Inhibition of DNA Methyltransferase by RG108 Promotes Pluripotency-Related Character of Porcine Bone Marrow Mesenchymal Stem Cells

Abstract

Mesenchymal stem/stromal cells (MSCs) have been identified in almost all adult human tissues and been used in numerous clinical trials for a variety of diseases. Studies have shown that MSCs would undergo cellular senescence when cultured over a long term, which is brought on by increased epigenetic modifications, including DNA methylation. However, the mechanism of MSCs senescence is not well studied. In this study, the effects of RG108, a DNA methyltransferase inhibitor (DNMTi), on senescence, apoptosis, and pluripotency gene expressions in porcine bone marrow (pBM)-MSCs were investigated. First, we determined the optimized dose and time of RG108 treatment in pBM-MSCs to be 10 μM for 48 hours, respectively. Under these conditions, the pluripotency genes (NANOG, POU5F1), the anti-senescence genes (TERT, bFGF), and the anti-apoptosis gene (BCL2) were increased, whereas the apoptotic gene (BAX) was decreased. RG108 protected against apoptosis when pBM-MSC induces apoptosis with H₂O₂ for 1.5 hours. We also found that RG108 significantly induced the expression of NANOG and POU5F1 by decreasing DNA methylation in gene promoter regions. These results indicate that an optimized dose of RG108 may promote the pluripotency-related character of pBM-MSCs through improving cellular anti-senescence, anti-apoptosis, and pluripotency, which provide a better cell origin for stem cell therapy.

Introduction

Stem cells are undifferentiated cells that can differentiate into more specialized cells. They can proliferate to produce more stem cells or differentiate into many different cell types in the body during development (Kobolak et al., 2016). There are three types of stem cells: embryonically derived stem cells, fetal-derived stem cells, and adult stem cells. Mesenchymal stem/stromal cells (MSCs) is one kind of adult stem cells and has been identified in almost all adult tissues (Crisan et al., 2009) since the colony-forming fibroblasts were discovered in bone marrow in the 1970s (Friedenstein et al., 1974).

MSCs have anti-inflammatory and immunomodulatory properties, they also repair damaged tissues through secretion of soluble bioactive molecules, including growth factors (Burlacu et al., 2013), anti-fibrotic factors (Dong et al., 2015), angiogenic factors (Kuchroo et al., 2015), and molecules that inhibit apoptosis and activate tissue-specific progenitor cells.

Bone marrow-MSCs (BM-MSCs) have been used in stem cell therapies for various diseases (Cho et al., 2010a). However, it needs a sufficient amount of stem cells for therapies, and stem cells are difficult to obtain owing to their poor isolation yield from donors. Thus, most researchers expand the yield of isolated stem cells under in vitro culture conditions to obtain a sufficient quantity of stem cells. However, previous studies have found that the long-term in vitro culture induced the cellular senescence in MSCs through telomere shortening and a decline of telomerase (TERT) activity (Donate and Blasco, 2011). Thus, an increasing number of cell divisions increase the risk of cellular senescence, resulting in a reduction in the therapeutic efficacy or failure of cell therapies (Cho et al., 2010b).

Recent studies have shown that the senescence phenotype is brought on by increased epigenetic modifications (Bork et al., 2010), such as DNA methylation. DNA methylation is an important epigenetic modification that occurs predominantly at CpG dinucleotides. It plays critical roles in a number of key genomic functions, such as gene imprinting X chromosome inactivation, genome stability, retrotransposon silencing, and gene inactivation in cancer (Bird, 2002).

DNA methylation is catalyzed by members of the DNA methyltransferase (DNMT) family. The DNMT family includes three main members, DNMT1, DNMT3a, and DNMT3b. DNMT1 is largely responsible for maintaining methylation patterns through DNA replication, whereas both DNMT3a and DNMT3b are de novo methyltransferases (Leonhardt et al., 1992; Okano et al., 1999). There is also growing evidence that the activities of DNMTs are associated with aging and aging-related disease (Johnson et al., 2012).

Collectively, these reports suggest that proper DNA methylation modification may improve the quality of BM-MSCs during in vitro long-term culture. In this study, we investigated the effects of RG108, a noncovalent DNMT inhibitor (DNMTi), which could bind at the micromolar range to the DNMT1 pocket site and blocks DNA binding (Zinn et al., 2007), on the anti-senescence, anti-apoptosis, and pluripotency gene expressions in porcine bone marrow (pBM)-MSCs during in vitro culture.

Materials and Methods

Ethics statement

All animal studies were conducted according to the experimental practices and standards approved by the Animal Welfare and Research Ethics Committee at Jilin University (Approval ID: 20101008-2).

