Three-Dimensional Electroconductive Hyaluronic Acid Hydrogels

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Three-Dimensional Electroconductive Hyaluronic Acid Hydrogels Incorporated with Carbon Nanotubes and Polypyrrole by CatecholMediated Dispersion Enhance Neurogenesis of Human Neural Stem Cells Jisoo Shin, Eun Jung Choi, Jung Ho Cho, Ann-Na Cho, Yoonhee Jin, Kisuk Yang, Changsik Song, and Seung-Woo Cho Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00568 • Publication Date (Web): 06 Sep 2017 Downloaded from http://pubs.acs.org on September 7, 2017

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Three-Dimensional Electroconductive Hyaluronic Acid Hydrogels Incorporated with Carbon Nanotubes and Polypyrrole by Catechol-Mediated Dispersion Enhance Neurogenesis of Human Neural Stem Cells Jisoo Shin1, Eun Jung Choi2, Jung Ho Cho1, Ann-Na Cho1, Yoonhee Jin1, Kisuk Yang1, Changsik Song2*, and Seung-Woo Cho1* 1

Department of Biotechnology, Yonsei University, Seoul 03722, Republic of Korea

2

Department of Chemistry, Sungkyunkwan University, Suwon 16419, Republic of Korea

*Corresponding authors: Prof. Seung-Woo Cho Department of Biotechnology, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 03722, Republic of Korea Tel: +82-2-2123-5662; Fax: +82-2-362-7265; E-mail: [email protected] Prof. Changsik Song

1 Environment ACS Paragon Plus

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Department of Chemistry, Sungkyunkwan University, 2066 Seobu-ro, Jangan-gu, Suwon 16419, Republic of Korea Tel: +82-31-299-4567; Fax: +82-31-290-7075; E-mail: [email protected]


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ABSTRACT

Electrically conductive hyaluronic acid (HA) hydrogels incorporated with single-walled carbon nanotubes (CNTs) and/or polypyrrole (PPy) were developed to promote differentiation of human neural stem/progenitor cells (hNSPCs). The CNT and PPy nanocomposites, which do not easily disperse in aqueous phases, dispersed well and were efficiently incorporated into catecholfunctionalized HA (HA-CA) hydrogels by the oxidative catechol chemistry used for hydrogel crosslinking. The prepared electroconductive HA hydrogels provided dynamic, electrically conductive three-dimensional (3D) extracellular matrix environments that were biocompatible with hNSPCs. The HA-CA hydrogels containing CNT and/or PPy significantly promoted neuronal differentiation of human fetal neural stem cells (hfNSCs) and human induced pluripotent stem cell-derived neural progenitor cells (hiPSC-NPCs) with improved electrophysiological functionality when compared to differentiation of these cells in a bare HACA hydrogel without electroconductive motifs. Calcium channel expression was upregulated, depolarization was activated, and intracellular calcium influx was increased in hNSPCs that were differentiated in 3D electroconductive HA-CA hydrogels; these data suggest a potential mechanism for stem cell neurogenesis. Overall, our bio-inspired, electroconductive HA hydrogels provide a promising cell-culture platform and tissue-engineering scaffold to improve neuronal regeneration.

KEYWORDS Electroconductive hydrogel, hyaluronic acid, catechol-mediated dispersion, human neural stem cell, neurogenesis


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INTRODUCTION

Electrical signals are critical cues for neuronal development, function, and maturation. Neurons communicate with other neurons, muscle cells, and gland cells using electrical signals via neural networks and synapses.1-2 Electrical signals are essential for neuronal cells to survive, differentiate, and activate neural circuits,3 and electrical activity plays an essential role in the early development of the nervous system by regulating expression profiles of genes related to voltage-gated ion channels.4 Thus, electrical stimulation with various electroconductive materials, such as graphene, polyaniline (PANI), carbon nanotube (CNT), polypyrrole (PPy), and poly(3,4-ethylenedioxythiophene) (PEDOT), has been used to promote neuronal differentiation and maturation of neural stem/progenitor cells (NSPCs).5-10 Although studies on the use of electrical cues to enhance NSPC neurogenesis have provided promising results, they have primarily been conducted on two-dimensional (2D) substrates, which do not emulate threedimensional (3D) in vivo environmental conditions. Because a 3D microenvironment with an extracellular matrix (ECM) has been shown to provide biochemical and biophysical cues for enhancing NSPC neurogenesis,11-12 we aimed to reconstitute an electroconductive environment under 3D ECM conditions. We hypothesized that hyaluronic acid (HA), which is abundant in brain ECMs, could be used as a base biomaterial for creating an electroconductive 3D environment. HA has been shown to bind to receptors on neurons and on NSPCs, such as CD44 and RHAMM, thus facilitating neuronal morphogenesis and NSPC differentiation in the central nervous system.13-15 Further, HA has been used widely as a functional biomaterial scaffold in neural tissue engineering, and numerous studies have demonstrated the use of an HA-based 3D culture system for maintaining and differentiating


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NSPCs.16-17 However, the beneficial effects of HA and electroconductivity have not yet been combined in a 3D system because non-functionalized conductive materials with high conductivity are difficult to disperse evenly in aqueous solutions.18-19 Thus, the ability to effectively disperse and incorporate highly electroconductive materials into a 3D HA hydrogel may provide an electrically active 3D ECM platform that promotes NSPC neurogenesis for neuronal tissue engineering. In this study, we present a bio-inspired HA-based electroconductive hydrogel, which provides a 3D electroconductive ECM environment for promoting neuronal differentiation and functional maturation of two types of human NSPCs (hNSPCs): human fetal neural stem cells (hfNSCs) and human induced pluripotent stem cell (hiPSC)-derived neural progenitor cells (hiPSC-NPCs). To create an electroconductive environment, two electroconductive materials, single-walled CNT and PPy, were incorporated into HA hydrogels by oxidative catechol chemistry, which was inspired by a mussel adhesive foot protein (Figure 1a).20-22 Because CNT and PPy are good electrical conductors with low impedance and high charge storage capacity,2325

a catechol-functionalized HA (HA-CA) hydrogel incorporated with CNT and/or PPy showed

lower impedance than a bare HA-CA hydrogel. Interestingly, oxidative catechol-mediated crosslinking to form the HA-CA hydrogel facilitated dispersion of oxidized PPy and/or CNT in the 3D HA hydrogel. Enhanced neuronal differentiation, electrophysiological functionality, and calcium influx were observed in 3D cultures of hfNSCs and hiPSC-NPCs within electrically conductive HA-CA hydrogels containing CNT and/or PPy (Figure 1b). Furthermore, we found that the 3D electroconductive HA environment affected hNSPC differentiation and functionality by upregulating expression and activation of depolarization of voltage-gated calcium channels.


