Determination of Electrical Stimuli Parameters To Transdifferentiate Genetically Engineered Mesenchymal Stem Cells into Neuronal or Glial Lineages

Abstract

This study investigated the effect of electrical stimuli parameters using graphene-based devices for the transdifferentiation of genetically engineered brain-derived neurotrophic factor (BDNF) hypersecreting mesenchymal stem cells (BDNF-MSCs) into neuronal or glial lineages. The results suggest that BDNF-MSCs have the tendency to transdifferentiate into both neuronal and Schwann cell (SC)-like phenotypes at lower voltages (25–50 mV). However, as the applied voltage changed from 25 to 100 mV at 50 Hz, the transdifferentiation of BDNF-MSCs yielded more into SC-like phenotypes and resulted in complete transdifferentiation into SC-like phenotypes at 100 mV and 50 Hz. With an increase in voltage to 100 mV, the complete transdifferentiation to SC-like phenotypes also resulted in enhanced paracrine activity leading to total secretion of nerve growth factor (NGF) up to 50 ng/mL with pronounced biological activity, causing neurite extension of 4 μm/cell on PC12-TrkB cells. Moreover, 90% of the transdifferentiated cells demonstrated significant myelination potential. The contact co-culture of BDNF-MSCs with adult hippocampal progenitor cells (AHPCs) in the presence of electrical stimuli resulted in differentiation of BDNF-MSCs into SC-like phenotypes accompanied by synergistic neurite extension of AHPCs. Overall, this study demonstrates the possibility of controlling simultaneous and spatial differentiation of MSCs into selected neuronal and glial lineages at desired ratios via changes in electrical stimuli through graphene-based devices and can contribute to the development of novel cell-based strategies for nervous system rescue and repair.

Lay Summary

This work evaluates the effect of different electrical stimuli conditions applied through inkjet-printed and laser-annealed graphene-based interdigitated circuits on the differentiation behavior of mesenchymal stem cells. Our results suggested that it is possible to spatially and locally control the differentiation of mesenchymal stem cells into final lineage type (glial or neuronal) by manipulating the electrical stimuli. The future work will include the control of stem cell differentiation and fate commitment in an in vivo model using electrical stimuli.

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Introduction

Peripheral nerve (PN) injuries affect 2.8% of trauma patients world-wide and annually over 200,000 PN surgeries are performed only in the USA [1,2,3]. Currently available clinical treatments, particularly for large PN injuries, have demonstrated incomplete recovery and poor functional outcomes in most of the cases leading to life-long physical, social and economic hurdles (US market for repair of transected peripheral nerves is ~ $1.3–1.9 billion annually) [1,2,3]. Autologous nerve grafts, the current gold standard for nerve regeneration treatments, have significant disadvantages such as the requirement of multiple surgeries, limited graft tissue, biological complexity, and donor site morbidity [1,2,3]. Implantation of Schwann cells (SC), the glial cells of the PN system, using biodegradable nerve guidance conduits (NGCs), has been considered as a promising strategy for PN repair [4]. However, the limited availability, donor site morbidity, and slow in vitro growth of SCs limit the clinical translation of this strategy [5,6,7]. As an alternative, in vitro transdifferentiated mesenchymal stem cells (tMSCs), possessing SC-like phenotypes, have recently been explored for PN regeneration [8,9,10,11,12,13,14,15] because of the ease of accessibility from different tissue resources such as bone marrow, their ability to expand in culture, and their multipotency. However, there are three main challenges that need to be addressed for the clinical transition of this strategy. These challenges include (1) precise control on the final fate of the implanted cell population during the in vivo regeneration period, (2) non-scalable differentiation protocols, and (3) design of multifunctional conduit with desired properties that mimics the complex extracellular matrix microenvironment [16,17,18,19]. Thus, addressing these challenges makes tMSC-based strategies a viable solution for PN injury repair.

To overcome these issues, we recently demonstrated the efficient transdifferentiation of MSCs into SCs via electrical stimulation using graphene-based substrates for the first time [20]. We have showed that inkjet-printed graphene circuits printed into the shape of interdigitated electrode (IDE) circuits are promising platforms to control the MSC transdifferentiation through electrical stimulation [20]. Several advantages of graphene-based materials, such as electrical and thermal conductivity, mechanical strength, chemical stability, and biocompatibility, make them well-suited material for the design of functional scaffolds/conduits that are capable of mediating cell growth, proliferation, and differentiation [21,22,23,24,25,26,27,28,29,30]. This builds on our previous works [12, 31,32,33,34] and demonstrates the potential of using electrical stimulation to control migration, alignment, and differentiation of neural stem cells [35, 36]. The transdifferentiation of MSCs into SC-like phenotypes, as a result of varying electrical stimulation (free from chemical induction) with graphene-based materials, has not yet been investigated in detail. We hypothesized that modulating the electrical stimuli applied to MSCs through specifically designed inkjet-printed graphene IDEs would result in diversification of the final phenotype, with exhibition of SCs as well as neuronal-like phenotypes.

