Cardiac Stem Cell Secretome Protects Cardiomyocytes from Hypoxic Injury Partly via Monocyte Chemotactic Protein-1-Dependent Mechanism

Cardiac stem cells (CSCs) were known to secrete diverse paracrine factors leading to functional improvement and beneficial left ventricular remodeling via activation of the endogenous pro-survival signaling pathway. However, little is known about the paracrine factors secreted by CSCs and their roles in cardiomyocyte survival during hypoxic condition mimicking the post-myocardial infarction environment. We established Sca-1+/CD31− human telomerase reverse transcriptase-immortalized CSCs (Sca-1+/CD31− CSCshTERT), evaluated their stem cell properties, and paracrine potential in cardiomyocyte survival during hypoxia-induced injury. Sca-1+/CD31− CSCshTERT sustained proliferation ability even after long-term culture exceeding 100 population doublings, and represented multi-differentiation potential into cardiomyogenic, endothelial, adipogenic, and osteogenic lineages. Dominant factors secreted from Sca-1+/CD31− CSCshTERT were EGF, TGF-β1, IGF-1, IGF-2, MCP-1, HGF R, and IL-6. Among these, MCP-1 was the most predominant factor in Sca-1+/CD31− CSCshTERT conditioned medium (CM). Sca-1+/CD31− CSCshTERT CM increased survival and reduced apoptosis of HL-1 cardiomyocytes during hypoxic injury. MCP-1 silencing in Sca-1+/CD31− CSCshTERT CM resulted in a significant reduction in cardiomyocyte apoptosis. We demonstrated that Sca-1+/CD31− CSCshTERT exhibited long-term proliferation capacity and multi-differentiation potential. Sca-1+/CD31− CSCshTERT CM protected cardiomyocytes from hypoxic injury partly via MCP-1-dependent mechanism. Thus, they are valuable sources for in vitro and in vivo studies in the cardiovascular field.


Introduction
Cardiac Sca-1+ cells showed cardiac stem cell (CSC) properties differentiating into cardiac and endothelial cells, as evidenced by in vitro and in vivo studies [1][2][3][4][5]. Knockdown of Sca-1 transcripts in CSCs resulted in significant inhibition of proliferation and survival through Akt [6]. Sca-1+ CSCs were significantly increased in the mouse heart seven days after acute myocardial infarction (AMI) [7,8], and they migrated from a niche to the infarct zone to repair damaged myocytes after myocardial infarction (MI) under hypoxic conditions [9]. Sca-1 knockout revealed cardiac defects in myocardial contractility and repair consistent with impaired resident CSC proliferative capacity [1,10]. A significant and lasting contribution of Sca-1-derived cells to cardiomyocytes during normal aging were found [11].
Collectively, previous studies have demonstrated that Sca-1+ CSCs are valuable sources for myocardial renewal in the pathophysiological process as well as in the aging process of murine adult hearts. However, Sca-1+ CSCs were found to represent only 2% of total heart cells [1]. Therefore, small numbers of Sca-1+ CSCs present in the adult murine heart and their limited proliferative potential during in vitro culture restrict their use for in vitro and in vivo studies.
Telomerase reverse transcriptase (TERT), a catalytic subunit of telomerase, serves a critical role in stem cell function and tissue homeostasis [12]. Several studies have demonstrated that primary stem cells inserted with the TERT gene have maintained long-term stemness in vitro and have been immortalized without chromosomal aberrations or characteristics of malignant transformation [13][14][15]. Recently, we also demonstrated that TERT-immortalized Sca-1+ adipose stem cells (ASCs TERT ) exhibit stem cell properties similar to those of primary ASCs [16]. Interestingly, TERT-expressing cells in adult hearts were associated with Sca-1 expression [6], indicating that Sca-1 is a valuable surface marker for CSCs exhibiting high TERT activity.
A number of studies have reported that stem cells secrete diverse cytokines, chemokines, and angiogenic and cardiogenic growth factors, resulting in improvement of cardiac function via activation of the endogenous signaling pathways [17,18]. We, and others [16,[19][20][21] have demonstrated that functional improvement and beneficial left ventricular (LV) remodeling by stem cell transplantation into animal models of AMI have been primarily achieved through paracrine actions rather than direct transdifferentiation of the transplanted cells. However, little is known about paracrine factors secreted by CSC and their roles in cardiomyocyte survival during hypoxic condition in vitro mimicking the post-infarcted myocardial microenvironment.
The aims of this study were to establish TERT-immortalized Sca-1+ CSCs that exhibit stem cell properties similar to those of primary Sca-1+ CSCs, to analyze paracrine factors secreted by the immortalized CSCs, and to elucidate their role on cardiomyocyte survival during hypoxia-induced injury.

