Direct-current electric field stimulation promotes proliferation and maintains stemness of mesenchymal stem cells

Mesenchymal stem cells (MSCs) have gained more attention in regenerative medicine than embryonic stem cells and induced pluripotent stem cells, which are constrained by ethical concerns and potential tumorigenicity [1]. MSCs provide a plentiful source of adult stem cells with the capacity for self-renewal and multidirectional differentiation [2]. Because of their abundance [3], simplicity of separation [4,5], pluripotency [6], immunosuppressive effects [7], antifibrotic properties [8] and angiogenic potential [9,10], MSCs are the perfect cell type for regenerative medicine. The isolation of adipose-derived MSCs (Ad-MSCs) can be achieved by the enzymatic digestion of adipose tissue that has been removed by liposuction or lipectomy [4]. Harvesting MSCs from adipose tissue can provide a larger proportion of MSCs compared with other stem cell sources [5] and donor site problems are minimal. Furthermore, Ad-MSCs can be safely transplanted into autologous or allogeneic bodies, with few graft allografts and foreign body reactions [11,12]. Also, there are no ethical concerns because they can be derived from autologous fat. Based on these benefits, Ad-MSCs provide a more effective and practical treatment option for tissue and organ transplantation.

Although current approaches to obtaining Ad-MSCs are reasonably effective, their therapeutic use is limited because of their limited available quantity [13] and the fact that cell therapy regimens normally require hundreds of millions of MSCs for each treatment. It requires approximately ten weeks to obtain enough cells by in vitro expansion before implantation. In addition, studies have shown that the ideal culture conditions to produce MSCs at a clinical scale vary with the age and characteristics of the patient and cellular senescence of MSCs in vitro, potentially leading to aging and age-related diseases [14,15]. Prolonged culture expansion of human MSCs (hMSCs) in vitro leads to a loss of differentiation potential [16]. In addition, the clinical outcomes of hMSCs transplantation remain variable [15], and the short survival rate of transplanted hMSCs and the regenerative potential of Ad-MSCs following transplantation are reduced [17,18]. The therapeutic efficacy of individual-derived MSCs must be enhanced due to their long expansion period, individual differences, low survival rate and limited regenerative capacity following transplantation, which is a challenge to their large-scale clinical use.

Electric fields (EFs) are required for cells to maintain dynamic homeostasis and to engage in a variety of biological phenomena ranging from embryonic development to tissue regeneration [19]. Biological processes, such as long-distance protein interactions, electron transfers during chemical reactions [20], embryogenesis [21], cell differentiation [22], growth [23], wound healing, tissue repair and remodeling [24,25], are influenced by endogenous EF, which exist in the cytoplasm and extracellular space [26,27]. The duration, intensity and orientation of these fields can also affect cell proliferation. Khaw et al. [28] devised a capacitive electrical stimulation device to apply 100 and 200 mV/mm uniform EFs to MSCs daily for 14 days, which increased the cell proliferation rate by fivefold. Martin et al. [29] found that osteoprogenitor cells stimulated with 125–500 mV/mm direct current (DC) for 3 hours every day for 3 days exhibited a higher propensity to proliferate and differentiate. Panda et al. [30] stimulated hMSCs with 100 mv/mm DC for 10 min each day for 21 days and discovered that their proliferation rate decreased, but osteogenic differentiation capacity was enhanced. Notably, EF can have either a stimulatory or an inhibitory effect, depending on the exposure conditions and cell type [31,32]. Nonetheless, there is evidence that EF signals play an important role during the cellular regeneration process [23]. Clinically, EF has been used to treat several diseases, including osteoporosis, chronic skin ulcers, cardiac arrhythmias and traumatic injury [33–36].

Based on these findings, EF stimulation can be used to stimulate cell regeneration. Because it does not require the addition of exogenous immunogenic biological substances or complex equipment, EF signaling stimulation offers many advantages [37,38]. Currently, in vitro electrical stimulation approaches, such as DCEF, are being used [39]. DCEF not only affects cell survival and proliferation, but also influences cell migration and morphology [40,41].

