Demethylating agent zebularine is reported to be capable of inducing differentiation of stem cells by activation of methylated genes, though its function in hepatocyte differentiation is unclear. p38 signal pathway is involved in differentiation of hepatocytes and regulating of DNA methyltransferases 1 (DNMT1) expression. However, little is known about the impact of zebularine on bone marrow mesenchymal stem cells (BMMSCs) and p38 signaling during hepatic differentiation. The present study investigated the effects of zebularine on hepatic differentiation of rabbit BMMSCs, as well as the role of p38 on DNMT1 and hepatic differentiation, with the aim of developing a novel strategy for improving derivation of hepatocytes. BMMSCs were treated with zebularine at concentrations of 10, 20, 50, and 100
Liver regeneration is critical for graft survival and competent organ function. Bone marrow mesenchymal stem cells (BMMSCs) have immense potential in the field of regenerative medicine for its multilineage potential. BMMSCs, therefore, are increasingly being considered in cell-based therapeutic strategies for liver regeneration. However, their low hepatic differentiation efficiency in vitro is a significant obstacle to hepatocyte transplantation. Current evidence suggests that epigenetic programming plays an important role in stem cell biology by maintaining pluripotency and promoting stem cell differentiation into more mature cell progenies [
Hepatocyte growth factor (HGF) played a critical role in the development and regeneration of the liver and induced expression of the hepatic phenotype in mesenchymal stem cells in vitro [
In this study, we investigated the effects of zebularine on DNA methyltransferases 1 (DNMT1) and hepatic differentiation of BMMSCs. Activation of p38 and its role on DNMT1 and hepatic differentiation were also analyzed. The objective is to come up with a novel strategy that would be highly potent in terms of differentiating the BMMSCs into hepatocytes while being least toxic to biological systems.
We purchased sheep anti-rabbit antibodies of CD29, CD34, CD44, CD45, and fluorescein isothiocyanate- (FITC-) labeled IgG secondary antibody from eBioscience (San Diego, CA). Sheep polyclonal anti-human antibodies to alpha-fetoprotein (AFP), albumin (ALB) and
The p38 inhibitor SB203580 was obtained from Merck (Germany). A stock solution of SB203580 was prepared in dimethyl sulfoxide (DMSO). SB203580 was diluted in Dulbecco modified Eagle medium (DMEM; Gibco, Rockville, MD) and was added to cell culture 1 h before stimulation with HGF and kept further during the exposure to this compound. A stock solution of zebularine for cellular assays was prepared in DMSO and then diluted in the optimal medium to the final concentrations. Indocyanine green (ICG) was obtained from Aladdin (Shanghai, China).
The biological study was approved by the Medicine Human Ethics Committee of the Second Xiangya Hospital of the Central South University. Rabbit bone marrow was obtained from three New Zealand rabbits (male, two-month-old, less than 1 kg in weight) obtained from the Laboratory Animal Unit of the Second Xiangya Hospital of the Central South University. BMMSCs were harvested from expansion culture of cell suspension and were propagated in DMEM supplemented with 10% fetal bovine serum (FBS; Gibco, Rockville, MD), 100 U/ml penicillin, and 100 mg/ml streptomycin. The third to fifth generation of cells were collected for study. BMMSCs were identified by the flow cytometric analysis of CD29, CD34, CD44, and CD45. The experiments were performed at least three times in cell strains from the three rabbits and obtained similar results.
Primary rabbit hepatocytes were isolated from anesthetised rabbits by in situ perfusion of the liver with 0.05% collagenase as previously described [
Approximately 1 × 106 cells of the third generation cells were harvested and resuspended in 1 ml phosphate-buffered saline (PBS). Cells were incubated with primary antibodies of CD29 (1 : 100), CD34 (1 : 100), CD44 (1 : 100), CD45 (1 : 100), and ALB (1 : 200) for 1 h at 4°C. Subsequently, the cells were washed with PBS for 3 times and incubated with FITC-conjugated secondary sheep antibody (1 : 200) for 40 min at 4°C.