In this study, we used genetically engineered, brain-derived neurotrophic factor (BDNF) hypersecreting MSCs (BDNF-MSCs) to demonstrate superior neurotrophic growth factor secretion and provide enhanced regeneration. In particular, we focused on BDNF, which contributes to the growth of regenerating axons into peripheral nerve grafts [37], and nerve growth factor (NGF), which helps to regulate axonal signals that control myelination [38]; secretion of both is upregulated in SCs. The effects of different electrical parameters applied using graphene-based devices on the transdifferentiation and paracrine activity of BDNF-MSCs was investigated. The obtained glial or neuronal cell types were assessed via detailed immunocytochemistry and gene expression analysis. The paracrine activity upon transdifferentiation was evaluated using ELISAs, while the biological activity of secreted growth factors was determined through the evaluation of neurite extension on PC12-TrkB cells co-cultured with transdifferentiated BDNF-MSCs (tBDNF-MSCs). In addition, co-cultures of BDNF-MSCs with adult hippocampal progenitor cells (AHPCs) in the presence of electrical stimuli was conducted to evaluate their capacity to promote neurite outgrowth from neurons differentiating from AHPCs. These efforts provide additional support for the potential benefit of electrical stimuli to control stem cell differentiation and develop cell-based strategies for nervous system rescue and repair.

Materials and Methods

Graphene Ink Formulation

Graphene ink was prepared similar to our previously reported protocols [20, 39, 40]. Briefly, 50 mL of inkjet printable ink was prepared by mixing 175 mg of completely reduced, pristine graphene (ACS GN1P0005, referred to throughout manuscript as graphene) and 175 mg of ethyl cellulose (Sigma-Aldrich) in cyclohexanone and terpineol (80/20%). Ethyl cellulose was chosen as a surfactant to increase suspension stability and viscosity in order to create a jettable ink. The colloidal suspension was then probe-sonicated (Sonics Vibra-cell VCX-750 ultrasonic processor) for 4 h with a 9-s pulse and 1-s rest at 70% amplitude. Probe sonicating was used to ensure that graphene flakes were completely exfoliated, that the graphene flake size was reduced in order to prevent printer clogging, and that the graphene flakes were evenly coated with the surfactant in the colloidal suspension. Moreover, the beaker was suspended in ice water to keep the temperature below 40 °C while probe sonicating.

Inkjet Printing Protocol

Before printing, the as-prepared ink was vortexed for 1 min to ensure an even colloidal suspension and then filtered through a 0.45-μm syringe filter to remove any remaining large graphene flakes that may cause printer clogging. The graphene ink was inkjet-printed onto polyimide film (Dupont Kapton 12 m thick) with a Fujifilm Dimatix Material Printer (DMP2800) using a 10-pL nominal drop volume nozzle. The waveform was optimized to print consistent spherical droplets with limited tails to increase the printing resolution (prevent satellite droplets) at a nozzle temperature of 40 °C. The graphene IDEs were printed (40 printer passes or layers) with a 30-μm drop spacing on a heated (60 °C) platen table. These printer parameters prevented ink pooling as well as the coffee ring effect to yield an even film deposition.

Thermal Annealing

Graphene IDE electrodes were thermally annealed (400 °C for 60 min, plus 30 min heat-up and cool-down times before and after respectively) in a tube furnace (MTI Corp., OTF-1200x). The annealing process was conducted under an inert atmosphere (forming gas, 95% argon and 5% hydrogen) to prevent graphene oxidation. This forming gas mixture was used to increase the electrical conductivity of the graphene and most likely helped remove solvents from the graphene as we have reported previously [40]. More specifically, as the graphene electrode temperature increases, the ethyl cellulose should carbonize and the graphene flakes should  “weld” together, both of which also increase electrical conductivity and adhesion. Moreover, the resultant thermally annealed printed graphene exhibited a smoother surface than the laser-annealed graphene and also was hydrophilic (Supplementary Information section Figs. S1S4).

Laser Annealing

As an alternative to thermal annealing, some graphene IDEs were laser annealed. Laser annealing is amenable to thermally sensitive substrates (i.e., paper or polymer films). A low-cost 1000-mW diode laser engraver (HTPOW) was used for laser annealing in a manner similar to our previous manuscripts [41, 42]. The diode laser emitted a blue-violet (405 nm) wavelength which did not harm the polyimide film substrate when performed under a fast raster rate (carving time 5 ms). Similar to thermal annealing, the laser carbonized surfactants, removed solvents, improved adhesion, and increased conductivity; however, the laser dramatically increases the surface roughness by nano-/micropatterning of the surface of the graphene to create a more hydrophobic surface (Supplementary Information section Figs. S1 and S2), similar to our previous protocols [39, 42,43,44].

BDNF-MSC Culture

Brain-derived neurotrophic factor (BDNF) hypersecreting Sprague Dawley rat MSCs (BDNF-MSCs) that can help with nerve regeneration were isolated and genetically engineered as described in our previous works [45, 46]. Briefly, bone marrow-derived rat mesenchymal stem cells were isolated and engineered to produce and secrete the neurotrophic factor BDNF using lentiviral vectors. The continuous and stable production in MSCs transduced with BDNF vectors was shown in our previous studies [45, 46]. BDNF-MSCs were grown and sub-cultured in maintenance medium (MM) composed of alpha minimum essential medium (α-MEM, Gibco BRL) supplemented with 20% fetal bovine serum (Atlanta Biologicals), 4 mM L-glutamine (Gibco BRL), and antibiotic-antimycotic (Invitrogen). The cells were incubated at 37 °C in a 5% CO2 environment and sub-cultured every 2–3 days when cells reached 80% confluency, as described in our previous works [45, 46]. Following thawing, the cells were used after the second sub-culture for the transdifferentiation experiments.

Transdifferentiation of BDNF-MSCs

For the electrical stimuli-based transdifferentiation, BDNF-MSCs (cell density of 1 × 105 cells per device) were seeded and grown on the graphene IDE in MM and electrical stimuli were applied as described in our previous work [20]. Briefly, following the cell seeding, the cells on graphene IDE circuits were incubated for 2 days to ensure cellular attachment and growth. Then, different electrical stimuli (25, 50, or 100 mV at 50 Hz) were applied for 10 min every day for 10 days using a potentiostat (CHI Instruments 600 Series) electrically wired to the graphene IDE circuits. The electrical stimuli application conditions were selected based on physiological electric fields [47], our previous work [36, 48, 49], and previous reports [28, 50,51,52,53,54]. The electrical fields on the graphene devices can also be seen in Supplementary Information section Fig. S5.

Paracrine Activity

Paracrine activity was evaluated to detect the amount of secreted neurotrophic growth factors from the transdifferentiated cells. During transdifferentiation, the MM media was collected and replaced with fresh MM every 2 days and stored frozen for ELISA analysis. The released nerve growth factor (NGF), glial cell-derived neurotrophic factor (GDNF), and brain-derived neurotrophic factor (BDNF) amounts were detected through corresponding ELISA kits (Abcam Rat Beta NGF ELISA kit ab100757 and Promega BDNF and GDNF Emax Immuno Assay Systems) by following the manufacturer’s procedure.

Immunocytochemistry

Immunocytochemical (ICC) analysis was conducted on control BDNF-MSCs (non-electrically stimulated cells) and electrically transdifferentiated BDNF-MSCs (tBDNF-MSCs) to characterize the degree of differentiation into SC-like phenotypes and neuronal-like phenotypes as described in our previous work [44]. Briefly, the cells were fixed in 4% paraformaldehyde for 20 min at room temperature and washed with PBS. Then, they were incubated in blocking buffer composed of 5% normal donkey serum (NDS, Jackson ImmunoResearch), 0.4% bovine serum albumin (BSA; Sigma), and 0.2% Triton X-100 (Fisher Scientific) in PBS for 1 h. A panel of primary antibodies, including glial cell markers of calcium binding protein Rab-α-S100 (1:500, Jackson ImmunoResearch), Mo-α-S100β (1:1000, Jackson ImmunoResearch), and low-affinity neurotrophin factor Rab-α-p75 (1:1000, Jackson ImmunoResearch), and neural cell markers of Mo-α-Tuj1 (1:2000, Jackson ImmunoResearch), Mo-α-GFAP (1:250, Jackson ImmunoResearch), and Mo-α-MAP2ab (1:250, Jackson ImmunoResearch), were applied and incubated overnight at 4 °C. After the incubation, the cells were rinsed with PBS and subsequently incubated with the corresponding secondary antibodies donkey-α-mouse-Cy3 (1:500, Jackson ImmunoResearch), donkey-α-rabbit-Cy3 (1:500, Jackson ImmunoResearch), and DAPI (1:50, Jackson ImmunoResearch) for nuclear staining. All the dilutions for primary and secondary antibodies were performed in the same blocking buffer. The cells were incubated for 90 min in the dark at room temperature and then rinsed with PBS before fluorescence microscopy imaging. At least 10 images were taken from different sites and the images were analyzed using the ImageJ image analysis software.

