DNA Methylation and miRNA Profiling of NSC Differentiation on Electrospun POMA Nanofibers

Research Article

Austin J Biosens & Bioelectron. 2016; 2(1): 1019.

DNA Methylation and miRNA Profiling of NSC Differentiation on Electrospun POMA Nanofibers

Augustus T Mercado1,2, Yui Whei Chen-Yang2,3, Ting Yu Chin1,3, Jui-Ming Yeh2,3* and Chung-Yung Chen1,3

¹Department of Bioscience Technology, Chung Yuan Christian University, Taiwan

²Department of Chemistry, Chung Yuan Christian University, Taiwan

³Center for Biomedical Technology, Chung Yuan Christian University, Taiwan

*Corresponding author: Jui-Ming Yeh, Department of Chemistry and Chung Yung Chen, Department of Bioscience Technology, Chung Yuan Christian University, No. 200, Chung-Pei Road, Chung-Li, 32023, Taiwan

Received: June 09, 2016; Accepted: July 20, 2016; Published: July 25, 2016


A genetic level assessment of Neural Stem Cell (NSC) differentiation towards a synthesized novel nanofibrous material is needed for its biomedical applications. NSCs differentiated on a neat electrospun Poly-O-Methoxyaniline (POMA) underwent a modified method of PCR Selective Suppression Hybridization (PSSH) to provide a DNA methylation profile. DNA fragments were sequenced and identified using BLAST. Then, identified genes were annotated using Gene Set Toolkit (Gestalt), Database for Annotation, Visualization and Integrated Discovery (DAVID) and Pathway Interaction Database (PID). Eleven genes (NCAM2, KIF5C, DMD, ELMO1, F2R, FZR1, HCN1, DAGLA, HSPB8, RAB38, and CMKLR1) corresponding to neural stem cell functions were methylated after differentiation on the nanomaterial. This indicates that POMA scaffold was efficient in enhancing differentiation of NSCs. Moreover, the miRNA profiles of NSC before and after differentiation were assessed by miRNA microarray. POMA nanofibers stimulated the expression of miR-1224, miR-204- 3p, miR-30c-1-3p, and miR-92-5p, as compared to a flat substrate (PDL).The differential upregulation of these miRNAs further enhanced the differentiation and proliferation potential of POMA as a scaffold for neuronal tissue engineering.

Keywords: NSC differentiation; Poly-o-methoxyaniline; DNA methylation; PCR Selective Suppression Hybridization; miRNA microarray


Neural tissue engineering aims to repair neural tissue through the use of biological tools such as normal or genetically engineered cells and creating Extracellular Matrix (ECM) equivalents along with potent synthetic tools such as biomaterial for scaffold design. Recent studies showed that Neural Stem Cells (NSCs) possess a great potential as main seed cells in nerve regeneration because of the potential therapeutic effect to a number of CNS disorders [1]. However, increasing evidence has shown that stem cell development requires a niche, which is a local microenvironment that houses stem cells and regulates their self-renewal and differentiation [2]. For this reason, the physical environment of the cells can affect its differentiation. Advanced nano-based polymers are being considered to be used as a substrate biomaterial for nerve engineering due to the simple fact that the nanostructure mimics more closely the ECM dimensions; thus, useful for promoting cell attachment, migration, and proliferation. Thus, there are a number of studies using different types of material as potential biomaterial for the growth and differentiation of NSCs.

Conducting polymers can be applied as biosensors, scaffolds for tissue engineering, neural probes, drug-delivery devices, and bioactuators [3]. Polyaniline is one of the oldest known conducting polymers that has been often studied due to its beneficial properties as a polymeric nanostructure [4]. Many have studied this material because of its low cost, high electrical conductivity and good environmental stability. A prepared neat electrospun poly(omethoxyaniline) (designated as POMA) was previously characterized [5] and used for efficient neural stem cell differentiation. Results of the cell viability assay, immunofluorescence staining, qRT-PCR and calcium image studies confirmed that POMA showed enhanced NSC attachment and accelerated differentiation [6].

However, stem cell differentiation is controlled by a complex pattern of gene regulation, which is governed by an array of cellular signaling pathways. It is believed that extracellular factors and intracellular process, including epigenetic modification, control cell fate specification and differentiation of NSCs [7]. These modifications include DNA methylation, histone modification and non-coding RNA expression, like miRNAs.