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MATERIALS AND METHODS

Preparation of Electroconductive HA-CA Hydrogels. The HA-CA conjugate was prepared by conjugating dopamine hydrochloride (Sigma, St. Louis, MO, USA) to HA (Mw = 200 kDa, Lifecore Biomedical, Chaska, MN, USA) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (TCI Co., Tokyo, Japan)/N-hydroxysulfosuccinimide (NHS) (Sigma) chemistry as previously described.20-22 The resultant HA-CA conjugate was confirmed using 1H nuclear magnetic resonance (NMR, Bruker 400 MHz, Bruker, Billerica, MA, USA). The degree of substitution was calculated by measuring the absorbance of the HA-CA conjugate solution at 280 nm wavelength. In this study, single-walled carbon nanotubes (SWNTs) were purchased from Unidym (Menlo Park, CA, USA), which were synthesized by a high-pressure carbon monoxide (CO) decomposition process (HiPCO). The SWNTs possess individual diameters of 0.8~1.2 nm and individual lengths of 100~1000 nm. SWNTs were received as powders and dispersed in polymer solution of HA-CA. To generate electroconductive HA hydrogels, CNTs were added to a 2% (w/v) HA-CA pre-gel solution in phosphate-buffered saline (PBS) at a final concentration of 1 mg/ml and were dispersed in the pre-gel solution by mildly sonicating (26 W) at 20% amplitude for 5 min. Undispersed CNTs were removed by centrifugation at 13,000 rpm for 30 min. Pyrrole (Py) was added to a CNT-dispersed HA-CA solution or to a bare HA-CA solution at a final concentration of 30 mM. Oxidative crosslinking of HA-CA to form an HA-CA hydrogel was carried out by the addition of a sodium periodate solution (NaIO4, Sigma) that was equimolar to the catechol group in the HA-CA conjugate. The incorporation of CNTs and PPy into the HA-CA hydrogel was confirmed by scanning electron microscope (SEM) (S4800, Hitachi, Tokyo, Japan) at a voltage of ~10–15 kV. Raman spectra for checking the interactions


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between HA-CA and electrical additives (CNT, PPy) were acquired on alpha 300R (WITec, Ulm, Germany) confocal Raman microscopy.

Swelling, Degradation, and Moduli of Electroconductive HA-CA Hydrogels. To measure swelling ratios, hydrogels were incubated in PBS at 37 °C for 1 week. At various timepoints, the swollen hydrogels were weighed after removal of excess water on the surface of the hydrogels, and the swelling ratio was calculated using the following equation: swelling ratio (%) = (Ws Wi) / Wi × 100, in which Ws is the weight of the hydrogel in the swollen state and Wi is the initial weight of the hydrogel before swelling.26 The degradation rates of the hydrogels were determined by measuring the weights of the degrading hydrogels at various timepoints during treatment with 100 U/ml hyaluronidase (Sigma).21 The elastic moduli of the hydrogels were measured in frequency sweep mode using a rotational rheometer (Bohlin Instruments, Worcestershire, UK).

Measurement of Impedance. Electrochemical impedance spectra of HA-CA hydrogels with varying amounts of CNT and PPy were measured using potentiostats (CompactStat, Ivium Technologies, Eindhoven, Netherlands). Electrochemical impedance spectroscopy measurements were performed at an amplitude of 10 mV and a frequency of 0.1–100,000 Hz with open-circuit voltage, and hydrogels were placed between indium tin oxide (ITO)-coated glass electrodes with 1 mm Teflon spacer.

Cell Culture and Encapsulation. The seeding and culture conditions for the expansion of hfNSCs were described previously.27 The Yonsei University School of Medicine provided the


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hfNSCs, and this study was carried out with the approval of the Research Ethics Committee of Yonsei University College of Medicine (Permit Number: 4-2003-0078). To maintain their capacity for self-renewal, hfNSCs were seeded at a density of 6.0 × 105 cells/ml and were cultured as neurospheres in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 (DMEM/F12) medium (Gibco BRL, Gaithersburg, MD, USA) with N-2 supplement (Gibco BRL), basic fibroblast growth factor (bFGF, 20 ng/ml, Sigma), and leukemia inhibitory factor (LIF, 10 ng/ml, Sigma) under humidified air with 5% CO2 at 37 °C. The hfNSCs that underwent 15–20 passages were used in further experiments. The Yonsei University School of Medicine provided the hiPSC cell line WT3, and the Institutional Review Board (IRB) of Yonsei University (Permit Number: 1040917-201510-BR229-01E) approved this study. The culture conditions for the maintenance of undifferentiated hiPSCs were described previously.28-29 Differentiation of hiPSCs into NPCs was carried out by the addition of 5 µM of dorsomorphin (DM) (Sigma) and 5 µM of SB431542 (Sigma) to media during embryonic body (EB) formation. EBs were attached to culture dishes coated with Matrigel (BD Biosciences, San Jose, CA, USA) and were cultured in neuronal induction medium containing DMEM/F12 medium (Invitrogen, Carlsbad, CA, USA) with N-2 supplement (Invitrogen) and non-essential amino acids (Invitrogen).30 After 6 days, hiPSC-NPCs appeared at the centers of attached EBs and were collected using a hooked Pasteur pipette.31 To encapsulate cells in a 3D hydrogel, cells were suspended in pre-gelation solution at 1.5 × 106 cells per 100 µl of gel and NaIO4 solution was added for gelation.

Viability and Differentiation of hfNSCs and hiPSC-NPCs. To evaluate cytotoxicity, cells were stained with a Live/Dead solution (Invitrogen) according to the manufacturer’s instructions.


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Fluorescently stained cells were observed using a confocal microscope (LSM 880, Carl Zeiss, Oberkochen, Germany), and the percentage of viable cells was determined by counting live cells and dead cells in fluorescent images. To evaluate the neuronal differentiation capacity of encapsulated hfNSCs and hiPSCNPCs in CNT and/or PPy-containing HA-CA hydrogels, hydrogels containing hfNSCs (single cells or neurospheres) were maintained under spontaneous differentiation conditions in DMEM/F12 medium without mitogenic factors. Hydrogels containing hiPSC-NPCs were maintained in neuronal induction medium containing DMEM/F12 medium supplemented with N-2 supplement and non-essential amino acids. Neuronal differentiation of hfNSCs and hiPSCNPCs was analyzed by immunocytochemistry and quantitative real-time polymerase chain reaction (qPCR) after 5 and 7 days in culture, respectively.

