Mesenchymal stem cells (MSCs) are multipotent stromal cells that have the ability to self-renew and to differentiate in to a wide variety of cells of the mesodermal, ectodermal, and endodermal lineages [ 18 ]. This is why they represent an important tool intended for cell therapy and regenerative medicine. However , the difference procedures may be long and not able to respond to the need. The improvement and optimisation of these procedures is of an important importance to ensure the differentiated cells in a reasonable time. Several studies have tested the effects of physical stimuli on the differentiation results. Altman et al. showed that the application of a cyclic mechanical stimulation (translational and rotational strain) to undifferentiated MSCs (embedded in a collagen gel) over a period of 21  times resulted in ligament cell lineage differentiation even without difference factors [ 19 ]. Ward and colleagues studied the result of the application of a 3– 5% tensile strain to some collagen I substrate and concluded that it stimulated osteogenesis of attached hMSCs [ 20 ]. Low-intensity ultrasounds, an additional form of mechanical stress, have been shown to enhance the chondrogenic difference [ 21 ]. Sun et al. demonstrated that zero. 1  V/cm electrical stimulation applied for 30  minutes each day for 10  days, on attached MSCs cultured along with osteoinductive factors, accelerated their osteodifferentiation [ 14 ].

Ca 2+ oscillations play an important role in the transduction of many bodily stimuli in the cells. For example , tissue strain and shear stress on mouse tibia are transduced by repeated Ca 2+ spikes in the osteocytes. The spikes frequency and amplitude depend on the mechanised magnitude [ 22 ]. Electromagnetic fields (EMFs) can also market MSCs differentiation to osteoblasts through Ca 2+ -related mechanisms. The exposition of MSCs in order to pulsed EMFs for 10  minutes every day results in the particular enhancement of osteogenesis early stages [ 23 ].

Ca 2+ oscillations are an universal mode of Ca 2+ signalling in both excitable [ 24 , 25 ], and non-excitable cells [ 26 ]. In non-excitable cells such as hMSCs, Ca 2+ oscillations are typically started by a receptor-triggered production of IP3 (after the joining of an agonist to the receptor) [ 27 ] as well as the subsequent Ca 2+ release from your ER [ 28 ], which stimulates the store-operated calcium mineral entry (SOCE) via the SOCCs [ 29 ]. It was demonstrated that the Ca 2+ oscillation regularity is different between undifferentiated MSCs and MSCs on path to differentiation and it differs between the various differentiated cell varieties [ 11 ]. According to Titushkin et al., the MSCs Ca 2+ oscillations are under control after the addition of neuroinductive factors, but reappear in under 7  days after neurodifferentiation [ 11 ]. Sun plus colleagues observed that multipotent MSCs present 8. 06  ±   2 . 64 Ca 2+ spikes per 30  minutes of observation, which this number decreases to 3. 66  ±   2 . 42 after 28  days of incubation with osteoinductive factors [ 14 ].

Within our study, we decided to analyse the effect of a type of brief electric pulses termed microsecond pulsed electric fields (μ sPEFs) on the spontaneous Ca 2+ oscillations of haMSCs because many of the physical stimuli are usually transduced by cytosolic Ca 2+ concentration rapid changes (oscillations) in the cells. The ultrashort pulses (one thousandth to one ten thousandth shorter compared to pulses of 100  μ s used in our study) called nanopulses or nanosecond pulsed electric fields (nsPEFs) have been used already to induce Ca 2+ bursts in the cell, which resulted from your electropermeabilization of the ER, the plasma membrane or each [ 13 , 30 ]. However , contrary to the 100-μ s electrical pulses, the technology to deliver nanopulses is not yet distribute in the laboratories and is not simple to use.