Chemicals

Chemicals and media were purchased from Sigma (St. Louis, MO), unless otherwise stated.

Cell culture and treatment

pBM-MSCs were isolated and purified from the bone marrow of a 1-week-old pig as previously described (Fang et al., 2003). The purified pBM-MSCs were seeded in a 24-well plate with density of 2.0 × 10⁴ cells per well and cultured in DMEM/F12 medium (containing 1 mM calcium) supplemented with 10% fetal bovine serum (FBS), 100 U/mL of penicillin sodium, and 100 μg/L of streptomycin sulfate in a humidified incubator with atmosphere of 5% CO₂ at 37°C. The pBM-MSCs were treated with various concentrations (5, 10, 20, and 50 μM) of RG108 for 48 or 72 hours.

MSC properties

To assess multipotency, the purified pBM-MSCs were cultured in adipogenic, osteogenic, and chondrogenic differentiation medium for 4 weeks in a humidified incubator with atmosphere of 5% CO₂ at 37°C as described previously (Rajaraman et al., 2013). To detect the differentiation, the cells were fixed with 4% paraformaldehyde and stained with 1% Oil Red O for adipogenesis, 4% Alizarin Red (pH 4.1) for osteogenesis, and 1% Alcian blue (pH 2.5) on paraffin-embedded sections (5 μm) of the micromass pellet for chondrogenesis. Stained cells were examined under a microscope (Nikon, Tokyo, Japan), and images were taken with 10 × objective lens.

Cell surface markers detection

The CD105, CD90, and CD44 were selected as positive surface markers, and CD14, CD34, and CD45 were selected as negative markers to identify pBM-MSCs with reference to a review by Bharti et al. (2016). Flow cytometry was used to detect the specific antigen expression of CD105 (10862-1-AP; Proteintech, China), CD90 (66766-1-Ig; Proteintech), CD44 (60224-1-Ig; Proteintech), CD45 (60287-1-Ig; Proteintech), CD34 (14486-1-AP; Proteintech), and CD14 (17000-1-AP; Proteintech) in pBM-MSCs.

pBM-MSCs were routinely digested, neutralized using 0.25% trypsin containing ethylenediaminetetraacetic acid, and counted using a hemocytometer to 1 × 10⁶ cells. The collected cells were resuspended in a 2 mL centrifuge tube, centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded. Diluted primary antibody was added to the centrifuge tube and incubated at 4°C for 1.5–2 hours. Diluted secondary antibody was added to the centrifuge tube, incubated at 4°C for 30 minutes, and centrifuged at 1500 rpm for 5 minutes. After adding 1 mL of cold phosphate-buffered saline (PBS) and washing twice, the cells were resuspended in 500 μL of PBS for testing.

Apoptosis analysis by flow cytometry

To assess apoptosis, the treated and untreated cell pellets were stained with Annexin V-APC/PI kit following the manufacturer's protocol (FA101-01; TransGen Biotech). Briefly, cells were trypsinized and resuspended in 100 μL binding buffer, 5 μL of Annexin V-APC solution was added to the cell suspension and incubated for 15 minutes at room temperature protected from light. After washing with the binding buffer, 5 μL of propidium iodide (PI) was added to the cells suspended in 200 μL binding buffer and cells were immediately examined by flow cytometry using BD FACS Canto™ II and analyzed with Flow Jo 7.6.3.

RNA isolation, cDNA preparation, and quantitative real-time polymerase chain reaction

The total RNAs from the pBM-MSCs were extracted using RNAiso Plus (TaKaRa, Shiga, Japan). The First-Strand cDNA Synthesis Kit (Promega, Madison, WI) was used to synthesize the first-strand cDNA, according to the manufacturer's instructions, immediately after RNA isolation.

The primers used for quantitative real-time polymerase chain reaction (qRT-PCR) analysis are listed in Table 1. The real-time PCR mix (20 μL) consisted of 2 μL of cDNA, 10 μL of SYBR Green Master Mix, 6.4 μL of RNase-free water, and 0.8 μL each of forward and reverse primers (10 pmol) for each gene. The program used for the amplification of all genes consisted of a denaturing cycle of 3 minutes at 95°C, 40 cycles of PCR (95°C for 10 seconds, 55°C for 45 seconds, and 95°C for 1 minute), a melting curve analysis consisting of 95°C for 1 minute, followed by 55°C for 1 minute, a step cycle starting at 55°C for 10 seconds with a 0.5°C/second transition rate, and cooling at 4°C. Relative gene expression data were analyzed using qRT-PCR and the 2^−▵▵CT method. The qRT-PCR analysis was performed three times for each sample.

