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Nanopillar Surface Topology Promotes Cardiomyocyte Differentiation through Cofilin-Mediated Cytoskeleton Rearrangement Ha-Rim Seo,†,‡ Hyung Joon Joo,†,‡ Dae Hwan Kim,§ Long-Hui Cui,† Seung-Cheol Choi,† Jong-Ho Kim,† Sung Woo Cho,∥ Kyu Back Lee,*,§,⊥ and Do-Sun Lim*,†,⊥ †

Department of Cardiology, Cardiovascular Center, College of Medicine and §School of Biomedical Engineering, College of Health Science, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul 02841, Republic of Korea ∥ Division of Cardiology, Department of Internal Medicine, Inje University College of Medicine, Seoul Paik Hospital, 9 Mareunnae-ro, Jung-gu, Seoul 04551, Republic of Korea S Supporting Information *

ABSTRACT: Nanoscaled surface patterning is an emerging potential method of directing the fate of stem cells. We adopted nanoscaled pillar gradient patterned cell culture plates with three diameter gradients [280−360 (GP 280/360), 200− 280 (GP 200/280), and 120−200 nm (GP 120/200)] and investigated their cell fate-modifying effect on multipotent fetal liver kinase 1-positive mesodermal precursor cells (Flk1+ MPCs) derived from embryonic stem cells. We observed increased cell proliferation and colony formation of the Flk1+ MPCs on the nanopattern plates. Interestingly, the 200−280 nm-sized (GP 200/280) pillar surface dramatically increased cardiomyocyte differentiation and expression of the early cardiac marker gene Mesp1. The gradient nanopattern surface-induced cardiomyocytes had cardiac sarcomeres with mature cardiac gene expression. We observed Vinculin and p-Cofilin-mediated cytoskeleton reorganization during this process. In summary, the gradient nanopattern surface with 200−280 nm-sized pillars enhanced cardiomyocyte differentiation in Flk1+ MPCs. KEYWORDS: Flk1-positive mesodermal precursor cells, gradient nanopattern plates, cardiomyocyte differentiation, cytoskeleton reorganization, nanoimprinting

1. INTRODUCTION

ships between changes in cell morphology and nanopillar surfaces. Fetal liver kinase 1-positive mesodermal precursor cells (Flk1+ MPCs) derived from embryonic stem cells (ESCs) are able to differentiate into multiple cell types, including endothelial cells, hematopoietic cells, smooth muscle cells, and cardiomyocytes.9,10 Cell lineage specification is regulated by various chemical inhibitors and cytokines in Flk1+ MPCs. For example, Flk1+ MPCs cultured on a Matrigel-coated dish treated with the ROCK inhibitor Y-27632 exhibit endothelial cell differentiation.11 Furthermore, cardiovascular progenitor cells are differentiated from Flk1+ MPCs treated with VEGF, FGF2, BMP4, and DKK1 on OP9 stromal cells.12 Matrigel and OP9 stromal cells promote both cytokine and physical stimulation of Flk1+ MPCs. These data suggest that physical stimulation by direct contact with Matrigel or OP9 cells plays a role in the induction of the directional differentiation of Flk1+ MPCs.

Cells adhere to neighboring cells or the extracellular matrix (ECM) in vivo and sense and respond to chemical and mechanical stimulation. The identification of cell-to-cell and cell-to-ECM associations is essential for comprehending the in vivo microenvironment niche.1 Nanotopography provides a powerful means of understanding the influence of the microenvironment and mimicking cell behavior in the ECM.2 Previous reports have suggested that a nanoscale topography that mimics growth factors, cytokines, and cell membrane protein structure can influence various cell behaviors and functions.3,4 For example, a nanopattern plate with grooves at 277 nm intervals elicits reduced cell adhesion and proliferation responses in astrocytes.5 A grooved nanopattern plate also improves cell body elongation and the alignment of smooth muscle cells.6 The cytoplasm of mouse preosteoblasts is absorbed by 200 nm diameter nanopillar plates, and F-actin expression increases in intensity.7 High directionality of filopodia extension has been reported in human osteoblasts on 130 nm diameter nanopillar plates formed by nanoimprinting.8 However, few studies have examined the relation© XXXX American Chemical Society