The haMSCs loaded with the fluorescent marker Fluo-4 asynchronously displayed periodic changes in their cytosolic Ca 2+ concentration detected by the periodic boost of their fluorescence. The videos of the haMSCs showed not all the cells presented Ca 2+ oscillations at a specific time. Some of the cells never shown oscillations during the recording time (20  minutes). This statement can be related to the fact that MSCs display Ca 2+ oscillations only during the phases G1 plus S of the cell cycle, and not all the cells within the visualisation area of an experiment were in those stages of the cell cycle. Ca 2+ oscillations increase the levels of cell cycle regulators such as cyclins A and E and probably control cell period progression and cell proliferation, via the regulation of cyclin levels (amongst other mechanisms) [ 31 ]. In addition , the particular haMSCs presenting Ca 2+ oscillations displayed them at different rhythms and frequencies: the particular oscillations were asynchronous, and at a given time, each cellular was in a different phase of a Ca 2+ oscillation and the oscillations frequencies displayed wide variants between cells. The average duration of an oscillation was about 2  minutes.

Electric signal of 100  μ s duration are already applied in lots of biotechnological and medical applications, notably in anticancer electrochemotherapy [ 32 ], tumour ablation [ 33 ], and cellular transfection [ 34 ]: by permeabilizing the plasma membrane layer temporally they allow the internalisation of non-permeant molecules appealing like drugs or nucleic acids. Classically, eight effective pulses of 100  μ s are used in biomedical sciences. The technology is spread in many laboratories and it is very simple to use. In our study, we applied a single electrical pulse because, when several pulses are delivered, signal repetition frequency may impact the efficacy of cellular membrane permeabilization as we have recently shown in our group [ 35 ]. When an electric pulse of 100  μ s i9000 was applied to the cells, an electrically induced Ca 2+ spike was observed synchronously in the given percentage of the cells (as a function from the electric field amplitude). Moreover, the electric field extravagance should be enough to cause the transmembrane voltage to achieve a permeabilization threshold. This threshold depends on the size from the molecule to be internalised. This is why, at 100  V/cm, that is a very low electric field amplitude, no Ca 2+ spike was observed because there was simply no permeabilization and hence no Ca 2+ entry. 120  V/cm was the lowest electric field extravagance at which some cells presented Ca 2+ spikes. This threshold is very low compared to additional ones previously reported, because the Ca 2+ has a small size compared to other classical guns such as yo-pro-1 iodide, propidium iodide or bleomycin which are those frequently used (Hanna et al., submitted). At 450  V/cm, 100% of the cells responded to the electric heartbeat. The origin of the Ca 2+ evoking the Ca 2+ spike was not usually the same: it could be the result of Ca 2+ entry from the external medium only, or the combination of California 2+ entry from cells outdoors and Ca 2+ release in the ER. Our group demonstrated recently that the μ sPEFs could permeabilize not only the cell membrane (as generally observed and stated) but also the internal organelles membranes (Hanna et al., submitted). The origin of the Ca 2+ spike depends on the electric field amplitude. When the latter is below 500  V/cm, the pulse permeabilizes only the plasma membrane (Hanna et al., submitted), as well as the Ca 2+ spike is mainly the result of Ca 2+ entry with the plasma membrane followed by an amplification due to Ca 2+ entry from the voltage-operated Ca 2+ channels (VOCCs), activated by the membrane layer depolarisation [ 36 ]. However , above 500  V/cm, the particular electric pulses will also affect the ER membranes, permeabilize all of them and cause Ca 2+ discharge from the ER (Hanna et al., submitted). Hence, the particular Ca 2+ spike at higher electric field amplitudes, resulted primarily of a massive California 2+ entry through the plasma membrane layer (due to the generation of larger pores at the increased field amplitudes) also followed by the external Ca 2+ entry through the VOCCs, the California 2+ release from the ER as well as the subsequent stimulation of the store-operated Ca 2+ channels (SOCCs) [ 37 ]. This explains exactly why the Ca 2+ spike caused by electric fields below 500  V/cm had a much the same shape to the spontaneous Ca 2+ oscillations with a gradual increase and same amplitude, while the Ca 2+ spike caused by electric fields above 500  V/cm had increased amplitude than the spontaneous oscillations, and presented a crisper rise (due to the abrupt penetration of Ca 2+ through the permeabilized membranes). At 600  V/cm, three different outcomes were observed, mainly because of four factors: first the distribution of the electric industry which results in field amplitude small differences in the area involving the two electrodes; second, the cells are heterogeneous in size plus according to Schwan equation [ 38 ], the electric industry impact will be different between the smallest and the largest cells; 3rd, the orientation of the cell with respect to the electric field outlines, which has an impact on the induced transmembrane potential that will result in the cell membrane permeabilization [ 39 ]; fourth, the positioning of the cells are in the cell cycle that could result in different responses to the pulses. However , at 750  V/cm, it seems that the electric field has exceeded a certain tolerance above which no more heterogeneity was observed and almost all the cells had their Ca 2+ oscillations inhibited.

