DMSO affects the morphology and pluripotency of mESCs
DMSO has been shown to regulate epigenetic modification by altering CpG methylation patterns in diverse E14 mESCs and tissues [31, 32], thus affecting their development and differentiation . To study the effects of DMSO on cell morphology and differentiation, E14 mESCs were exposed to diverse concentrations of DMSO for 96 h. mESCs were collected after 4 days of culture in the presence of diverse concentrations of DMSO (0.1%, 0.5%, 1.0%, or 2.0%) or with the fundamental pluripotency factor LIF as a control for self-renewal and pluripotency. Colony growing as a round and dome shape could be seen under control conditions, whereas cells growing without LIF no longer formed these typical pluripotent colony. On the other hand, higher levels of differentiation were apparent, resulting in fewer colonies without defined borders. Rather, cultures grown without LIF in the presence of 1.0% and 2.0% DMSO produced well-defined, round colonies and exhibited a reduced number of differentiated cells in comparison with the differentiation control (without LIF) (Fig. 1A).
To determine whether DMSO influences pluripotency, we stained mESCs with AP. Differentiation of mESCs is characterized by the loss of AP staining and the emergence from a flattened cellular morphology . In the present study, mESCs treated with DMSO for 96 h were resembling the control (with LIF), showing deep AP staining and a similar colony morphology (Fig. 2). Given that pluripotent cells express high levels of AP, we also performed an AP assay to monitor the number of AP-positive red colonies [35, 36] (Fig. 2). The differentiation control (without LIF) showed a low rate of AP-positive colonies, while cells growing without LIF in the presence of DMSO had a higher rate of AP-positive colonies. Treatment with 1.0% DMSO showed the highest rate of AP-positive colonies.
DMSO regulates the expression of mESC pluripotency genes in a concentration-dependent manner
To determine whether DMSO influences mESC differentiation, cells were treated with DMSO and the effect on differentiation in the absence of LIF was evaluated. mESCs were exposed to various concentrations of DMSO (0.1%, 0.5%, 1.0%, or 2.0%) or LIF as a control for 96 h, and then collected after 4 days of culture. The expression levels of pluripotency markers such as Oct4, Sox2, and Lin28 were analyzed by qRT-PCR (Fig. 3A–C). Treatment with 0.1%, 0.5%, and 1.0% DMSO increased Oct4, Sox2, and Lin28 mRNA levels after 4 days in a dose-dependent manner. Treatment with 2.0% DMSO, however, resulted in a significant decrease in Oct4, Sox2, and Lin28 mRNA expression levels after 4 days of culture.
mRNA expression levels of markers of endoderm, mesoderm and ectoderm were also examined by qRT-PCR (Fig. 4A–D). mRNA levels of the endoderm marker Foxa2 decreased significantly when mESCs were treated with 0.5% or 2.0% DMSO (Fig. 4A). mRNA levels of another endoderm marker, Sox17, also decreased in a dose-dependent manner following DMSO treatment (0.1%, 0.5%, and 1.0%) (Fig. 4B). Treatment with 2.0% DMSO, however, significantly increased Sox17 mRNA levels in comparison with those of the control. The mRNA levels for the mesoderm marker Hand1 and the ectoderm marker β-tubulin 3 also decreased in a dose-dependent manner with DMSO treatment (Fig. 4C, D). On the whole, we find out pluripotency markers were differentially expressed, suggesting that DMSO biases differentiation towards the three germ layers.
DMSO also affects the mRNA levels of genes involved in active DNA methylation
To evaluate the effect of DMSO on the active DNA methylation of mESCs, qRT-PCR was used to assess mRNA expression levels of two members of the TET oncogene family: Tet1 (Tet oncogene 1) and Tet2 (Tet oncogene 2), following DMSO treatment. Expression of these genes after 4 days of culture increased in a dose-dependent manner; however, expression was downregulated by 2.0% DMSO treatment (Fig. 1A, B). mRNA expression of the third member of the TET family, Tet3, was increased significantly in a dose-dependent manner by DMSO treatment. These findings suggest that regulating the expression of the TET oncogene family members is critical for proper differentiation and methylation of mESCs.