Immunocytochemistry. Cells within HA hydrogels were fixed with 2% formaldehyde (Sigma) for 1 h, were membrane permeabilized with 0.5% triton X-100 (Wako, Osaka, Japan) for 1 h at room temperature, were incubated with 5% bovine serum albumin (Wako) for 2 h at room temperature to avoid nonspecific binding of antibodies, and then were incubated with the target primary antibody for 48 h at 4 °C. The target primary antibodies used were mouse anti- neuronal class III β-tubulin (Tuj1) (Cell Signaling Technology, Danvers, MA, USA), rabbit antimicrotubule-associated protein 2 (MAP2) (Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-glial fibrillary acidic protein (GFAP) (Millipore, Temecula, CA, USA), and goat anti-L-type voltage-gated Ca2+ channel (Cav1.2) (Santa Cruz Biotechnology). After primary antibody binding, the cells in the hydrogels were washed three times with PBS at room temperature and were then incubated overnight at 4 °C with secondary antibodies, such as Alexa Fluor 488-


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conjugated goat anti-mouse IgG (Invitrogen), Alexa Fluor 594-conjugated goat anti-rabbit IgG (Invitrogen), and Alexa Fluor 594-conjugated rabbit anti-goat IgG (Invitrogen). Nuclei were stained with 4ʹ,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA). The immunofluorescently stained cells were observed with a confocal microscope (LSM 880).

Quantitative Real-Time Polymerase Chain Reaction (qPCR). For qPCR analysis, encapsulated cells were collected from hydrogels by enzymatic degradation with 100 U/ml of hyaluronidase. Total RNA was extracted from isolated cells using a MiniBEST universal RNA extraction kit (TaKaRa, Shiga, Japan). cDNA was prepared from the extracted RNA by reversetranscription using a PrimeScriptTM strand cDNA synthesis kit (TaKaRa). qPCR was performed with the synthesized cDNA as the template using a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA), TaqMan Fast Universal PCR Master Mix (Applied Biosystems), and TaqMan gene expression assays (Nestin: Hs00707120_s1, octamer-binding transcription factor 4 (Oct4): Hs00742896_s1, Nanog: Hs02387400_g1, Tuj1: Hs00801390_s1, MAP2: Hs00258900_m1, oligodendrocyte transcription factor 2 (Olig2): Hs00258900_m1, GFAP: Hs00909238_g1, 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase): Hs00263981_m1, and Cav1.2: Hs00167681_m1). Gene expression values were calculated using the comparative Ct method and were normalized to an endogenous housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH: Hs02758991_g1).

Calcium Influx Imaging. For calcium imaging, hiPSC-NPCs encapsulated in HA-CA hydrogels were incubated with a Ca2+-sensitive indicator, Fluo-4 AM (Invitrogen), for 1 h at 37 °C and then


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for 30 min at room temperature. Time-series imaging was then performed using a confocal microscope (LSM 880) to observe changes in intracellular Ca2+ levels in response to 300 µM glutamate (Sigma). The fluorescence intensity of each cell was defined by the Region of Indices (ROIs) collected with the Carl Zeiss software ZEN (Carl Zeiss). All of the fluorescence values obtained throughout the time-series imaging were normalized to the fluorescence value obtained from the first image. The normalized changes in fluorescence (∆F/F) were plotted to express the changes in the intracellular Ca2+ influx.

Statistical Analysis. Statistical significance was determined using the unpaired Student′s t-test in GraphPad Prism 6 software (GraphPad Software, San Diego, CA, USA). Values were shown as mean ± standard deviation. We considered *p < 0.05 and **p < 0.01 statistically significant in this study.

RESULTS AND DISCUSSION

Preparation and Characterization of Electroconductive HA-CA Hydrogels. The electroconductive HA-CA hydrogel was prepared by dispersing 1 mg/ml of CNT and/or 30 mM of Py into an HA-CA pre-gel solution. The synthesis of HA-CA conjugate was confirmed by NMR analysis (Supplementary Figure S1). HA-CA hydrogel with 7.4% catechol substitution was used for the study. Due to poor CNT solubility, non-functionalized CNTs with high conductivity do not easily disperse in aqueous polymer solutions and thus, have rarely been used to generate electrically conductive hydrogels, especially for cell encapsulation.18, 32-33 Significant efforts have been made to increase CNT solubility by modifying CNTs with various functional


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groups, including hydroxyl, carboxyl, and amino groups,34-36 but conventional processes for CNT functionalization damage samples and induce CNT defects, which lead to reduced conductivity.37-38 In contrast, in our system, the catechol functional group appears to facilitate dispersion of non-functionalized CNTs into 3D HA-CA hydrogels via hydrophobic and π–π interactions.39-40 Indeed, CNTs were well-dispersed in a pre-gel HA-CA conjugate solution after 5 min of mild and short sonication (Figure 2a), which may preserve the original CNT and HA backbone structures. Severe conditions for sonication can give damages to the CNTs. Thus, the power and duration of sonication along with temperature should be controlled to render CNTs well-dispersed without damages.41 In our experiments, the CNT dispersion was conducted at an ice-bath under a relatively low power of sonication (26 W). Thus, we assume that the dispersed CNTs maintained their intrinsic properties without significant damages by sonication. According to several literatures that utilized the similar sonication conditions to those in our study,42 SWNTs showed high quality of van Hove singularities, indicating little damages to nanotubes from sonication, and individual nanotubes could be well-dispersed by this protocol. The resulting solutions were further purified by centrifugation (13,000 rpm, 30 min), so that some large agglomerates were removed. In contrast, CNTs did not disperse in a pre-gel solution of methacrylated HA (HA-ME) conjugate, which is a conventional HA hydrogel commonly used in biomedical engineering applications (Figure 2a). Gelation of HA-CA with electroconductive materials was induced by adding the oxidizing agent NaIO4, as described previously.21-22 We found that the catechol functional group promoted polymerization of Py via oxidative crosslinking to form the HA-CA hydrogel43 and dispersed PPy via hydrogen bonding and non-covalent hydrophobic and π–π interactions.10, 44-45 Upon induction of gelation, the yellowish Py turned dark brown when added to the HA-CA