Materials & methods

Cell isolation, culture & passaging

Ad-MSCs were harvested from adipose tissue following liposuction surgery. The principles outlined in the Declaration of Helsinki were followed. Human adipose tissues obtained from liposuction were soaked in ice-cold phosphate-buffered saline (PBS; Biological Industries, CT, USA), washed three times with PBS, shredded into a crumbly form and digested with 10 mg collagenase I (Gibco™, NY, USA) in DMEM/F12 (Gibco, NY, USA) (1:1) medium containing 2% double antibiotic (Gibco, NY, USA) and filter sterilized. Digestion was performed while shaking manually at 37°C for 1 hour. Tissue clumps were removed by filtration through a 70-μm filter membrane. The membrane was washed with PBS and the single nucleated cells were collected by centrifugation at 500 g for 5 min to yield Ad-MSCs. The cells (2000 cells/cm²) were seeded in DMEM (Gibco, NY, USA) in culture flasks containing 10% fetal bovine serum (FBS; Gibco, NY, USA). Cells were then suspended in 10 ml of growth medium (DMEM/F12, 10% CS, 2% double antibodies) and incubated in 10-cm dishes. After 12–24 hours, the medium was removed and the cells were washed with PBS to remove unadhered cells, tissue clumps and debris. The medium was changed every 3 days until 90% confluence of the cells. The Ad-MSCs were incubated with 0.25% trypsin-EDTA (Biological Industries, CT, USA) then passaged or seeded into 6-well plates at a density of 1 × 10⁵/well and cultured with growth medium. Ad-MSCs from passages 3–5 were used for this research.

Authentication of Ad-MSCs

After DCEF stimulation, the MSCs phenotype was characterized by flow cytometry. Next, 1 × 10⁵ Ad-MSCs were incubated with monoclonal antibodies CD90, CD105, CD45, CD73, CD14, CD31 and CD34 (Becton Dickinson, NJ, USA) for 40 min at 4°C protected from light. Flow cytometry (LSRF Ortessa, BD Biosciences, USA) was used to record and analyze the data. At least 1 × 10⁴ cells per run were analyzed.

Differentiation of MSCs

For MSCs with or without EF treatment, adipogenic differentiation was induced in DMEM/F12 medium supplemented with 10% FBS, 1% penicillin-streptomycin, 500 μM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, 10 μM insulin and 200 μM indomethacin (Sigma, LA, USA). After 2 weeks, two groups of MSCs were fixed in 4% paraformaldehyde and stained with Oil Red O (Sigma, LA, USA) to observe lipid droplets. Cultured MSC cells in DMEM-HG supplemented with 10% FBS, 1% penicillin-streptomycin, 10 nM dexamethasone, 50 μM ascorbic acid 2-phosphate (Sigma, LA, USA) and 10 nM β-glycerophosphate (Sigma, LA, USA) were used to induce osteogenic differentiation. After 2 weeks, the MSCs were fixed in 4% paraformaldehyde and stained with Alizarin red (Sigma, LA, USA) to examine mineralized matrix apposition.

DC electrical stimulation chamber

The in vitro electrical stimulation method proposed by Mobini et al. [37,41] was enhanced and optimized by exposing Ad-MSCs to EF signals to induce cell proliferation. This in vitro electrical stimulation tool works with changes in the electrodes and the presence of an electric current generates electrothermal effects that evaporate the medium, which may have additional electrothermal effects on the cells. Therefore, the device was improved by using a DC and an electrode plate to generate a uniform EF to eliminate the current throughout the experiment. The device is suitable for various biological and biomedical studies requiring bioelectric field stimulation.

The device consisted of gold-plated electrodes that are attached to the top cover of the cell culture chamber and fit standard 6-well cell culture plates (128 mm × 85 mm × 22 mm; inner diameter: 33.78 mm; growth area: 8.96 cm²) for easy manipulation and sterilization and to reduce evaporation of the medium. Each electrode plate group consisted of 13 gold-plated electrodes of 1-mm diameter running in parallel. To prevent harmful material released during electrical stimulation from interfering with the experimental results, each set of electrode plates was sealed with polymethylmethacrylate. The electrode points protruding from the lid were soldered to parallel circuits of silver-plated copper wires and connected to a standard power supply with a banana connection. By adjusting the power supply, the amount of electricity and its application pattern could be modified. Before the experiment, the device was sterilized in 75% ethanol for 2 h in a calcium-free solution, washed in DPBS (Gibco, USA), then exposed to UV radiation overnight.