Received: January 31, 2017 Accepted: May 8, 2017

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DOI: 10.1021/acsami.7b01555 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

cm2 in cardiomyocyte differentiation medium with complete α-MEM supplemented with 10 μM Y-27632 (688000, Calbiochem), 1 μg/mL EW-7197 (N-[[4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1H-iMediumazol-2-yl]methyl]-2-fluoroaniline, S7530, Selleckchem), 400 μM Trolox (238813, Sigma), and 3 μg/mL Cyclosporin A (489551, Novartis). After seeding on the gradient nanopattern plates, the Flk1+ MPCs were treated with NucBlue Live ReadyProbes Reagent (live 4′,6-diamidino-2-phenylindole (DAPI); R37605, Molecular Probes), and phase contrast images were obtained at Nanoday 2, 4, and 6 using a DMI3000 B microscope (LEICA). 2.3. Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR). The total mRNA (mRNA) from the samples was extracted on postseeding Nanoday 2 and 4 with TRIzol (TR-118, MRC) according to the manufacturer’s protocol. The concentration of extracted mRNA was measured using a NanoDrop spectrophotometer (ND-1000, Thermo Fisher Scientific), and 500 ng of mRNA was used for complementary DNA synthesis by supplementation with M-MLV reverse transcriptase (28025-013, Invitrogen) at 37 °C for 50 min in a 20 μL volume. qRT-PCR was performed using SYBR Green Mixture (170-8880, Bio-Rad), and the results were recorded using an MYiQ2 Detection System (Bio-Rad). The quantification of relative gene expression levels was based on ΔΔCt values normalized to GAPDH values. The primer list is presented in Supporting Information Table 1. 2.4. Immunofluorescence Staining. The cells were washed twice with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde (P6148, Sigma) dissolved in PBS for 20 min. The fixed cells were permeabilized with 0.5% Triton X-100 in PBS and blocked with 5% goat serum (26050-088, GIBCO) in 0.1% Triton X100 (X-100, Sigma) containing PBS (PBST) at room temperature for 1 h. The cells were incubated at room temperature for 3 h in a humid chamber with the following primary antibodies: rabbit antiphosphohistone-H3 (PHH3; 06-570, Millipore), mouse anti-Ki67 antibody (M7240, DAKO), rabbit anticleaved caspase 3 (9664, Cell Signaling Technology), mouse anti-Mesp1 (NBP1-51613, Novus Biologicals), mouse anti-α-SMA (F3777, Sigma), rabbit anti-Flk1 (2479, Cell Signaling Technology), rabbit anti-Cofilin (ab42824, Abcam), rabbit antiphospho-Cofilin (p-Cofilin; 3313, Cell Signaling Technology), mouse anti-α-actinin (A7811, Sigma), mouse anti-Vinculin (V9131, Sigma), mouse anti-Cx43 (C8093, Sigma), and mouse anti-cTnT (RVC2, DSHB) diluted with PBST. The cells were washed twice in PBST and incubated for 2 h at room temperature with the following secondary antibodies: Alexa Fluor 488 Phalloidin (A12379, Molecular Probes), Alexa Fluor 488 chicken antirabbit IgG (A21441, Molecular Probes), Alexa Fluor 594 goat antimouse IgG (A11005, Molecular Probes), and Alexa Fluor 594 goat antirabbit IgG (A11012, Molecular Probes). Nuclei were stained with 1 μg/mL of DAPI (D9542, Sigma). The stained cells were mounted using a fluorescent mounting solution (S3023, DAKO). Immunofluorescence images were acquired using a fluorescence microscope (BX61, Olympus) and a confocal fluorescence microscope (LSM700, Carl Zeiss). 2.5. TUNEL Assay. Cells were fixed with 1% paraformaldehyde for 15 min on ice for the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The TUNEL assay was performed using an APO-BrdU TUNEL Assay Kit (MP23210, Invitrogen) according to the manufacturer’s instructions. Fluorescence images were acquired using an Olympus fluorescence microscope (BX61, Olympus). 2.6. Scanning Electron Microscopy (SEM). The Flk1+ MPCs that had adhered to the GP 200/280 and Flat surfaces were fixed overnight with 2.5% glutaraldehyde (G5882, Sigma) at 4 °C. Each sample was washed twice with PBS and treated with 1% osmium tetroxide (201030, Sigma) at room temperature for 2 h. After washing twice with PBS, the samples were dehydrated using an ethanol concentration gradient (50−100%). Finally, the samples were freeze dried and coated with platinum for 5 min. The cells were examined by FE-SEM (JSM6701, JEOL). 2.7. Flow Cytometry. Cells were harvested using 0.25% trypsin/ ethylenediaminetetraacetic acid (25200-072, GIBCO) and resuspended in 2% fetal bovine serum in PBS at a concentration of 0.5 × 106 cells per 100 μL. The endogenous green fluorescence proteinexpressing cells were analyzed using a flow cytometer (FACS Caliber;

