Differentiation of brown adipose-derived stem cells into cardiomyocyte-like cells is regulated by a combination of low 5-azacytidine concentration and bone morphogenetic protein 4

Abstract

Adipose-derived stem cells (ADSCs) could be an ideal candidate for seed cells to regenerate damaged heart tissue. This study examined and compared the cardio-myogenic differentiation efficacy of neonatal rat brown ADSCs (rbADSCs) treated with either 5-azacytidine (5-AZA), bone morphogenetic protein 4 (BMP4), or lower doses of both molecules. Briefly, by investigating the protein expression of cardiac-specific markers (i.e., cardiac troponin-I, α-sarcomeric actinin, sarcoplasmic reticulum Ca²⁺-ATPase, and connexin 43), our data indicated that rbADSCs could be differentiated into cardiomyocyte-like cells by all three treatments. By quantitatively measuring the number of cells with positive staining for the above markers, we found that the low-dose combined treatment showed higher differentiation efficiency compared to standard dose 5-AZA and BMP4 treatment. Similarly, the expression levels of these proteins as determined by western blotting were higher in the low-dose combination group than in the standard dose 5-AZA and BMP4 groups. Also, the combined strategy maintained the decreased cell viability caused by cytotoxicity of 5-AZA, probably through reducing the ratio of apoptotic rbADSCs. Furthermore, the extracellular regulated protein kinase (ERK) signaling pathways participate in the differentiation process, but the observed effects between the BMP4 and 5-Aza treatments are quite different.

Introduction

Cardiovascular disease (CVD) is a leading cause of death worldwide [1]. The main functional components of the heart, cardiomyocytes (CMs), are terminally differentiated cells with rare proliferation capacity [2,3]. Once damaged, CMs will be replaced with fibroblasts, which in turn generate irreversible regression of cardiac function. Recently, stem cell therapy has been regarded as a potential candidate for curing CVD, through substituting functional healthy tissue for the lost cardiac muscle, thus improving cardiac function and survival rate [4].

Among these stem cells, mesenchymal stem cells (MSCs) have been widely applied for the superiorities of immunogenicity-free, self-renewal ability, and multi-differentiation potential [5]. Adipose-derived stem cells (ADSCs) are one type of MSCs, that resemble bone marrow stromal cells (BMSC) in phenotype and gene expression profiles [6]. ADSCs might be more suitable to regenerate heart since they can be attained from more sources and are much more abundant at the same weight compared to BMSCs [7]. However, the cardiomyogenic differentiation potentials of ADSCs from white and brown adipose tissue are distinct. Compared to white ADSCs, brown ADSCs could be differentiated into cardiomyocytes with higher efficiency, and the transplantation of brown ADSCs into infarct border zone of an acute myocardial infarction model in rat showed decreased infarction area and improved left ventricular function compared to that of white ADSCs [8]. These data indicate that brown ADSCs might be more helpful than the widely used white ADSCs, when used in the cardiac regenerative medicine. Nevertheless, the above results were based on cells from adult rat; the detailed information of brown ADSCs from neonatal rat has been seldom mentioned before.

Transplantation of defined cardiac lineage has shown to be more beneficial compared to uncommitted stem cells, since they led to better myocardiac regeneration, improved short-term and long term cardiac function [9,10], stimulated angiogenesis in peri-infarct areas [11], and ameliorated cardiac mechanical and electrophysiological functions associated with lower incidence of induced ventricular tachyarrhythmias (VT) and decreased conduction velocity [12]. Nevertheless, safe and effective strategies for cardiomyotic differentiation are still not well understood, and require further exploration.

