Role of Bioelectricity During Cell Proliferation in Different Cell Types

Most living organisms possess varying degrees of regenerative capabilities but how these regenerative processes are controlled is still poorly understood. Naturally occurring bioelectric voltages (like Vmem) are thought to be playing instructive role in tissue regeneration, as well as embryonic development. The different distribution of ions on the either side of the cell membrane results in intra- and extra-cellular voltage differences, known as membrane potential or Vmem. The relationship between Vmem and cell physiology is conserved in a wide range of cell types and suggests that Vmem regulation is a fundamental control mechanism for regeneration related processes e.g., proliferation and differentiation. In the present study we measured Vmem in three different cell types (human osteogenic sarcoma cell line (OSC), rat bone marrow derived mesenchymal stem cells (BM-MSC), and rat dermal fibroblasts) and characterized the relationship between their Vmem and proliferation. In order to find out if Vmem controls proliferation, or visa-versa, we blocked and then unblocked Na+/K+-exchanging ATPase using ouabain and measured the proliferation. Our results demonstrate that Vmem can be pharmacologically manipulated to control proliferation in certain cell types like BM-MSC. Taken together, it is clear that control of bioelectrical properties in non-excitable cells could prove to be potentially a useful tool in regenerative medicine efforts.


INTRODUCTION
Current reconstructive treatments aimed at restoring normal form and function to diseased, injured or missing tissues and/or organs use a patient's own tissues, tissues and organs transplanted from donors, or prosthetic devices. While these treatments enjoy varying degrees of success, they are often associated with drawbacks such as limited donor availability, infection, immunological rejection, and high costs (Mao and Mooney, 2015). In contrast, regenerative therapies could potentially restore normal tissue form and function, without these drawbacks (Levin and Stevenson, 2012;Bessonov et al., 2015;Tyler, 2017). While most living organisms possess varying degrees of regenerative capabilities, the signals that control these processes are still poorly understood. Naturally occurring bioelectric signals have been shown to play an important role in tissue regeneration, as well as embryonic development (Gurtner and Chapman, 2016;Tyler, 2017).
Bioelectricity originates at the cell membrane from a constant imbalance in charge between the intra-and extracellular compartments, caused by the passage of ions (Na + , K + , Ca 2+ , Cl − , etc..) through different types of ion pumps and channels. The different distribution of these ions on either side of the cell membrane results in intra-and extracellular voltage differences, known as membrane potential or V mem . Such a balance is maintained via passive and active ion transport through various ion channels and transporters located within the membrane (Sundelacruz et al., 2009). Membrane potential forces ions to passively move in one direction: positive ions are attracted by the "negative" side of the membrane and negative ions by the "positive" one (Hammond, 2015). If we suppose that there is no concentration gradient for any ions (there is the same concentration of each ion in the extracellular and intracellular media), ions will diffuse according to membrane potential only positively charged ions, the cations Na + , Ca 2+ and K + , will move from the extracellular medium to the intracellular one according to membrane potential. In contrast, anions (Cl − ) will move from the intracellular medium to the extracellular one. V mem is expressed relative to the extracellular environment so that a cell is "depolarized" when its V mem is less negative, while a cell is "hyperpolarized" when its V mem is more negative (Cervera et al., 2016;Erndt-Marino and Mariah, 2016). Accordingly, V mem values of rapidly proliferating embryonic and tumor cells, generally have high "depolarized" V mem values, whereas nonproliferating, terminally differentiated somatic cells, such as, skeletal muscle cells, neurons and fibroblasts typically have low "hyperpolarized" V mem values as shown in Figure 1 (Binggeli and Weinstein, 1986;Chernet and Levin, 2013;Levin et al., 2019;Sundelacruz et al., 2019).
The relationship between V mem and -cell physiology is conserved in a wide range of cell types (precursor and mature cells; proliferative and quiescent cells; normal and cancerous cells) and suggests that V mem regulation is a fundamental control mechanism for regeneration related processes e.g., proliferation and differentiation (Sundelacruz et al., 2008(Sundelacruz et al., , 2009. From this arises the intriguing possibility of being able to control a cell's proliferative and/or regenerative capabilities by manipulating its V mem . In other words, by increasing the V mem of a normally non-proliferative fibroblasts, one could stimulate it to proliferate. Or conversely, by lowering the V mem of a tumor cell one could reduce its proliferation and arrest its growth. In the present study we measured V mem in three different cell types (osteogenic sarcoma cell line (OSC), bone marrow derived mesenchymal stem cells (BM-MSC), and fibroblasts) whose normal proliferative states are relatively high, medium and low, in order to characterize the relationship between their V mem and proliferation. In order to investigate whether V mem controls proliferation, or vise-versa, we blocked and then unblocked Na + /K + -exchanging ATPase and measured the proliferation.