Analysis of methylation states in genomic DNA has provided insights into biological phenomena as disparate as genomic imprinting, human disease, and atypical floral morphologies [8]. Bisulfite treatment can be used to determine the methylation states of individual cytosines in DNA. When bisulfite-treated DNA is amplified by PCR, 5mC on the template strand pairs with guanine on the newly synthesized strand; converted cytosine, which is uracil, pairs with adenine. The methylation patterns of individual DNA molecules therefore can be inferred from the sequences of sub-cloned PCR products. Conversely, differentially methylated DNA fragments can be selected through PCR Selective Suppression Hybridization (PSSH). It is an adapted and modified technique known as Suppression Subtractive Hybridization [9]. PSSH is a PCRbased technique to analyze methylation states in the genomic DNA. This is used to selectively amplify target DNA fragments, which are differentially methylated, and simultaneously suppress non-target DNA amplification. The method is based on suppression PCR effect wherein long inverted terminal repeats attaches to DNA fragments that can selectively suppress amplification of undesirable sequences in PCR procedures.

The DNA methylation and miRNA expression profiles of neural stem cell differentiation towards a synthetic and inorganic microenvironment have never been elaborately discussed. The objective of this study is to identify the DNA methylation changes and miRNA expression levels triggered by extrinsic mechanical signals of POMA for regulating the proliferation and differentiation of NSCs. Genomic DNA of NSCs underwent PSSH to have a DNA methylation profile and identify differentially methylated genes after differentiation. Additionally, the effect of the substrate on the miRNA expression was compared with PDL. The data obtained from this study will help in discovering the related mechanisms associated with NSC differentiation on a biomaterial. These would aid in the assessment of the biocompatibility of the material for neural tissue engineering, thereby its potential for regenerative medicine.

Materials and Methods

Preparation of poly (o-methoxyaniline)

First, 0.164 mol of o-methoxyaniline was added into 400 mL of 2.0 M CaCl2 in HCl and cooled to about 0 °C by stirring in an ice bath. Then, 0.038 mol of ammonium per sulfate, which served as an oxidant, was dissolved in 100 mL of 2.0 M CaCl2 in HCl solution at 0°C and it was then mixed with the o-methoxyaniline/CaCl2/HCl solution by stirring for 12 h. Subsequently, an intense blue-green precipitate of POMA was collected and washed on a funnel fitted with filter paper (90 mm diameter, Advantec No. 7 filter paper). The final product was washed usually five times with 1.0 MHCl (aq) to remove any grey salts. Finally, the precipitate was dedoped by stirring with 500 mL of 1.2 M NH4OH (aq) for 48 h. The Emeraldine Base (EB) of POMA was obtained by filtering and drying at a temperature of 60 °C under vacuum for at least 24 h. The yield of POMA was typically 20%. The EB of the POMA powder was dissolved in a co-solvent system of THF/DMF (50:50 v/v) as an electrospinning solution in the concentration range 1–6 wt%. This solution was placed in a plastic syringe (Terumo, 5 ml), and the POMA fibers were produced by electrospinning, controlling specific parameters (Q, H and V). The electrical field used for electrospinning was generated by a variable high-voltage power supply (Matsusada, AU-40R0.75), which can apply voltages as high as 40 kV. The solution was fed by a syringe pump (KD Scientific Model 200) with the feeding rate tuned for conjugated polymer solutions (Q = 0.02, 0.03 and 0.04 mL min-1). A metallic needle was connected to a high-voltage source set at 15 or 20 kV, and nozzle-to-collector distances of 8, 10, 12 and 14 cm were set to collect the electrospun fiber mat samples. Finally, the collected mat was dried in an oven for 1 h at a temperature of 100 °C.

Cell culture and genomic DNA extraction

Pregnant Sprague-Dawley rats were purchased from the National Laboratory Animal Center (Taiwan, ROC). All animal operations received were in humane care according to the Guidelines for Care and Use of Experimental Animals [10]. This study was also approved by the Animal Research Ethics Board of Chung Yuan Christian University (Taiwan, ROC).

NSCs were isolated from the brains of Sprague-Dawley rat embryos on day 14-15. The embryo brains were dissected, cut in to pieces and treated with digesting solution containing 30 mg/ ml papain, 50 mM EDTA, 2 mg/ml cysteins, and 150 mM CaCl2.