Several methods have been used to transdifferentiate adipose-derived stem cells into cardiomyocytes, including co-culturing with neonatal rat cardiomyocytes [13], or treating cells with compounds such as 5-azacytidine(5-AZA), dimethylsulfoxide, and phorbolmyristate acetate (PMA) [9,14,15], or adding one or several growth factors into culture medium [16]. However, these methods have several drawbacks, i.e. cytotoxic properties and relatively low differentiation efficacy. Recent study has demonstrated that the combination of cytotoxic agents 5-AZA and growth factors TGFβ1 at low concentration could enhance the cardiomyogenic differentiation efficiency of BMSCs associated with reduced cell death [17]. However, the results varied for different MSC types, since our pre-experiments suggested the effect of TGFβ1 on ADSC was quite low and the combination of the two did not reach the above efficacy. Luckily enough, we found that BMP4 might be advantageous in inducing ADSCs to become CMs. BMP4 is another component in the transforming growth factors β superfamily, that promotes cardiac differentiation of BMSCs, cardiac progenitors, and iPS [18,19]. However, BMP4 is unable to induce structural and functional mature cardiomyocytes from ADSCs alone [20], but promotes cell growth at low doses [21]. Thus, low-dose combination of BMP4 and potent drug 5-AZA, that inhibit cell growth [22], should be more powerful in cardiomyogenic induction while maintaining cell growth.

There are several signaling mechanisms that mediate the differentiation process of BMP4, including SMAD-mediated signaling, activation of MAPK14 (p38 MAPK), PIK3 (PI3 kinase), RAS, and MAPK8 (JNK) [23]. It has been demonstrated that the Erk signaling pathway mediates the 5-AZA-induced differentiation process [24]; nevertheless, whether this signaling pathway is involved in BMP4-induced or 5-AZA+BMP4 -induced differentiation process remains unclear. In the present study, the effects of pERK signaling pathways in the 5-AZA- and/or BMP4-mediated differentiation process were investigated.

Materials and methods

All experimental procedures were performed under approval from the Animal Care and Experiment Committee of Shanghai Jiao Tong University School of Medicine.

Cell isolation and culture

rbADSCs were isolated from subscapular brown adipose tissue of Sprague-Dawley rats at the age of 1-2 days old as described previously [25]. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco), 100 IU/ml penicillin (Gibco) and 0.1 mg/ml streptomycin (Gibco) in a humidified atmosphere containing 5% CO₂/95% air at 37°C. The cells were subcultured when reaching 90% confluence and incubated till fourth passage before further testing.

Flow cytometric analysis

For surface immunophenotype assay, rbADSCs were washed with PBS, and were then incubated in 2% (v/v) FBS/PBS (FBS, Gibco; PBS, Hyclone) containing or FITC-conjugated or PE-conjugated antibody for 30 min at 4°C. The mouse IgG was used as reference.

The used antibodies included: CD31-PE, CD29-FITC (BD Pharmingen, San Jose, CA, USA). Corresponding isotype-matched controls were used in all flow cytometric staining procedures. Flow cytometric analysis was carried out on a FACS Calibur (BD, FACSCanto).

Differentiation of rbADSCs into osteoblasts, adipocytes and chondrocytes

Osteogenic potential

rbADSCs (20000/cm²) at third passage were grown in osteogenic differentiation medium [α-MEM medium supplemented with 10% FBS, 0.2 mM ascorbic acid (Sigma), 10-8M dexamethasone (Sigma), 10 mM β-glycerol phosphate (Sigma)] for 4 weeks. After the induction time, cells were washed with PBS for three times and stained with Alizarin Red-S (40 mM, pH=4.1, Sigma). Each sample was run in triplicate.

Adipogenic potential

rbADSCs (25000/cm²) at passage 4 were incubated in DMEM medium containing 10% FBS until reaching 80% confluence. Then rbADSCs were subjected to rat adipogenic differentiation medium (Cyagen Biosciences Inc, China) following the manufacturer’s instructions. After three weeks’ induction, cells were loaded with Oil Red-O-staining, and the lipid accumulation within the cells was analyzed (n=3).

Chondrogenic potential

rbADSCs (2.5×10⁵) per well were centrifuged at 450g for 10 min. The cell pellets were carefully resuspended in chondrogenic medium (Cyagen Biosciences Inc, China) using a 15 ml centrifuge tube. The medium was refreshed every 3 days. The induction process lasted for 21 days. The resultant micromasses were collected and proceeded for paraffin sectioning, H&E and Alcian Blue staining. Each sample was run in triplicate.