RT-PCR Analysis

To identify the expression of the selected genes, RT-PCR analysis was conducted. Briefly, total RNA was extracted from transdifferentiated BDNF-MSCs using TRI Reagent (Molecular Research Center) by following manufacturer’s instructions. The cDNA was synthesized from the total RNA using iScript™ reverse transcription super mix (BioRad) according to the supplier’s instructions. RT-PCR analysis was conducted using the primer sequences for selected genes using GoTaq® green master mix by following manufacturer’s instructions. The primer sequences, obtained from IDT-DNA Technologies, are listed in Table S1 in Supplementary Information section. The RT-PCR amplification conditions were 95 °C for 30 s (denaturation), 56 °C for 1 min (annealing), and 72 °C for 1 min (extension) for a total of 30 cycles. For each RT-PCR experiment, an initial denaturation of 95 °C for 2 min and final extension of 72 °C for 1 min was applied. Amplification products were run in 2.0% agarose gel electrophoresis and visualized by ethidium bromide staining.

Co-culture of BDNF-MSCs with PC12-TrkB Cells

To evaluate the biological activity of the secreted growth factors, the BDNF-MSCs were co-cultured with PC12-TrkB cells. PC12-TrkB cells were grown in RPMI-1640 media supplemented with 10% horse serum (HS) and 5% FBS at 37 °C under 5% CO2 atmosphere and sub-cultured every 2 days until reaching 70–80% of confluency. Then, PC12-TrkB cells (density of 1 × 104 cell/cm2) were seeded on mouse laminin coated 6-well plates and incubated for 24 h to allow attachment and growth. After the incubation, the electrically transdifferentiated tBDNF-MSCs on the graphene devices (1 × 105 cells per device) were placed on trans-well membrane inserts (Corning trans-well inserts) and integrated to 6-well plates to provide non-contact co-culture conditions, where the seeded PC12-TrkB cells were below the membrane inserts, while the tBDNF-MSCs with graphene devices in the inserts were cultured above. The cells were maintained in 3 mL of co-culture media composed of 70% PC12-TrkB cell culture media and 30% of MM. The cells were co-cultured for 3 days at 37 °C under 5% CO2 atmosphere. During the incubation, electrical stimuli (100 mV at 50 Hz for 10 min/day) was applied to tBDNF-MSCs, while no electrical stimuli were applied to the PC12-TrkB cells. For this purpose, graphene devices with tBDNF-MSCs were removed from the trans-well membrane inserts and placed in another petri dish containing co-culture media. The electrical stimulus was applied for 10 min and then the graphene devices with tBDNF-MSCs were placed back to the trans-well membrane inserts (see Fig. S6 for an image of experimental setup). Therefore, the PC12-TrkB cells were not exposed to any electrical stimuli that can bias the results. Co-culture of tBDNF-MSCs with PC12-TrkB cells and culture of PC12-TrkB cells alone with no electrical stimuli was conducted as controls. At the end of the experiment, an ICC analysis using primary antibody Tuj1 and secondary Alexa Fluor 488 (1:1000, Molecular Probes-Life Technologies) was conducted and neurite extension from the PC12-TrkB cells were visualized using fluorescence microscopy. The neurite extension of PC12-TrkB cells for various conditions was imaged using the ImageXpress Micro high content imaging system (Molecular Devices) and quantified through the neurite outgrowth module of the MetaXpress software (Molecular Devices) as mentioned in our previous studies [31, 33].

