Impedance-based real-time monitoring of neural stem cell differentiation
Pubblicato online: 06 ott 2021
Pagine: 34 - 49
Ricevuto: 23 dic 2020
DOI: https://doi.org/10.2478/joeb-2021-0006
Parole chiave
© 2021 Shah F. J., Caviglia C., Zór K., Carminati M., Ferrari G., Sampietro M., Martínez-Serrano A., Emnéus J. K., Heiskanen A. R., published by Sciendo
This work is licensed under the Creative Commons Attribution 4.0 International License.
Parkinson’s disease (PD) is a progressive neurodegenerative disorder mainly affecting the motor functions due to dysfunctional or dying dopamine (DA) producing neurons in substantia nigra pars compacta [1]. Currently, the applied therapeutic approaches include, e.g., L-3,4-dihydroxyphenylalanine (L-DOPA) medication, administration of a DA receptor agonist, and electrical deep brain stimulation in the subthalamic nucleus. All of them are symptomatic treatments with several limitations and implicated by side effects causing motor response oscillations as well as L-DOPA induced dyskinesia [2]. To restore DA production in PD patients, one of the suggested therapeutic approaches is cell replacement therapy (CRT), i.e. transplantation of cells that acquire dopaminergic properties in the brain [1,2]. Stem cell lines of different origins have been exploited and investigated as relevant cellular sources for CRT in PD. The stem cells can be further categorized into embryonic stem cells, neural stem cells (NSCs), induced neural stem cells, mesenchymal stem cells, and induced pluripotent stem cells, each type having both advantages and disadvantages. NSCs from fetal mesencephalon are considered to be one of the potential sources of transplantable cells [1,2]. However, NSCs must be differentiated into dopaminergic neurons prior to transplantation in order to avoid inefficient transplantation as well as possible tumorigenicity [2]. In addition, the purity and yield of cells having dopaminergic phenotype are critical for successful stem cell-based CRT. For these reasons,
Several biochemical assays are available to monitor and characterize stem cell differentiation
Having the diverse applications of stem cells on the horizon, the need for non-invasive real-time characterization may comprise, e.g., high-content and high-throughput screening for quality and safety assessment in large-scale production of stem cells for CRT and drug development, as well as for disease modeling, pharmacological studies, and toxicity assessment [10]. Real-time bright-field and epifluorescence imaging as well as molecular beacon-based monitoring of mRNA appearance can provide valuable information about changes in cellular morphology and gene expression, respectively [11, 12, 13]. However, especially electrical impedance measurements, pioneered by Giaever and Keese [14], have become well-established as a label-free real-time monitoring technique that has found many applications in monitoring of complex cellular behavior, such as cell motility [15], adhesion and spreading [16], proliferation and cytotoxicity [17,18] as well as receptor activation [19,20]. Additionally, due to the increased interest for studying stem cell differentiation [4], impedance monitoring has proved to be a useful and powerful tool also in real-time monitoring of this cellular processes, revealing mechanisms, the study of which otherwise requires laborious end-point assays [8, 9, 10,21,22].
Two human mesencephalic fetal neural stem lines, LUHMES [23,24] and hVM1 Bcl-XL [25,26], developed for applications concerning the pathogenesis and CRT in PD, have been thoroughly characterized at different stages of differentiation in terms of their gene expression and ability to acquire dopaminergic phenotype [12,13,23, 24, 25, 26, 27, 28, 29]. In this study, we demonstrate how impedance-based multi-parameter analysis can serve as a tool for distinguishing proliferating cell populations from ones undergoing differentiation. We also present a comparison between the widely used data analysis approach to monitor changes in normalized impedance magnitude with time and equivalent circuit analysis. The impedance-based findings are correlated with fluorescence microscopic visualization of morphological changes during the progress of proliferation and differentiation.
All experiments were performed using a previously reported impedance measurement setup (Figure 1) comprising a micromilled poly(methyl methacrylate) cell culture unit (600 μL well for cell culturing) [17], having a microelectrode array (MEA) chip with 12 interdigitated electrodes (IDEs – 12 digits: length 500 μm; width and gap 10 μm), fabricated in a previously published UV lithographic process including e-beam evaporation of metals (150 nm of Au on a 10 nm Ti adhesion layer; 500 nm silicon nitride passivation layer to define active electrode areas and contact pads) [30], a miniaturized custom-made 12-channel bipotentiostat and data acquisition software [31].