To demonstrate the effectiveness of the EF signal delivery device, the EF was applied to Ad-MSCs for 3 days and its effects on cell proliferation and stemness were assessed. All tests were performed in triplicate. Briefly, human Ad-MSCs were cultured at a density of 2 × 10⁴ cells/cm² to 80% confluence and then expanded for three generations. The fourth-generation cells were seeded into 6-well cell culture plates at a density of 5 × 10³ cells/cm² and incubated at 37°C in a 5% CO₂/3% oxygen atmosphere. After inoculation, the cells were cultured in growth medium. After 1 day, the culture plate cover was changed to the EF device cover (with electrodes). A 4.4 V DC voltage was applied to generate a uniform DCEF of 200 mV/mm in the cell culture medium of the stimulation group. Cells were exposed to the DCEF for 1 hour/day for 3 days and subsequently compared with the control group. After completing the experiment, the equipment was cleaned and sterilized in preparation for usage the next day. The cells were washed twice with PBS 24 hours before the end of the experiment. Prior to the next EF stimulation, the medium was changed and the cells were photographed under a microscope.

Protocol for electrical stimulation

The magnitude of the EF strength in the cell surrounding is dependent upon the amount of DC voltage. According to the uniform EF formula: U = V/d (U: EF magnitude; V: DC voltage; d: electrode plate distance), the current electrical stimulation device may be considered a uniform EF. The current device stimulation mode (200 mV/mm, 1 hour) was selected as an efficient strategy for cell proliferation.

Cell counting

Cell counting was performed using an automated cell counter (RWD, Shenzhen, China). MSCs were digested with 0.25% trypsin from each well every day. The cells were resuspended in DMEM and the cell number for each group was counted.

Cell cycle analysis

Cell cycle analysis was performed by cell cycle and apoptosis detection kits (Beyotime Institute of Biotechnology, Shanghai, China). MSCs were digested with 0.25% trypsin and washed twice with PBS after treatment. The cells were resuspended in 75% cold ethanol and fixed overnight at 4°C with 75% ethanol. The following day, the cells were rinsed and disseminated with cold PBS to avoid aggregation. Propidium iodide (PI) staining solution was added to the cell pellet (0.5 ml/tube). After mixing, the cells were incubated at 37°C for 30 min in the dark. Subsequently, flow cytometry (Agilent Technologies, CA, USA) was used to examine the cell cycle pattern.

Cell proliferation analysis

Cell proliferation was determined using a cell counting kit (CCK-8; Beyotime, Shanghai, China) and Taipan Blue staining. Following treatment with or without EF, the cells were collected, seeded at a density of 2000 cells/well in 96-well plates and incubated in 5% CO₂ at 37°C overnight. CCK-8 solution (10 μl/well) was added to each well and incubated in 5% CO₂ at 37°C for 3 h. Finally, the optical density was measured at 450 nm using a microplate reader (Victor 3; Perkin Elmer, Inc).

Quantitative real-time PCR

After EF stimulation, the cells were harvested by TRIzol (Invitrogen, NY, USA) and disrupted in QIAshredder columns (Invitrogen, NY, USA). RNA was extracted using the RNeasy kit (Invitrogen, NY, USA) following the manufacturer's instructions. To produce cDNA, a first-strand cDNA synthesis kit (Invitrogen, NY, USA) was utilized. To validate the PCR assay, real-time PCR was performed with cDNA and repeated three times for each gene on SYBR Green (Roche, Basler, Switzerland). Primers for Bcl-2, BAX, cyclin D1, C-myc and β-actin (housekeeping gene) were provided by QuantiTectPrimer Assay (Qiagen, Duesseldorf，GER). The Step-One system (Applied Biosystems, NY, USA) was applied for real-time PCR.