Isolation of Sca-1+ CSCs from Adult Myocardium
Primary Sca-1+ CSCs were isolated after collagenase treatment followed by magnetic activated cell sorting (MACS) from mouse adult myocardium. Flow cytometry showed that Sca-1+ CSCs were more than 86% pure ( Figure 1A). In confocal microscopic analysis, intense Sca-1 signals were shown in MACS-purified Sca-1+ CSCs with a fibroblast-like morphology ( Figure 1A). For phenotypic characterization of Sca-1+ CSCs, cells were immunostained with antibodies to stem cells or cell lineage markers. Sca-1+ CSCs strongly expressed CD44 and CD106 ( Figure 1B). They also moderately expressed CD29 and CD71. Interestingly, primary Sca-1+ CSCs were heterogeneous with respect to CD31 expression. Some primary Sca-1+ CSCs were negative for CD31 but others were positive for CD31. In accordance with our observation, previous studies [3,[5][6][7][8] also reported that cardiac Sca-1+/CD31´cells exhibit multipotent differentiation potential in vitro and their therapeutic potential in experimental myocardial infarction models, whereas cardiac Sca-1+/CD31+ cells showed endothelial-like characteristics.

Establishment of Human TERT (hTERT)-Immortalized Sca-1+ CSC Lines
To establish hTERT-immortalized mouse Sca-1+ CSC lines, a retroviral vector carrying hTERT-internal ribosome entry site (IRES)-green fluorescent protein (GFP) was constructed ( Figure 2A). Retroviruses were produced in 293GPG packaging cells by transfection with a retroviral vector carrying hTERT-IRES-GFP ( Figure 2B). MACS-purified Sca-1+ CSCs isolated from adult heart tissue were infected with retroviruses harboring hTERT-IRES-GFP, and then selected in a 10-cm culture dish containing puromycin during a three-week subculture period. Then, hTERT-immortalized Sca-1+ CSCs were further selected at a single cell level by limiting dilution in 96-well cell culture plates based on their GFP expression, morphology, and stem cell marker expression ( Figure 2C and Table S1). Two putative CSC clones (Clones #8 and #17) were finally selected ( Figure 2D and Table S1).
dish containing puromycin during a three-week subculture period. Then, hTERT-immortalized Sca-1+ CSCs were further selected at a single cell level by limiting dilution in 96-well cell culture plates based on their GFP expression, morphology, and stem cell marker expression ( Figure 2C and Table S1). Two putative CSC clones (Clones #8 and #17) were finally selected ( Figure 2D and Table S1).

Evaluation of Stem Cell Potency of hTERT-Immortalized Sca-1+ CSC Lines
Phenotypic characterization of finally selected two hTERT-immortalized Sca-1+ CSC lines was further evaluated by immunostaining and flow cytometry with different cell surface antibodies. Two CSC lines were positive for CD29, CD44, CD71, and CD106 ( Figure 3A,B and Figure S1A,B), showing phenotypic characteristics similar to those of primary Sca-1+ CSCs. Interestingly, one clone (Clone #8) was negative for CD31 ( Figure 3A,B), but the other was positive for CD31 ( Figure S1A,B). The two clones were designated as Sca-1+/CD31´CSCs hTERT and Sca-1+/CD31+ CSCs hTERT , respectively. Given the different phenotypic characteristics of the two cell lines, we selected Sca-1+/CD31´CSCs hTERT for further studies because Sca-1+/CD31´cells, but not Sca-1+/CD31+ cells were shown to possess more potent CSC properties [3,7,8].