Cytotoxicity of zebularine was evaluated by trypan blue exclusion test prior to the experiment. Six concentrations (0, 10, 20, 50, 100, and 200
HGF (60
Cells in each group were collected on days 7, 14, and 21. The total RNA was harvested using Trizol according to instructions of the manufacturer (Invitrogen, Carlsbad, CA). After RNA gel electrophoresis, the qualified RNA was subjected to reverse transcription using the PrimeScript RT reagent kit (Takara, Shiga, Japan). Polymerase chain reaction conditions were initial denaturation at 95°C for 10 min, followed by 40 cycles at 95°C for 5 s and 60°C for 30 s. Amplification of the housekeeping gene
Cell proteins were extracted using lysis buffer (20 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) supplemented with phosphatase inhibitors (1 mM sodium vanadate, 50 mM NaF) and protease inhibitors (0.1% phenylmethylsulfonyl fluoride; Complete protease inhibitor, Roche). After being boiled in Laemmli sample buffer, 10 mg of protein extracts was subjected to SDS-PAGE gel. Next, proteins were transferred to a polyvinylidene difluoride membrane by a Bio-Rad gel-blotting apparatus (Bio-Rad, Hercules, CA, USA). The blots were blocked with 5% nonfat dry milk at room temperature for 1 h and then incubated overnight with the desired primary antibodies at 4°C. After being washed thrice for 10 min each with TBST, peroxidase was detected by chemiluminescence and visualized by exposure to X-ray films (Kodak, USA). Quantification of Western blot products was performed by a laser densitometry (Bio-Rad, USA) and presented as a ratio between optical density of target protein band and
After being rinsed in PBS, cells cultured on coverslips in each group were fixed in 40 g/l paraformaldehyde, permeabilized by triton X-100, and blocked in phosphate-buffered saline buffer supplemented with 5% bovine serum albumin. Cells were labeled with primary sheep anti-rabbit antibodies against ALB (1 : 100) for 1 h at 4°C. After washing, cells were incubated with FITC-conjugated secondary antibodies for ALB. DAPI was used as a nuclear counterstain. Cells were visualized using a fluorescence microscope (Axio Vert 200, Zeiss, Germany).
ICG was dissolved in 0.01 M PBS to produce a 5 mg/ml stock solution and was diluted in optimal medium to a final concentration of 1 mg/ml. The seeded scaffolds were immersed in the ICG solution and incubated for 30 min at 37°C. After being washed using PBS, the cells were finally examined and imaged microscopically.
The differentiated BMMSCs were incubated with 1 ml medium containing 5 mM NH4Cl (Sigma) for 24 h at 37°C on days 0, 7, 14, and 21. After incubation, the supernatants were collected and the urea concentrations were analyzed using a colorimetric assay kit (GBD Corporation, San Diego, CA). The content of urea was presented in terms of pg protein/cell/day.
All data are presented as means ± SD. Statistical analyses were performed with SPSS 19.0 (Chicago, IL, USA). Comparisons between results from multiple treatment groups were performed using one-way analysis of variance (ANOVA), and LSD test was performed as a post hoc test to show individual differences. Statistical significance was defined as
The BMMSCs are characterized by expression of CD29 but negative for CD45. To address the cell phenotyping, flow cytometry analysis of CD29 and CD45 was performed. The cells showed high expression of CD29 (93.99% ± 3.21%) and CD44 (90.57% ± 3.03%) while there was no obvious expression of CD34 (3.08% ± 1.66%) and CD45 (3.81% ± 1.95%, Figure
Positive expression rates of BMMSC surface marker antigens CD29, CD44, CD45, and CD34 and toxicity of zebularine on BMMSCs. Asterisk indicates
Cell viability in 200
AFP, a characteristic marker of hepatocyte during embryonic development and fetal stages, was recruited when hepatic transdifferentiation was studied. Noninduced BMMSCs expressed little AFP mRNA. Treatment with HGF in the absence of zebularine (0
Zebularine promoted the mRNA expression of AFP and ALB. Noninduced BMMSCs and primary rabbit hepatocytes (PRHs) were used as negative or positive controls. (a) On day 7, noninduced BMMSCs expressed little AFP; increased AFP mRNA expression was observed in 0 and 10
Differentiation of BMMSCs was also traced up by determining expression for ALB, a typical marker for mature hepatocytes. The mRNA expression of ALB increased on day 14 and reached a peak on day 21 in the 0
To further quantify the effects of zebularine on ALB expression, Western blot assays were performed. Noninduced BMMSCs expressed little ALB. The protein expression of ALB increased on day 14 and reached a peak on day 21 in the 0
Zebularine promoted protein expression of ALB in BMMSCs and attenuated activation of the p38 signal pathway induced by HGF. (a) The protein expression of ALB increased on day 14 and reached a peak on day 21 in the 0
To address the involvement of p38 in hepatic differentiation, phospho-specific antibody for the activated form of p38 was used in Western blot analyses of cell lysates. BMMSCs were stimulated with zebularine and/or HGF; the p-p38 level was measured at various time points from 0 to 60 min. HGF increased the expression of phosphorylated p38 in cultured BMMSCs. However, addition of zebularine significantly suppressed p38 pathway activation in the cells stimulated with HGF (
Cells were treated with zebularine, SB203580, and zebularine + SB203580 in the presence of HGF. Expression level of DNMT1 protein was also measured via Western blot (Figure
Zebularine and SB203580 inhibited expression of DNMT1 and upregulated expression of ALB in BMMSCs. Noninduced BMMSCs and primary rabbit hepatocytes (PRH) were used as negative or positive controls. HGF increased the expression of DNMT1. HGF-induced expression of DNMT1 was inhibited by zebularine and SB203580. ALB protein expression was regulated by SB203580 and zebularine. The peak of expression of ALB in the induced groups was lower than that in primary rabbit hepatocytes. Asterisk indicates
We also found that ALB protein expression was upregulated by SB203580 (
Recruitment of ALB in the cytoplasm of BMMSCs on the 21st day. (a) Noninduced BMMSCs, (b) HGF, (c) HGF + SB203580, (d) HGF + zebularine, (e) HGF + zebularine + SB203580, and (f) primary rabbit hepatocytes. Cells analyzed by immunofluorescence with ALB (green) as a hepatocyte marker and DAPI to stain nuclear (blue). There were (18.67 ± 6.19)%, (26.71 ± 7.31)%, (70.04 ± 6.09)%, (71.82 ± 5.67)%, and (89.36 ± 5.38)% positive cells in HGF, HGF + SB203580, HGF + zebularine, HGF + zebularine + SB203580, and primary rabbit hepatocyte groups, respectively.