Recent studies have shown that nanopattern plates are able to regulate various stem cell behaviors and have roles in stem cell lineage specification and differentiation. Nanopattern plates with 100 or 150 nm pillars influence the self-renewal, adhesion, and spreading of human embryonic stem cells.13 Mesenchymal stem cells have the potential to differentiate into osteoblasts on a nanotopography that imitates the extracellular matrix via the α1β1 integrin signaling pathway.14 Nanofiber structures with diameters of 100−300 nm have the potential to promote the proliferation and differentiation of osteoblasts.15 Furthermore, nanopattern plates with grooves at 650 nm intervals increase osteogenic and adipogenic differentiation from mesenchymal stem cells.16 These studies have demonstrated a relationship between nanotopography and the differentiation of stem cells into neural or osteogenic cells. However, the biophysical role and underlying mechanism of the effect of the nanopillar surface on Flk1+ MPCs during embryonic stem cell differentiation remains unclear. In this study, we compared the differentiation of Flk1+ MPCs on nanopattern plates with that on conventional Flat-pattern plates (Flat). The nanopattern plates comprised nanoscaled pillars with three diameter gradients, i.e., 280−360 (GP 280/ 360), 200−280 (GP 200/280), and 120−200 nm (GP 120/ 200).

2. EXPERIMENTAL SECTION 2.1. Fabrication of Gradient Nanopattern Plates. Methods for fabricating gradient nanopattern plates have been described in the literature.17 Gradient nanopattern plates were produced with pillar sizes divided into three groups, i.e., 280−360 (GP 280/360), 200−280 (GP 200/280), and 120−200 nm (GP 120/200). The pillar interval was fixed at 440 nm. Gradient nanopattern polystyrene (PS) scaffolds were fabricated by thermal imprinting with anodic aluminum oxide (AAO) molds containing nanosized pores using a nanoimprinting device. Aluminum plates (ultrapure grade, Goodfellow, Canada) were refined using a perchloric acid solution (perchloric acid:pure ethanol = 1:4, v/v). Refined aluminum plates were anodized in a mixed deionized water−methanol−phosphoric acid solution (deionized water:methanol:phosphoric acid = 59:40:1, v/v). The porous AAO mold was coated with heptadecafluoro-1,1,2,2-tetrahydrodecyl-trichlorosilane monomers. Finally, 35 mm × 20 mm PS polymer blocks were heated to 165 °C for 10 min and pressed on the AAO mold for 2 min. The PS scaffold was detached from the AAO molds at room temperature. The PS scaffold was coated with plasma and thoroughly dried before use. The gradient nanopattern was verified by field emission scanning electron microscopy (FE-SEM, JSM6701, JEOL), and the gradient nanopattern plate was coated with platinum for 5 min. 2.2. Mouse ESC (mESC) Culture and Differentiation. The EMG7 mouse ESCs from routine cultures were a generous gift from Jun K. Yamashita (Kyoto University). The EMG7 cells expressed endogenous green fluorescence protein (eGFP) under the control of α-myosin heavy chain promoter.18 Briefly, EMG7 cells were maintained on 0.1% gelatin-coated Flat plates at a density of 7.6 × 103 cells/cm2 in Glasgow’s minimal essential medium containing leukemia inhibitory factor (LIF; ESG1107, Millipore). To obtain Flk1+ MPCs, EMG7 cells were cultured on 0.1% gelatin-coated Flat plates at a density of 4.3 × 103 cells/cm2 in 10% (v/v) fetal bovine serum containing LIF-free alpha-minimal essential medium (complete αMEM). After differentiation on day 4.5, Flk1+ MPCs were sorted using an autoMACS separator (Miltenyi Biotec) with biotin-conjugated antimouse Flk1 (13-5821-82, eBioscience) and streptavidin MicroBeads (130-048-101, Miltenyi Biotec). The Flk1+ MPCs were subsequently plated on 0.1% gelatin-coated gradient nanopattern plates at a density of 1.4 × 104 cells/cm2 and cultured in complete αMEM. To induce cardiomyocytes, Flk1+ MPCs were cultured on 0.1% gelatin-coated gradient nanopattern plates at a density of 4 × 104 cells/ B


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Figure 1. Flk1+ MPCs promoted colony formation on the gradient nanopattern plate. (a) Schematic diagram of Flk1+ MPC purification from embryonic stem cells and seeding on the gradient nanopattern plates. (b) Representative phase contrast images of Flk1+ MPCs on the Flat and gradient nanopattern plates at Nanoday 2 and 4. Nuclei were stained with live DAPI (blue). Scale bar = 50 μm. (c and d) Quantification of the number of cells and colonies for Flat and gradient nanopattern plates at Nanoday 4. (e and f) Quantification of the coverage of the colony area and the perimeter of the colonies for Flat and gradient nanopattern plates at Nanoday 4. All experiments n = 5. *p < 0.05. anti-GAPDH (1:10000; G8795, Sigma) antibodies at 4 °C overnight. The membranes were then washed three times with TBST and incubated with a horseradish peroxidase-conjugated secondary antibody (1:7000; Santa Cruz Biotechnology) in TBST at room temperature for 1 h. Chemiluminescence was visualized using ECL Plus reagents (32132, Thermo Fisher Scientific) and recorded on Xray film. 2.9. Statistical Analysis. The results are presented as means ± standard deviation (SD). Significant differences in means between groups were evaluated using the Student−Newman−Keuls test. Significance was set at p < 0.05 in comparisons with the Flat plates. All statistical analyses were performed using version 3.5 of the SigmaStat software package (SPSS, Chicago, IL, USA)

