successfully observed vertical sections of embryonic stem cells. This vertical analysis of the stereoscopic colony emphasized structural features such as the dome shape of naïve pluripotent stem cells. This method could have the potential for analysis of three-dimensional structures and histological preservation in cultured cells.

Polyploid amphibians and fishes occur naturally in nature, while polyploid mammals do not. For example, tetraploid mouse embryos normally develop into blastocysts, but exhibit abnormalities and die soon after implantation. Thus, polyploidization is thought to be harmful during early mammalian development. However, the mechanisms through which polyploidization disrupts development are still poorly understood. In this study, we aimed to elucidate how genome duplication affects early mammalian development. To this end, we established tetraploid embryonic stem cells (TESCs) produced from the inner cell masses of tetraploid blastocysts using electrofusion of two-cell embryos in mice and studied the developmental potential of TESCs. We demonstrated that TESCs possessed essential pluripotency and differentiation potency to form teratomas, which differentiated into the three germ layers, including diploid embryonic stem cells. TESCs also contributed to the inner cell masses in aggregated chimeric blastocysts, despite the observation that tetraploid embryos fail in normal development soon after implantation in mice. In TESCs, stability after several passages, colony morphology, and alkaline phosphatase activity were similar to those of diploid ESCs. TESCs also exhibited sufficient expression and localization of pluripotent markers and retained the normal epigenetic status of relevant reprogramming factors. TESCs proliferated at a slower rate than ESCs, indicating that the difference in genomic dosage was responsible for the different growth rates. Thus, our findings suggested that mouse ESCs maintained intrinsic pluripotency and differentiation potential despite tetraploidization, providing insights into our understanding of developmental elimination in polyploid mammals.