Co-culture of BDNF-MSCs with AHPCs

To evaluate the influence of BDNF-MSCs and electrical stimuli on the differentiation of adult hippocampal progenitor cells (AHPCs, provided by F. Gage, Salk Institute, La Jolla, CA), a contact co-culture of BDNF-MSCs with AHPCs in the presence and absence of electrical stimuli was conducted. BDNF-MSCs were grown and sub-cultured as described above. For this experiment, we used green fluorescence protein (GFP) expressing AHPCs similar to our previous study [55] to distinguish AHPCs from BDNF-MSCs. AHPCs were grown and sub-cultured in Dulbecco’s modified Eagle’s medium/Ham’s F-12 (DMEM/F-12, 1:1; Omega Scientific) supplemented with 2.5 mM L-glutamine, 1 × N2 supplement (Gibco BRL), and 20 ng/mL basic fibroblast growth factor (human recombinant bFGF; Promega Corporation). For contact co-cultures, BDNF-MSCs were plated onto graphene devices previously coated with poly-L-ornithine (100 μg/ml in sterile water) and laminin (10 μg/ml in Earle’s Balanced Salt Solution) at a density of 1 × 105 cells per device. After cellular attachment and growth of BDNF-MSCs on the devices for 1 day, AHPCs were plated onto the monolayers of BDNF-MSCs at the same cell density (1 × 105 cells per device). The cells were maintained in co-culture media, consisting of AHPC differentiation media and BDNF-MSC MM at a ratio of 50:50. Following the cell seeding, attachment, and growth, electrical stimuli of 100 mV at 50 Hz for 10 min/day was applied for 10 days while the cells were in contact co-culture to enable simultaneous transdifferentiation. As controls, AHPCs and BDNF-MSCs were plated separately in the same co-culture medium at their respective densities. In addition, a contact co-culture of the cells without any electrical stimuli was also used as another control. Cells were maintained at 37 °C in a 5% CO2 atmosphere. Co-culture media was refreshed every 2–3 days. At the end of the experiment, the ICC tests along with the quantification analysis were conducted for the selected markers. In addition, neurite outgrowth from the AHPCs was evaluated using the ImageJ software.

Statistical Analysis

ANOVA analysis by Tukey’s method with 95% confidence interval was used to evaluate the statistical significance. For each analysis, at least three independent experiments were conducted (data sets with n ≥ 3), and results were presented as average ± standard deviation.

Results

BDNF-MSCs seeded on graphene devices were easily attached, grown and proliferated on the device surface. The transdifferentiation of BDNF-MSCs on graphene devices was conducted by applying different electrical stimuli, free from any additional chemical induction paradigm. We selected 25, 50, and 100 mV at 50 Hz to investigate the effect of voltage parameters on the transdifferentiation behavior of BDNF-MSCs to build on our previous work. Our purpose was to observe if changing the electrical conditions can modulate the transdifferentiation of BDNF-MSCs into different glial or neuronal lineages. Our immunocytochemical results indicated that 10% of the cells subjected to 25 mV stimulation were immunolabeled with MAP2ab and Tuj1 neuronal markers showing neuronal-like behavior while the remaining 90% depicted immunolabeling with SCs markers, S100, S100β, and p75, indicating transdifferentiation into SC-like phenotypes (Fig. 1a, b). As the applied voltage increased to 50 mV, we observed a decrease in the tendency to transdifferentiation into neuronal phenotypes, which is accompanied by an increase in transdifferentiation to SC-like phenotypes. The MAP2ab and Tuj1 immunolabeled cells decreased to 5%, while the percentages of cells labeled with SC markers increased up to 95%. As we further increased the voltage to 100 mV, complete transdifferentiation of BDNF-MSCs into SC-like phenotypes was noted (Fig. 1 a, b). The control cells, growing on the graphene substrates but not exposed to any electrical stimuli, did not show any transdifferentiation tendency to any neural cell lineage (Fig. 1a, b). In our previous studies [12, 20, 33], we did not observe any significant expression of a common glial marker, glial fibrillary acidic protein (GFAP). However, at lower voltages, below 50 mV, a significant increase in GFAP expression was noted. These tests were conducted on both laser and thermally annealed devices. Although the structures of laser annealed, and thermally annealed graphene are different (thermally annealed are more hydrophilic and less rough than the laser annealed as indicated in the Supplementary Information section Figs. S1S3, we did not observe statistically significant differences in the immunocytochemistry results. Therefore, the rest of the experiments were conducted with the laser-annealed devices, which are more hydrophobic and rougher, to be consistent with our previous study [20].

Fig. 1
figure1

a Immunocytochemistry results showing the effect of different electrical stimuli conditions (25, 50, and 100 mV at 50 Hz) on the transdifferentiation of BDNF-MSCs into SC-like or neuronal phenotypes. Control group: BDNF-MSCs not treated with electrical stimuli. Immunolabeling was conducted with the following glial cell markers—GFAP, s100, s100β, and p75—and neuronal cell markers—MAP2ab and Tuj1. Staining with red (Cy3) and nuclear staining with blue (DAPI). The initial cell density was 1 × 105 cell per device and fluorescent images were acquired after 10 days of transdifferentiation. b Quantitative analysis of immunolabeling for the selected neuronal and glial markers. Same letters and asterisks represent statistically significant differences (p < 0.05). Immunocytochemistry results for MBP marker. c The control group not treated with electrical stimuli. d BDNF-MSCs transdifferentiated into SC-like phenotypes using electrical stimuli of 100 mV at 50 Hz for 10 min/day for 10 days. Initial cell density, 1 × 105 cell per device. Almost 90% of the cells in the treatment group showed MBP staining

SCs are known to provide a myelin sheath and participate in remyelination in response to injury. Therefore, it is vital to have the myelination property for the transdifferentiated stem cells to function properly. Our results in Fig. 1c, d indicate that tBDNF-MSCs, having SC-like phenotypic properties, showed significant immunolabeling (90% ± 5.5) for myelin basic protein (MBP). This result demonstrates the functional transdifferentiation of the cells using electrical stimuli.