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hydrogel, but did not change color when added to the HA-ME hydrogel (Figure 2b); this indicates that oxidative polymerization of Py and dispersion of the resulting PPy only occurred in the HA-CA hydrogel due to its catechol group. As a result of efficient dispersion and even distribution of CNTs and/or PPy within the HA-CA pre-gel solution, the HA-CA hydrogel turned a carbon black color upon gelation (Figure 2c). SEM revealed an even distribution of nanotubes adhered to the internal networks of the HA-CA hydrogel (Figure 2d). We could estimate the width and length of CNTs dispersed in HA-CA hydrogel (40 nm in width and 4 µm in length) (Figure 2d), indicating that there might be negligible fracture of CNTs and both individual CNTs and bundles of CNTs remained intact after sonication. In SEM image, PPy was found to grow into clusters of spherical particles within HA-CA hydrogel construct. It seems that clustered PPy molecules are connected each other, which can strengthen internal network with hydrogel portions (Figure 2d). In the case of pyrrole, it underwent polymerization simultaneously during oxidative gelation, so we can assume safely that almost all of pyrrole would be retained in the HA hydrogel. However, in the case of CNTs, there may be some portions that are not dispersed well and removed after centrifugation. When we tried to measure the weight of the “undispersed” CNT portion of the HA-CA/CNT solution (feeding: 1 mg/ml), 0.2 mg of CNTs could be collected out of 1 ml solution, which suggests the concentration of dispersed CNTs would be 0.8 mg/ml. In the UV-vis spectra of the solution of HA-CA/CNT (data not shown), we were able to observe slightly the van Hove singularities, which suggests that CNTs were dispersed individually to some extent, but since the sonication power was not high enough, some bundles of CNTs might remain in solutions. There have been several methods to improve dispersion of CNTs in aqueous solution by using chemical modification and surfactants. For example, PEGylated CNTs can be used to


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improve dispersion in aqueous solutions without damaging the structure,46 but this approach requires additional chemical steps for CNT functionalization and dispersion. Common surfactants have also been utilized to disperse SWNTs in water, such as sodium dodecyl sulfate (SDS), sodium cholate, and cetyltrimethylammonium bromide,47 but the presence of surfactants in hydrogel may cause cytotoxicity and affect cellular behaviors of stem cells in hydrogel construct. In contrast, our dispersion strategy based on oxidative catechol chemistry allows for highly efficient dispersion of CNTs in 3D HA hydrogel construct via non-covalent interactions of CNTs such as π-π bonding between catechol group in HA-CA hydrogel and SWNTs.48 Therefore, our approach is believed to provide the simplest method to avoid the necessity of chemical conjugation and additives for efficient dispersion which causes cytotoxicity, induces unwanted cellular behaviors, and requires additional procedures. As we already described above, HA-CA was found to mediate more efficiently CNT dispersion in 3D hydrogel compared to HA-ME, probably due to π-π interactions. Actually, there are several published papers which have mentioned that the catechol moiety could help CNTs well-dispersed in aqueous solutions via π-π interactions.49-50 After gelation, we suspect that the catechol moiety in the HA-CA hydrogel may sustain its π-π interactions and secondary interactions with electroconductive additives (CNTs and PPy). To check this, we have measured Raman spectra of the HA-CA/SWNT hydrogels with a laser excitation at 532 nm (Supplementary Figure S2). Although the base lines were distorted due to scattering from the amorphous structure of the hydrogels, Raman spectra clearly showed the slight red-shift of the graphitic (G) peak of SWNTs dispersed into HA-CA hydrogel (SWNT-HA-CA: 1588 cm-1) distinguished from the peak of SWNTs dispersed with SDS (SWNT-SDS: 1592 cm-1),51 which indicates π-π interactions between catechol and SWNT in the HA-CA hydrogel. Additionally, the


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G peak of SWNTs in the HA-CA hydrogel with both additives (SWNTs and PPy) (SWNT/PPyHA-CA) was further shifted to 1576 cm-1, which indicates that there are certain secondary interactions between SWNTs and PPy. Therefore, mechanical properties (e.g., elastic modulus) and electroconductivity of HA-CA hydrogels supplemented with electroconductive additives may be highly influenced by π-π interactions and secondary bonding between catechol group, CNT, and PPy. Incorporation of the electroconductive motifs, CNT and PPy, significantly changed the physical, mechanical, and degradation behaviors of the HA-CA hydrogels. The swelling ratio of a HA-CA hydrogel was measured by calculating the change in the wet weight of the hydrogel after 1 week of incubation in PBS at 37 °C (Figure 2e, f). After 1 week of swelling, HA-CA hydrogels did not show significant weight loss or volume shrinkage, regardless of whether electroconductive materials were incorporated (Figure 2e, f). Swelling reached equilibrium at day 2 for all the hydrogels tested and remained at equilibrium for 7 days (Figure 2f). Compared with the non-electroconductive HA-CA hydrogel without CNT or PPy (CNT 0/PPy 0: 90.1 ± 14.0%), the electroconductive hydrogels incorporated with CNT and/or PPy showed reduced swelling on day 7 (CNT 1/PPy 0: 45.6 ± 4.8%, CNT 0/PPy 30: 27.1 ± 7.7%, and CNT 1/PPy 30: 32.1 ± 6.2%; Figure 2f). CNT may reinforce the HA polymer network in the 3D HA-CA hydrogel via π–π bonds between CNT and the catechol moiety of HA-CA, which may lead to an increase in the crosslinking density of the HA-CA hydrogel.39 Because the oxidative crosslinking process that induces HA-CA hydrogel formation also initiates oxidative polymerization of Py to PPy,52-53 the additional polymerized moieties that interact with the catechol groups in HA-CA may also increase crosslinking of the HA-CA hydrogel.54-55 We tested this hypothesis by