Table 1.

Primers Used in the Quantitative Real-Time Polymerase Chain Reaction Analysis

Name	Primer sequences (5′–3′)
GAPDH	F-ACCTGCCGCCTGGAGAAACC
GAPDH	R-GACCATGAGGTCCACCACCCTG
POU5F1	F-CTTCACCACCCTGTACTCCTC
POU5F1	R-GCTTCTCTCCCTAGCTCACC
NANOG	F-CCTACAATCCAGCTCTTTGG
NANOG	R-CTCAGGCATTGGTGAAGATT
TERT	F-CATGGAAAACAGACTGTCCCCG
TERT	R-TCCTCCACAGGGAAGTTCACG
bFGF	F-AAGGAGTGTGTGCTAACCGT
bFGF	R-CTGCCCAGTTCGTTTCAGTG
BCL2	F-CAAGCCTTCAACCATTATCTCAGT
BCL2	R-GGGGGGTAAAGAAAACAGCAT
BAX	F-AGAAGCGGGTTAGAAGAGTGGA
BAX	R-GCCTGAATGAAACGGGTGAC

Sodium bisulfite genomic sequencing

For bisulfite sequencing, the genomic DNA was extracted from the pBM-MSCs using a TIANamp Genomic DNA Kit (Tiangen, Beijing, China) and subjected to bisulfite conversion using an EZ DNA Methylation-Direct Kit (Zymo Research, Los Angeles, CA) according to the manufacturer's instructions. The bisulfite-modified DNA was subjected to PCR, and the PCR primer sequences are listed in Table 1. The purified PCR fragments were then cloned into a PMD™18-T vector for sequencing (TaKaRa, Japan). At least 10 clones per gene were sequenced.

Western blotting analysis

pBM-MSCs were collected and treated with RIPA lysis buffer including 1 mM phenylmethanesulfonyl fluoride (PMSF). The protein samples were fractionated on a Biofuraw™ Precast Gel (Tanon, Shanghai, China) and electrically transferred onto a polyvinylidene difluoride membrane. Next, the membranes were blocked with 5% nonfat milk and incubated overnight at 4°C with a primary antibody POU5F1 (ab 18976; Abcam), NANOG (ab 77095; Abcam), and GAPDH (10494-1-AP; Proteintech). The membranes were washed three times with Tris-HCl buffer including Tween (TBST) and incubated for 1–2 hours at room temperature with a secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit IgG and donkey anti-goat from Proteintech). Finally, the membranes were washed three times with TBST and visualized by using a Tanon 5200 automatic fluorescence/chemiluminescence imaging analysis system (Tanon).

Statistical analysis

The data were analyzed by a one-way analysis of variance, using Statistics Production and Service Solution Software (SPSS, version 16.0). Significant differences of percentage were determined by chi-square test. Differences were considered significant at the value of P < 0.05 and highly significant at the value of P < 0.01.

Results

Isolation and identification of pBM-MSCs

The pBM-MSCs were isolated and cultured for passage and showed fibroblast-like shape (Fig. 1A). The flow cytometry results showed that almost all the isolated cells were positive for the MSC markers, the percentage of CD105, CD90, and CD44 (positive surface markers) is 100%, 99.1%, and 100%, and the percentage of CD45, CD34, and CD14 (negative markers) is 2.93%, 2.16%, and 1.76%, respectively (Fig. 1B). We also analyzed the multipotency of the isolated cells by directed induction differentiation experiments and found that the isolated cells have the abilities for adipogenesis, osteogenesis, and chondrogenesis (Fig. 1C).

FIG. 1.

Isolation and identification of pBM-MSCs. (A) Primary separation of pBM-MSCs. (a) Primary separation at day 3. (b) Primary separation at day 7. (c) Primary separation at day 14. (B) Flow cytometry analysis of bone marrow MSC surface markers. (a) 100% of the cells were positive for CD105. (b) 99.1% of the cells were positive for CD90. (c) 100% of the cells were positive for CD44. (d) 1.93% of the cells were negative for CD45. (e) 2.16% of the cells were negative for CD34. (f) 1.76% of the cells were negative for CD14. (C) The analysis of multipotency of the isolated cells. (a) Induced differentiation of osteoblasts and identification by Alizarin red staining, which is shown as a dark stain. (b) Induced differentiation of adipocytes and identification by Oil red O staining, which is shown as a dark stain. (c) Induced differentiation of chondrocytes and identification by Alcian blue staining, which is shown in gray. pBM-MSCs, porcine bone marrow mesenchymal stem cells.