MATERIALS AND METHODS
All experiments were performed in accordance with guidelines established by our animal care and oversight committed at the Johann Wolfgang Goethe University in Frankfurt am Main, according to German animal welfare act §4 and EU Act 2010/63 for the protection of laboratory animals.

Cell Preparation and Culture
Rat BM-MSC were purchased from Cyagen Biosciences (Santa Clara, CA, USA Cat. No. RASMD-01001), fibroblasts were extracted from the skin of already euthanized rats (Seluanov et al., 2010) received from different project (German animal welfare act §4 and EU Act 2010/63), and human OSC cell line was purchased from the DMSZ-Cell bank (Braunschweig, Germany, Cell line: SAOS-2). All cells were stored in liquid nitrogen at −196 • C, then, on the day of the experiment they were thawed, cultured, and expanded to reach the desired number. To achieve the appropriate number, cells were cultured until they reached 80% confluency and then expanded over 6-8 passages. Cells were then seeded in normal cell growth medium (Dulbecco's Modified Eagle Medium, GlutaMAX 1 g/L D-Glucose, 10% Fetal Calf Serum, and 1% Penicillin/Streptomycin (10 U/ml), all obtained from GibcoR (Gaithersburg, MD, USA), in 6-well cell culture plates (TPP, Trasadingen, Switzerland) at a density of 50,000 cell/cm 2 . All cells were cultured for 14 days in a humidified incubator at 37 • C with 5% CO 2 , and culture medium was changed every 3 days.
Na + /K + -Exchanging ATPase Blocker Na + /K + -exchanging ATPase blocking was achieved using ouabain (10 µM, Sigma-Aldrich) which was added to the medium from a fresh stock solution in distilled water. This blocking effect was reversed (unblocked) by washing the cells 5 times with 1X PBS.

V mem Measurements
To visualize and measure V mem changes at predetermined measurement time points (0, 3, 5, 7, 10, and 14 days) during proliferation, cells were dyed with the anionic voltage-sensitive dye, Bis-(1,3-diethylthiobarbituricacid) trimethine Oxonol [DiBAC 4 (3), Invitrogen, Carlsbad, CA, USA], whose uptake by cells is voltage dependent. Higher dye uptake is seen in more depolarized cells (Adams and Levin, 2014;Bhavsar et al., 2019). V mem changes were visualized and measured using fluorescence microscopy, as described by Adams and Levin (2014). For each measurement a fresh solution of 10 mM DiBAC 4 (3), in DMSO was prepared and diluted to 0.5 mM in Hank's Buffered Salt Solution (HBSS, Invitrogen, Carlsbad, CA, USA). After adding the dye, the cells were left for 30 min in an incubator at 37 • C, then washed two times using PBS at room temperature and imaged using a Nikon Eclipse Ti-E Inverted Microscope (Nikon, Tokyo, Japan). The DiBAC 4 (3) dye was excited with a 420 nm light and the fluorescence images were captured at 520 nm by a non-descanned photomultiplier tube, controlled by NIS Element Software. The captured images were saved as bright field (BF) images and for every BF image, a flatfield image (FF) (made by defocusing the image) and a dark field (DF) image (made FIGURE 1 | Depolarized (above) and Hyperpolarized (below) cells with ion channels shown schematically. The inward-rectifying channels (inward arrows) favor hyperpolarization while the outward-rectifying channels (outward arrows) favor depolarization. Cells physiological state is coinciding with its V mem, whereby highly proliferative (cancer, fertilized eggs, stem) cells are depolarized and mature, terminally differentiated (muscle, fibroblasts, neurons) cells are hyperpolarized. Image modified from Cervera et al. (2016) and Levin et al. (2019).
by closing the shutter) were taken. These three images were later used for corrections and analysis (described in detail in Bhavsar et al., 2019). All samples were imaged on the same day to minimize time dependent variations. Since fluorescence intensity was quantified for each image, the gain, exposure time, and offset settings of the microscope were kept constant over the duration of each experiment.

Cell Proliferation
To measure cell proliferation, cell number was evaluated using PicoGreen assay according to the manufacture's protocol (Quant-iTTM PicoGreen, ThermoFisher, Germany) at days 0, 3, 5, 7, 10, and 14. Briefly, cells were washed two times with PBS, treated with lysis buffer (400 mM potassium phosphate buffer, 2% Triton X100, 10 mM EDTA, pH 7.0), and cell lysates were used for DNA content measurements. A serial dilution of a known number of cells was lysed with lysis buffer and used to create a calibration curve showing the correlation between cell number and fluorescence. This latter procedure allowed us to indirectly determine the number of cells in the cultured wells via a calibration curve and measurement of DNA content through fluorescence of Pico-green.

Cell Viability
To measure cell viability, AlamarBlue assay was performed at days 0, 3, 5, 7, 10, and 14. The AlamarBlue Assay incorporates an oxidation-reduction (REDOX) indicator that changes color in response to chemical reduction of growth medium resulting from cell growth. As cells being tested grow, innate metabolic activity results in a chemical reduction of AlamarBlue (resazurin) to resorufin. AlamarBlue assay was performed according to the manufacturer's protocol (AlamarBlue R Cell proliferation assay Kit, BIORAD). Briefly, the culture medium was completely aspirated from the wells and cells were washed twice with sterile PBS. One milliliter of fresh medium was added along with 100 µl of AlamarBlue reagent. Additionally, wells containing medium and AlamarBlue reagent only (no cells) were used for blank measurements. Cells and blank samples were incubated for 4 h (37 • C, 5% CO 2 ). After 4 h incubation, three aliquots (100 µl) of each sample were pipetted in a 96 well plate and absorbance was measured at 570 and 600 nm using plate reader (Infinite 200PRO Tecan, München, Germany). Absorbance mean values of triplicates for each sample were calculated and the percentage of Alamar blue reduction was calculated using a formula described in the manufacturer's protocol (AlamarBlue R Cell proliferation assay Kit, BIORAD).

Data Analysis and Statistics
All experiments were performed in triplicate and the data is presented as the box and whisker plots unless otherwise indicated. The distribution of the data was checked using Shapiro-wilk test (p < 0.05 = non-parametric, p > 0.05 = parametric). The statistical significance of differences between the groups and time points was analyzed by non-parametric Friedman test and a Bonferroni corrected p < 0.05 was used to indicate statistical significance. The p-values are indicated on the plot using asterisks ( * p < 0.05, * * p < 0.01). Statistics were calculated using the software Bias 11.03 (Epsilon-Verlag, Darmstadt, Germany).

V mem Profiles of Different Cell Types
V mem was measured in the three different cell types using voltagesensitive dye DiBAC 4 (3) at different time points, as shown in Figure 2. V mem values (fluorescence intensity) of BM-MSC increased from day 0 to day 10, significant at day 3 and 10 (p < 0.05). However, V mem values significantly decreased (p < 0.01) at day 14. V mem in fibroblasts, was constant during the entire time course. V mem values in OSC increased, throughout, from day 0 to 14, significant at day 3, 7, 10, and 14 day 10 (p < 0.05) (see Supplementary Table 1 for details).

Cell Proliferation and V mem Measurements
Cell proliferation and V mem in all three cell types were measured using PicoGreen assay and DiBAC 4 (3) voltagesensitive fluorescent dye, respectively. Further, to determine the correlation, if any, between the cell proliferation and V mem , non-parametric Spearman correlation analysis was performed (Figures 3A-C). A moderate correlation between the cell number and V mem was observed in BMMSC (ρ = 0.42) and in OSC (ρ = 0.48). However, no correlation (ρ = −0.2) was found  between the cell number and V mem in fibroblasts ( Figure 3B) (see Supplementary Table 2 for details).
Na + /K + -Exchanging ATPase Blocking and Cell Proliferation Na + /K + -exchanging ATPase was blocked, using ouabain, in all three cell types. During the blocking period, cell proliferation and viability were measured using PicoGreen and AlamarBlue, respectively. In the case of BM-MSC, the control group showed a significant increase (p < 0.01) in proliferation (cell number) from day 0 through 14, while cells treated with ouabain, showed neither increase nor decrease in the cell number ( Figure 4A, left graph). In addition, BM-MSC V mem , blocked with ouabain, showed a significant reduction (p < 0.01) in cell metabolic activity at all the time points (Figure 4A, right graph). In contrasts, fibroblasts treated with ouabain, showed an increase in the cell number (significant at day 10 and day 14, p < 0.01) compared to their respective controls ( Figure 4B). OSC treated with ouabain, showed a significant (p < 0.01) decrease in the cell number, especially at days 3, 5, 7, and 14 ( Figure 4C, left graph). In addition, in ouabain treated OSC metabolic activity was significantly reduced (p < 0.01) at days 0, 3, 7, 10, and 14 ( Figure 4C, right graph). The negligible negative values seen in OSC treated with Ouabain ( Figure 4C, right) at later time points is due to the cytotoxic effect of Ouabain and drastic reduction in the cell number at later time points (Figure 4C, left graph). Apparently, there is almost no cells in these group at Day 10, 14 timepoints and therefore it is only % reduction of the culture medium. The calculations of % of reduction is always made against the negative control (medium only) samples and obtained negative values could be result of differences among mediums (pH) in samples and negative control wells since AlamarBlue is influenced by the pH of the cell growth medium (Rampersad, 2012) (see Supplementary Tables 3, 4 for details).