Experimental design of cardiomyogenic differentiation

According to the previous researches, rbADSCs were respectively given standard dose 5-AZA (10 μmol/L) [13], standard BMP4 (10 ng/ml) [20], low dose combination of the two (5 μmol/L 5-AZA + 5 ng/ml BMP4), and neither of the two drugs group. The protein expression of cardiomyocyte-specific markers were tested to certify the existence of cardiomyocyte-like cells. As 5-AZA could inhibit ADSCs growth [22], while BMP4 promoted cell growth at low dose [21], the cell viability was examined through a cell proliferation assay. Furthermore, the underlying mechanisms for distinct cell viability among different groups were tested through cell apoptosis analysis.

Immunofluorescence staining

Immunofluorescence staining was performed to identify cardiomyocyte-like cells through cardiac-specific protein expression. Briefly, following 3 weeks induction, rbADSCs of the four different groups were fixed in 4% paraformaldehyde (Sigma) for 20 min at room temperature. After washing with PBS for three times, the cells were permeabilized with 0.2% TritonX-100 for 15 min and then blocked with goat serum for 70 min at room temperature. Subsequently, rbADSCs in different treated groups were incubated with α-sarcomeric actinin (dilution 1:400, sigma), cTnI (dilution 1:400, Abcam, Britain), SERCA2α (dilution 1:200, Santa Cruz Biotechnology, USA), and Cx43 (dilution 1:300, Sigma) at 4°C for 24 h. After three washes with PBS, the cells were stained with Alexa Fluor 488 or Alexa fluor-594 conjugated secondary antibodies at room temperature in the dark. Finally, the nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) for 15 minutes at room temperature in the dark. The coverslips were mounted with Mounting Medium (Beyotime) to glass slides. The images were captured using a Leica confocal laser scanning microscope (Leica Microsystems) and were analyzed with Adobe Photoshop (Adobe Systems).

Western blot analysis

To verify the existence of cardiomyogenic differentiation, western blot analysis was performed to analyze the protein expression of cardiac-specific markers in the four different treatment groups. Briefly, cells were washed and lysed with SDS sample buffer and scraped from a 6-well plate. Protein samples of similar amount were separated on 10% SDS polyacrylamide gels and transferred to membranes. For protein detection, membranes were incubated with primary antibodies including troponin I (dilution 1:1000, Abcam, Cambridge, UK), α-sarcomeric actinin (dilution 1:1000, Sigma, USA), SERCA2α (dilution 1:800, Santa Cruz Biotechnology, USA), Connexin 43 (Cx43) (dilution 1:1000, Sigma, USA), pERK (dilution 1:2000, CST, USA), total ERK (1:2000, CST, USA), GAPDH (dilution 1:10,000, Santa Cruz Biotech, USA), and β-actin (1:10,000, Santa Cruz Biotechnology, USA). Protein expression was analyzed using the Odyssey system (LI-COR Biosciences, USA).

Cell proliferation assay

Cell proliferation was measured using the Alamarblue assay (Gibco, USA) according to the manufacturer’s instructions. Briefly, cells were seeded in a 96-well plate (5000 cells per well). Cell proliferation was measured on 1, 3, 5 and 7 days post-treatment. The medium containing 5-AZA was disposed of after 24 hours incubation, and then changed into basic DMEM medium or supplemented with BMP4 in the medium. 100 μl basic medium containing 10% Alamarblue staining solution was added to each well, and incubated for 4 hours at 37°C. Fluorescence was detected at 570 nm and 600 nm using a microplate reader (Biotek, USA). The experiment was run in triplicate.

