Tetraploid embryonic stem cells can contribute to the development of chimeric fetuses and chimeric extraembryonic tissues

Abstract

Our study examined the in vivo chimeric and survival capacities of chimeras created by injecting tetraploid embryonic stem cells (ESCs) expressing green fluorescent protein (GFP) into diploid embryos. At 3.5 days post-coitum (dpc) and 4.5 dpc, the tetraploid ESCs were able to contribute to the inner cell mass (ICM) just as diploid ESCs tagged with GFP. At 6.5 dpc, 8.0 dpc and 10.5 dpc, the tetraploid ESCs manifested in the same location as the diploid ESCs. The GFP cells in the extraembryonic tissues and fetuses of tetraploid ESC chimeras were tetraploid as determined by fluorescence activated cell sorting (FACS). Furthermore, tetraploid ESCs contributed to the development of the placenta, embryolemma and umbilical cord at 13.5 dpc and 16.5 dpc; however, very less GFP cells were found in the fetuses of tetraploid ESC chimeras. We further found that the proliferation of tetraploid ESCs was slower than that of diploid ESCs. In addition, the relative mRNA expression in the three germ layers and the trophoblast was abnormal in the EBs of tetraploid ESCs compared with diploid ESCs. In short, slower proliferation and abnormal differentiation potential of tetraploid ESCs might be two of the reasons for their poor survival and chimeric capacities.

Figure 1

Characterization of 4.5 dpc tetraploid blastocysts. (A) Differential expression of NANOG in diploid and tetraploid embryos at the 4.5 dpc blastocyst stage. Bar scale = 20 μm. (B) Percentages of different groups of blastocysts. Diploid blastocysts and tetraploid blastocysts were differentially stained with EPI, and total cells were individually classified into four groups according to the number of EPI-positive cells (0%, 0–5%, 5–10% and 10%). 0% represented no epiblast cells in the blastocyst, 0–5% represented the percentage of epiblast cells in the blastocyst greater than 0% and less than or equal to 5%. 5–10% represented the percentage of epiblast cells in the blastocyst greater than 5% and less than or equal to 10%. 10%- represented the percentage of epiblast cells in the blastocyst greater than 10%. (C) The methylation status of the Oct4 promoter showed differentially methylated regions in diploid and tetraploid blastocysts. (D) The colonies of diploid and tetraploid ESCs were round and three-dimensional. Bar scale = 200 μm.

Figure 2

Characterization of tetraploid ESCs. (A) Tetraploid ESCs were positive for AP staining. Bar scale = 200 μm. (B) Immunocytochemical staining of OCT4, SOX2 and SSEA1 in tetraploid ESCs. Bar scale = 10 μm. (C) Hematoxylin and eosin staining of teratoma sections of tetraploid ESCs. Left: blood vessel of endothelium (ectoderm); middle: muscle (mesoderm); right: gut-like epithelium (endoderm). Scale bars = 50 μm. (D) Karyotype analysis of diploid and tetraploid ESCs. (E) Methylation status of the Oct4 promoter showed methylated regions in diploid and tetraploid ESCs. (F) Relative mRNA expression levels of pluripotent genes in diploid and tetraploid ESCs. Different superscripts represent statistically significant differences between groups (P < 0.05).

Figure 3

The chimeric capacity of diploid and tetraploid ESCs. (A) Diploid ESC and tetraploid ESC chimeric embryos at 2.5 dpc, 3.5 dpc and 4.5 dpc. Bar scale = 100 μm. (B) Diploid ESC and tetraploid ESC chimeras at 6.5 dpc (Bar scale = 100 μm), 8.0 dpc (Bar scale = 200 μm) and 10.5 dpc (Bar scale = 2 mm). (C) The percentage of survival conceptus and chimeric conceptus at 6.5 dpc, 8.0 dpc and 10.5 dpc. Different superscripts represent statistically significant differences between groups (P < .05).

Figure 4

Proliferation and differentiative potential of diploid and tetraploid ESCs. (A) Proliferation of diploid and tetraploid ESCs. (B) Cell diameter of diploid and tetraploid ESCs. (C) FACS analysis of cell cycle differences between diploid and tetraploid ESCs. (D) Relative mRNA expression levels of cell division-related genes in diploid and tetraploid ESCs. (E) Relative mRNA expression levels of three germ layer and trophectoderm genes in 3 d and 5 d diploid and tetraploid EBs. Different superscripts represent statistically significant differences between groups (P < 0.05).