Cellular uptake and releasing of ICG, which determines the liver-specific metabolic function, were used to identify differentiated hepatocytes in vitro. Induced cells were positive for ICG after incubation in ICG solution for 30 min (Figure
ICG metabolism and urea production. BMMSC-derived cells were positive for ICG after incubation in ICG solution for 30 min (a). ICG in the differentiated cells was released after 6 h (b). Zebularine treatment induced urea production in the cells (c). Asterisk indicates
Though hepatocyte transplantation is considered to be an alternative to orthotopic liver transplantation, the shortage of donor cells for hepatocyte transplantation has not been resolved. Therefore, the derivation of hepatocytes from stem cells is of value in the creation of an unlimited source of donor cells for hepatocyte transplantation therapy. Nowadays, autologous BMMSCs have been widely applied in the liver repair, but its efficacy is often limited by the low transdifferentiation rate [
By activation of methylated genes in stem cells, DNMTi serve as prime candidates for cellular differentiation agents. The effect of zebularine on inhibiting DNA methylation was demonstrated in mammalian stem cell lines [
Previous studies have demonstrated that treatment of BMMSCs by zebularine resulted in the expression of cardiac-specific proteins in vitro [
The balance between proliferation and differentiation as well as between apoptosis and cell survival is required for DNMTi to promote liver regeneration. Indeed, inhibited HGF signaling, disrupted hepatocyte proliferation, and hepatocyte apoptosis increased the susceptibility of the liver to failure [
p38 is also involved in regulating of DNMT expression, though its role is often controversial in different cell types and in different laboratories. For example, reports revealed that phosphorylated p38 induces toxic injuries through mediating DNMT1, DNMT3a, and DNMT3b expressions [
Graphical abstract. The increase in the level of DNMT1 and phosphorylated p38 in BMMSCs was restrained by zebularine. Zebularine leads to remarkable increment in hepatic differentiation of BMMSCs, partly through regulation of HGF-induced p38 activation.
There are several questions that remain unanswered. For instance, the maturation degree and substitution function of hepatocyte-like cells from BMMSCs still fell short of primary rabbit hepatocytes. The concentration and induction time needs further improvement in order to obtain better transdifferentiation rate. Besides, we used BMMSCs from rabbit in vitro to explore the potential of zebularine as the differentiation inducer; potential response between zebularine and cells in vivo has not been investigated. Our next research interests may involve the therapeutic potential of hepatocyte-like cells induced by zebularine in vivo in a rabbit model of liver injury, as well as induction of BMMSCs derived from human. Further studies are needed to investigate how zebularine restrains p38 and the detailed interactions between its downstream targets.
In conclusion, we demonstrated that zebularine significantly increases the expression of hepatic-associated marker proteins in BMMSCs and enhances the urea production and ICG metabolic function of these cells. Zebularine exerts its demethylating effect through inhibition of DNNT1, partially by interfering with p38 MAPK signaling. The results obtained from this study would serve as an attempt in the therapeutic march to enhance regeneration of injured liver.
The data used to support the findings of this study are available from the corresponding author upon request.
The authors declare that there is no conflict of interest regarding the publication of this paper.
This study was supported by the National Nature Science Foundation of China (nos. 81571784, 30870695), the Foundation of Hunan Province and Technology Department, China (no. 2015SF2020-4), and the Foundation of Hunan Provincial Development and Reform Commission, China (no. 201583).