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

Abstract

Alterations in ploidy tend to influence cell physiology, which in the long-term, contribute to species adaptation and evolution. Polyploid cells are observed under physiological conditions in the nerve and liver tissues, and in tumorigenic processes. Although tetraploid cells have been studied in mammalian cells, the basic characteristics and alterations caused by whole genome duplication are still poorly understood. The purpose of this study was to acquire basic knowledge about the effect of whole genome duplication on the cell cycle, cell size, and gene expression. Using flow cytometry, we demonstrate that cell cycle subpopulations in mouse tetraploid embryonic stem cells (TESCs) were similar to those in embryonic stem cells (ESCs). We performed smear preparations and flow cytometric analysis to identify cell size alterations. These indicated that the relative cell volume of TESCs was approximately 2.2-2.5 fold that of ESCs. We also investigated the effect of whole genome duplication on the expression of housekeeping and pluripotency marker genes using quantitative real-time PCR with external RNA. We found that the target transcripts were 2.2 times more abundant in TESCs than those in ESCs. This indicated that gene expression and cell volume increased in parallel. Our findings suggest the existence of a homeostatic mechanism controlling the cytoplasmic transcript levels in accordance with genome volume changes caused by whole genome duplication.

Fig. 1.

Morphology of mouse tetraploid embryonic stem cells (TESCs) and mouse diploid embryonic stem cells (ESCs). Like ESCs, TESCs formed typical round-shaped colonies with clear boundaries. TESC colonies stained positive for the control ESC-positive marker alkaline phosphatase (AP). Representative images are shown. Scale bar, 50 µm.

Fig. 2.

Flow cytometric analysis of cell cycle distribution using propidium iodide staining. (A) Flow cytometry DNA histograms of different TESC and ESC lines. (B) Analysis of subpopulations in G1/G0, S, and G2/M phases. No significant differences were detected between the ESCs and TESCs for each phase. Data represent mean ± SD.

Fig. 3.

Relative size measurement of TESCs and ESCs by flow cytometry. (A) Density plots of TESCs and ESCs stained with propidium iodide. (B) Debris-excluded histograms (FSC-A vs. cell number) for ESCs (blue) and TESCs (red). Average FSC values are shown. FSC = forward scatter.

Fig. 4.

Measurement of the cell area in fixed TESCs and ESCs. (A) Single ESCs and TESCs stained with Giemsa or hematoxylin-eosin (HE). Scale bar, 10 µm. Representative images are shown. (B) Cell area of single ESCs (n = 165) and TESCs (n = 135). Data represent mean ± SD. * P < 0.05.

Fig. 5.

Relative gene expression in TESCs and ESCs. (A) Schematic representation of gene expression analysis. Total RNA was extracted from 2 × 10⁵ TESCs or ESCs and 1.2 × 10⁸ copies of λ polyA RNA were added as external standard. Reverse transcription and quantitative real-time PCR (qRT-PCR) were performed. (B) Relative expression levels of housekeeping genes, Gapdh and Actb. (C) Relative expression levels of pluripotency marker genes, Nanog and Oct3/4. Data represent the mean ± SD (n = 6). * P < 0.05, ** P < 0.01.