Figure 1
Impedance measurement setup:
Prior to cell seeding in the cell culture device for impedance-based assays, each MEA chip was cleaned using the previously described two-step method, including a chemical (10 min in a mixture of 25% H2O2 and 50 mM KOH) and electrochemical (potential sweep in 50 mM KOH between −200 mV and −1200 mV) step [32]. Sterilization of the culture well was done by a 20-minute treatment with 500 mM NaOH followed by thorough rinsing with PBS [17]. By using the abovementioned cleaning procedure, each microelectrode chip could be reused in three experiments.
Lund human mesencephalic (LUHMES) [23,24] and human ventral mesencephalic (hVM1 Bcl-XL) [25,26] neural stem cell lines were generated using human ventral mesencephalic tissue from 8 and 10 weeks old fetuses, respectively. Both cell lines were immortalized using the v-
Both cell lines were cultured in Nunc flasks that were pre-coated with Geltrex® (ThermoFisher, A1413301), diluted 1:100 in sterile PBS, at 37 °C for 1 h. After the coating, Geltrex® was removed and cells were seeded and cultured at 37 °C in a humidified incubator (5% CO2 in air) using Dulbecco’s modified Eagle’s medium/F12 with GlutaMax (DMEM/F12/GlutaMax) containing 0.5 % AlbuMax-I, 1 % Penicillin/Streptomycin (P/S), 1 % N2 supplement (all purchased from Thermofisher Scientific), as well as for LUHMES: 40 ng/ml human recombinant basic fibroblast growth factor (hrbFGF, R&D systems, USA), and for hVM1 Bcl-XL: 20 ng/ml hrbFGF; 20 ng/ml human recombinant epidermal growth factor (hrEGF, R&D systems, USA).
Different cell densities (30,000 cells/cm2, 60,000 cells/cm2 and 120,000 cells/cm2) were seeded onto a Geltrex® pre-coated electrode array chip. For differentiation experiments, 24 hours after cell seeding the culture medium was replaced by differentiation medium prepared in DMEM/F12/GlutaMax containing 0.5 % AlbuMax-I, 1 % P/S, 1 % N2 supplement, 1 mM cyclic adenosine monophosphate (cAMP, Sigma-Aldrich, USA), as well as for LUHMES: 1 μg/ml tetracycline (Sigma-Aldrich, USA), 40 ng/ml human recombinant glial cell line derived neurotrophic factor (hrGDNF, R&D systems, USA) and for hVM1 Bcl-XL: 2 ng/ml hrGDNF. During differentiation, half of the medium was changed every second day.
End-point staining was performed to evaluate viability of the cells. A stock solution of 1 mg/ml Calcein AM (Sigma Aldrich, USA) was prepared in DMSO (Sigma Aldrich, USA) and diluted to 2 μg/ml in PBS prior to use. Staining was performed for 10 min at 37 °C. The cells were further incubated in fresh medium for 10 min to allow de-esterification of AM esters. Fluorescence microscopy images were acquired using an LSM 700 confocal laser scanning microscope (Carl Zeiss AG, Göttingen, Germany) and the ZEN lite software (Carl Zeiss AG, Göttingen, Germany). The employed laser light source provided the 488 nm excitation wavelength. Emission was monitored at 516 nm.
Impedance spectra (30 data points in the frequency range from 100 Hz to 100 kHz with the averaging time of 2 s) were recorded continuously at a time interval of 4 h on each of the 12 IDEs of an electrode array chip over the entire experimental period. The applied sinusoidal potential was set to 200 μV. The impedance measurements were performed using the bipolar sensing configuration facilitated by IDEs to achieve higher sensitivity as previously demonstrated [33].
Changes in the recorded impedance were presented using the dimensionless parameter, Cell Index (CI), which represents the maximum value of the background subtracted normalized impedance based on Equation (1) [34],
where |
Equivalent circuit-based data analysis was done using EchemAnalyst software (V. 6.10) from Gamry Instruments (Warminster, USA) by fitting the data to the appropriate equivalent circuit models using nonlinear least squares regression. To secure sufficient cell density on the analyzed electrodes, equivalent circuit-based data analysis was performed only for impedance spectra acquired on electrodes that showed at least 10 % increase in CI during the first 24 h.