western blot analysis

Protein expression of the Sox-2, Oct-4 and Nanog transcription factors was analyzed after EF stimulation. The cells were resuspended in cell lysis buffer (Thermo Scientific, USA) and sonicated. A BCA protein quantification kit (Thermo Scientific, USA) quantified protein in the supernatants after centrifugation. Proteins (90 μg) from each group were mixed with Laemmli sample buffer and boiled for 10 min at 100°C. SDS-PAGE and polyvinylidene difluoride membrane blotting separated proteins; western blot was performed using anti-Oct-4 (Abcam, UK), anti-Sox-2 (Abcam, UK), anti-Nanog (Abcam, UK) and anti-β-actin (Abcam, UK) antibodies. Primary antibodies were incubated overnight at 4°C on the membranes. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h after vigorous washing. The blots were developed by an enhanced chemiluminescence detection system (Siemens, Germany).

Statistical analysis

All experiments were repeated three times. A one-way analysis of variance was performed followed by Tukey's post-hoc test using Stats Direct software; p < 0.05 and p < 0.01 were considered significant and highly significant, respectively.

Results & discussion

Ad-MSCs maintain normal morphology after stimulation with DCEF

The Ad-MSCs obtained from adipose tissue were adherent fibroblast-like cells that grew as elongated, adherent fibroblast-like cells even after three days of EF stimulation (Figure 1A). For cell surface antigen expression, the cells were assessed for MSC markers before and after EF treatment. Greater than 90% of the adipose MSCs were positive for CD73, CD90 and CD105 and negative for CD34, CD31 and CD45 in waveforms before EF stimulation and 3 days after treatment, which was similar to untreated control Ad-MSCs (Figure 1B).

Ad-MSCs maintain ability to differentiate after EF stimulation & exhibit higher expression of stemness markers

In vitro differentiation assays demonstrated the differentiation of Ad-MSCs into osteoblasts and adipocytes before and after electrical stimulation (Figure 2A). Western blot analysis revealed higher expression of Nanog, Oct-4 and Sox2 in Ad-MSCs with EF treatment compared with the untreated Ad-MSCs (Figure 2B).

Compared with unstimulated Ad-MSCs, proliferation of Ad-MSCs significantly increased following electrical stimulation

EF is expected to promote the proliferation of MSCs. Therefore, cell proliferation was measured on days 1, 2 and 3 after EF stimulation and the proliferation of Ad-MSCs after EF stimulation was significantly higher compared with that of unstimulated Ad-MSCs (p < 0.05; Figure 3A). On day 2, the proliferation of EF-stimulated Ad-MSCs was significantly higher compared with that of other EF-stimulated and unstimulated Ad-MSCs (p < 0.05).

Cell cycle analysis indicates more cells in S phase after EF compared with unstimulated cells

To determine the effect of EF on the cell cycle, a PI-based cell cycle analysis was performed before EF and on days 1 and 2 following EF (Figure 3B). After EF stimulation, the numbers of cells in the S and G2 phases, but not G1, were markedly higher in Ad-MSCs on days 1 and 2 compared with unstimulated Ad-MSCs (p < 0.05). The fraction of cells in the S phase, which represents the DNA synthesis phase, was increased. Compared with unstimulated Ad-MSCs, there were more cells in the S and G2 phases and significantly fewer in G1.

Quantitative PCR analysis reveals increased gene expression in EF-treated cells compared with unstimulated cells

Changes in the expression of genes associated with cell proliferation were measured in EF-treated Ad-MSCs. Ad-MSCs in the EF-stimulated and control groups were collected and real-time PCR was used to measure the expression of Bcl-2, Bax, Cyclin D1 and C-myc (Figure 3C). The results indicated that Bcl-2, cyclin D1 and c-myc were upregulated, whereas Bax was downregulated, in the EF-stimulated group compared with the control group (p < 0.05).

