Another face Of DMSO: Effects of dimethyl sulfoxide (DMSO) on the pluripotency and differentiation capacity of mouse embryonic stem cells

Background Mouse embryonic stem cells (mESCs) go through self-renewal in the existence of the cytokine leukemia inhibitory factor (LIF). LIF is add to the mouse stem cells culture medium, and its removal results in fast differentiation. Dimethyl sulfoxide (DMSO) is one of the most general used solvents in drug test. Methods We exposed 4-day mESC cultures to different concentrations of DMSO (0.1%, 0.5%, 1.0%, and 2.0%) to identify the safest dose exhibiting efficacy as a solvent. mESCs grown under general pluripotency conditions in the absence of LIF were treated with DMSO. In addition, as a control for differentiation, mESCs were grown absence of LIF. Results DMSO downregulated the mRNA expression level of pluripotency markers. Moreover, DMSO reduced the mRNA expression levels of ectodermal marker (β-tubulin3) and a mesodermal marker (Hand1) and endodermal markers (Foxa2 and Sox17) in mESCs. These results indicate that DMSO treatment enhances the pluripotency and disrupts the differentiation of mESCs. We also show that members of the Tet oncogene family are critical to inhibiting the differentiation and methylation of mESCs. Conclusion DMSO is appropriate to sustain the pluripotency of mESCs in the absence of LIF, and that mESCs can be sustained in an undifferentiated state using DMSO. Therefore, DMSO may, in part, function as a substitute for LIF.

renewal. Pluripotency markers (Oct4, Sox2, Lin28, Nanog), and alkaline phosphatase (AP) are significant for sustaining the cells' pluripotency and self-renewal capacity [2][3][4]. mESCs obtained from preimplantation embryos can be cultured in an undifferentiated state, and are considered an suitable model for preimplantation toxicity test. ESCs have been generally used in the study of early embryonic state of pluripotent cell and development [5][6][7][8].
On removal of leukemia inhibitory factor (LIF) and feeder cells grown on non -adhesive dishes, mESCs differentiate into cells of three primary germ layers: the endoderm, mesoderm, and ectoderm. LIF is indispensable for maintaining mESC pluripotency.
Removal of LIF allows stem cells to differentiate, but these cells retain their proliferative potential and pluripotency [9]. Therefore, LIF is used in the culture of mESCs. LIF is necessary to sustain stem cells in an undifferentiated state. Though, genetic manipulation of ESCs enables LIF-independent growth, particularly by over-expression of the gene Nanog [10]. LIF is added to stem cell culture medium to decrease inherent differentiation [11].
DNA methylation is a stable epigenetic mark that is critical for diverse biological processes including gene and transposon silencing, imprinting, and the suppression of retrotransposons [12]. DNA demethylation come about in the early embryo and the germ cells [13,14], and may be mediated by the ten eleven translocation Tet family of enzymes (Tet1,2,3) [15][16][17], which convert 5-methylcytosine (mC) to 5-hydroxymethylcytosine (hmC) [18]. Tet enzymes have been extensively studied in mESCs [19][20][21][22]. Here, we report that the addition of DMSO to mESCs promotes Tet activity, leading to a increase in the number of mESCs.
In the present study, we developed a novel and robust method for the differentiation of mESCs into the three germ layers and evaluation of their methylation potential by the 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 selfrenewal 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 [34]. 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.

Discussion
ESCs have properties like pluripotency, self-renewal and continuous proliferation [2,[37][38][39]. For such properties, the mESCs can be maintained by the cytokine LIF, and LIF is regularly added to the culture of mESCs. Conversely, if LIF is not added, it brings the differentiation of mESCs. Because of such LIF properties, a lot of researchers have conducted the studies related to the embryonic stem cells, focused on such LIF properties with great interest.
DMSO is generally solvent for many different kinds of organic substance, and it is widely used for cryopreserve of cell as cryoprotectant. In particular, in case of culturing ESCs, DMSO regulates the differentiation of cell. This research came to conduct this study in order to examine the effect of solvent like DMSO on in vitro development [40]. Especially, this study was really designed to investigate the effect of DMSO on the E14 mESCs and mouse embryonic body mEBs. The deduced result in this study showed the deeper understanding of the effect of DMSO on the early embryonic development. The treatment of DMSO increased the expression of pluripotency markers at mRNA level of E14 mESCs and decreased the expression of germ layer markers under mEBs condition. Overall, according to our data, this study has found out that DMSO played an important role in regulating the pluripotency of mESCs and differentiation of mEBs through the regulation of expression for pluripotency markers (Oct4, Sox2, Lin28, and Nanog) related to the pluripotency [41,42].
It is important to understand the molecular mechanisms that control pluripotency and selfrenewal in stem cells for application in the fields of developmental biology, regenerative medicine, and cancer biology. Pluripotency is sustained by a synergistic interaction between extrinsic stimulus and intrinsic circuitry, which allow for sustentation of an undifferentiated and self-renewing state. Notwithstanding, despite recent study, the precise mechanisms regulating differentiation and self-renewal remain unclear. LIF is widely used for culturing the mESCs and IPSs and inhibiting the differentiation of cell by paracrine signal while especially LIF stimulates the ESCs self renewal at mESCs [43][44][45].
The use of feeder cells is replaceable by adding LIF to culture solution, and serum is replaceable with BMP. Since such discovery, the condition of ESCs culture has greatly improved [46]. However, it is still difficult situation in the mechanism that DMSO affects mESCs, but this study has shown that DMSO could maintain pluripotency at mESCs without LIF.  Table 1.

Statistical analysis
Values are expressed as means ± standard errors (S. E.) of the mean. Statistically significant differences between the DMSO treats(0.1%, 0.5%, 1.0%, or 2.0%) and controls were analyzed by ANOVA, followed by Dunnett's multiple comparisons test. All tests of statistical significance were two-sided, and p-values <0.05 were considered to indicate statistical significance.

Consent for publication
Not applicable.

Availability of data and materials
All data generated or analysed during this study are included in this published article.
12 and approved the manuscript, and ensure that this is the case.   Figure 1 Effects of DMSO on mESC morphology. E14 mESCs were exposed to DMSO, and cell morphologies were observed. Magnification, 40×. Cells were cultured with various concentrations of DMSO (0.1%, 0.5%, 1.0%, or 2.0%) and for controls (with LIF or without LIF) for 96 h. Effects of DMSO on alkaline phosphatase staining and activity in mESCs. Cells were cultured with various concentrations of DMSO (0.1%, 0.5%, 1.0%, or 2.0%) and for controls (with LIF or without LIF) for 96 h. Effects of DMSO on differentiation capacity and expression of markers of three germ layers markers in mESCs. mRNA levels of endodermal markers (Foxa2, Sox17) and mesodermal (Hand1) and ectodermal (β-tubulin 3) markers were determined by quantitative real-time PCR using GAPDH as an internal control.
Each data point was normalized to the control (with LIF), and the means ± S.E. from three independent experiments are presented. Indicates significant difference when the values were compared to that of the control (p < 0.05).

Figure 5
Effects of DMSO on DNA methylation and expression of TET oncogenes in mESCs.
Tet1 (Tet oncogene 1), Tet2 (Tet oncogene2), and Tet3 (Tet oncogene3) mRNA levels were determined by quantitative real-time PCR using GAPDH as an internal control. Each data point was normalized to the control (with LIF), and the means ± S.E. from three independent experiments are presented. Indicates significant difference when the values were compared to that of the control (p < 0.05).

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