For each experiment, the acquired impedance data were analyzed and averaged. Each experiment was repeated at least twice. Data are presented as average ± standard error of mean (s.e.m.) unless otherwise stated. Comparisons between means were performed using t-test (Prism 9, GraphPad Software, San Diego, CA, USA). Significant differences between means were indicated by asterisk: p < 0.05 (*); p < 0.01 (**); p < 0.001 (***), p < 0.0001 (****).
The conducted research is not related to either human or animal use.
In cell-based applications, impedance measurements were originally demonstrated using a configuration with small sensor electrodes and a large distant counter electrode [14].
This format is still widely used in many studies based on either custom-made or commercial setups. Ehret
The most usual approach to present changes in impedance is to use either normalized impedance magnitude [15] or Cell Index (CI) [34], which is background subtracted normalized impedance magnitude based on the frequency that provides the highest sensitivity. In most studies related to, for instance, toxicological evaluations and also drug testing, this approach provides sufficient information on the time-course of cellular responses.
Impedance data, comprising the magnitude and phase angle, can be presented, aside from the normalized magnitude, using the real and/or imaginary component of impedance evaluated in the complex impedance plane (Nyquist format), complex admittance plane (inverse of impedance), or complex capacitance (angular frequency normalized admittance) plane (also denoted as complex dielectric permittivity plane) [37]. By presenting changes in any of these parameters with time can provide additional information of cellular interaction with the electrode surface and, hence, how the cellular functions and integrity are affected by different chemicals or biochemical processes. This approach was applied by, for instance, Bagnaninchi and Drummond in their study on differentiation of adipose-derived stem cells [9]. If recording complete impedance spectra at different time points, the possible third approach for analyzing impedance data in cell-based applications is to use an equivalent circuit model that describes the spectral behavior and is relevant for the cellular system. Although not as usual as presentation of normalized impedance as a function or time, this approach has been used in many investigations to obtain more details regarding cellular behavior.
In the study presented here, our focus was to obtain information that would allow differentiation between proliferating and differentiating neural stem cells. We evaluated the potentials of both normalized impedance presentation and equivalent circuit analysis, both of which are presented below.
The LUHMES cell line is one of the most commonly used immortalized cell lines as a model for Parkinson’s disease [38]. The cells express v-
Initially, the correlation between cell density and CI was investigated by continuous impedance monitoring of LUHMES cells for 5 days at three different cell densities (30,000 cells/cm2, 60,000 cells/cm2, and 120,000 cells/cm2) under proliferation and differentiation conditions. The cells were seeded on the electrodes at time zero. Figure 2 shows growth and differentiation profiles of LUHMES cells at all three cell densities. For proliferating cells at 30,000 cells/cm2 and 60,000 cells/cm2 (Figure 2A and B), a steady increase in CI was observed over the 5-day period. For the highest cell density, 120,000 cells/cm2 (Figure 2C), the CI increased and levelled off upon reaching confluent cell layer followed by a drop, indicating oversaturation of the culture well and spontaneous detachment of the cells. The corresponding behavior has been previously reported for cancer cells and explained as immediate complete cell coverage on the electrode surface causing weak adhesion of the cells [17].
Figure 2
Cell Index vs. time for LUHMES cells: Initial cell density (cells/cm2)
Differentiation of LUHMES cells was initiated at day 1. The CI for the differentiating cells followed initially the same pattern as for the proliferating cells. On day 2, the CI for differentiating cells started to decrease and continued to decrease over time until it leveled off around day 4, reaching approximately the same level for all the cell densities. Based on microscopic imaging, this decrease in CI could be attributed to the morphological changes of the cells during differentiation (Figure 3). The choice of the optimal cell density for subsequent experiments and equivalent circuit-based data analysis was determined based on the observed behavior of the CI for both proliferating and differentiating cells at the three cell densities. At the density of 60,000 cells/cm2, proliferation could be monitored throughout the entire 5-day period without any decrease whereas differentiation caused a clear change in the CI at a distinct time point. Hence, this was considered as the most optimal density for LUHMES cells.