Unblocking Na + /K + -Exchanging ATPase in BM-MSC
In order to unblock Na + /K + -exchanging ATPase, reversing the blocking effect of ouabain, BM-MSC were first treated with ouabain until day 3 and then washed five times using 1X PBS. The cell number and metabolic activity were visualized and measured at days 0, 3, 5, and 7 using PicoGreen and AlamarBlue, respectively. During the blocking phase, cells displayed significant (p < 0.05) reduction in number at day 3 with reduced (though not significant) cell metabolic activity (Figures 5A,B). After unblocking (washing with PBS), cell numbers were increased at days 5 and significantly at (p < 0.01) day 7. In addition, cell metabolic activity was significantly (p < 0.01) increased at day 7 (Figures 5A,B) (see Supplementary Table 5 for details).

DISCUSSION
The complex processes of tissue development, healing and regeneration involve multiple cellular activities like, proliferation, migration, adhesion and differentiation, all of which are, at least partially, regulated by V mem related bioelectric signaling (Sundelacruz et al., 2008(Sundelacruz et al., , 2009Levin et al., 2019). In this study, we characterized membrane potential (V mem ) profiles for BM-MSC, fibroblasts, and OSC during proliferation using the voltage-sensitive fluorescent dye DiBAC 4 (3). We observed that changes in V mem and proliferation coincided, and that by blocking and unblocking Na + /K + -exchanging ATPase we were able to control proliferation in BM-MSC. We saw that as the cells began to proliferate, their V mem values shifted from lower to higher (depolarization), and when they stopped proliferating, their V mem shifted from higher to lower values (hyperpolarization). These observations coincided with those of others in the literature, which indicate that V mem depolarization is required for both G1/S phase and G2/M phase transitions during cell proliferation (Sundelacruz et al., 2008;Blackiston et al., 2009;Yang and Brackenbury, 2013). In our experiments, OSC showed a consistent increase in V mem during the course of proliferation. Reports in the literature suggest that cancer cells tend to be more depolarized than other cell types, due to a higher concentration of Na + /K + -exchanging ATPase (Yang and Brackenbury, 2013) and higher intracellular Na + levels in comparison to other cell types (Camero et al., 1980;Sparks et al., 1983). This could explain the increase in V mem we observed throughout the course of proliferation. On the other hand, we saw that fibroblasts V mem displayed a pattern of hyperpolarized during the course of proliferation. This could FIGURE 5 | Graph of blocked and unblocked Na + /K + -exchanging ATPase of BM-MSC and proliferation. (A) BM-MSC proliferation (cell number) measured during blocking (ouabain) at days 3 and 5, and after unblocking (washing) at days 5 and 7. (B) BM-MSC cell metabolic activity (% reduction of Alamar blue) measured during blocking (ouabain) of Na + /K + -exchanging ATPase at days 3 and 5 and unblocking (washing) at days 5 and 7. Asterisks indicate degree of significant differences between groups at the same time points, *p < 0.05, **p < 0.01. be due to a lower number of voltage-gated Na+ channels present in dermal fibroblasts (Estacion, 1991), leading to lower concentrations of intracellular Na + , lower proliferative capacity and hyperpolarized V mem .
Pharmacological blocking of ion channels has been a popular method to study V mem function. Using small molecule drugs to target specific ion channels of cells allows the precise control of a given cells' V mem profile (Blackiston et al., 2009;Levin et al., 2019). Another advantage of pharmacological blocking over other methods (knockout, RNAi or morpholinos) is this approach can reveal membrane potential function per se, which is not necessarily dependent on any one particular gene (Blackiston et al., 2009). In this study we used ouabain, that affects V mem by specifically blocking Na + /K + -exchanging ATPase or the sodiumpotassium ion pumps in the cell membrane. Ouabain is a plant derived cardiac glycoside that was traditionally used in Africa as an arrow poison for hunting (Schoner, 2000). Ouabain blocks Na + /K + -exchanging ATPase by binding to the α-subunit at the external end of the ion permeation pathway and inhibits its ion exchange pump activity. Once ouabain binds to this enzyme, the pump ceases to function, leading to an reducing the activity of sodium-potassium ion pump which pumps one calcium ion out of the cell and three sodium ions into the cell (Shen et al., 2019).