Becton Dickinson). The data were analyzed using CellQuest Pro software (Becton Dickinson). 2.8. Western Blotting. Flk1+ MPCs induced with cardiomyocyte differentiation medium on Flat and GP 200/280 gradient nanopattern plates for 1 day were washed twice with PBS and lysed with 1 mM phenylmethylsulfonyl fluoride (P7626, Sigma) containing 1× cell lysis buffer (9803, Cell Signaling Technology). A quantitative analysis of the samples was performed using Bradford assay dye reagent (5000006, Bio-Rad). The sample protein (20 μg) was boiled in 1× loading dye for 5 min and subjected to electrophoresis on a 12% sodium dodecyl sulfate-polyacrylamide gel. After transfer to a polyvinylidene fluoride membrane (ISEQ00010, Millipore), the protein was blocked in 5% bovine serum albumin containing 1× TBST (a mixture of Trisbuffered saline and Tween 20; WH400028806, 3M) at room temperature for 1 h. The membranes were incubated with antiIntegrin α5 (1:1000; 4705, Cell Signaling Technology), anti-p-Cofilin (1:1000; 3313, Cell Signaling Technology), anti-Cofilin (1:1000; ab42824, Abcam), anti-p-MLC2 (1:1000; 3671, Cell Signaling Technology), anti-MLC2 (1:1000; 10906-1-AP, Proteintech), and

3. RESULTS AND DISCUSSION 3.1. Gradient Nanopattern Plates Induce Flk1+ MPC Colony Formation. A gradient nanopattern plate is a powerful tool for understanding the changes in stem cell fate by C


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Figure 2. Phenotypic comparison of gene expression in Flk1+ MPCs between Flat and gradient nanopattern plates. qRT-PCR analysis of the expression of endothelial cell markers (Flk1 and CD144), a smooth muscle cell marker (α-SMA), a cardiac cell marker (Mesp1), and hematopoietic cell markers (Runx1, Gata2, and Fli1) in Flk1+ MPCs on Flat or GP 280/360, GP 200/280, and GP 120/200 gradient nanopattern plates at Nanoday 2 and 4. Three independent experiments were performed. *p < 0.05. (b) Immunofluorescence analysis of Mesp1 (red) and DAPI (blue) in cells on Flat or GP 200/280 gradient nanopattern plates at Nanoday 4. White arrowheads indicate Mesp1-expressing cells. Scale bar = 50 μm. (c) αSMA (green) and Flk1 (red) immunofluorescence images between Flat and GP 200/280 gradient nanopattern plates at Nanoday 4. Nuclei were stained with DAPI (blue). Scale bar = 50 μm.

suggest that the gradient nanopattern plate plays an important role in the mechanotransduction of stem cells. Several studies have indicated an association between the specific topography of nanopillars and stem cell differentiation via changes in cell morphology. In human embryonic stem cells, a nanopattern surface comprising pillars of 100 nm diameter resulted in randomly distributed punctate focal adhesion over the entire cell spread area.13 A 120 nm pillar nanopattern stimulates osteogenesis, with increased BMPR2 expression in mesenchymal stem cells, via ERK activity.19 Moreover, nanotopography is an important source of three-dimensional organ cultures.20 Our results are consistent with those of prior research and show that 120−360 nm gradient nanopattern plates significantly enhance the proliferation and colony formation of Flk1+ MPCs compared with Flat plates. Furthermore, the observed colonies

mimicking attachment and adhesion to other cells or the ECM. We sorted Flk1+ MPCs from the differentiated EGM7 cells and cultivated them on gradient nanopattern plates to investigate morphological changes in the Flk1+ MPCs. The sorting efficiency of the Flk1+ MPCs was greater than 97%. The gradient nanopattern plate surface, which was manufactured using PS scaffolds, is shown in a schematic diagram (Figure 1a). We cultured the purified Flk1+ MPCs and seeded them on a gradient nanopattern plate for 4 days; an increase in the number of cells resulted in the formation of colonies. Gradient nanopattern plates comprising pillars with diameters of 120− 360 nm enhanced cell proliferation and generated a colony-like form of Flk1+ MPCs (Figure 1b and 1c). The number of the colonies was generally 3-fold higher on the gradient nanopattern plates than on the Flat plate (Figure 1d). These results D