Sca-1+/CD31´CSCs hTERT CM Reduces Hypoxia-Induced Cardiomyocyte Apoptosis Partly via MCP-1-Dependent Mechanism
To define which paracrine factors secreted from Sca-1+/CD31´CSCs hTERT are responsible for protecting HL-1 cardiomyocytes from hypoxic cell death, we chose MCP-1 as a candidate among 21 growth factors and inflammatory cytokines because it was the most dominant factor secreted by Sca-1+/CD31´CSCs hTERT . We generated siRNA duplex against MCP-1 to inhibit endogenous MCP-1 expression in Sca-1+/CD31´CSCs hTERT . Real-time polymerase chain reaction (PCR) analysis showed that the silencing efficiency in MCP-1 siRNA-transfected Sca-1+/CD31´CSCs hTERT was approximately 80% after a 48 h transfection ( Figure 5A). To further elucidate which paracrine factors are affected in MCP-1 siRNA-transfected Sca-1+/CD31´CSCs hTERT , a total of 21 growth factors and inflammatory cytokines from Sca-1+/CD31´CSCs hTERT CM after transfection of negative control (NC) siRNA or MCP-1 siRNA were analyzed using mouse cytokine antibody arrays, and were subjected to densitometry. Expectedly, we observed that MCP-1 protein expression was significantly reduced by 57% in Sca-1+/CD31´CSCs hTERT CM by MCP-1 siRNA transfection compared to that of the NC siRNA ( Figure 5B,C). Interestingly, we found that IL-6 protein expression was also significantly reduced by 20% in MCP-1 siRNA-transfected Sca-1+/CD31´CSCs hTERT CM compared to that of the NC siRNA after a 48 h transfection ( Figure 5B,C).

Discussion
The hTERT gene was used to immortalize Sca-1+ CSCs to minimize the occurrence of genetic and phenotypic instabilities caused by viral oncogenes, such as the simian virus 40 T antigen, adenoviral E1A/E1B genes, and human papillomavirus 16 E6/E7 genes [13]. A retroviral vector encoding hTERT-IRES-GFP was constructed to direct gene expression from the immediate early promoter of cytomegalovirus and GFP expression through the IRES to prevent adverse effects on TERT activity due to the GFP sequence. In this study, we found that overexpression of ectopic hTERT was sufficient to immortalize mouse adult CSCs sustaining similar phenotypic characteristics and multi-differentiation potential more than 100 PDs to primary CSCs. Sca-1+/CD31− CSCs hTERT still exhibited strong GFP activity even after long-term in vitro culture exceeding ~100 PDs, indicating that these are very useful for monitoring cell movement or location.
A number of studies have also demonstrated that various adult stem cells inserted with the TERT gene have maintained long-term stemness in vitro and have been immortalized [13][14][15][27][28][29]. Freire et al. [30] also showed that Sca-1+ CSCs immortalized by the mTERT gene showed robust selfrenewal capacity while preserving a stable phenotype in long-term culture using a similar approach to ours. Recently, we also established hTERT-immortalized mouse ASC TERT [16], demonstrating the efficacy of TERT gene-mediated immortalization. In contrast, several studies have reported that hTERT alone was not sufficient for immortalization of human ASCs [31] or CD34+ human cord blood cells [32], revealing the necessity of co-transduction of viral genes with the TERT gene. These