RG108 induced the expressions of POU5F1 and NANOG in pBM-MSCs

To evaluate the concentration effects of RG108, pBM-MSCs were treated with 0, 5, 10, 20, and 50 μM of RG108 for 48 or 72 hours. The expression of POU5F1 was assessed by qRT-PCR, and the maximum expression was identified to be in 10 μM for 48 hours in RG108-treated pBM-MSCs (Fig. 2A) (P < 0.01). Therefore, treatment with RG108 at 10 μM for 48 hours was for the further research. Treatment with RG108 also increased the expression of NANOG in pBM-MSCs (P < 0.05).

FIG. 2.

RG108 induced the expressions of POU5F1 and NANOG in pBM-MSCs. (A) Optimization of RG108 treatment. (a) The expressions of POU5F1 in pBM-MSCs treated with RG108 (0–50 μM) were assessed by qRT-PCR. (b) The expressions of POU5F1 in pBM-MSCs treated with RG108 at 10 μM for 48 hours and 72 hours were assessed by qRT-PCR. (B) Treatment with RG108 increased the expressions of POU5F1 and NANOG in pBM-MSCs. (a and b) The expressions of POU5F1 and NANOG in pBM-MSCs treated with RG108 at 10 μM for 48 hours were assessed by qRT-PCR. (c) The expressions of POU5F1 and NANOG at the protein level assessed by immunoblot analysis. (C) DNA methylation situations in promoter regions of POU5F1 and NANOG genes measured by BSP-PCR. qRT-PCR, quantitative real-time polymerase chain reaction; BSP-PCR, bisulfite sequencing polymerase chain reaction. *p < 0.05; **p < 0.01.

Then, we analyzed the DNA methylation situations in promoter regions of POU5F1 and NANOG genes by bisulfite sequencing polymerase chain reaction (BSP-PCR). The results showed that RG108 decreased the DNA methylation levels in promoter regions of POU5F1 (56.4%) and NANOG (36.7%) when compared with the control group (POU5F1: 71.4%; NANOG: 42.2%) (Fig. 2C). To confirm the qPCR results at the protein level, Western blotting was performed with antibodies against POU5F1, NANOG, and GAPDH (Fig. 2B).

RG108 prevented cellular senescence in pBM-MSCs

Studies have shown that MSCs cultured over a long term will undergo cellular senescence. So, we analyzed the influence of RG108 on the expressions of anti-senescence genes TERT and bFGF by qRT-PCR. The results showed that transcripts of TERT and bFGF genes were significantly increased in RG108-treated pBM-MSCs (Fig. 3A and B) (P < 0.05).

FIG. 3.

RG108 prevented cellular senescence in pBM-MSCs. (A) The expressions of TERT in pBM-MSCs treated with RG108 at 10 μM for 48 hours were assessed by qRT-PCR. (B) The expressions of bFGF in pBM-MSCs treated with RG108 at 10 μM for 48 hours were assessed by qRT-PCR. *p < 0.05.

RG108 prevented cellular apoptosis in pBM-MSCs

We analyzed the influence of RG108 on the expressions of anti-apoptosis gene BCL2 and apoptosis gene BAX2 by qRT-PCR. We found that RG108 significantly increased the BCL2 expression (P < 0.05) and decreased the BAX2 expression (P < 0.05) in RG108-treated pBM-MSCs (Fig. 4A). Then, we examined the protective effect of RG108 on apoptosis. RG108-pretreated pBM-MSCs were exposed to H₂O₂ (0–1.5 μM) for 2 hours and subjected to cell apoptosis assay. The results showed that RG108 significantly decreased the apoptosis rate when H₂O₂ concentration was 0.5 μM (P < 0.05) and 1.0 μM (P < 0.05). However, RG108 could not protect pBM-MSCs against apoptosis when H₂O₂ concentration was 1.5 μM (P > 0.05) (Fig. 4B).

FIG. 4.

RG108 prevented cellular apoptosis in pBM-MSCs. (A) The expressions of anti-apoptosis gene BCL2 (a) and apoptosis gene BAX2 (b) were assessed by qRT-PCR. (B) The protective effect of RG108 on apoptosis. The cell apoptosis assay by flow cytometry after exposure of pBM-MSCs to H₂O₂ (0–1.5 μM) (a). The statistical analysis of the flow cytometry data (b). *p < 0.05.