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Figure 3. Gradient nanopattern plate promotes cardiac differentiation from Flk1+ MPCs. (a) Schematic diagram of cardiac differentiation of Flk1+ MPCs cultured on a gradient nanopattern plate in cardiomyocyte differentiation medium. (b) Representative SEM images of Flk1+ MPCs on the GP 200/280 gradient nanopattern plate at Nanoday 1. Scale bar = 1 μm. (c) Representative phase contrast images of Flk1+ MPCs on a Flat and gradient nanopattern plate in cardiomyocyte differentiation medium at Nanoday 1, 2, 4, and 6. Differentiated cardiomyocytes are indicated by eGFP (green). Nuclei were stained with live DAPI (blue). Scale bar = 50 μm. Specific induction of cardiomyocytes from Flk1+ MPCs on gradient nanopattern plate at Nanoday 2. (d−f) Quantitative analysis of the number of colonies and colony coverage after Nanoday 1 based on the area and perimeter of the colonies in Flat and GP 200/280 gradient nanopattern plates in cardiomyocyte differentiation medium at Nanoday 4. n = 7. *p < 0.05. (g) Flow cytometry analysis of the number of eGFP-positive cardiomyocytes. (h) Quantitative flow cytometry analysis of the number of eGFP-positive cells on Flat and GP 200/280 gradient nanopattern plate in cardiomyocyte differentiation medium at Nanoday 4. n = 3. *p < 0.05.

had a significantly higher coverage area (3.92 ± 1.02-fold) and perimeter (2.20 ± 0.49-fold) after cultivation on gradient nanopattern plates than on the Flat plate (Figure 1e and 1f). These results suggest that the Flk1+ MPCs recognized the gradient nanopattern plate surface. Next, we evaluated the viability of Flk1+ MPCs on the gradient nanopattern plates. Cell proliferation was determined based on the percentage of PHH3-positive cells. The values were 13.0 ± 3.4% (Flat), 21.5 ± 2.2% (GP 280/360), 23.2 ± 1.2% (GP 200/280), and 23.0 ± 2.9% (GP 120/200). The proliferation of Flk1+ MPCs on the gradient nanopattern plates was significantly higher than on the Flat plate (Figure S1a,b).

The number of Ki67-positive cells was also significantly higher on the gradient nanopattern plates than on the Flat plate (Figure S1c,d). Cell apoptosis was quantified based on the percentage of cleaved caspase 3-positive cells. The values were 4.0 ± 0.9% (Flat), 4.8 ± 0.8% (GP 280/360), 4.3 ± 1.4% (GP 200/280), and 4.7 ± 1.6% (GP 120/200) (Figure S2a,b). The TUNEL assay data showed that the populations of apoptotic cells on the gradient nanopattern plates did not differ significantly from the population on the Flat plate (Figure S2c,d). The apoptosis rates of the Flk1+ MPCs on the gradient nanopattern plates were not significantly higher than the apoptosis rate on the Flat plate. Therefore, surfaces with E


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Figure 4. Differentiated cardiomyocytes from Flk1+ MPCs on gradient nanopattern plates expressed mature cardiac-specific markers. (a) eGFP (green) and cTnT, Cx43, and α-actinin (red)-stained Flk1+ MPCs on the GP 200/280 gradient nanopattern plate in cardiomyocyte differentiation medium at Nanoday 4. Nuclei were stained with DAPI (blue). Three independent experiments were performed. Scale bar = 20 μm. (b) qRT-PCR analysis of the expression of mature cardiac cell markers (α-MHC, cTnT, cTnI, and Nkx2.5) in Flk1+ MPCs in cardiomyocyte differentiation medium on Flat and GP 200/280 gradient nanopattern plates at Nanoday 4. Three independent experiments were performed. *p < 0.05.

groove widths ranging from 350 to 2000 nm.21 A pore nanopattern plate with pore diameters of 200 nm promotes pancreatic differentiation from human pluripotent stem cells compared with plates with diameters ranging from 130 to 230 nm.22 Although a direct correlation between the nanopillar surface and increased Mesp1 expression was not fully addressed, the gradient nanopattern plate with pillars of 200−280 nm diameter had the potential to induce the differentiation of cardiomyocytes from Flk1+ MPCs. Our results suggest that a nanotopography with 200−280 nm pillars promotes the differentiation of Flk1+ MPCs into cardiomyocytes. 3.3. Combined with Growth Factors and Other Chemical Stimulants, the Gradient Nanopattern Plate Induces More Rapid and Robust Cardiomyocyte Differentiation. We attempted to induce cardiomyocyte differentiation directly using Cyclosporin A-containing cardiomyocyte differentiation medium. An α-MHC promoter-eGFP construct was inserted into EMG7 cells to induce cardiomyocyte differentiation. The differentiated cardiac cells expressed eGFP strongly, which demonstrated the generation of eGFPpositive cardiomyocytes from Flk1+ MPCs. The specific protocol to induce cardiomyocytes from Flk1+ MPCs is described schematically in Figure 3a. The Flk1+ MPCs recognized the GP 200/280 gradient nanopattern plate and produced filopodia, as shown in the SEM image (Figure 3b). Beating cardiomyocytes were found on the gradient nanopattern plate as early as Nanoday 2. The cells cultivated on both Flat and gradient nanopattern plates established beating cardiomyocytes at Nanoday 4 and 6 (Figure 3c). The number of eGFP-positive colonies from Flk1+ MPCs was significantly higher on the GP 200/280 gradient nanopattern plate than on the Flat plate. Furthermore, the eGFP-positive cell coverage and perimeter were significantly higher on the GP 200/280 gradient nanopattern plate than on the Flat plate (Figure 3d− f). Flow cytometry analysis showed that the percentage of