Discussion
The hTERT gene was used to immortalize Sca-1+ CSCs to minimize the occurrence of genetic and phenotypic instabilities caused by viral oncogenes, such as the simian virus 40 T antigen, adenoviral E1A/E1B genes, and human papillomavirus 16 E6/E7 genes [13]. A retroviral vector encoding hTERT-IRES-GFP was constructed to direct gene expression from the immediate early promoter of cytomegalovirus and GFP expression through the IRES to prevent adverse effects on TERT activity due to the GFP sequence. In this study, we found that overexpression of ectopic hTERT was sufficient to immortalize mouse adult CSCs sustaining similar phenotypic characteristics and multi-differentiation potential more than 100 PDs to primary CSCs. Sca-1+/CD31´CSCs hTERT still exhibited strong GFP activity even after long-term in vitro culture exceeding~100 PDs, indicating that these are very useful for monitoring cell movement or location.
A number of studies have also demonstrated that various adult stem cells inserted with the TERT gene have maintained long-term stemness in vitro and have been immortalized [13][14][15][27][28][29]. Freire et al. [30] also showed that Sca-1+ CSCs immortalized by the mTERT gene showed robust self-renewal capacity while preserving a stable phenotype in long-term culture using a similar approach to ours. Recently, we also established hTERT-immortalized mouse ASC TERT [16], demonstrating the efficacy of TERT gene-mediated immortalization. In contrast, several studies have reported that hTERT alone was not sufficient for immortalization of human ASCs [31] or CD34+ human cord blood cells [32], revealing the necessity of co-transduction of viral genes with the TERT gene. These results suggest that immortalization of primary cells via overexpression of TERT alone could be cell type-dependent.
In the current study, we investigated the effect of MCP-1 silencing in Sca-1+/CD31´CSCs hTERT CM on CoCl 2 -or H 2 O 2 -induced cardiomyocyte apoptosis because MCP-1 was the most dominant factor in Sca-1+/CD31´CSCs hTERT CM. Although we significantly reduced both MCP-1 mRNA and protein expression levels by MCP-1 siRNA-transfection in Sca-1+/CD31´CSCs hTERT , but a large amount of MCP-1 was still secreted from Sca-1+/CD31´CSCs hTERT ( Figure 4A-C). MCP-1 siRNA-transfected Sca-1+/CD31´CSCs hTERT CM still showed significant reductions (78% in CoCl 2 -induced hypoxic injury model, and 85% in H 2 O 2 -induced oxidative stress model, respectively) in the proportions of apoptotic HL-1 cardiomyocytes compared to NC siRNA-transfected Sca-1+/CD31Ć SCs hTERT CM ( Figure 5D-G). Therefore, failure in an increase of the proportion of apoptotic HL-1 cardiomyocytes to the level of NC siRNA-transfected Sca-1+/CD31´CSCs hTERT CM could be due to partial silencing of MCP-1 in Sca-1+/CD31´CSCs hTERT . Our findings are in line with previous studies that MCP-1 significantly decreased hypoxia-induced cell death in cardiomyocytes [36,37]. Martire et al. [38] demonstrated that cardiac overexpression of MCP-1 in transgenic mice mimics ischemic preconditioning through stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) 1/2 activation, suggesting that a permanent activation of SAPK/JNK1/2 pathway in MCP-1 transgenic mice could be involved in the development of cardiac resistance against ischemia. Furthermore, MCP-1 played a critical role in neuroprotection against in rat primary midbrain neurons [39], and protected the kidney during the acute inflammatory response following renal I/R injury [40]. Additional studies will be required to further elucidate precise mechanism(s) by which Sca-1+/CD31´CSCs hTERT -secreted MCP-1 leads to an increase in cardiomyocyte survival during hypoxia-induced injury.
Interestingly, we also found that MCP-1 silencing in Sca-1+/CD31´CSCs hTERT resulted in a significant reduction of IL-6 protein expression level. Morimoto et al. [47] revealed that a combination of IL-6 with MCP-1 synergistically stimulated and sustained STAT3 activation in cardiomyocytes. Furthermore, marked myocardial IL-6 secretion, STAT3 activation, and LV hypertrophy were observed after MI in transgenic mice overexpressing MCP-1, thereby resulting in the prevention of LV dysfunction and remodeling after MI [47]. Liu et al. [48] reported that anti-apoptotic effect of MCP-1 in fibroblasts was eliminated in the presence of anti-IL-6 neutralizing antibody. These results suggest that MCP-1-mediated pro-survival signaling was achieved via activation of IL-6 signaling.

Construction of a Retroviral Vector Encoding hTERT-GFP and Production of Retroviruses
We generated the pLPCX-hTERT-IRES-GFP vector, a BglII-SalI fragment containing hTERT cDNA was amplified by PCR using pCI-neo-hEST2 (Addgene, Cambridge, MA, USA) as a template. The BglII-SalI digested PCR fragment containing hTERT cDNA was inserted into the BglII-SalI site of pIRES2-GFP vector (BD Biosciences). Finally, the BglII-ClaI digested PCR fragment containing hTERT-IRES-GFP cDNA was finally inserted into the BglII-ClaI site of the pLPCX vector (BD Biosciences). The detailed cloning strategy of a retroviral vector encoding hTERT-IRES-GFP and production of retroviruses expressing pLPCX-hTERT-IRES-GFP in 293GPG packaging cell line [49] was performed as previously described [16].