Discussion

BM-MSCs have multipotential capacities to differentiate toward osteocyte, chondrocyte, adipocyte, and myocyte (Uccelli et al., 2008), performing an important role in disease therapy, wound healing, and regenerative medicine (Bajada et al., 2008). Pigs exhibit similar structure and function to those of humans and have been widely used as a valuable model in biomedical research such as tissue engineering and cell therapy (Swindle et al., 2012). Although BM-MSCs have been used in stem cell therapies for various diseases (Cho et al., 2010a), it is difficult to obtain a sufficient amount of stem cells owing to their poor isolation yield from donors. Thus, the use of BM-MSCs for cell-based therapy requires their expansion in culture conditions that support homogenous growth and maintain self-renewal and multipotency.

However, studies have shown that MSCs cultured over a long term will undergo cellular senescence (Donate and Blasco, 2011). Senescence is brought on by increased epigenetic modifications (Bork et al., 2010), as results in the formation of senescence-associated heterochromatin foci (Johnson et al., 2012), which can also be initiated by DNA methylation. Thus, in this study, we investigated whether the treatment with RG108, a noncovalent DNMTi, could improve pluripotency-related character of pBM-MSCs or not during a long-term culture in vitro.

POU5F1 and NANOG are two major pluripotency-related genes and play important roles in maintaining of pluripotency and multipotency (Silva et al., 2009). Treatment with RG108 could improve the pluripotency of mice-induced pluripotent stem cells through increasing the expressions of POU5F1 and NANOG caused by the DNA methylation decrease in the promoter regions (Pasha et al., 2011). Thus, we first analyzed the effects of RG108 on the expressions of POU5F1 and NANOG in pBM-MSCs. The results indicated that treatment with RG108 significantly increased the expressions of POU5F1 and NANOG in pBM-MSCs.

Previous study reports that RG108 significantly induced the expression of TERT by blocking DNA methylation at the TERT promoter region in human BM-MSCs (Oh et al., 2015). Our previous research shows that treatment with RG108 increases the expressions of POU5F1 and NANOG in bovine parthenogenetic blastocyst by decreasing the DNA methylation levels in promoter regions of these two genes (Zhang et al., 2015). So, we examined the DNA methylation levels in promoter regions of POU5F1 and NANOG genes in pBM-MSCs. We found that RG108 treatment decreased the DNA methylation levels in promoter regions of POU5F1 and NANOG genes in RG108-treated pBM-MSCs.

These results indicated that RG108 increased the expressions of POU5F1 and NANOG through decreasing the DNA methylation levels in promoter regions of POU5F1 and NANOG genes. The high expressions of POU5F1 and NANOG genes could improve the multipotential capacities of pBM-MSCs when cultured in vitro for a long term.

Senescence and apoptosis are two main obstacles that prevent the pBM-MSCs for a long-term culture in vitro (Donate and Blasco, 2011). Previous study showed that long-term cultured MSCs, with increases in both the expression of the anti-senescence protein and the percentage of aneuploidy cells, were rescued by transduction of the hTERT gene (Estrada et al., 2013). In our study, we analyzed the senescence and apoptosis in RG108-treated pBM-MSCs. As reported in human BM-MSCs, the expressions of anti-senescence genes TERT and bFGF were significantly increased in RG108-treated pBM-MSCs. The increased expression of TERT in RG108-treated pBM-MSCs might result from the decreased DNA methylation level at TERT promotor regions as in human BM-MSCs.

Inhibition of DNMTs with RG108 blocked completely the increase in 5-methycytosine and the apoptosis of motor neurons in mice (Chestnut et al., 2011). And our results showed that RG108 significantly increased the anti-apoptosis gene BCL2 expression and decreased the apoptotic gene BAX expression in pBM-MSCs. Treatment with RG108 also protected pBM-MSCs against apoptosis when they were exposed to H₂O₂, indicating that RG108-treated pBM-MSCs may have acquired protective effects against the damage induced by oxidative stress as previous report (Oh et al., 2015). The improvement of anti-apoptosis ability may be caused by the decrease of the global genome DNA methylation levels (Chestnut et al., 2011) or the activation of some unknown signal pathways (Gurung et al., 2015).

In summary, this study indicated that an optimized dose of RG108 may promote the pluripotency-related characters of pBM-MSCs through improving the cellular anti-senescence, anti-apoptosis, and pluripotency, which provide a better cell origin for stem cell therapy.

Footnotes

Author Disclosure Statement

The authors declare they have no competing financial interests.

Funding Information

This research was funded by the National Key R&D Program of China, grant number 2017YFA0104400, and Program for Changjiang Scholars and Innovative Research Team in University, grant number IRT_16R32.

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