nanoscale morphology could be effective intermediate carriers of physical stimuli to stem cells. 3.2. Early Cardiac Marker Mesp1 Is Upregulated on the GP 200/280 Gradient Nanopattern Plate. We examined the gene expression levels of various cell-specific markers to determine whether cell differentiation from Flk1+ MPCs was induced on the gradient nanopattern plates. The qRT-PCR results showed a dramatic increase in the expression of the early cardiac lineage marker Mesp1 on the GP 200/280 gradient nanopattern plate at Nanoday 2 (2.64 ± 0.64-fold) and 4 (2.17 ± 0.43-fold). On the basis of a gene expression analysis of Flk1+ MPCs, there was significant upregulation of the early cardiomyocyte-specific gene Mesp1 exclusively on the GP 200/ 280 gradient nanopattern plate. However, the GP 200/280 gradient nanopattern plate suppressed endothelial cell markers Flk1 and CD144, the smooth muscle cell marker α-SMA, and hematopoietic cell markers Runx1, Gata2, and Fli1 in the Flk1+ MPCs (Figure 2a). The immunofluorescence images show that the Flk1+ MPCs on the GP 200/280 gradient nanopattern plate expressed Mesp1 to a greater extent than those on the Flat plate at Nanoday 4. In particular, α-SMA and Flk1 expression levels were low on the GP 200/280 gradient nanopattern plate compared with those on the Flat plate (Figure 2b and 2c). The early cardiomyocyte marker Mesp1 was highly expressed on the GP 200/280 gradient nanopattern (200−280 nm) plates. However, in other cell lineages, including endothelial, smooth muscle, and hematopoietic cells, Mesp1 expression was reduced on the gradient nanopattern plates, and there was no significant difference in expression levels between the GP 280/360 and the GP 120/200 gradient nanopattern plates and the Flat plate. These results indicate that a specific pillar size is necessary for cardiomyocyte induction. For example, a groove nanopattern plate with groove widths of 800 nm generates human-induced pluripotent stem cell-derived cardiomyocytes with longer sarcomeres compared with those generated on plates with F


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Figure 5. Flk1+ MPCs on gradient nanopattern plate have more filopodia and greater complexity. (a) Representative SEM images of Flk1+ MPCs on Flat or GP 200/280 gradient nanopattern plate at Nanoday 1. White arrowheads indicate protruding filopodia. Scale bar = 10 μm. (b) Quantification of the number of filopodia per cell. n = 50, *p < 0.05. (c) Immunofluorescence images of Flk1+ MPCs on Flat and GP 200/280 gradient nanopattern plate in cardiomyocyte differentiation medium at Nanoday 1. Cells were incubated with primary antibodies followed by F-actin (green), Vinculin (red), and DAPI (blue). Scale bar = 25 μm.

eGFP-positive cardiomyocytes from Flk1+ MPCs was significantly higher on the GP 200/280 gradient nanopattern plate (13.89 ± 0.24%) than on the Flat plate (7.38 ± 0.07%) (Figure 3g and 3h). These results demonstrate that a gradient nanopattern plate with 200−280 nm pillars supports cardiomyocyte differentiation from Flk1+ MPCs to a greater extent than Flat plate. Cyclosporin A is a well-known chemical that induces cardiogenic progenitors with the potential for cardiomyocyte differentiation.23 It significantly enhances the expression of cardiomyocyte lineage markers but inhibits the expression of hematopoietic and endothelial lineage markers during embryoid body formation in P19 cells.24 We used both a gradient nanopattern plate and Cyclosporin A-containing cardiomyocyte differentiation medium for the direct induction of cardiomyocytes from Flk1+ MPCs. On the basis of our results, 7−15% of the Flk1+ MPCs successfully differentiated into cardiomyocytes on both Flat and GP 200/280 gradient nanopattern plates using cardiomyocyte differentiation medium. These results suggest that the effective differentiation of Flk1+ MPCs into cardiomyocytes can be synergistically augmented through both physical (nanopattern plate) and chemical (Cyclosporine A) stimuli. 3.4. Gradient Nanopattern Plates Induce Mature Cardiomyocyte Differentiation. Next, we characterized differentiated cardiomyocytes from Flk1+ MPCs cultivated on the GP 200/280 gradient nanopattern plate. The immunofluorescence images showed that the differentiated cardiomyocytes expressed endogenous eGFP that overlapped with the expression of cTnT or Cx43. In particular, α-actinin expression