Figure 3
Fluorescence microscopy images of live stained (Calcein AM) LUHMES cells in growth medium (GM) and differentiation medium (DM). Initial cell density 60,000 cells/cm2. Time in days after cell seeding. (Scale bars: 50 μm).
Additionally, we acquired images of live stained (calcein AM) LUHMES cells during days 3-5 (Figure 3) to confirm that impedance monitoring did not affect cell viability. On day 3, no obvious changes in the morphology of the calcein stained cells could be observed. However, on day 4, the differentiating cells showed decrease in size and increased formation of neural projections. On day 5, all the differentiating cells showed manifestation of a similar morphology with several long processes protruding from the cell soma, whereas the proliferating cells started to become round and overgrow each other.
Based on the CI profile, an early discrimination between growing and differentiating cells was possible (Figure 2). As early as day 2, i.e. 1 day after induction of neural differentiation, the behavior of differentiating cells started to divert from that of the proliferating cells. On day 3, the CI value for the proliferating cells had increased to 0.9, whereas the corresponding value for the differentiating cells had decreased to 0.3. The CI profiles for proliferating and differentiating LUHMES cells, shown in Figure 2A and B, present cellular behavior that is similar to what has been shown previously by Bagnaninchi and Drummond for adipose-derived stem cells differentiating to either osteoblasts or adipocytes [9]. For the cells that underwent osteogenesis, impedance at 64 kHz continued to increase steadily after induction of differentiation.
On the other hand, adipogenesis led to decrease in the recorded impedance soon after induction of adipogenesis and later on leveled off at values slightly above the initial impedance at the time of cell seeding. In that study, phase-contrast imaging indicated that osteogenesis led to decreased cell size and tightened cell-cell junctions, whereas adipogenesis caused the opposite effect. Based on fluorescence microscopy in our study, images shown in Figure 3, the decrease in CI could be attributed to the decrease in cell size and formation of neural projections, both of which contribute to an increase in the free electrode area. However, on day 3, the changes in the cellular morphology that can be seen in microscope images are still very weakly manifested. Hence, the overall conclusion is that impedance monitoring was able to reveal changes in the behavior of LUHMES cells clearly earlier than microscopy. Comparison between our study and differentiation of adipose-derived stem cells [9] shows clearly that although the normalized impedance or the recorded changes in impedance magnitude may behave similarly for different cell lines, the underlying reasons in terms of cell morphology and cell-cell contacts may differ.
The hVM1 Bcl-XL cell line has been differentiated into functional A9 type substantia nigra dopaminergic neurons that exert morphological and functional properties similar to the ventral mesencephalon primary neurons [41]. This cell line has also been transplanted in parkinsonian animals showing amelioration of motor functions in short- and longterm studies [42,43].
Prior to performing impedance monitoring of proliferating and differentiating hVM1 Bcl-XL cells, the optimal cell density was determined by performing preliminary 36 h long impedance monitoring of proliferating cells seeded at two different densities, 60,000 cells/cm2 and 120,000 cells/cm2. As shown in the Supplementary Material Figure S-2, the CI for 120,000 cells/cm2 showed a clear increase immediately after cell seeding and reached the value of over 0.5 after 36 h, whereas the CI for 60,000 cells/cm2 indicated a long lag phase and weak increase after that. Hence, 120,000 cells/cm2 was chosen for the subsequent experiments.
Impedance monitoring of proliferating and differentiating hVM1 Bcl-XL cells was performed for 10 days (Figure 4). The cells were seeded at day 0 and at day 1 the differentiation was induced. For both conditions, the CI had only minor differences. The maximum CI was reached in about 2 days followed by a decrease until the end of the 10-day period. Throughout the whole period, the CI vs. time plots were considerably overlapping.
Figure 4
Cell Index vs. time for hVM1 Bcl-XL cells: Initial cell density 120,000 cells/cm2. Proliferating (blue) and differentiating (red) cells. Time in days after cell seeding. (Error bars: s.e.m., n = 6).
In parallel with impedance monitoring, hVM1 Bcl-XL cells were stained with calcein AM in order to evaluate the viability and morphological changes of the cells during the measurements (Figure 5). Live imaging confirmed that both proliferating and differentiating hVM1 Bcl-XL cells were unaffected by the measurements.