In our experiments, we found that using ouabain to block Na + /K + -exchanging ATPase significantly decreased proliferation and reduced cell metabolic activity in BM-MSC. In order to confirm this finding, we subsequently unblocked the Na + /K + -exchanging ATPase, reversing Ouabain's effect and restoring proliferation and cell metabolic activity to their pre-blocked levels. This finding demonstrates that, by increasing or decreasing these cell's V mem , in this case using pharmacological blockers, one can control their proliferative capacity. In summary, our results suggest that pharmacological blocking/unblocking of Na + /K + ATPase may provide a proproliferative environment in therapeutic tissue engineering applications where BM-MSC are used. Ouabain, that blocks Na + /K + ATPase, is commonly used to treat congestive heart failure and supraventricular arrhythmias (Wu et al., 2015), however, its use systematically, in tissue engineering (TE) applications is unlikely. The growing field of TE, employs a large range of novel strategies, of which local ouabain application might be considered. For example, pre-treating cells in-vivo before transplanting them into a defect (Carpizo et al., 2008). Another approach could be to delivery ouabain into a defect incorporated in modified scaffolds, which release the drug locally and in a controlled manor (Kretlow et al., 2007;Garg et al., 2012;Sengupta and Prasad, 2018). Further animal studies are needed to determine whether ouabain's effects are maintained in-vivo in these conditions, and to demonstrate proof-of-concept using these approaches.
In dermal fibroblasts we found that using ouabain to block, initially (days 0-7) had no effect on proliferation, and then at days 10 and 14 blocking Na + /K + -exchanging ATPase in the cells caused an increase in proliferation, compared to controls. This finding coincides with those of others who reported that lower concentration of ouabain can induce proliferation in several different cell types, including fibroblasts (Orlov et al., 1999;Isaev et al., 2000;Nguyen et al., 2007;Winnicka et al., 2010). These authors propose that at lower concentrations ouabain can activate mitogen-activated protein kinase (MEK) and extracellular signal-regulated kinases (ERK) pathways, which in turn results in the expression of genes that are involved in cell growth and cell proliferation. Since ouabain can readily activate proliferation in dermal fibroblasts, it may have a direct impact on the dermal equivalents and bilayer skin substitutes which are used to treat a range of different chronic non-healing wounds in clinical settings (Wong et al., 2007).
We found that in OSC, blocking Na + /K + -exchanging ATPase with ouabain induced cell death at the later time points (days 7, 10, 14). In a similar study, Chou et al. (2018) investigated the effect of ouabain on apoptotic cell death of human osteosarcoma-derived U-2 OS cells. Based on their finding they suggest that blocking Na + /K + -exchanging ATPase with ouabain induced S-G2/M phase cell-cycle arrest in osteosarcoma cells, which in turn induced apoptotic cell death by activating the caspase-dependent and -independent pathways, accompanied also by mitochondrial dysfunction. Our findings support this and provide important insight into the cytotoxic effects of ouabain on OSC. It is noteworthy that many studies have shown the potential of ouabain as a therapeutic agent against various cancers (Winnicka et al., 2008;Liu et al., 2013;Pongrakhananon et al., 2013;Chen et al., 2014;Xiao et al., 2017;Shen et al., 2019). Our findings confirm this showing that blocking Na + /K + -exchanging ATPase with ouabain decreased osteosarcoma cell proliferation, suggesting it could potentially be used to treat osteosarcoma.
Our in-vitro results demonstrate that V mem can be pharmacologically manipulated to control proliferation in certain cell types like BM-MSC. If reproduced in in-vivo models this may be used to regulate specific cell behaviors in cell-based clinical therapies to optimize their effectiveness.

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the article/Supplementary Material.

AUTHOR CONTRIBUTIONS
MB conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, and assisted in preparing the manuscript for publication. LL conceived and designed the experiments, contributed reagents/materials/analysis tools, and assisted in preparing the manuscript for publication. KC assisted in preparing the manuscript for publication. JB edited and corrected the manuscript. All authors contributed to the article and approved the submitted version.

FUNDING
This study was supported in part by the Friedrichsheim Foundation (Stiftung Friedrichsheim) based in Frankfurt/ Main, Germany.