Another indication of successful transdifferentiation with functional outcomes is detectable levels of secreted neurotrophic factors. tBDNF-MSCs possessing SC-like phenotypes are also capable of secreting neurotrophic factors similar to endogenous Schwann cells. Our results in Fig. 2 indicate that as we increased the voltage from 25 to 100 mV, we detected a significant increase in NGF secretion via ELISA which was significantly higher than the untreated control group. This is also another indication of the successful transdifferentiation of BDNF-MSCs into SC-like phenotypes. For the case of BDNF, the decrease in the voltage and simultaneous tendency to differentiate into neuronal lineages resulted in a decrease in the secreted BDNF amount. However, the amount of BDNF secreted remained similar for the case of 100 mV and control. We were not able to detect any significant glial cell-derived neurotrophic factor (GDNF) secretion on our experiments.

Fig. 2
figure2

ELISA test results showing the effect of different electrical stimuli conditions (25, 50, and 100 mV at 50 Hz) on the paracrine activity of BDNF-MSCs transdifferentiated into SC-like phenotypes. Control group: BDNF-MSCs not treated with electrical stimuli. Initial cell density: 1 × 105 cells per device. Incubation time: 10 days. All cases have statistically significant difference (p < 0.05), except GDNF secretion and BDNF secretion for 50 mV, 100 mV, and control

To investigate the biological activity of the released neurotrophic factors from the tBDNF-MSCs transdifferentiated on graphene devices, a co-culture experiment with PC12-TrkB cells was conducted. We specifically selected PC12-TrkB cells since they are responsive to BDNF and NGF that stimulates neurite outgrowth in PC12-TrkB cells. The co-culture experiment includes the following groups: PC12-TrkB cells alone as control with no electrical stimuli, PC12-TrkB cells co-cultured with BDNF-MSCs with no electrical stimuli, and PC12-TrkB cells co-cultured with tBDNF-MSCs with electrical stimuli (100 mV at 50 Hz) as indicated in Fig. 3. The results show that the control PC12-TrkB cells without any electrical stimuli or co-cultured with BDNF-MSCs displayed few neurite extensions, whereas the co-culture with BDNF-MSCs triggered neurite extensions in PC12-TrkB cells (Fig. 3a, b). However, co-culture of PC12-TrkB cells with tBDNF-MSCs further accompanied by electrical stimuli (which is only applied to tBDNF-MSCs but not to the PC12-TrkB cells) led to a significant increase in neurite extension (Fig. 3c). The quantification of neurite extension for the indicated cases via MetaExpress analysis revealed that PC12-TrkB neurite outgrowth reached 5 μm per cell for the PC12-TrkB cells co-cultured with tBDNF-MSCs and treated with electrical stimuli extension (Fig. 3d). This result is in accordance with the findings for neurotrophic factor secretion of tBDNF-MSCs represented in Fig. 2.

Fig. 3
figure3

Bioactivity of neurotrophic growth factors (NGF, BDNF, and GDNF) secreted from BDNF-MSCs transdifferentiated into SC-like phenotypes on PC12-TrkB cells neurite extension. a PC12-TrkB cell control, no electrical stimuli. b PC12-TrkB cells co-cultured with BDNF-MSCs, no electrical stimuli. c PC12-TrkB cells co-cultured with tBDNF-MSCs with electrical stimuli (100 mV at 50 Hz). d Quantitative analysis of neurite outgrowth of PC12-TrkB cells at different conditions (p < 0.05). PC12-TrkB cell density, 1 × 104 cells/cm2. BDNF-MSC cell density, 1 × 105 cells per device. Incubation time, 3 days

A detailed RT-PCR analysis further supported the ICC and paracrine activity results identifying the expression of selected genes following the transdifferentiation of the BDNF-MSCs. RT-PCR analysis showed that expressions at the mRNA levels of S100, S100β, NGF, and Krox20 were significantly increased as the applied voltage increased from 25 to 100 mV (Fig. 4). This supports the previous ICC and paracrine activity results suggesting that 100 mV at 50 Hz was the optimum condition to transdifferentiate BDNF-MSCs into SC-like phenotypes. The higher expression of S100 and S100β ensures the SC-like lineage while the higher expression in NGF and Krox20 demonstrates the functional transdifferentiation. Krox20 is known to control myelination in the peripheral nervous system [56] and expression of this gene upon transdifferentiation indicates the myelination function of tBDNF-MSCs as endogenous SCs. Similar to the paracrine activity results, we did not observe a significant change in BDNF expression. Moreover, contrary to the ICC results, we were not able to detect any p75 expression in RT-PCR analysis.

