Intermittent electrical stimuli for guidance of human mesenchymal stem cell lineage commitment towards neural-like cells on electroconductive substrates
Introduction
Endogenous electric fields serve as an important physiological cue in many of the biological processes, right from maintaining cellular homeostasis to embryonic development to healing [1], [2], [3]. The disruption of these electric field results in abnormality of morphogenesis [4]. In order to understand its crucial role in nervous system development and regeneration, clinicians have used electric stimulation in restorative therapy to recover the lost functions, following spinal cord injury [5]. In this backdrop, several in vitro findings have shown the role of exogenous electromagnetic fields in regulating various cellular events such as cell-surface receptor accumulation, cytoskeletal reorganization, cell shape changes, preferential alignment, change in intracellular calcium ion level, transmembrane channel activation, etc. [6], [7].
Similar to other cell types (endothelial cells, fibroblast, muscle cells, nerve cells etc.) [2], electric field exposure to stem cells also plays a crucial role in eliciting appropriate stem cell response. For instance, Lim et al. [8] recorded a change in expression of osteogenesis related genes and differentiation of human alveolar bone-derived mesenchymal stem cells (hABMSCs), when exposed to extremely low frequency pulsed electromagnetic fields (ELF-PEMFs) [8]. In a similar study by Hronik-Tupaj et al. [1], a delayed but an increase in osteogenic gene marker expression was recorded within 10 days in hMSCs exposed to a 20 mV/cm 60 kHz electric field. The authors also hypothesized the possible mechanisms of how electric stimulation affects cell differentiation, which includes altering membrane potential through hyperpolarization and depolarization, modification of ion channels including density and distribution of receptors, calcium channel activation, and up regulation of ERK pathway. Besides, direct current (DC) electric field (EF) has been proved to guide neurite growth and migration of neurons and other types of cells [9]. Hammerick et al. [10] observed the orientation and migration of multipotent adipose derived stromal cells (mASCs) to stronger electric field in a dose dependent manner due to the activation of MAPK, PI3K and ROCK signaling pathways. Another instance of electric field effects on stem cells is the study on the differentiation of embryonic stem cells to cardiomyogenic lineage, that resulted due to an increase in intracellular ROS generation [11].
Considering the biological significance of electric stimulus, electroconductive substrates have been developed to support tissue growth as well as to allow for localized delivery of electrical signals. Electroconductive materials seem to have enormous potential in biomaterials due to its tendency to control space, level and duration of stimulus and its capacity to aid tissue repair. Therefore, to improve the regenerative therapies involving stem cells, it is necessary to understand and determine the effect of electroconductive substrates and electric field stimuli on stem cell fate processes. Recently, Prabhakaran et al. [12] carried out electrical stimulations of nerve stem cells cultured on conducting PLLA/PANI nanofibrous polymeric scaffolds. Nerve stem cells exposed to a steady potential of 1.5 V for 1 h displayed higher neurite extensions, which facilitated accelerated growth, differentiation and regeneration of nerve. This study clearly illustrates the application of electrical impulse as target signals for neural differentiation, even in the absence of differentiation growth factors [12].
Stem cell-based tissue engineering is one of the recent approaches in regenerative medicine that aims at restoring or enhancing tissue and organ function [13]. In particular, mesenchymal stem cells (MSCs) are considered as competent cell source for tissue engineering applications, in terms of immune compatibility, ease of isolation and its potency to generate multiple differentiated progenies [14], [15], [16]. Besides their capacity to self-renew, proliferate and differentiate into chondrocytes, osteoblasts, and adipocytes [17], it can differentiate into skeletal [18] and cardiac muscle cells [19], hepatocytes [20], and neural cells [21], [22] when stimulated using specific biological and chemical inducers. Although questionable, this validates its tendency to overcome its germ layer commitment to differentiate into somatic cells of non-mesodermal origin [21]. Traditionally, stem cell differentiation was controlled using genetic and molecular mediators (e.g., growth factors, transcription factors). However, in recent years, the findings have demonstrated the response of stem cells to the physical cues presented by the matrix, though the intrinsic mechanisms involved have not been well understood [1], [23]. This has generated significant interest, since directing stem cell growth and lineage commitment by the physical signals from the engineered construct can act as potential therapeutic target in regenerative medicine and tissue engineering [23]. A multitude of external factors that were identified to have a direct role in determining stem cell fate were matrix stiffness [24], roughness [25], surface chemistry [26], nanotopography [27], biofunctionalization [28], nanoscale textures (pores, grooves, tubes, pillars) [29], [30], [31] etc. By precisely tuning some of these microenvironmental cues, the growth and differentiation of stem cells can be induced locally, thereby avoiding usage of toxic biochemical reagents or soluble factors. In this regard, studies on conductivity effect in eliciting appropriate stem cell response have seldom been reported till date [32]. Bio-interactive matrices having inherent conductivity and electroactivity can serve as a suitable platform for guiding the differentiation of stem cells into electro-responsive cells such as neural or muscle cells. The underlying electrical conductivity of the substrates enables cell–cell cross talks as well as cell–substrate interactions thereby modulating cellular adhesion and differentiation [33], [34]. This serves as an attractive option of providing electrical cues locally to investigate the response of stem cells to microenvironment cues [34].