Generation of Immortalized Sca-1+ CSCs
MACS-sorted Sca-1+ CSCs were plated at 2ˆ10 5 cells in 6-cm culture dishes in Dulbecco's Modified Eagle's Medium (DMEM)-low glucose (LG) supplemented with 10% FBS and 100 U/mL penicillin/streptomycin (P/S). Cells were infected with retroviruses harboring pLPCX-hTERT-IRES-GFP at 60% confluence for three days. The cells were selected in medium against 0.5 µg/mL puromycin by repeated sub-culturing at a 1:3 ratio three times per week during a three-week subculture period in 10-cm culture dishes. For clonal analysis, the selected cells were plated in 96-well plates at one cell per 100 µL by limiting dilution in DMEM-LG supplemented with 10% FBS and 100 U/mL P/S, as described previously [6]. Briefly, wells containing one cell per well were only selected by visual inspection 24 h after plating, and were further cultured for 12 days. Among 20 clones derived from a single cell, two clones were finally selected based on microscopic examination of morphology, proliferation, GFP expression, and hTERT expression. . Fluorescence images were obtained using a TEFM Epi-fluorescence system attached to an inverted microscope (Olympus, Tokyo, Japan) or were acquired with a confocal fluorescence microscope (LSM710, Carl Zeiss, Oberkochen, Germany).

Phenotypic Characterization of Sca-1+ CSCs by Flow Cytometry
Primary CSCs and hTERT-immortalized Sca-1+ CSC lines were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. The cells were subsequently incubated for 20 min at 4˝C with the following primary antibodies: CD14, CD29, CD31, CD34, CD44, CD45, CD71, CD90, CD106, CD117, CD133, and Sca-1. After washing twice with PBS + 2% FBS, cells were incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-rat antibodies (e-Bioscience) for 15 min at 4˝C. For control experiments, the cells were stained with secondary antibodies only. After washing twice with PBS + 2% FBS, thirty thousand cells for each sample were analyzed on a FACSCalibur flow cytometer (BD Biosciences). Data were analyzed using CellQuest Pro software (BD Biosciences).

Differentiation Potential of Sca-1+ CSCs
Primary CSCs and Sca-1+/CD31´CSCs hTERT were plated at a density of 1~2ˆ10 4 cells/mL in 24-well plates containing 0.1% (w/v) gelatin-coated glass coverslips. Cells were cultured in DMEM-LG supplemented with 10% FBS and 100 U/mL P/S for 2-3 days. Cardiomyogenic differentiation of primary CSCs and Sca-1+/CD31´CSCs hTERT was induced by incubation in DMEM-LG supplemented with 10% FBS, 100 U/mL P/S, and 1 µM 5-azacytidine (Sigma-Aldrich) for 21 days. Cultures were maintained by media exchange every 3~4 days. Endothelial differentiation of primary CSCs and Sca-1+/CD31´CSCs hTERT was induced by incubation in 60% DMEM-LG and 40% MCDB-201 (Sigma-Aldrich), supplemented with 1ˆinsulin-transferrin-selenium, 1ˆlinoleic acid-BSA, 10´8 M dexamethasone, 10´4 M ascorbic acid 2-phosphate (all from Sigma-Aldrich), and 100 U/mL P/S plus 20 ng/mL VEGF (R and D Systems, Minneapolis, MN, USA) for 21 days. Cultures were maintained by media exchange every 3-4 days. To assess cardiac or endothelial differentiation, the cells were fixed with 4% paraformaldehyde in PBS for 10 min, washed with PBST, and permeabilized with 0.1% Triton X-100 in PBS for 30 min. Cells were washed with PBST and blocked for nonspecific binding by incubation in 5% NGS in PBST for 30 min. Then, the cells were incubated overnight at 4˝C with the following primary antibodies: anti-MLC (Sigma-Aldrich), anti-cTnI (Abcam, Cambridge, UK), anti-cardiac troponin T (cTnT; Developmental Studies Hybridoma Bank, Iowa City, IA, USA), and anti-vWF (DAKO). After washing three times with PBST, the cells were stained with Alexa Fluor 488-or 594-conjugated secondary antibodies (all from Molecular Probes) for 30 min, and washed three times in PBST. For control experiments, the cells were stained with secondary antibodies only. Nuclei were stained with DAPI (Sigma-Aldrich). The cells were mounted with fluorescent mounting medium (DAKO). Fluorescence images were obtained with a TE-FM Epi-Fluorescence system attached to an inverted microscope (Olympus). Adipogenic differentiation of primary CSCs and Sca-1+/CD31Ć SCs hTERT was induced by incubation in DMEM-LG supplemented with 5% FBS and 100 U/mL P/S, 1 µM dexamethasone, 10 µg/mL insulin, 100 µM indomethacin, and 0.5 µM methyl-isobutylxanthin (all from Sigma-Aldrich) for 10 days. Culture media were changed every three days. Adipogenic differentiation was assessed on day 10 using Oil Red O (Sigma-Aldrich) stain as an indicator of intracellular lipid accumulation. The cells were fixed with 4% paraformaldehyde in PBS for 20 min, washed with 60% isopropanol, and stained with 0.3% Oil Red O solution in 60% isopropanol for 10 min. After washing three times with water, cells were de-stained in 100% isopropanol for 15 min. Osteogenic differentiation of primary CSCs and Sca-1+/CD31´CSCs hTERT was induced by incubation in culture medium with 1 µM dexamethasone, 10 mM glycerophosphate, and 50 µM ascorbic acid (all from Sigma-Aldrich) for 21 days. Osteogenic differentiation was determined by Alizarin Red S (Sigma-Aldrich) staining.