revealed highly organized sarcomere formation in the differentiated cardiomyocytes (Figure 4a). The qRT-PCR results showed that cardiomyocyte markers, such as cTnI, cTnT, αMHC, and Nkx2.5, were more highly expressed in Flk1+ MPCs grown on GP 200/280 gradient nanopattern substrates than in those grown on Flat substrates (Figure 4b). Therefore, Flk1+ MPCs exhibit extensive differentiation to mature cardiomyocytes on gradient nanopattern plates with 200−280 nm diameter pillars. 3.5. Cytoskeleton Reorganization Is Associated with Nanopillar-Induced Cardiomyocyte Differentiation. Finally, we confirmed the mechanism of cardiomyocyte differentiation from Flk1+ MPCs based on the association between the nanopillar and a cytoskeleton marker. The SEM images show that the Flk1+ MPCs on the GP 200/280 gradient nanopattern plate had filopodia that had a more complicated form and protruded further than those on the Flat plate (Figure 5a). The number of filopodia per cell was significantly higher in the Flk1+ MPCs on the GP 200/280 gradient nanopattern plate than in those on the Flat plate (Figure 5b). The immunofluorescence images show that Vinculin was more highly expressed in the Flk1+ MPCs on the GP 200/280 gradient nanopattern plate than in those on the Flat plate (Figure 5c). Furthermore, Cofilin expression was enriched within plasma membrane of Flk1+ MPCs and colocalized with Vinculin in Flk1+ MPCs (Figure 6a). The expression of p-Cofilin was significantly higher in the Flk1+ MPCs on the GP 200/280 gradient nanopattern plate and was colocalized with Vinculin expression (Figure 6b). The immunoblotting images show that G


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Figure 6. Cytoskeleton rearrangement is crucial for cardiomyocyte differentiation from Flk1+ MPCs. (a) Immunofluorescence images of Cofilin (green) and Vinculin (red) in either Flat or GP 200/280 gradient nanopattern plates at Nanoday 1 with cardiomyocyte differentiation medium. Nuclei were stained with DAPI (blue). Scale bar = 25 μm. (b) Immunofluorescence images for p-Cofilin (green) and Vinculin (red) in Flat and GP 200/280 gradient nanopattern plates at Nanoday 1 cultured in cardiomyocyte differentiation medium. Nuclei were stained with DAPI (blue). White arrowheads indicate colocalization of p-Cofilin and Vinculin. Scale bar = 25 μm. (c) Representative Western blotting images of Integrin α5, p-Cofilin, Cofilin, p-MLC2, and MLC2 expression in Flk1+ MPCs on Flat and GP 200/280 gradient nanopattern plate in cardiomyocyte differentiation medium after Nanoday 1. GAPDH was used as an endogenous control. Three independent experiments were performed. (d) Representative magnified images of F-actin (green)-stained Flk1+ MPCs on the GP 200/280 gradient nanopattern plate in cardiomyocyte differentiation medium at Nanoday 1. Nuclei were stained with DAPI (blue). White arrowheads indicate fragmented F-actin. Three independent experiments were performed. Scale bar = 10 μm.

We also added Y-27632, a ROCK inhibitor, to the cardiomyocyte differentiation medium. ROCK signaling is crucial for F-actin-mediated cytoskeleton reorganization. Inhibition of ROCK signaling by Y-27632 in bovine oocytes results in a reduction of cortical actin and decreases p-Cofilin and p-MLC levels.25 Metalloproteinase activation occurs via the renovation of the microfilament cytoskeleton in cardiac fibroblasts and is connected to increased cardiac cell migration.26 Our results showed that the F-actin in the Flk1+ MPCs was fragmented on the Flat plate, but stress fibers were maintained in the Flk1+ MPCs on the gradient nanopattern

the expression of p-Cofilin was higher on the GP 200/280 gradient nanopattern plate than on the Flat plate (Figure 6c). The F-actin expression pattern demonstrated that actin stress fibers were retained and exhibited clustering in the Flk1+ MPCs cultivated on the GP 200/280 gradient nanopattern plate compared with those on the Flat plate (Figure 6d). Therefore, gradient nanopattern plates with 200−280 nm diameter pillars induce the inhibition of F-actin disassembly and revive cytoskeleton reorganization via the transduction of p-Cofilin (Figure 7). H