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evaluating the degradation profiles and elastic moduli of the HA-CA hydrogels modified with CNT and PPy. HA-CA hydrogel degradation profiles were generated by enzymatic degradation with hyaluronidase to breakdown the HA backbone. We found that the rate of biodegradation of HACA hydrogels was reduced upon the incorporation of electroconductive nanomaterials. HA-CA hydrogels containing CNT and/or PPy degraded more slowly than the bare HA-CA hydrogel (Figure 2g). Hydrogels containing CNT (CNT 1/PPy 0 and CNT 1/PPy 30) displayed slower degradation rates than CNT-free hydrogels (CNT 0/PPy 0 and CNT 0/PPy 30; Figure 2g) because the non-degradable CNTs likely reinforced the internal structural networks. HA-CA hydrogels with PPy (CNT 0/PPy 30) also showed slow degradation compared with PPy-free HACA hydrogel (CNT 0/PPy 0; Figure 2g) probably due to reinforcement of the crosslinking network by PPy incorporation. Although CNTs cannot be degraded by hyaluronidase, they can be degraded by neutrophil enzymes, such as myeloperoxidase, in vivo,56 thus CNT-incorporated hydrogels may be used as an implantable scaffold for in vivo applications. In terms of biodegradation of CNTs, Kagan et al. have reported that SWNTs catalytically degraded by human neutrophil enzyme myeloperoxidase.56 After incubation of short carboxylated CNTs with human myeloperoxidase and H2O2 at 37ºC for 24 hours, the suspension turned translucent, indicating that CNTs totally degraded.56 In myeloperoxidase-driven biodegradation process, human neutrophils biodegraded 30% and 100% of CNTs in vitro after incubation for 6 and 12 hours, respectively. To a lesser degree, human macrophages with lower level of myeloperoxidase also biodegraded 13% and 50% of CNTs after incubation for 12 and 48 hours, respectively.56 Furthermore, they revealed that biodegrading CNTs did not induce pulmonary inflammatory response in mice, suggesting that CNTs may be a potential material for in vivo applications when


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appropriate concentration was applied.56 In our study, we showed that HA-CA hydrogel containing CNTs completely degraded within 48 hours upon hyaluronidase treatment (Figure 2g). All components in our electroconductive HA-CA hydrogel can degrade completely by enzymatic reactions in vivo, indicating the potential utility of HA-CA composite for biomedical applications. Electroconductive HA-CA hydrogels incorporated with CNT and/or PPy exhibited increased mechanical properties when compared to non-conductive HA-CA hydrogels. The elastic moduli, measured in frequency sweep mode, of HA-CA hydrogels incorporated with CNT and/or PPy increased from 0.44 ± 0.01 kPa (CNT 0/PPy 0) to 1.33 ± 0.34 kPa (CNT 1/PPy 0) or 2.14 ± 0.14 kPa (CNT 0/PPy 30), respectively (Figure 2h, i). The HA-CA hydrogel that incorporated both CNT and PPy had greater elastic modulus (CNT 1/PPy 30: 3.17 ± 0.56 kPa) than the hydrogels that incorporated a single electroconductive material (Figure 2i). These data demonstrated that CNT and PPy improved the physical and mechanical properties of the HA-CA hydrogels and altered the HA-CA gel degradation profiles by reinforcing internal polymer networks. In the case of CNTs, they have been widely used to reinforce polymer structures owing to their own mechanical properties.57 For effective reinforcement, efficient dispersion of CNTs is the one of the critical requirements.57 Thus, catechol-mediated dispersion of CNTs via non-covalent hydrophobic and π-π interactions may contribute to reinforce modulus of HA-CA hydrogel by increasing various bonding and interactions between CNTs and HA-CA polymer backbone. In addition, oxidative polymerization of Py to PPy within HA-CA hydrogel could form additional internal network via hydrogen bonding and non-covalent hydrophobic and π-π interactions between PPy and catechol groups, resulting in increased elastic modulus of HA-CA hydrogel. The Raman spectra of HA-CA hydrogel with electrical additives (CNT and PPy)


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shown above (Supplementary Figure S2) also indicated additional secondary interactions between CNTs and PPy in HA-CA hydrogel, further contributing to increased modulus of HACA hydrogel.

Electrical Properties of Electroconductive HA-CA Hydrogels. The electroconductivity of HA-CA hydrogels incorporated with CNT and/or PPy was evaluated by measuring impedance at different frequencies using an ITO-coated glass electrode. The even distribution of CNTs in the HA-CA hydrogels increased the electroconductivity of the HA-CA hydrogels. Oxidative catechol crosslinking to form HA-CA hydrogels induced oxidative polymerization of Py to PPy, which further contributed to the increased electroconductivity of the HA hydrogels.10, 52 At frequencies greater than 100 Hz, there were no differences in the impedances between the ITO substrate and all of the hydrogel groups.58 At lower frequencies of ~10 Hz, the impedances of the HA-CA hydrogels were reduced in proportion to increases in CNT (0 – 1 mg/ml) and PPy (0 – 50 mM) (Figure 3a, b). As expected, the HA-CA hydrogel containing both CNT (1 mg/ml) and PPy (30 mM) exhibited the lowest impedance, and therefore, the greatest electroconductivity (Figure 3c, d). Since HA hydrogel matrix with the additives (CNT and/or PPy) is electrically conductive and the fluid in the gel is also ionically conductive, our hydrogel system could show improved electrical conductivity to promote neuronal differentiation of hfNSCs. The analysis of the impedance changes according to the weight fractions of conductive additives (CNT and PPy) and HA-CA (Supplementary Figure S3) showed that as the weight fraction of PPy increases, the impedance decreases with an almost linear relationship. However, in the case of CNTs, the impedance values showed the minimum at the weight fraction of ~0.05 (1 mg/ml of CNTs) and electroconductivity seemed to be saturated over this point because the dispersion stability of


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CNTs decreased in 2% (w/v) HA-CA system. Because conductive substrates and scaffolds can enhance neuronal differentiation and maturation by providing shortcuts for effective electrical signal transmission between cells,9, 23, 59-60 the HA-CA hydrogel incorporated with CNT and PPy is expected to provide a highly electroconductive 3D ECM environment that facilitates the functional neuronal differentiation of hNSPCs. In our study, the surface of CNT and PPy might be passivated by insulated layers formed through interaction with HA-CA, affecting interaction of CNT and PPy with stem cells. However, efficient dispersion and even distribution of CNT and PPy in 3D environment of HA-CA hydrogel would increase the effects of electroconductive materials on stem cell differentiation. In addition to direct effects of CNT and PPy as electrical conductors, they could also affect indirectly stem cell differentiation via alteration in mechanical and biophysical properties.