To our knowledge, this is the first study using a modified EF device to analyze the effect of a DCEF on Ad-MSCs in vitro. Ad-MSCs treated with a DCEF at an intensity of 200 mV/mm for 1 h/d for a total of 3 days had significantly higher proliferation rates compared with untreated cells; however, their morphology did not change considerably after EF stimulation (Figure 1A), whereas differentiation capacity remained intact. We used this device to expose Ad-MSCs to EF signals to induce cell proliferation. The in vitro electrical stimulation device was prepared without exterior changes near the electrode plate and without electrothermal effects that could evaporate the medium in the culture dish from the absence of current, thus preventing additional electrothermal effects that may affect the cells. A uniform EF was generated by means of a DC and electrode plates, thus eliminating the effect of the electric current on the whole experimental apparatus. This device is suitable for various biological and biomedical studies exploring bioelectric field stimulation effects and their applications.

Ad-MSCs were selected to evaluate the impact of DC fields as they are one of the primary cell types engaged in wound healing. We examined Ad-MSCs, which were positive for CD73, CD90 and CD105, but negative for CD34, CD31, CD14 and CD45 expression (Figure 1B). We also examined the differentiation ability of the cells before and after EF treatment and discovered that they all developed into a distinct MSC lineage (Figure 2A). Pluripotency markers, such as Oct-4, Sox-2 and Nanog, are crucial for renewal and differentiation capabilities. western blot analysis revealed higher expression of Nanog, Oct-4 and Sox2 in EF-treated Ad-MSCs compared with the untreated cells (Figure 2B). In addition, the proliferation of Ad-MSCs after EF stimulation was significantly higher compared with that of unstimulated Ad-MSCs (p < 0.05; Figure 3A). After 3 days of EF treatment, more cells entered the G2 and S phases of the cell cycle, whereas fewer cells entered the G0/G1 phase. EF-stimulated cells exhibited a higher proliferation rate than unstimulated Ad-MSCs (Figure 3B) Finally, quantitative PCR revealed that Bcl-2, cyclin D1 and C-myc were upregulated, whereas Bax was downregulated, in the EF-stimulated group compared with the control group (p < 0.05; Figure 3C).

Several EF stimulation treatments have been clinically utilized to treat skin wounds [42–44]. Many studies have claimed effectiveness and the results of these diverse experiments have been thoroughly reviewed elsewhere. However, many studies [45] in this area are replete with poor and uncontrolled experiments where different types of metal electrodes were directly inserted into the wound bed or cell culture medium. Secondary electrochemical effects of O₂ or H+ released by metal ions or electrodes can affect the tissue pH within the wound, which are not separate from the primary effects of EF [46]. Unfortunately, our understanding of the cell biology of EF-induced wound healing remains limited. Thus, a more complete understanding of the effects of EF treatment is needed to identify the ideal stimulation conditions for applying a DCEF to heal skin wounds.

A large number of in vitro studies are centered on the effects of EF stimulation on cells [47,48]. In the present study, the EF supplied by the developed device significantly increased the proliferation rate of MSCs compared with unstimulated cells [49]. Furthermore, the greater working surface of this design not only provides a larger sample volume but also enables studies that incorporate 3D cultures. This device may be a useful tool for examining the effects of EF on diverse cell types in vitro. Finally, exposing cells to EF using this device is simple, repeatable, adjustable, affordable and may accommodate various experimental methods.

Conclusion

Ad-MSCs treated with a DCEF at 0 or 200 mV/mm for 1 h/d for 3 days using this customized bioreactor exhibited a markedly higher proliferation rate and retained their stemness phenotype.

Future perspective

The expanded working surface in this design not only allows for a larger sample volume but also enables future studies involving 3D cultures. This device may be beneficial for exploring the effects of EF on various cell types in vitro. Finally, exposing cells to EF with this device is straightforward, repeatable, tunable, inexpensive and may be utilized for a variety of experiments. This study revealed a unique perspective on Ad-MSC proliferation, which is imperative for tissue engineering and regenerative medicine. Because this device does not require the addition of exogenous immunogenic biological substances or complex equipment, EF stimulation can be used to stimulate cell regeneration.