Tetraploid Embryonic Stem Cells Maintain Pluripotency and Differentiation Potency into Three Germ Layers

Abstract

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.

Effects of whole genome duplication on cell size and gene expression in mouse embryonic stem cells

“…In this study, we used three previously established and characterized cell lines: control diploid ESCs, ESC#1, #2, and, #3; and tetraploid ESCs, TESC#1, #2 and, #3 [15]. TESC lines maintained their initial morphology and positive AP staining even after 25 passages (Fig.…”

“…Establishment of ESCs and TESCs has been described previously [15]. ESCs and TESCs were seeded on mitomycin C-treated mouse embryonic fibroblasts (MEFs) in ESGRO complete serum-free medium (Merck Millipore, Billerica, MA, USA) supplemented with 20% KnockOut serum replacement (Life Technologies Japan, Tokyo, Japan), 100 μg/ml penicillin, and 50 μg/ml streptomycin.…”

“…Thus, tetraploid cells produced by either method are not suitable for physiological characterization in mammals. We have previously established mouse tetraploid embryonic stem cells (TESCs) from a single tetraploid blastocyst produced by electrofusion [15]. TESCs can be also created by transferring two somatic cell nuclei into an enucleated single-celled embryo using specific nuclear transfer techniques [16].…”

“…TESCs can be also created by transferring two somatic cell nuclei into an enucleated single-celled embryo using specific nuclear transfer techniques [16]. The karyotypes of TESCs are more homogeneous and do not present any aberration in cell division or chromosomal defects due to highly stable chromosomes [15]. However, the relative proliferation rate of TESC lines is significantly lower than that of ESC lines [15].…”

“…The karyotypes of TESCs are more homogeneous and do not present any aberration in cell division or chromosomal defects due to highly stable chromosomes [15]. However, the relative proliferation rate of TESC lines is significantly lower than that of ESC lines [15]. Thus, even though TESC lines were successfully created using an established method, differences in proliferation rates compared to those of ESCs persisted.…”