Fig. 4
figure4

RT-PCR test results showing the effect of different electrical stimuli conditions (25, 50, and 100 mV at 50 Hz) on the expression of selected genes in BDNF-MSCs transdifferentiated into SC-like phenotypes. Control group: BDNF-MSCs not treated with electrical stimuli. Initial cell density, 1 × 105 cell per device. Incubation time, 10 days

In our previous comparative proteomics study [32], we addressed significantly regulated proteins and potential pathways upon transdifferentiationa via chemical induction. Among these proteins, ILK, CTGF, and CAV-1 were identified as significantly downregulated upon transdifferentiation. ILK is known for radial sorting of axons, SC remyelination and stimulating neurite outgrowth via NGF in the peripheral nervous system. Similar to our previous proteomics results, the expression of ILK gene was downregulated upon electrical transdifferentiation. SCs are also known to inhibit central nervous system myelination and secrete connective tissue growth factor (CTGF). We observed that CTGF expression was also downregulated in RT-PCR analysis upon electrical transdifferentiation, suggesting cell impact on myelination (Fig. 4). We also observed a significant downregulation of Cav-1 gene expression in the tBDNF-MSCs, which is consistent with a decrease of CAV-1 in the distal nerve stump after axotomy when SCs return to their pre-myelinating phenotype (Fig. 4). Particularly, the expression of CTGF and CAV-1 significantly downregulated as we increase the voltage to 100 mV, which suggests SC-like functional transdifferentiation of BDNF-MSCs (Fig. 4).

We also investigated the synergetic interactions of BDNF-MSCs that are in contact co-culture with AHPCs in the presence of electrical stimuli. The contact co-culture has no negative or positive effect on successful transdifferentiation of BDNF-MSCs into SC-like phenotypes (Fig. 5). Almost 90% of the BDNF-MSCs transdifferentiated into SC-like phenotypes. However, the contact co-culture significantly improved the neurite extension and differentiation of AHPCs. In the absence of electrical stimuli, AHPCs maintained their proliferation and showed neurite extension (14.5 μm per cell) during the contact co-culture with BDNF-MSCs (Fig. 5). On the other hand, the application of electrical stimuli during the contact co-culture with BDNF-MSCs affected AHPC attachment and proliferation and caused a decrease in the AHPC cell number. Nevertheless, the differentiation and neurite extension of AHPCs showed a significant increase (31.4 μm per cell) (Fig. 5). AHPCs alone also showed a positive response to electrical stimuli by extending their neurites; however, this was further enhanced by co-culture with BDNF-MSCs and electrical stimuli (Supplementary Information section Fig. S7).

Fig. 5
figure5

Immunolabeling of BDNF-MSCs alone or in contact co-culture with AHPCs in the presence or absence of electrical stimuli. Initial BDNF-MSC density, 1 × 105 cells per device. Initial AHPC density, 1 × 105 cells per device. Electrical stimuli, 100 mV at 50 Hz for 10 min/day for 10 days. Selected markers: s100, s100β, p75, and Tuj1

Discussion

The fabricated graphene circuits provided a multifunctional platform for the attachment, growth, proliferation, and differentiation of BDNF-MSCs, similar to our previous work with regular unmodified MSCs [20]. BDNF-MSCs provided significantly higher BDNF secretion compared to the non-genetically engineered MSCs while possessing similar cell viability, proliferation, and migration [57]. It was observed that BDNF-MSCs were able to easily attach to the surface of the post-processed (either laser or thermal annealing), inkjet-printed graphene circuits without the need for deposition of any conventional extracellular matrix (ECM) substrate (e.g., laminin) enhancing cellular attachment [27, 58]. This could be due to the ππ interactions between the aromatic amino acids in the cell membrane and the graphene layers [27, 59, 60] or the hydrophobic nature of graphene [25, 27] that facilitates physicochemical interactions mediating the immobilization of ECM proteins and enhancing cellular attachment and proliferation [27, 28]. The easy attachment, growth, and proliferation of BDNF-MSCs on graphene substrates facilitate the transdifferentiation of these cells via electrical stimuli.

