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

<div><p>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.</p></div

Development of Morula Embryo Aggregates from Injections of <i>In Vitro</i> Cultures of EGFP-Expressing TESCs.

<p>Morphology, fluorescence, and merged images of aggregated embryos at the blastocyst stage.</p

Expression and Epigenetic Analysis of TESCs.

<p>(<b>A</b>) Relative expression of pluripotency markers by quantitative real-time RT-PCR analysis at passage 10. All data represent the mean and SEM (n = 3). *<i>P</i> < 0.05. (<b>B</b>) Analysis of DNA methylation profiles. DNA methylation levels in <i>Oct3/4</i> and <i>Nanog</i> genes were determined by Bio-COBRA in TESCs and ESCs. All data represent the mean and SEM (n = 3). *<i>P</i> < 0.05. (<b>C</b>) Immunostaining of pluripotency markers. DAPI was used to stain DNA. A representative figure is shown. Scale bar, 50 μm.</p

Production and Characterization of Tetraploid Embryonic Stem Cells (TESCs).

<p>(<b>A</b>) Diagram of production of TESCs with elecrofused diploid embryos. Two or three embryos were seeded onto a mouse embryonic fibroblast (MEF) feeder layer. (<b>B</b>) Flow cytometry analysis of DNA content after 10 passages. A representative figure is shown. (<b>C</b>) Karyotyping analysis of metaphase chromosome spreads. Tetraploid ESCs normally had 80 chromosomes, while control diploid ESCs had 40 chromosomes at passage 10. A representative figure is shown. (<b>D</b>) Proliferating TESCs formed typical round-shaped mouse ESC colonies with clear boundaries similar to control ESCs. TESC colonies also stained positive for the control ESC-positive marker alkaline phosphatase (ALP). A representative figure is shown. (<b>E</b>) Growth rates. The relative cell number was based on the cell number at day 0 (2 × 10⁴ cells). All data represent the mean and SEM (n = 3). *<i>P</i> < 0.001. Scale bar, 100 μm.</p

The Differentiation Potential of TESCs.

<p>(<b>A</b>) Morphology of embryoid bodies generated from TESCs and ESCs. Scale bars, 100 μm. (<b>B</b>) Relative expression of various cell lineage marker genes in embryoid bodies generated from TESCs and ESCs by quantitative real-time RT-PCR analysis. All data represent the mean and SEM (n = 3). *<i>P</i> < 0.05. (<b>C</b>) Teratoma formation by TESCs. Scale bars, 50 μm.</p

Primer sequence in COBRA.

<p>Primer sequence in COBRA.</p

Primer Sequences.

<p>Primer Sequences.</p