Figure 5
Fluorescence microscopy images of live stained (Calcein AM) hVM1 Bcl-XL cells in growth medium (GM) and differentiation medium (DM). Initial cell density 120,000 cells/cm2. Time in days after cell seeding. (Scale bars: 50 μm)
When evaluating the morphological changes during the 10-day period, at first glance, both proliferating and differentiating cells appeared to behave similarly. Under both conditions, the cell density was clearly increasing, and toward the end of the period the cells were growing on top of each other. Previous experience of the behavior of hVM1 Bcl-XL cells has indicated that proliferation still continues after replacement of the growth medium by differentiation medium, and if the cell population, proliferating or differentiating, becomes overconfluent, the cells start to lose their adherence on the growth substrate. At a closer look, toward the end of the 10-day period, the population of differentiating cells was, however, characterized by formation of a dense network of neural projections. Hence, the observed decrease in the CI after day 2, albeit apparently similar as indicated by the partially overlapping graphs, must in the case of differentiating cells have been caused by a combination of cell detachment and increased formation of neural projections. On the other hand, in the case of proliferating cells the cause was predominantly cell detachment due to overcrowding.
The results presented in the above section demonstrated that the calculated CI values allowed for distinction between proliferating and differentiating LUHMES cells first about 48 h after cell seeding (about 24 h after induction of neural differentiation). However, based on CI, the status of hVM1 Bcl-XL cells could not be evaluated. As has been shown in previous applications related to stem cell differentiation, equivalent circuit analysis could provide more detailed information regarding the cellular status [9,10,21,22]. The data that was used above for CI-based analysis, was further evaluated to find a suitable equivalent circuit model capable of describing the behavior of the two different neural stem cell lines under proliferation and differentiation conditions. The goal of the performed equivalent circuit analysis was to provide an early distinction between proliferating and differentiating cells.
For the study of epithelial and endothelial cells, Wegener
In later studies, the parallel circuit of a resistor (Rcell) and capacitor (Ccell), originally presented by Wegener
When characterizing the behavior of neural stem cells that are subjected to conditions that either promote proliferation or induce differentiation, the initial cell density is far below that of a confluent monolayer, as described above. The same is also valid when formation of neural projections becomes pronounced. The consequence is that at an early stage the cell layer does not fully cover the electrodes, which could make assignment of an equivalent circuit model more difficult than in studies that are based on a confluent monolayer of cells [19,44]. In order to find a suitable equivalent circuit model that could describe the cellular behavior, we used data recorded from IDEs that had a layer of proliferating stem cells that was as confluent as possible. Preliminary tests using an equivalent circuit comprising a resistor in series with a constant phase element (CPE) combined with a parallel circuit of a capacitor and resistor, indicated that the data could be fit. However, when using that equivalent circuit, analysis of spectra acquired during an entire proliferation and differentiation experiment indicated that it was not fully possible to distinguish between proliferating and differentiating hVM1 Bcl-XL cells. In the case of LUHMES cells, the distinction was possible to the same extent as shown above for CI-based data presentation.
Our preliminary data analysis, using an equivalent circuit model comprising Rextra in series with the parallel combination of Rcell and Ccell (Figure 6A), indicated, though, that for both stem cell lines, disregarding whether the impedance spectra had been acquired during an early or late stage during proliferation/differentiation, the behavior of the cells could be described. A CPE was assigned for the electrode-electrolyte interface impedance and the bulk medium conductivity was described by Rmedium. The additional series resistor, Rsys, accounted for the characteristic resistance of the measurement system, including the possible contributions of interfacing to the miniaturized impedance analyzer.
Figure 6
Equivalent circuit models for analysis of impedance spectra acquired
To determine the cell specific equivalent circuit parameters (Rextra, Rcell, Ccell), impedance spectra acquired in the absence of cells were first analysed using a simplified model (Figure 6B). For each electrode, the determined ZCPE, Rmedium and Rsys were kept constant during the subsequent analysis of spectra that were acquired in the presence of cells. Figure 7 shows typical impedance spectra (presented as Bode plots for (A) impedance magnitude and (B) phase angle) acquired in the absence (electrode) and presence of LUHMES cells together with the resulting curve fitting. Impedance spectra were acquired in the range from 100 Hz to 100 kHz.