Flow cytometric analysis of cell apoptosis

Cell apoptosis was determined through flow cytometric analysis. After 5 days incubation, rADSCs were digested with trypsin-EDTA and washed with PBS. 5×10⁵ cells, and were then resuspended in 100 μL Annexin V binding buffer, stained with 5 μL Annexin V APC and incubated for 15 min in dark. After disposing of unbound stain, cells were resuspended in 200 μL binding buffer, and subsequently stained with 5 μL Propidium Iodide (PI) for 5 min at room temperature in the dark. Finally, all samples were then loaded on the flow cytometry (BD, FACSCanto). All experiments were run in triplicate, and results were calculated as the mean ± s.d.

Statistical analysis

All data are displayed as mean ± SEM. Multiple group comparisons were performed using one-way ANOVA and Bonferroni post-hoc test provided by GraphPad Prism 7 software. All p-values were two-sided and P < 0.05 was considered significantly different.

Results

The morphology and phenotypes of rat brown adipose-derived stem cells

The freshly isolated adipose tissue cells displayed ununiform morphology, and with extended culture time, cells began to adopt a more uniform fibroblast-like character, like BMSCs (Figure 1A). Moreover, after three weeks culture, CD29 expression was found in the most of the rbADSCs (95.2% ± 0.8), while CD31 was rarely observed (8.92% ± 0.56) (Figure 1C).

Figure 1.

Phenotype of rat brown adipose-derived stem cells (rbADSC). A. Morphology of rbADSCs at different timepoints: rbADSCs gradually appeared with fibroblast-like shape on the 3rd day post-plating. B. Multi-lineage differentiation potency of rbADSC: differentiate to osteocytes, adipocytes and chondrocytes. C. Immunophenotype markers (CD29, CD31) expression in rbADSCs after three weeks of culturing, measured using flow cytometry.

When dealing with specific compounds, rbADSCs at passage 4 or 5 underwent osteogenic, adipogenic, and chondrogenic differentiation, as confirmed by specific staining assays. The rbADSCs’ multi-potent differentiation capabilities were proven in at least three independent samples (Figure 1B). In sum, the rbADSCS used in the present study were a kind of mesenchymal stem cell, with multipotent differentiation ability, which in turn could be differentiated into desired cell types by other agents.

The differentiation efficiency of combination 5-AZA and BMP4 treatments

As shown in Figure 2A, the fluorescence intensities of the four stained cardiac-specific markers were low without BMP4 or 5-AZA induction. The staining increased with the addition of 10 ng/mL BMP4 or 10 μM 5-AZA, and was further enhanced by treatment with 5 μM 5-AZA and 5 ng/mL BMP4.

Figure 2.

The protein expression of cardiac-specific markers analyzed by immunofluorescence staining assays. A. Immunofluorescence staining of cardiac-specific marker proteins cardiac troponin-I (cTnl), α-sarcomeric-actinin (α-SA), connexin43 (Cx43), and endoplasmic reticulum calcium ATPase2α (Serca2α) proteins in cells treated with 10 µM 5-azacytidine (10AZA), 10 ng/mL bone morphogenetic protein 4 (10BMP4), or 5 µM 5-AZA + 5 ng/mL BMP4 (5A + 5B), or no treatment (control). Scale Bar=75 µm. B. Quantitative analyses of cardiac-specific marker-positive cells in the four experimental groups after 21 days incubation. *P < 0.05 versus other groups.

Quantitative analyses showed that the number of cells positive for cardiac troponin 1 (cTnI), sarcoplasmic or endoplasmic reticulum calcium ATPase2α (Serca2α), connexin 43 (Cx43), and α-sarcomeric-actinin (α-SA) were noticeably higher in the combined treatment group compared to the 5-AZA, BMP4, and control groups (all P < 0.05). Furthermore, the proportions of α-SA- and Cx43-positive cells in the 5-AZA group were significantly higher than those of the BMP4 group (Figure 2B) (P < 0.05).

Protein expression levels of cTn1, α-SA, Cx43, and SERCA2α as determined by western blotting

To further verify the differentiation of rbADSCs into cardiomyocyte-like cells, we evaluated the expression levels of cTnI, α-SA, Cx43, and Serca2α proteins using western blot analyses after 3 weeks incubation. The cTnI, α-SA, Cx43, and Serca2α protein expression levels in the three experimental groups were significantly higher when compared to the control group (P < 0.05). Moreover, the strongest expression of the four proteins was observed in the low-dose combination group (P < 0.05). Furthermore, cTnI and Cx43 levels in the 5-AZA group were higher compared to the BMP4 group (P < 0.01), whereas the levels of the other two proteins were similar (Figure 3A, 3B).

Figure 3.

The protein levels of cardiac-specific markers analyzed using western blots. A. The protein levels of cardiac-specific markers troponin-I (cTn1), α-sarcomeric actinin (α-SA), connexin 43 (Cx43), and Serca2α after 3 week incubation in the control cells or those treated with 10 ng/mL bone morphogenetic protein 4 (10BMP4), 10 μM 5-azacytidine (10AZA), or 5 μM 5-AZA + 5 ng/mL BMP4 (5A + 5B). β-actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as loading controls. B. Semi-quantitative analyses of cTnI, α-SA, Cx43, and Serca2α levels in the different cell treatment groups. *P < 0.05 versus other groups.

Cell proliferation and apoptosis of the four treatment groups

The results of cell proliferation assays in the treated rbADSCs at different time points (1, 3, 5, and 7 days of incubation) are shown in Figure 4A. Stable proliferation was observed in all groups with relatively slow increase in cell viability observed at Day 5. Throughout the study, cell proliferation in the BMP4 and low-dose combination groups was insignificantly lower than control group (P > 0.05), while the values in the 5-AZA group were significantly lower compared to control and BMP4 group (all P < 0.05). Notably, cells treated with the combination of 5-AZA and BMP4 at low doses maintained better cell viability over time compared to those treated with 5-AZA alone (all P < 0.05).

Figure 4.

Cell proliferation and apoptosis of the four treatment groups. A. Analyses of the proliferation of rat brown adipose-derived stem cells (rbADSCs) treated with 10 µM 5-azacytidine (10AZA), 10 ng/mL bone morphogenetic protein 4 (10BMP4), or 5 µM 5-AZA and 5 ng/mL BMP4 (5A + 5B) after 1, 3, 5, and 7 days incubation. Results are the mean of four independent samples. *Denotes significant differences from the control group. #denotes significant differences from the 5A + 5B group. B. rbADSC apoptosis was determined by flow cytometry in the different groups after 5 days incubation. Cell apoptosis was assessed by annexin V and propidium iodide staining. Results are representative of three independent experiments. C. The percentage of early apoptotic and late apoptotic/necrotic cells (Q2 + Q3) in the different groups. *Denotes significant differences between groups.

To explore the mechanism of the distinct cell proliferation among different groups, we further examined cell apoptosis on day 5 using flow cytometry. Briefly, the percentage of early apoptotic and late apoptotic/necrotic cells in the 5-AZA group (24.03 ± 0.88%) was significantly higher compared to the control (8.82 ± 0.31%), BMP4 (11.01 ± 0.32%), and combination (15.94 ± 0.58%) groups (all P < 0.05) (Figure 4B and 4C). In addition, the apoptotic percentage of the combination group was higher than that of the control and BMP4 groups, while it was significantly lower compared to the 5-AZA group. Moreover, there was no significant difference in apoptosis percentages between the control and BMP4 groups.

The mechanism of 5-AZA- and BMP4-induced cardiomyocyte differentiation

To investigate the pathways involved in the differentiation process, the activation of the MAPK signaling pathway was assessed by measuring the phosphorylation levels of a MAPK pathway member, extracellular regulated protein kinase (ERK) 1/2. The ratio of phospho-ERK1/2 (p-Erk1/2) to total-ERK1/2 (t-Erk1/2) protein levels in the three experimental groups were higher than those of the control group (P < 0.05) (Figure 5A, 5B), and the ratio was highest in the 5-AZA group, indicating that the ERK pathway was involved in differentiation.