Cytotoxicity of Electroconductive HA-CA Hydrogels. To verify the applicability of electrically conductive HA-CA hydrogels for neural tissue engineering, cytotoxicities of HA-CA hydrogels incorporated with CNT and/or PPy were examined with hfNSCs. Live/dead staining was used to determine the viability of encapsulated hfNSCs within HA-CA hydrogels (Figure 4 and Supplementary Figure S4). Immediately after encapsulation of single cell hfNSCs, no significant cytotoxicity was found (Figure 4a, b), but at day 3 after encapsulation, the viability of single cell hfNSCs was reduced in the HA-CA hydrogels containing electroconductive materials, especially in the PPy-containing hydrogels (CNT 0/PPy 30 and CNT 1/PPy 30; Figure 4a, b). When hfNSCs were cultured as neurospheres in the hydrogels, the cells were highly viable immediately after encapsulation and remained viable 5 days after hfNSC neurosphere encapsulation (Figure 4c) demonstrating that hfNSCs are biocompatible with the


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electroconductive HA-CA hydrogels. CNT concentration above 2 mg/ml seemed to show significant cytotoxicity (Supplementary Figure S4). Considering both cytotoxicity and electrical conductivity, we chose 1 mg/ml CNT and 30 mM PPy which showed high electrical conductivity while less cytotoxic to cells. Although most of the conductive hydrogels described until now are not feasible as 3D cell culture platforms because of cytotoxicity,61 our electroconductive HA-CA hydrogels can provide biocompatible 3D environments with electrical stimulation and can ensure a degree of cell viability during the extended time 3D cultures required for stem cell differentiation.

Enhanced Neurogenesis of hfNSCs and hiPSC-NPCs in Electroconductive HA-CA Hydrogels. Electroconductive HA-CA hydrogels incorporated with CNT and/or PPy promoted neuronal differentiation of hNSPCs in 3D cultures. Single cell hfNSCs encapsulated in HA-CA hydrogels incorporated with CNT and/or PPy were induced to spontaneously differentiate by maintaining the hfNSCs in differentiation media lacking mitogenic factors, such as bFGF and LIF. Immunocytochemical staining of single differentiated hfNSCs in HA-CA hydrogels containing PPy (CNT 0/PPy 30 and CNT 1/PPy 30) 5 days after induction of differentiation revealed increased expression of Tuj1, a neuronal marker (Figure 5a). Expression of Nestin, an undifferentiated neural stem cell (NSC) marker, was reduced in hfNSCs cultured in HA-CA hydrogels containing CNT and/or PPy (Figure 5b). These data indicate accelerated differentiation of hfNSCs in electroconductive HA-CA hydrogels. qPCR analysis confirmed that the incorporation of PPy into HA-CA hydrogels increased expression of Tuj1, a neuronal differentiation marker, and Olig2, an early marker for motor neuron differentiation, but reduced expression of GFAP, an astrocyte marker, in hfNSCs (Figure 5c). Interestingly, expression of


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CNPase, a functional oligodendrocyte marker, increased in the presence of CNT, but was reduced in the presence of PPy (Figure 5c). Because Olig2 can also serve as a marker to determine oligodendrocyte lineage,62 we will investigate the differentiation of stem cells into oligodendrocytes in our electroconductive HA hydrogels. Collectively, our data suggest that our electroconductive HA-CA hydrogel has the potential to promote hNSPC neurogenesis and to modulate differentiation into neurons, astrocytes, or oligodendrocytes by adjusting CNT and PPy concentrations. We found that our electroconductive hydrogels accelerated neuronal differentiation of hfNSC neurospheres, as well as single cell hfNSCs. hfNSC neurospheres that differentiated in electrically conductive 3D HA-CA hydrogels incorporated with CNT and/or PPy exhibited highly extended Tuj1-positive neurite growth when compared to stem cell neurospheres that differentiated in bare HA-CA hydrogel (Figure 6a). Similar to the results from the single cell differentiated hfNSCs, expression of Nestin and the stem cell self-renewal markers, Oct4 and Nanog, were reduced in hfNSC neurospheres in conductive HA-CA hydrogels containing CNT and/or PPy (Figure 6b, c). Expression of the neuronal markers Tuj1, MAP2, and early motor neuron marker (Olig2) increased in hfNSC neurospheres in electrically conductive HA hydrogels containing PPy (CNT 0/PPy 30; Figure 6d). Our data showed that PPy was more effective than CNT in directing hfNSC differentiation into neuronal lineage, irrespective of single cell hfNSCs or cluster hfNSCs. The efficacy of electrically conductive HA-CA hydrogels to improve cellular differentiation was also evaluated with reprogrammed pluripotent stem cells. Many recent studies have evaluated the therapeutic ability of hiPSCs to regenerate neuronal tissue.63-65 Neural rosettes, which are a type of NPC, were differentiated from hiPSCs and cultured in conductive


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HA-CA hydrogels for further analyses. We found that electroconductive HA-CA hydrogels accelerated the differentiation of hiPSC-NPCs. Upon differentiation in 3D HA-CA hydrogels containing CNT and/or PPy and after being cultured for 7 days in differentiation media, hiPSCNPCs exhibited highly extensive and elongated neurites that expressed Tuj1 and MAP2 (Figure 7a). Expression of Oct4, Nanog, and Nestin was reduced in hiPSC-NPCs that were cultured in HA-CA hydrogels containing CNT (CNT 1/PPy 0 and CNT 1/PPy 30) when compared to expression in bare HA-CA hydrogels (Figure 7b, c). qPCR confirmed increased expression of Tuj1 and MAP2 in electroconductive hydrogels (Figure 7d). We also found that CNT was more effective than PPy in promoting neuronal differentiation of hiPSC-NPCs. CNT and PPy may be differentially effective for hfNSC and hiPSC-NPC differentiation because hiPSC-NPCs and hfNSCs have different electrical properties, such as membrane capacitance and cytoplasm conductance. hiPSC-NPCs and hfNSCs with different intrinsic electroresponsive properties may require different optimal electrical conductivities for survival and neuronal differentiation, and thus, may have differential preferences towards PPy and CNT.66-68 In this study, hNSPC neurogenesis in electroconductive HA-CA hydrogels was more affected by the electroconductivity of the hydrogel rather than the mechanical properties of the hydrogel. Numerous studies have demonstrated the importance of the mechanical properties of the substrates and scaffolds used in regulating stem cell differentiation.69-71 The chemistry of CNT and PPy, which alters mechanical and biophysical properties of the hydrogel, can also have significant effects on neuronal differentiation of hNSPCs. Generally, softer substrates or matrices have been shown to be more efficient and suitable for inducing neuronal differentiation of stem cells than stiffer substrates or matrices.72-74 According to these previous literatures, if mechanical modulus is considered only, CNT and/or PPy-incorporated HA-CA hydrogels showing higher


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elastic modulus compared to bare HA-CA hydrogel would impede neurogenesis of hNSPCs. Actually, there were some cases showing slight reduction or no increase in neuronal gene expression of stem cells in electrically conductive hydrogel groups (e.g., Tuj1 expression of single cell hfNSCs in CNT 1/PPy 0 and CNT 1/PPy 30 groups in Figure 5c, MAP2 expression of neurosphere hfNSCs in CNT 1/PPy 30 group in Figure 6d) probably due to the offsetting effect of increased elastic modulus. However, neuronal differentiation of hNSPCs was mostly upregulated in electrically conductive HA hydrogel groups, except for above-mentioned three cases. Although the elastic moduli of the electrically conductive hydrogels increased upon the incorporation of CNT and/or PPy (Figure 2i), we observed that neuronal differentiation of hNSPCs was further enhanced in CNT and/or PPy-containing HA-CA hydrogels. Thus, we believe that the conductivity of the hydrogel may have a greater influence on neurogenesis of encapsulated NSPCs than the mechanical modulus of a hydrogel with a stiffness range of 0.5 – 3 kPa (Figure 2i).

Increase in Calcium Ion Channel Expression and Depolarization-induced Calcium Influx in hiPSC-NPCs Cultured in Electrically Conductive HA-CA Hydrogels. Lastly, we elucidated how increased electrical conductivity enhanced neuronal differentiation of hiPSCNPCs cultured in 3D HA-CA hydrogels. Previous studies on the relationship between electrical activity/calcium currents generated by environmental cues and neurogenesis have demonstrated that the electrical activities of NSCs during early developmental stages trigger expression of genes encoding voltage-gated ion channels, thus elevating the intracellular Ca2+ ion level, which is essential for neurogenesis and for providing feedback to regulate neuronal development.4 Celliot et al. revealed that CNT-induced dendritic calcium electrogenesis resulted in boosted


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action potentials in neurons.23 Based on these data, we hypothesized that the conductivity of CNT and PPy in HA-CA hydrogels would promote neuronal differentiation by increasing expression of voltage-gated Ca2+ channels, especially L-type voltage-gated Ca2+ channel (Cav1.2), whose expression strongly correlates with neuronal differentiation of NSCs.75 We measured gene and protein expression of Cav1.2 in hiPSC-NPCs by immunocytochemistry and qPCR analysis and found that hiPSC-NPCs differentiated within electrically conductive HA-CA hydrogels showed increased expression of Cav1.2, especially within CNT-containing hydrogels (e.g., CNT 1/PPy 0 and CNT 1/PPy 30; Figure 8a, b). Upregulated expression of Cav1.2 Ca2+ channels led to improved depolarization-induced Ca2+ ion influx into hiPSC-NPCs that were differentiated in electrically conductive 3D HA-CA hydrogels. To confirm the enhanced depolarization of hiPSC-NPCs in HA-CA hydrogels containing CNT and PPy, electrophysiological responses of hiPSC-NPCs to a neurotransmitter, glutamate, were visualized using a fluorescent calcium indicator, Fluo-4 AM (Figure 8c). Upon exposure to glutamate stimulation, the increased intracellular Ca2+ ion influx through functional Ca2+ channels was detected in hiPSC-NPCs encapsulated within conductive HA-CA hydrogels (CNT 1/PPy 30) as an increase in the fluorescence intensity of Fluo-4 (Figure 8c, d) relative to cells encapsulated in bare HA-CA hydrogels (CNT 0/PPy 0; Figure 8c, d). These data support our hypothesis that the highly conductive 3D environment provided by the HA-CA hydrogels containing CNT and PPy promotes neurogenesis of human stem cells by increasing expression of functional Ca2+ channels (Cav1.2) and elevating intracellular Ca2+ levels as illustrated schematically in Figure 8e.

CONCLUSIONS


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In summary, we introduced electroactive nanomaterials, CNT and PPy, into a 3D in vivo-like environment to promote neuronal differentiation of hNSPCs. Enhancing hNPSC differentiation is required to develop neurodegenerative disease models and a source of cells for transplantation. However, the current methods do not lead to fully mature cells and require a long time for differentiation. The data presented here describe the electrical conductivity and the biocompatibility of electroactive CNT and PPy to promote neuronal differentiation of clinically relevant hNSPCs. Dispersion and incorporation of CNT and PPy into HA-CA hydrogels, which was facilitated by the catechol functional groups in the HA-CA hydrogels, improved the electrochemical properties of the HA hydrogels. In addition, neuronal differentiation of hfNSCs and hiPSC-NPCs in 3D culture was accelerated more efficiently in the HA-CA hydrogels containing CNT and/or PPy than in the bare HA-CA hydrogel without electroconductive motifs. Incorporation of CNT and PPy allowed for spatial control of conductivity and the 3D bioinspired HA-based environment rescued the viability of hNSPCs. We showed that the conductive 3D environment provided by the CNT/PPy-containing HA-CA hydrogels was beneficial for neuronal differentiation of hNSPCs by showing that calcium channel expression was upregulated, depolarization was activated, and intracellular calcium influx was increased in human stem cells that were stimulated with electrical signals, and these outcomes are indicative of stem cell neurogenesis. To the best of our knowledge, this study is the first account of hNSPC differentiation guided by 3D electroconductive HA hydrogels incorporated with CNT and PPy, and this bio-inspired electroconductive HA hydrogel provides a useful culture platform and tissue-engineering scaffold for stem cell-mediated neuronal regeneration.


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ASSOCIATED CONTENT Author Information Corresponding Authors *Correspondence and requests for materials should be addressed to S.-W.C. ([email protected]) and C. Song. ([email protected]). Author Contributions J.S., E.J.C., and J.H.C. performed the experiments. A.-N.C. and K.Y. provided the cells. S.-W.C. and C.S. designed and supervised the project. J.S., Y.J., C.S., and S.-W.C. wrote the manuscript.

ACKNOWLEDGMENTS This work was supported by grants (2015R1A2A1A15053771, 2015M3A9B4071076, 2013K1A3A7A03078216, and 2017R1A2B3005994) from the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning.

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Figure 1. Schematic illustration of a mussel-inspired electroconductive 3D HA-CA hydrogel for NSC engineering. (a) Catechol-assisted incorporation of CNT and PPy within 3D HA-CA hydrogel networks. (b) Enhanced neurogenesis of NSCs within electrically conductive HA-CA hydrogel networks.


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Figure 2. Characteristics of electroconductive HA-CA hydrogels. Images of HA-ME and HA-CA solutions with (a) CNTs before and after sonication and with (b) Py before and after gelation. Red dashed circles in (a) and (b) represent CNTs and Py, respectively. (c) Images of bare HA-CA hydrogel formation (top) and CNT/PPy-containing HA-CA hydrogel formation (bottom). (d) SEM images of HA-CA hydrogels with CNTs (upper) and PPy (lower). The red arrowheads indicate incorporated CNTs within the HA-CA hydrogel network, scale bars = 10 µm. (e) Optical images of HA-CA hydrogels before (day 0) and after swelling (day 7). (f) Swelling ratios of HA-CA hydrogels upon incubation at 37 °C in PBS (n = 4). (g) Hyaluronidase-mediated degradation profiles of HA-CA hydrogels (n = 4). (h) Rheological analysis of the elastic moduli of HA-CA hydrogels was performed using the frequency sweep mode. (i) The average elastic moduli of the hydrogels at 1 Hz (n = 4, **p < 0.01 versus CNT 0/PPy 0, ++p < 0.01 versus CNT 0/PPy 30, ##p < 0.01 versus CNT 1/PPy 0).


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Figure 3. Electroconductivity of HA-CA hydrogels incorporated with CNT and/or PPy. (a-c) The impedances of 1-mm-thick hydrogels decreased with increased concentrations of CNT and PPy. (d) The impedance values at 1 Hz and 10 Hz of HA-CA hydrogels with conductive additives. The electroconductivity of the HA-CA hydrogels was higher when they contained both CNT and PPy.


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Figure 4. Cytotoxicity of electroconductive HA-CA hydrogels. (a) Images of live/dead stains of single cell hfNSCs in conductive hydrogels at day 0 and 3, scale bar = 50 µm. (b) Imagebased quantification of live/dead stains of single cell hfNSCs in conductive hydrogels at day 0 and 3 (n = 4–6, **p < 0.01 versus CNT 0/PPy 0, ++p < 0.01 versus CNT 0/PPy 30, ##p < 0.01 versus CNT 1/PPy 0). (c) Images of live/dead stains of hfNSC neurospheres in conductive hydrogels at day 0 and 5, scale bar = 50 µm.


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Figure 5. Differentiation of single cell hfNSCs within electroconductive HA-CA hydrogels 5 days after encapsulation. (a) Double-immunofluorescent staining of hfNSCs in hydrogels with a neuronal marker (Tuj1; green) and an astrocyte marker (GFAP; red), scale bar = 50 µm. qPCR analysis to quantify expression of (b) Nestin (an undifferentiated NSC marker), (c) Tuj1 (a neuronal marker), Olig2 (an early motor neuron marker), GFAP (an astrocyte marker), and CNPase (an oligodendrocyte marker). The levels of relative gene expression were normalized to the CNT 0/PPy 0 group (n = 3–4, *p < 0.05 and **p < 0.01 versus CNT 0/PPy 0, +p < 0.05 and ++p < 0.01 versus CNT 0/PPy 30, #p < 0.05 and ##p < 0.01 versus CNT 1/PPy 0).


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Figure 6. Differentiation of neurosphere hfNSCs within electroconductive HA-CA hydrogels 5 days after encapsulation. (a) Immunofluorescent staining of hfNSC neurospheres in hydrogels 5 days after encapsulation with a neuronal marker (Tuj1), scale bar = 50 µm. qPCR analysis to quantify expression of (b) Oct4 and Nanog (self-renewal stem cell markers), (c) Nestin (an undifferentiated NSC marker), (d) Tuj1 and MAP2 (neuronal markers) and Olig2 (an early motor neuron marker). The levels of relative gene expression were normalized to the CNT 0/PPy 0 group (n = 3–4, *p < 0.05 and **p < 0.01 versus CNT 0/PPy 0, +p < 0.05 and ++p < 0.01 versus CNT 0/PPy 30, #p < 0.05 and ##p < 0.01 versus CNT 1/PPy 0).


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Figure 7. Differentiation of hiPSC-NPCs within electroconductive HA-CA hydrogels 7 days after encapsulation. (a) Double-immunofluorescent staining of hiPSC-NPCs in hydrogels with neural markers (Tuj1; green and MAP2; red), scale bar = 50 µm. qPCR analysis to determine gene expression of (b) Oct4 and Nanog (self-renewal stem cell markers), (c) Nestin (an undifferentiated NSC marker), (d) Tuj1 and MAP2 (neuronal markers). The levels of relative gene expression were normalized to the CNT 0/PPy 0 group (n = 3–4, *p < 0.05 and **p < 0.01 versus CNT 0/PPy 0, +p < 0.05 versus CNT 0/PPy 30).


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Figure 8. Voltage-gated calcium channel ion expression and glutamate-responsive calcium influx in hiPSC-NPCs that differentiated within electroconductive HA-CA hydrogels. (a) Immunostaining of L-type voltage-gated Ca2+ channel (Cav1.2) in hiPSC-NPCs that differentiated within hydrogels, scale bar = 50 µm. (b) qPCR analysis of gene expression of Cav1.2 in hiPSC-NPCs. The level of relative gene expression in each group was normalized to the CNT 0/PPy 0 group (n = 3). (c) Intracellular Ca2+ influx in hiPSC-NPCs within a bare HACA hydrogel (CNT 0/PPy 0) and within electroconductive HA-CA hydrogels (CNT 1/PPy 30) before and after glutamate stimulation, scale bar = 20 µm. (d) Time-course of calcium influx into representative cells. The representative cells are shown within white dashed circles in the (c) images. (e) Schematic illustration showing how the increased conductivity of HA hydrogels enhances neuronal differentiation of NPCs: i) CNTs and PPy enhance expression of L-type voltage-gated Ca2+ channels (Cav1.2) in NPCs, ii) expression of the Cav1.2 channel increases


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Ca2+ influx, iii) the increased influx of calcium upregulates expression of genes related to neurogenesis, and iv) neuronal differentiation of NPCs is promoted.


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