Previous studies in the literature have mostly used combined chemical and electrical stimuli to control stem cell transdifferentiation [19]. Although this approach is promising, it is difficult to maintain precise control of chemical induction in a complex in vivo environment. With this motivation, we aimed to eliminate the requirement for chemical stimuli, which is mostly based on using expensive chemicals and growth factors, and use the advantages of sole electrical stimuli to control stem cell transdifferentiation [19]. The sole use of electrical stimuli also has the potential to enable the control of stem cell transdifferentiation and fate commitment in a complex in vivo environment. Our results suggest that chemical stimuli can be completely eliminated, and sole electrical stimuli can dominate the stem cell transdifferentiation. Based on our results, it can also be seen that changing the applied voltage can be further used to determine the final transdifferentiated state and desired cell lineage of the initial stem cell population. For instance, we observed that the lower voltages triggered modest transdifferentiation to neuronal phenotypes, while the higher voltages promoted complete transition to SC-like phenotypes. This brings about the possibility of achieving spatial, local, and selective control of stem cell transdifferentiation via electrical stimuli which is not possible with chemical stimuli-based transdifferentiation approaches applied in bulk. Moreover, most of the chemical stimuli-based transdifferentiation procedures take longer times (around 15 days) to obtain the desired SC-like phenotypes [9, 10, 33, 61]. However, we found out that the electrical condition of 100 mV at 50 Hz applied for 10 min/day for 10 days can be considered as an optimal condition to achieve complete transdifferentiation to SC-like phenotypes. Compared to our previous tests, which achieved the SC-like phenotypes from MSCs by applying the same electrical conditions for 15 days, we were able to achieve SC-like phenotypes from BDNF-MSCs by using the same electrical conditions for 10 days. This could be attributed to the genetically engineered nature of BDNF-MSCs or influence of electrical stimuli.

Our ICC, ELISA, and RT-PCR results along with the co-culture experiments with PC12-TrkB cells suggested the successful and functional transdifferentiation of BDNF-MSCs into SC-like phenotypes using electrical stimuli on graphene-based devices. Several different physiochemical mechanisms could potentially play roles in transdifferentiating BDNF-MSCs. Possible molecular signaling pathways mediating the transdifferentiation could be through promoting the upregulation of key neural gene expression in focal adhesion kinase (FAK) signaling or mitogen-activated protein kinase signaling (p38) pathways [22, 23, 28, 58, 62, 63]. Additional or synergistic mechanisms could act via altering cellular membrane potential through hyperpolarization and depolarization, modification of ion channels, activation of calcium channels, and upregulation of the ERK pathway [64,65,66]. Other potential reasons could be the activation of various signaling pathways such as MAPK, PI3K, and ROCK [51, 67] and the increase in intracellular ROS generation [51, 68].

It was previously reported in the literature that the BDNF-MSCs can also promote the morphological differentiation of neuronal-like as well as oligodendrocyte-like brain stem/progenitor cells [55]. In addition, the positive response of neuronal cells to the electrical stimuli has also been known. Considering this, we demonstrated the synergistic effect of BDNF-MSCs co-cultured with AHPCs and electrical stimuli to develop novel strategies for nervous system rescue and repair. This synergistic approach suggested that it is possible to simultaneously control the BDNF-MSC transdifferentiation along with neurite extension of AHPCs upon electrical stimuli, which could potentially be used for neural regeneration not only for peripheral nervous system but also for central nervous system rescue and repair.

Conclusions

Building upon our previous studies, this work revealed the significance of electrical parameters in controlling the transdifferentiation of BDNF-MSCs into neuronal or glial lineages. This electrical stimuli-based approach eliminates the limitations of conventionally used chemical induction-based, bulk transdifferentiation approaches. Furthermore, it may potentially provide spatial and precise in situ-in vivo transdifferentiation and allow control over the fate commitment of implanted stem cell populations paving the way for implementation of flexible graphene electrodes at the injury site providing electrical simulation for nerve regeneration.

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Acknowledgments

SKM would like to thank Dr. Robert Langer, on the occasion of his 70th birthday, for all his inspiration and support. The authors acknowledge the generous funding support by the Roy J. Carver Charitable Trust under award number 15-4615, US Army Medical Research and Materiel Command under contract W81XWH-11-1-0700, as well as by the Iowa State University College of Engineering and Department of Mechanical Engineering. The authors are also grateful to the Carol Vohs Johnson Chair for the additional funds in support of this work.

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Uz, M., Hondred, J.A., Donta, M. et al. Determination of Electrical Stimuli Parameters To Transdifferentiate Genetically Engineered Mesenchymal Stem Cells into Neuronal or Glial Lineages. Regen. Eng. Transl. Med. 6, 18–28 (2020). https://doi.org/10.1007/s40883-019-00126-1

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Keywords

  • Electrical stimuli
  • Stem cell differentiation
  • Glial and neuronal lineage
  • Neural regeneration
  • Flexible electronics