WST-1 Proliferation Assay
Sca-1+/CD31´CSCs hTERT were plated at a density of 1ˆ10 3 cells/well in a 96-well plate. Cells were cultured in Mesencult mesenchymal stem cell (MSC) Basal Medium supplemented with 10% Mesencult MSC Stimulatory Supplements (StemCell Technologies Inc., Vancouver, BC, Canada) and 100 U/mL P/S. The cells were analyzed at days 1, 2, 3, 4, 5, and 6 using a WST-1 assay (Roche Applied Science, Mannheim, Germany). In brief, the cells were incubated at a concentration of 10 µM of WST-1 for 2 h. The cells were then incubated until color development was sufficient for photometric detection. The reaction product was quantified by measuring absorbance using an ELISA reader (Molecular Devices, Sunnyvale, CA, USA) at 440 and 690 nm. Data were analyzed using SoftMax ® Pro quantification of absorbance analysis software (Molecular Devices).

Apoptosis Assay of HL-1 Cardiomyocytes
Sca-1+/CD31´CSCs hTERT were cultured on six-well plates at a density of 5ˆ10 4 cells/well and transfected with 50 nM of MCP-1 siRNA duplexes (5 1 -CACAACCACCTCAAGCACT-3 1 ) or NC siRNA (all from Bioneer) using Lipofectamine RNAiMAX (Invitrogen) for 48 h as suggested by the manufacturer. For CoCl 2 -(Sigma-Aldrich) or H 2 O 2 -induced hypoxia, HL-1 cells, a cardiomyocyte cell line that continuously divides and spontaneously contracts while maintaining a differentiated cardiac phenotype [50] was used. HL-1 cardiomyocytes were seeded at 6ˆ10 5 cells in fibronectin (12.5 mg/L)-gelatin (0.02%) coated six-well culture dishes and allowed to reach~80% confluence in Claycomb medium (Sigma-Aldrich) supplemented with 10% FBS (Invitrogen), 2 mM L-glutamine, 0.1 mM norepinephrine, 0.3 mM ascorbic acid, and 100 U/mL P/S in a humidified 37˝C/5% CO 2 incubator. The cells then were treated with 150 µM CoCl 2 or 200 µM H 2 O 2 for 24 h in Mesencult MSC Basal Medium supplemented with 2% FBS and 100 U/mL P/S in the presence of MCP-1 siRNA-transfected Sca-1+/CD31´CSCs hTERT CM or NC siRNA-transfected Sca-1+/CD31´CSCs hTERT CM. Annexin V (AV) and propidium iodide (PI) staining were performed using a FITC Annexin V Apoptosis Detection Kit II (BD Biosciences) according to the manufacturer's instructions, and then flow cytometric analysis was performed. Viable cells were counted using a hemocytometer after staining with 0.2% trypan blue (Invitrogen) to reveal the dead cells.

Statistical Analysis
All statistical values are expressed as the mean˘standard deviation (SD). Significant differences between means were determined using the Student's t-test or by analysis of variance followed by the Student-Newman-Keuls test. Statistical significance was set at p < 0.05.
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/17/ 6/800/s1. experiments. Hyung Joon Joo assisted with analyzing and interpreting data, and provided technical support. Soon Jun Hong and Do-Sun Lim designed the research, analyzed the data and critical revision of manuscript for important intellectual content. All authors read and approved the final manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.