Conducting polymers like polypyrrole, polythiophene, poly(3-methylthiophene), polyaniline, poly(3,4 ethylene dioxythiophene) have been extensively investigated for its biological and medical applications, as they exhibit stability, cytocompatibility and flexibility in processing [35], [36]. The conductivities of such polymers can be enhanced up to 12 orders of magnitude from 10−10 S/cm to 102 S/cm, depending on the polymer, the type and extent of doping [37]. Amidst various classes of conducting polymers, polyaniline (PANI) is one of the most promising materials for tissue engineering application and has been chosen as the model system for this study. Being an excellent matrix supporting growth and proliferation of cardiac myoblasts [38] and nerve cells [39], [40] with enhanced neurite extensions [41], it is also proven to be locally and systemically non-toxic in vivo [42]. A study by Jun et al. [43] highlights the potential of incorporating PANI in PLCL matrix to produce electrically conductive composite fibers for the differentiation of C2C12 skeletal myoblasts. Though, significant enhancement in myoblast proliferation was not achieved on these substrates, the fibers containing PANI could promote differentiation with longer myotube formation.
Given the potential role of substrate conductivity and endogenous electric field in exerting physical cues, the present study attempts to understand the stem cell behavior on substrates with varying conductivity in the presence of external electric stimuli. In particular, the morphology of hMSCs on PANI films with tunable conductivity was closely examined to determine the impact of external electric field on stem cell differentiation. Immunocytochemical and RT-PCR analysis of neural markers was also evaluated to check if substrate conductivity induces a switch in lineage commitment to neural-like cells. While a range of material characterization techniques, i.e. XPS, Nanoindentation and four point probe method were used to characterize surface chemistry/physical properties of doped PANI substrate, a host of biochemical analysis, including MTT, flow cytometry, RT-PCR and immunocytochemistry were employed to probe the cellular response.
Section snippets
Synthesis of PANI
The synthesis of PANI was based on the oxidation of aqueous solutions of aniline by ammonium peroxydisulfate (APS) at −30 °C [44]. Briefly, freshly distilled aniline (Merck, India) was dissolved in 1 m Hydrochloric acid (HCl) (S.D. Fine Chem. Ltd, India) and maintained at −30 °C ± 1 °C in a cryocool immersion cooler CC100-II, with iso-propanol as the cooling agent. Likewise, a mixture of APS (Merck, India) [(NH4)2S2O8] solution, mixed in 1 m HCl and 6 m LiCl (S.D. fine Chem. Ltd, India) was
PANI Substrate characterization
The wide spectrum scan of undoped and doped PANI showed characteristic C1s and N1s peaks along with an extra Cl2p signal in the doped PANI. The C1s peak (Fig. 1a and b) was deconvoluted into 4 signals, all arising from sp2 carbons attributed to the aromatic carbon, carbon attached to the amine nitrogen, carbon bonded to imine nitrogen and a minor signal due to C–O bond. As shown in Fig. 1a, the corresponding C1s peaks in the undoped PANI appear at 284.2, 284.6, 285.6 and 287.0 eV, respectively.
Discussion
The use of conducting polymers as scaffolds for tissue regeneration can serve as attractive means to deliver electric signals for manipulating the cell growth and differentiation non-invasively, in a controlled and localized manner. This can be accomplished without altering the culture medium composition in vitro or by addition of chemical/growth mediators, that can later affect the neighboring healthy tissues in vivo [57]. Considering these biological aspects, we sought to analyze whether the
Conclusions
We have developed conducting platforms using PANI films for assessing the ability of hMSCs to respond to the electric field stimulated culture medium without any biochemical inducers. The distinctive morphological change as well as enhanced cytoskeletal elongation of the cultured hMScs is significantly influenced by the intermittent delivery of weak electric field stimulation (100 mv/cm) at regular interval of 24 h in culture. The use of such culture protocol subsequently triggered the
Acknowledgments
The authors thank Dr. Praveen C Ramamurthy, Materials Engineering, IISc for the useful discussion and for allowing to synthesize PANI in his lab. The authors are also grateful to Prof. Maneesha Inamdar of JNCASR for her valuable suggestions regarding stem cell culture. One of the authors, GT would like to thank Ranjith K for his assistance during PANI synthesis and Sterin NS for his help in conductivity study. The authors also thank MNCF facility members, CeNSE, IISc for their assistance in
References (71)
- et al.
Regulation of mesenchymal stem cell adhesion and orientation in 3D collagen scaffold by electrical stimulus
Bioelectrochemistry
(2006) - et al.
Endogenous electrical currents and voltage gradients in Xenopus embryos and the consequences of their disruption
Dev Biol
(1994) - et al.
Implanted spike wave electric stimulation promotes survival of the bone marrow mesenchymal stem cells and functional recovery in the spinal cord injured rats
Neurosci Lett
(2011) - et al.
The role of electro-osmosis in the electric-field-induced movement of charged macromolecules on the surfaces of cells
Biophys J
(1981) - et al.
In vitro effects of direct current electric fields on adipose-derived stromal cells
Biochem Biophys Res Commun
(2010) - et al.
Electrospun conducting polymer nanofibers and electrical stimulation of nerve stem cells
J Biosci Bioeng
(2011) - et al.
Neuronal differentiation of human mesenchymal stem cells: changes in the expression of the Alzheimer's disease-related gene seladin-1
Exp Cell Res
(2006) - et al.
Neural differentiation of human mesenchymal stem cells: evidence for expression of neural markers and eag K+ channel types
Exp Hematol
(2006) - et al.
Control of stem cell fate by physical interactions with the extracellular matrix
Cell Stem Cell
(2009) - et al.
Matrix elasticity directs stem cell lineage specification
Cell
(2006)