Then, the dissected tissue were treated with DNase I, added into 10% horse serum and collected by centrifugation. The neural stem cells were expanded in Dulbeco’s Modified Eagle Medium (DMEM) supplemented with N2, 20 ng/ml Epidermal Growth Factor (EGF), 10 ng/ml basic Fibroblast Growth Factor (bFGF) and the following antibiotics: 0.5% penicillin and 1% streptomycin. Cells were allowed to grow in a CO2 incubator at 37 °C, 5% CO2 and 95% humidity. The number of live cells was counted by trypan blue exclusion assay in a hemocytometer. The electrospun nanofibers on the block of glass were exposed to UV radiation overnight. The cells were seeded on the nanofibers placed in a petri dish at a density of 1 x 106 cells per dish and cultured with DMEM-F12 medium containing 1% N2 supplement. Cells were allowed to grow and proliferate for two days before 0.5 mM of dcAMP was added. Cell adhesion, proliferation, and differentiation were assessed using a Phase Contrast Light microscope. Cells were harvested after 7 days of differentiation and genomic DNA extraction was done using the Phenol-chloroform method. The genomic DNA was extracted on the seventh day from the cultivated cells with and without dbcAMP. The range of the DNA concentration obtained was between 300-450 ng/μl.

Bisulfite conversion of DNA

Bisulfite treatment was performed using the Qiagen® Epitech Bisulfite kit in accordance to the manufacturer’s protocol. A reaction mixture consisted of 1 ng ~ 2 μg of DNA solution, 85 μL of bisulfite mix, 35 μL DNA protect buffer, and diluted to 140 μL with RNase-free water. Bisulfite DNA conversion was conducted using a thermal cycler (ABITM, GenAmp PCR system 9700) with the following conditions: 5 minutes at 95°C, 25 minutes at 60°C, 5 minutes at 95°C, 85 minutes at 60°C, 5 minutes at 95°C, and 175 minutes at 60°C. Converted DNA was cleaned up using EpiTect® Spin Columns provided with the kit. Uracil residues of bisulfite-converted DNA samples (1~2 μg) were cleaved using 1~5U of USERTM Enzyme NEB® (NEB, M5505S) suspended in 1X T4 ligase buffer and incubated at 37°C in a water bath. Uracils were replaced with biotin-dCTP (InvitrogenTM) by incubation at 37°C for 30 minutes with other dNTPs. The reaction was stopped by incubating the mixture at 75°C for 20 minutes. To reestablish the phosphodiester bonds of double-stranded biotinylated DNA, the mixture was suspended in 1x T4 DNA ligase buffer for a total volume of 70 μl with 350 units of T4 DNA ligase (TAKARA) and incubated at 4°C overnight or longer for successful ligation.

Separation of biotinylated DNAs

Dynabeads®MyOneTM Streptavidin (InvitrogenTM, 650.01) with a stock concentration of 10mg/mL and a binding capacity of ~20μg double-stranded biotin-labeled DNA per mg Dynabeads® was initially washed with 1X BW buffer according to the manufacturer’s instructions. Washings were done repeatedly as deemed necessary using an Easy 50 EasySep® Magnet (STEMCELL TECHNOLOGIES) to separate the supermagentic Dynabeads® from the buffer and preservatives. Dynabeads® were then resuspended in 2X BW buffer to a final concentration of 5μg/μL. For optimum immobilization, an equal volume of biotin-labeled DNA suspended in water is added to the resuspended Dynabeads® to reduce the NaCl concentration in the buffer from 2M to 1M. Incubation was done at room temperature with gentle rotation for 15 minutes. Biotin-labeled DNA bound to Dynabeads® was separated by three-minute incubations in the Easy 50 EasySep® Magnet and washed with 1X BW buffer. Washed supernatant was collected in a separate tube and labeled as methylated DNA from differentiated cells. The immobilized biotin-labeled DNA was dissociated from Dynabeads® by incubation at 90°C in 95% form amide and 10mM EDTA for two minutes. Released biotin-labeled DNA contained the unmethylated DNA from undifferentiated NSCs. Methylated and unmethylated samples were desalted using Amicon® Ultra-0.5 50K centrifugal filters (MILLIPORE Corp.) according to manufacturer’s protocol. To eliminate the single-stranded extensions, biotin-labeled DNA (0.1 μg/μl) was suspended in 1X Mung Bean nuclease buffer with 1U of Mung Bean Nuclease (NEB®,M0250S) per μg DNA at 30°C for 30 minutes.

PCR Selective Suppression Hybridization (PSSH)

Figure 1 shows the schematic diagram on principle of PCR Selective Suppression Hybridization (PSSH). The methylated and unmethylated DNA from NSCs was ligated with Ad1 and Ad2 adapters, respectively. Adapter sequences were adapted from Diatchenko et al. [9] and were custom-synthesized by Genomics® BioSci& Tech, Taiwan. The adapter sequences used are as follows: