Elsevier

Biomaterials

Volume 35, Issue 24, August 2014, Pages 6219-6235
Biomaterials

Intermittent electrical stimuli for guidance of human mesenchymal stem cell lineage commitment towards neural-like cells on electroconductive substrates

https://doi.org/10.1016/j.biomaterials.2014.04.018Get rights and content

Abstract

In the context of the role of multiple physical factors in dictating stem cell fate, the present paper demonstrates the effectiveness of the intermittently delivered external electric field stimulation towards switching the stem cell fate to specific lineage, when cultured in the absence of biochemical growth factors. In particular, our findings present the ability of human mesenchymal stem cells (hMSCs) to respond to the electric stimuli by adopting extended neural-like morphology on conducting polymeric substrates. Polyaniline (PANI) is selected as the model system to demonstrate this effect, as the electrical conductivity of the polymeric substrates can be systematically tailored over a broad range (10−9 to 10 S/cm) from highly insulating to conducting by doping with varying concentrations (10−5 to 1 m) of HCl. On the basis of the culture protocol involving the systematic delivery of intermittent electric field (dc) stimulation, the parametric window of substrate conductivity and electric field strength was established to promote significant morphological extensions, with minimal cellular damage. A time dependent morphological change in hMSCs with significant filopodial elongation was observed after 7 days of electrically stimulated culture. Concomitant with morphological changes, a commensurate increase in the expression of neural lineage commitment markers such as nestin and βIII tubulin was recorded from hMSCs grown on highly conducting substrates, as revealed from the mRNA expression analysis using Reverse Transcriptase–Polymerase Chain Reaction (RT-PCR) as well as by immune-fluorescence imaging. Therefore, the present work establishes the key role of intermittent and systematic delivery of electric stimuli as guidance cues in promoting neural-like differentiation of hMSCs, when grown 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)

  • X. Hu et al.

    The influence of elasticity and surface roughness on myogenic and osteogenic-differentiation of cells on silk-elastin biomaterials

    Biomaterials

    (2011)
  • J.E. Phillips et al.

    Human mesenchymal stem cell differentiation on self-assembled monolayers presenting different surface chemistries

    Acta Biomater

    (2010)
  • L. Ferreira et al.

    New opportunities: the use of nanotechnologies to manipulate and track stem cells

    Cell stem Cell

    (2008)
  • M.R. Doran et al.

    Defined high protein content surfaces for stem cell culture

    Biomaterials

    (2010)
  • K.S. Brammer et al.

    Hydrophobic nanopillars initiate mesenchymal stem cell aggregation and osteo-differentiation

    Acta Biomater

    (2011)
  • R. Olivares-Navarrete et al.

    Direct and indirect effects of microstructured titanium substrates on the induction of mesenchymal stem cell differentiation towards the osteoblast lineage

    Biomaterials

    (2010)
  • E.A. Ostrakhovitch et al.

    Directed differentiation of embryonic P19 cells and neural stem cells into neural lineage on conducting PEDOT-PEG and ITO glass substrates

    Arch Biochem Biophys

    (2012)
  • N.K. Guimard et al.

    Conducting polymers in biomedical engineering

    Prog Polym Sci

    (2007)
  • P. Humpolicek et al.

    Biocompatibility of polyaniline

    Synt Met

    (2012)
  • I. Jun et al.

    The stimulation of myoblast differentiation by electrically conductive sub-micron fibers

    Biomaterials

    (2009)
  • R. Cruz-Silva et al.

    Comparative study of polyaniline cast films prepared from enzymatically and chemically synthesized polyaniline

    Polymer

    (2004)
  • J. Xiang et al.

    Templated growth of polyaniline on exfoliated graphene nanoplatelets (GNP) and its thermoelectric properties

    Polymer

    (2012)
  • I. Titushkin et al.

    Regulation of cell cytoskeleton and membrane mechanics by electric field: role of linker proteins

    Biophys J

    (2009)
  • J. Settleman

    Tension precedes commitment-even for a Stem Cell

    Mol Cell

    (2004)
  • K.J. Gilmore et al.

    Skeletal muscle cell proliferation and differentiation on polypyrrole substrates doped with extracellular matrix components

    Biomaterials

    (2009)
  • C.-C. Teng et al.

    The inhibitory effect of CIL-102 on the growth of human astrocytoma cells is mediated by the generation of reactive oxygen species and induction of ERK1/2 MAPK

    Toxicol Appl Pharmacol

    (2012)
  • K. Jin et al.

    Induction of neuronal markers in bone marrow cells: differential effects of growth factors and patterns of intracellular expression

    Exp Neurol

    (2003)
  • G. Thrivikraman et al.

    Substrate conductivity dependent modulation of cell proliferation and differentiation in vitro

    Biomaterials

    (2013)
  • S. Jain et al.

    Vertical electric field stimulated neural cell functionality on porous amorphous carbon electrodes

    Biomaterials

    (2013)
  • M. Hronik-Tupaj et al.

    Osteoblastic differentiation and stress response of human mesenchymal stem cells exposed to alternating current electric fields

    Biomed Eng Online

    (2011)
  • K.R. Robinson

    The responses of cells to electrical fields: a review

    J Cell Biol

    (1985)
  • M.R. Cho et al.

    Transmembrane calcium influx induced by ac electric fields

    FASEB J

    (1999)
  • K. Lim et al.

    Effects of electromagnetic fields on osteogenesis of human alveolar bone-derived mesenchymal stem cells

    Biomed Res Int

    (2013)
  • J.F. Feng et al.

    Guided migration of neural stem cells derived from human embryonic stem cells by an electric field

    Stem Cells

    (2012)
  • H. Sauer et al.

    Effects of electrical fields on cardiomyocyte differentiation of embryonic stem cells

    J Cell Biochem

    (1999)
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