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was associated with these nanopillar-induced cardiomyocyte differentiation processes. The gradient nanopattern plate supported the proliferation of Flk1+ MPCs and the induction of cardiomyocytes. In particular, the gradient nanopattern plate with pillars of 200−280 nm diameter contributed to the differentiation of mature cardiomyocytes. The gradient nanopattern plate provided a large quantity of differentiated cardiomyocytes. The differentiated cardiomyocytes formed sarcomeres and exhibited high cTnI expression. Moreover, pCofilin-mediated F-actin disassembly is a possible cause of cytoskeleton reorganization and cardiomyocyte differentiation. Cardiovascular diseases is a major cause of death worldwide and accounts for more than 0.6 million deaths per year in the United States.32,33 The results of this study may facilitate the massive production of mature cardiomyocytes for applications in cell therapy. Furthermore, differentiated cardiomyocytes from gradient nanopattern plates can be utilized for myocardium cell implantation therapy.

Figure 7. Gradient nanopattern plates induce F-actin dynamics. Illustrative summary of differentiation response of Flk1+ MPCs on gradient nanopattern plates. Scale bar = 200 nm.

plates with 200−280 nm diameter pillars after treatment with cardiomyocyte differentiation medium. Integrin expression did not differ significantly between Flat and gradient nanopattern plates; however, p-Cofilin expression was significantly higher in the Flk1+ MPCs on the gradient nanopattern plate. Our results demonstrate that nanopillar stimulation maintains p-Cofilin expression, which induces actin assembly in Flk1+ MPCs. MLC2 and the phosphorylated form were not detected in the Flk1+ MPCs on the gradient nanopattern plate at Nanoday 1. Therefore, we infer that ROCK activity was maintained, despite the Y-27632 to the cardiomyocyte differentiation medium, on the gradient nanopattern plate, and p-Cofilin-mediated actin cytoskeleton reorganization increased. Vinculin is initially expressed throughout the cell surface and closely associated with F-actin dynamics. Our results showed that Vinculin expression in the Flk1+ MPCs was higher on the gradient nanopattern plate than on the Flat plate. These results are similar to those of a previous study of normal human dermal fibroblasts.27 Furthermore, the Flk1+ MPCs on the gradient nanopattern plate had far more filopodia compared with those on the Flat plate. These results suggest that cells can recognize the nanosized pattern or a pillar surface and subsequently express Vinculin. We observed F-actin clustering in the Flk1+ MPCs on the gradient nanopattern plate. F-actin is an indicator of the cell response to the nanosized surface.28 An increase in the level of Vinculin is associated with the increased phosphorylation of Cofilin, colocalization with F-actin, and inhibition of F-actin disassembly.29−31 These results suggest that the gradient nanopattern plate increases Vinculin and pCofilin transduction and induces the cytoskeleton rearrangement of Flk1+ MPCs. Our understanding of cardiomyocyte differentiation, in which a physical stimulation-related signaling mechanism contributes to mature cardiomyocytes, remains limited. However, increased Vinculin expression is crucial for Factin clustering, and it induces the inhibition of F-actin disassembly, suggesting that the gradient nanopattern plate surface promotes cytoskeleton reorganization.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b01555. Analysis of cell proliferation via PHH3 and Ki67 immunofluorescence staining, analysis of cell apoptosis via cleaved caspase 3 immunofluorescence staining, TUNEL assay, and list of qRT-PCR primers (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Phone: +82-2-3290-5655; Fax: +82-2-929-8044; E-mail: [email protected]. *Phone: +82-2-920-5445; Fax: +82-2-927-1478; E-mail: [email protected]. ORCID

Do-Sun Lim: 0000-0001-5751-5177 Author Contributions ‡

H.-R.S. and H.J.J.: These authors made equal contributions to this work. K.B.L. and D.-S.L.: These authors took equal responsibility for supervising this work and are joint corresponding authors. Funding

This research was supported by a Basic Science Research Program through the National Research Foundation of Korea (NRF-2014R1A2A2A03007861 awarded to K.B.L. and NRF2016R1D1A1B03930758 awarded to D.-S.L.) funded by the Ministry of Education. Notes

The authors declare no competing financial interest. ⊥ K.B.L. and D.-S.L.: These authors took equal responsibility for supervising this work and are joint corresponding authors.

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ACKNOWLEDGMENTS We thank Jun K. Yamashita for providing the EMG7 cells. We are grateful to Li-Hua Huang for technical assistance.

4. CONCLUSION The principal findings of the present study were as follows: (1) the gradient nanopattern plate in which the nanopillar diameter was 200−280 nm upregulated the early cardiomyocyte marker Mesp1 in embryonic stem cell-derived Flk1+ MPCs; (2) the plate enhanced mature cardiomyocyte differentiation when combined with growth factors and other chemical stimulants; and (3) p-Cofilin-mediated actin cytoskeleton reorganization

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