Figure 5.

The ERK pathway in cardiomyocyte-like cell differentiation. A. The protein expression of Serca2α, t-ERK, and p-ERK in the control, 10 μM 5-azacytidine (10AZA), 10 ng/mL bone morphogenetic protein 4 (10BMP4), and 5 μM L5-AZA plus 5 ng/mL BMP4 (5A + 5B) groups, with or without 10 μM U0126 treatment. Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) was used as a loading control. B. Semi-quantitative analysis of p-Erk/t-Erk levels in the above four treatment groups without U0126. C. Semi-quantitative analysis of p-Erk/t-Erk levels in the four treatment groups with and without U0126. D. Semi-quantitative analysis of Serca2α levels in the different treatment groups.

We examined the effect of a specific inhibitor of the ERK pathway, U0126, in each treatment group. Upon adding U0126, the ratios of p-Erk1/2 to t-Erk1/2 protein levels in the 5-AZA and low-dose combination groups were significantly decreased. In contrast, the ratios of p-Erk1/2 to t-Erk1/2 protein levels in the BMP4 group was moderately increased after addition of U0126 (Figure 5A, 5C).

Serca2α was taken as a representative cardiomyocyte-marker that was used to survey the relation of cardiomyogenic differentiation and the activation of ERK pathway. As shown in Figure 5A and 5D, Serca2α levels were significantly reduced in the 5-AZA and low-dose combination groups after adding U0126. On the contrary, adding U0126 to the BMP4 group increased Serca2α expression. These results suggested that the ERK pathway was involved in the differentiation process in all cases, but played different roles in the BMP4 and 5-AZA treatment groups; other pathways might therefore be involved in the differentiation process.

Discussion

There are two types of adipose tissues present in humans and mammalian species: white adipose tissues that function in storage of metabolic energy, and brown adipose tissues (BAT) that dissipate energy for thermogenesis [26]. BAT in infants is abundant but gradually disappears with increasing age [27]. The main elements in adipose tissues are adipocytes and stromal vascular fractions, which contain heterogeneous cells including preadipocytes, fibroblasts, ADSCs, macrophages, endothelial cells, and lymphocytes [7]. Our data, which were based on a non-uniform cell morphology, confirmed that freshly isolated cells were a mixture of other cells, which displayed uniform fibroblast-like characteristics following the extended culturing times.

The characterization of MSCs is based on cell morphology, immune phenotyping, and proteomic assays. A widely accepted MSC immune phenotyping method, proposed by the International Society for Cell Therapy, uses an examination of positive expression of CD29, CD105, CD73, and CD90 [28], combined with negative expressions of hematopoietic lineage markers CD34, CD45, CD19, CD14 (CD11b), and HLA-DR [28,29]. So the CD29-positive and CD31-negative cells used in our study could be identified as one of the MSC, consistent with previous research [11,30]. Furthermore, the identification of rbADSC differentiation through the adipogenic, chondrogenic, and osteogenic lineage pathways satisfied another essential criterion to be classified as a multi-potent differentiated cell type [1]. Thus, cells derived from the BAT, that abundantly distributed in the subscapular position of neonatal rat, proved to be one type of MSCs that could be induced into specific CMs.

In our preliminary research, cells were divided into groups as control, normal dose 5-AZA, normal dose BMP4, half dose 5-AZA, half dose BMP4. The efficiencies of cardiomyogenic differentiation were as following: normal dose 5-AZA > half dose 5-AZA > normal dose BMP4 > half dose BMP4 > control. The result was based on the analysis of gene expression of cardiac-specific markers such as cTnI, α-SA through reverse transcription PCR. Moreover, on the final timepoint of induction, cell numbers in low dose 5-AZA groups were significantly lower than other groups, which was further decreased in the normal dose 5-AZA group. In consideration of efficiency and cell growth, combination of 5-AZA and BMP4 at low dose was assumed to be more potent and safe for further experiments. To demonstrate this, cardiomyogenic differentiation efficiency and cell proliferation were analysed and compared with control, normal BMP4, and normal 5-AZA groups.