Figure 7
Example of typical Bode plots for an electrode and the same electrode 48 h after seeding of 60,000 LUHMES cells/cm2:
The number of decades in the spectra is limited and can influence the accuracy of the determined equivalent circuit parameters. As mentioned above, impedance spectra recorded using IDEs comprise the contribution of the interface impedance of both sides. The same applies to the influence of adhering cells. The equivalent circuit model described above is a generalization where the parameters have the contribution of cells on both sides of an IDE. Based on the applied frequency range and simplified equivalent circuit model, only comprising a collective impedance contribution instead of separating both sides of an IDE, the resulting equivalent circuit analysis serves as a tool for distinction between different cellular states.
Figure 8 presents a summary of the variation of the three cell specific parameters over time (5 days) for both proliferating (A) and differentiating (B) LUHMES cells. For each parameter, day 1 represents the first 24 h after cell seeding. In the case of differentiating cells, it also corresponds to the time of medium change to induce differentiation.
Figure 8
Summary of the cell specific equivalent circuit components (Rcell, Rextra, Ccell) for
For proliferating LUHMES cells, Rcell showed an initial increase (from day 1 to day 3), reaching a maximum value at day 3. Subsequently, Rcell decreased continuously, reaching at day 5 a level that was only slightly above that of day 1. Rextra increased significantly until it levelled off after day 4. Ccell, on the other hand, did not undergo any statistically significant changes during the culture period. The initial increase in Rcell (until day 3) can be explained by the increased cell coverage and consequent formation of cell-cell contacts between the adjacent cells. Analogously, the increasing Rextra reflects the increasing number of strongly adhering cells. On the other hand, from day 3 to day 5, the contribution of Rcell differs significantly from that of Rextra. By closer inspection of microscopic images (Figure 3), the behavior of Rcell and Rextra may be explained. The increased number of proliferating cells led to formation of intercellular spaces (i.e. partial disruption of the initially formed tight cell-cell contacts), decreasing Rcell, whereas the entire cell layer remained, nevertheless, strongly adherent on the electrodes, contributing to the continuously increasing Rextra.
In terms of Rcell and Ccell, the behavior of proliferating LUHMES cells was similar to what has been reported by Seidel
Differentiating LUHMES cells showed an initial decrease in Rcell, leveling off after day 2, i.e. one day after the induction of differentiation, and showed only minor fluctuations during the remaining differentiation period. Rextra reached a maximum at day 2 followed by a continuous decrease until day 5. The greatest decrease was observed between day 3 and day 4. Ccell showed the same behavior that was observed for proliferating cells; no statistically significant changes occurred during the 5-day period. Although the induction of differentiation (at day 1) slows down proliferation and initiates formation of neural projections, some degree of proliferation has been observed until day 2.
The observed decrease in Rcell until day 2 can be attributed to the combination of these changes: The cell coverage on the electrodes still slightly increased; however, the initiated formation of neural projections resulted in morphological changes that widened the intercellular spaces, resulting in the net decrease in Rcell. The initial increase in Rextra until day 2 can plausibly be explained by the continued proliferation, which still increased the number of adherent cells. Since the initial formation of neural projections was slow, the observed net effect was an increase in Rextra. The subsequent decrease in Rextra clearly correlates with the increased formation of the neural projections, which do not strongly adhere on the electrodes. Microscopic images (Figure 3) also show that the formation of neural projections significantly increased from day 3 to day 4, explaining the strong decrease in Rextra. Furthermore, general observations on LUHMES cell cultures with advanced differentiation indicated that the entire cell layers became weakly adherent. This means that aside from an increased amount of neural projections, the decrease in Rextra could be additionally contributed to by loosely adherent cell bodies. Comparison between proliferating and differentiating LUHMES cells indicates that the behavior of Rextra has a similar profile as CI for the two conditions. However, in combination with the additional information provided by Rcell, it is clear that equivalent circuit analysis provides more detailed information of the cellular behavior and allows faster discrimination between proliferating and differentiating cells.
The observed overall behavior of LUHMES cells in terms of Rcell and Rextra was comparable to what has been reported by Seidel
Figure 9 presents a summary of the variation of the three cell specific parameters over time (10 days) for proliferating (A) and differentiating (B) hVM1 Bcl-XL cells. Day 1 represents the first 24 h after cell seeding, and in the case of differentiating cells, also corresponds to the time of medium change to induce differentiation.