The California 2+ spikes obtained looked like the particular Ca 2+ spikes induced simply by even shorter electric pulses (the so-called nanopulses, of the duration of e. g. 10  ns, that can straight affect the cell internal membranes such as those of the endoplasmic reticulum [ 30 ]). In our study, an electrically induced Ca 2+ spike along with properties very near to the oscillations did not inhibit the latter, might be applied at any time of an oscillation without interfering with the last mentioned, and after the pulse, the oscillations continued normally on their own rhythm, and with their normal shape. Nevertheless, the high-amplitude Ca 2+ spike inhibited the Ca 2+ oscillations. The particular percentage of cells that were unable to display further California 2+ oscillations increased proportionally using the electric field amplitude of the inhibitory electric pulse. The particular Ca 2+ oscillations inhibition can last some minutes or be longer, to reach many minutes, also depending on the electric field amplitude of the inhibitory electric pulse.

The inhibited of the Ca 2+ oscillations for a while allowed us to study the state of the cell during this period, to find out whether the cell was in a “ refractory state” or even if it was still able to respond to an electric pulse. The use of several pulses after a 900  V/cm pulse inhibition demonstrated that the cells could still react to the pulse plus present Ca 2+ spikes with various amplitudes depending on the applied electric field amplitude.

Last but not least, the use of one 100-μ s electric pulse did not affect the cellular viability 24  hours after treatment (Fig.  

6

), even if applying a much stronger electric field than the one utilized to inhibit the calcium oscillations for several minutes. Moreover, in a prior study [

16

] we showed that haMSCs maintain their stem cell phenotype after being exposed to also stronger electric field conditions (eight electric pulses associated with 100  μ s at 1500  V/cm and at the 1Hz frequency). More precisely:

  1. 1 .

    Seven days right after exposure, MSCs showed similar stem cell surface antigens expression compared with non-treated MSCs.

  2. 2 .

    Four days right after exposure to the eight electric pulses, MSCs were classy in osteogenic and adipogenic differentiation media and effectively differentiated into osteoblasts and adipocytes.

  3. 3.

    Eleven times after exposure to the electric pulses, MSCs cultures produced similar cell growth rates compared with non-treated MSCs civilizations.

The electrically induced Ca 2+ spikes can have wide effects on the Ca 2+ oscillations frequency, amplitude and duration. While the nanosecond pulses allowed only to add a Ca 2+ spike to the oscillations [ 30 ], the microsecond pulses display more complex effects. The electrical field amplitude allowed us either to induce the Ca 2+ spike without influencing the oscillations, or to inhibit the Ca 2+ oscillations and impose on the cell California 2+ oscillations at a desired rate of recurrence and amplitude. It is worth noting that the microsecond technologies is cheaper than the nanosecond one, it is more accessible in the laboratories, and simple generators can deliver this. These considerations add further value to the observations documented here. Taking into account that each type of differentiated cell from MSCs has its own Ca 2+ vacillation characteristics, and that these oscillations may play an important part in the decision of MSCs fate, the μ sPEFs constitute a novel useful tool to control the oscillations and the differentiation of MSCs.