Several cardiac-specific markers, including cTnI, α-SA, Serca2α, and Cx-43 were used in this study. By combining these marker proteins [17], we achieved appropriate representation of cardiomyogenic properties. As expected, a combination of BMP4 and 5-AZA at low doses stimulated higher numbers of marker-positive cells compared to the normal doses of 5-AZA or BMP4 alone. Similarly, the levels of the four proteins assessed through western blotting were higher in the low-dose combination group than in the 5-AZA and BMP4 groups. In addition, the 5-AZA group showed higher differentiation efficiency than the BMP4 group. In view of the observed differences, the low-dose combination treatment achieved higher differentiation efficacy than 5-AZA alone, suggesting that 5-AZA and BMP4 synergistically interacted with each other.

5-AZA could efficiently induce MSCs or ADSCs or other stem cells into CMs [22,17,24], consistent with our research, but it had the adverse effect of inhibiting cell growth [22]. However, the effect of BMP4 on human MSC viability was dose-dependent: high doses (100 ng/mL) reduced cell proliferation and increased apoptosis levels, whereas low doses (0.01-10 ng/mL) decreased apoptosis levels and increased cycling cells [21]. Consistent with the previously published research, we found that 10 ng/mL BMP4 maintained stable increase in cell proliferation and had no obvious negative effect on cell apoptosis; while 10 µM 5-AZA downregulated cell proliferation and increased the apoptotic cell ratio. However, when combining the two drugs at low dose the effects turned out to be moderate, with improved cell proliferation and less apoptosis compared to the normal 5-AZA group, and were similar to those of the BMP4 and control groups, suggesting that the combination strategy might counteract the cytotoxic effects of 5-AZA. At the same time, based on these data, we concluded that 10 µM 5-AZA significantly increased cell apoptosis and decreased the proportion of cycling cells, which in turn, caused a significant decrease in cell numbers.

The mechanism underlying BMP4- and 5-AZA-induced cardiomyocyte differentiation is not well understood. The roles of several signaling pathways have been proposed, including the MAP3K7/MAP3K7IP1 (Tak1/Tab1), PIK3 (PI3 kinase), MAPK1 (ERK), RAS, and MAPK8 pathways [23]. Our results suggested that the ERK pathway components were activated during 5-AZA, BMP4, and combination treatment-induced differentiation. Upon inhibition by U0126, p-Erk/t-Erk ratio and Serca2α expression in the 5-AZA group decreased to levels that were similar to those observed in the control group. The expression in the low-dose combination group decreased, but was higher than that of the control group; however, they were elevated in the BMP4 group. The results indicated that the ERK pathway played a significant role in 5-AZA- and 5-AZA combined with BMP4-induced differentiation processes, but was not as important in the BMP4-induced differentiation process. Furthermore, other pathways might be involved in the combination and BMP4 treatment groups, which should be clarified in further studies.

Conclusions

The results of the present study demonstrated that treatment with a combination of 5-AZA and BMP4 at low concentrations restored rADSC viability and maintained higher differentiation capacity when compared to the 5-AZA alone treatment. Moreover, the underlying mechanism of BMP4 and 5-AZA induction involved the ERK pathway. Therefore, the combination of 5-AZA and BMP4 can be a safe and effective approach to differentiate rADSCs into cardiomyocyte-like cells. Additional studies should be conducted to further investigate the specific mechanism underlying the combination of low-dose 5-AZA and BMP4 treatment, and the clinical applications of these cardiomyocyte-like cells in vivo.

Disclosure of conflict of interest

None.

Abbreviations

ADSCs

adipose-derived stem cells

BMP4

bone morphogenetic protein 4

CM

cardiomyocyte

cTnI

cardiac troponin I

Cx43

connexin 43

α-SA

α-sarcomeric-actinin

Serca2α

sarcoplasmic reticulum Ca2⁺-ATPase

5-Aza

5-azacytidine