Figure 9
Summary of the cell specific equivalent circuit components (Rcell, Rextra, Ccell) for
For proliferating hVM1 Bcl-XL cells, Rcell showed large fluctuations rather than any significant trend of increasing or decreasing values. On the other hand, Rextra decreased continuously until day 10 (the greatest decrease was observed between day 2 and day 3) while Ccell slightly increased until day 7, then leveling off. Microscopic imaging (Figure 5) during proliferation showed that the cell coverage on the electrodes continuously increased both laterally and vertically, i.e. while the area covered by the proliferating cells increased the cells also piled up on each other forming a multilayer. This led concomitantly to morphological changes manifested as more rounded and smaller cell bodies. The relatively constant, albeit fluctuating, Rcell can be understood as a contribution of multiple effects. The morphological changes caused by the increased lateral cell density decreased the tightness of the cell-cell contacts (increased gaps between the cells), leading to decreased Rcell.
The voids formed between the cells were partially filled by the cells that grew on top of them. The combination of the two opposing effects may have plausibly resulted in the fluctuating Rcell. Additionally, observations during cell proliferation indicated that the older the cultures became the more the entire cell layers were prone to detachment. This phenomenon may have contributed to an increased distance between the cells and the electrode surfaces, leading to the gradual decrease in Rextra. The small, albeit statistically significant, increase in Ccell can be explained in combination with the gradually decreasing Rextra due to the observed tendency of the cell layers to detach.
During differentiation of hVM1 Bcl-XL cells, Rcell remained relatively constant during the first 3 days followed by a gradual decrease until day 10. Rextra showed fluctuations until day 4 followed by a period of decrease, finally leveling off at day 6, albeit showing fluctuations. In the end of the 10-day period, the overall level of Rextra remained significantly higher than that of the proliferating cells. Ccell increased significantly until day 7, after which it started to show signs of leveling off. The overall level of Ccell increased to much higher values than for proliferating cells. hVM1 Bcl-XL cells continued proliferation after day 1 when the medium was changed to induce differentiation [25,26]. This can also be seen in the microscopic images shown in Figure 5. This may explain the initial period of relatively constant value of Rcell as was seen in the case of proliferating cells. The following decrease in Rcell is clearly caused by the starting formation of neural projections that create voids between the cells. Although the cells still continued to proliferate a few days after induction of differentiation, the formed cell layer did not become as highly packed and was not equally prone to detachment as in the case of proliferating cells. On the contrary, even the neural projections seemed to adhere on the electrodes. These observations can directly explain the smaller decrease in Rextra in comparison with the proliferating cells. The behavior of Rcell and Rextra, explained above, combined with the fact that the formation of neural projections increased the cell membrane to cell volume ratio can also explain the observed increase in Ccell similarly as has been reported for differentiating pNSC2 cells [10].
Comparison between proliferating and differentiating hVM1 Bcl-XL cells indicates that all the three cell specific parameters have a different behavior. Hence, equivalent circuit analysis clearly allows effective and early discrimination between the two conditions unlike the CI-based analysis, which was essentially unable to distinguish between them.
Using impedance-based monitoring, we present the first characterization of two human mesencephalic fetal neural stem lines (LUHMES and hVM1 Bcl-XL) that have been developed for the study of the pathogenesis of Parkinson’s disease and its treatment by cell replacement therapy (CRT). The widely adopted impedance magnitude analysis was able to provide a general distinction between proliferating and differentiating LUHMES cells, whereas in the case of hVM1 Bcl-XL cells no distinction was possible. Hence, the presented results highlight the value of equivalent circuit-based analysis as an excellent tool to distinguish between proliferating and differentiating neural stem cells when impedance magnitude does not provide sufficient information. Such data analysis allows to elucidate the changes in the cellular behavior related to cell morphology, cell-cell contacts, and adhesion to the growth substrate (electrodes). Moreover, our findings demonstrate that impedance monitoring provides such information much earlier than microscopic imaging. This study demonstrates the importance of impedance assays in providing additional insight into the differentiation process of stem cells and forms a basis for the development of future screening assays. Such impedance-based assays in stem cell biology can exactly be aimed at development of stem cell lines for CRT, elucidation of protocols for epigenetic control of stem cells, and drug screening.