Graphene Sheet-Induced Global Maturation of Cardiomyocytes

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Graphene Sheet-Induced Global Maturation of Cardiomyocytes Derived from Human Induced Pluripotent Stem Cells Jiaxian Wang, Chang Cui, Haiyan Nan, Yuanfang Yu, Yini Xiao, Ellen Poon, Gang Yang, Xijie Wang, Chenchen Wang, Lingsong Li, Kenneth Richard Boheler, Xu Ma, Xin Cheng, Zhenhua Ni, and Minglong Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b08777 • Publication Date (Web): 18 Jul 2017 Downloaded from http://pubs.acs.org on July 20, 2017

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ACS Applied Materials & Interfaces

Graphene Sheet-Induced Global Maturation of Cardiomyocytes Derived from Human Induced Pluripotent Stem Cells

Jiaxian Wang,†,‡,△ Chang Cui,†,‡ Haiyan Nan,§ Yuanfang Yu,§ Yini Xiao, ⊥ Ellen Poon,# Gang Yang,‡ Xijie Wang,|| Chenchen Wang,◊ Lingsong Li,◊ Kenneth Richard Boheler,# Xu Ma,△ Xin Cheng, Zhenhua Ni,*,§ and Minglong Chen*,‡ ⊥

‡

Division of Cardiology, The First Affiliated Hospital of Nanjing Medical University,

Nanjing 210029, China △National

Center for Human Genetics, National Research Institute for Family

Planning, Beijing 100081, China §

Department of Physics, Southeast University, Nanjing 211189, China

⊥

State Key Laboratory of Cell Biology, Shanghai Institutes for Biological Sciences,

Chinese Academic of Sciences, Shanghai 200031, China #

Stem Cell and Regenerative Medicine Consortium, School of Biomedical Sciences,

Li Ka Shing Faculty of Medicine, University of Hong Kong, Pokfulam 999077, Hong Kong ||

National Shanghai Center for New Drug Safety Evaluation and Research, Shanghai

201210, China ◊

SARI Center for Stem Cell and Nanomedicine, Shanghai Advanced Research

Institute, University of Chinese Academy of Sciences, Shanghai 201210, China

Keywords: graphene, cardiomyocytes, biomimetic surface, cardiac differentiation, human induced pluripotent stem cells

ACS Paragon Plus Environment


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Abstract: Human induced pluripotent stem cells (hiPSCs) can proliferate infinitely. Their ability to differentiate into cardiomyocytes provides abundant sources for disease

modeling,

drug

screening

and

regenerative

medicine.

However,

hiPSC-derived cardiomyocytes (hiPSC-CMs) display a low degree of maturation and fetal-like properties. Current in vitro differentiation methods do not mimic the structural, mechanical, or physiological properties of the cardiogenesis niche. Recently, we present an efficient cardiac maturation platform that combines hiPSCs monolayer cardiac differentiation with graphene substrate which is a biocompatible and superconductive material. The hiPSCs lines were successfully maintained on the graphene sheets, and were able to differentiate into functional cardiomyocytes. This strategy markedly increased the myofibril ultrastructural organization, elevated the conduction velocity and enhanced both the Ca2+ handling and electrophysiological properties in the absence of electrical stimulation. On the graphene substrate, the expression of connexin 43 increased along with the conduction velocity. Interestingly, the Bone morphogenetic proteins signaling was also significantly activated during early cardiogenesis, confirmed by RNA sequencing analysis. Here, we reasoned that graphene substrate as a conductive biomimetic surface could facilitate the intrinsic electrical propagation, mimicking the microenvironment of the native heart, to further promote the global maturation of hiPSC-CMs. Our findings highlight the capability of electrically active substrates to influence cardiomyocyte development. We believe that application of graphene sheets will be useful for simple, fast and scalable maturation of regenerated cardiomyocytes.


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1. INTRODUCTION Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent a potential unlimited source for disease modeling, cardiotoxicity screening and myocardial repairment1-3. Although a significant amount of efforts have been invested in developing highly efficient biochemical differentiation protocols for deriving hiPSC-CMs4-7, it is commonly accepted that their functional properties are immature in multiple aspects, such as slow conduction of electrical impulses, underdeveloped sarcomeres and poor electrophysiological properties7-9. These immature phenotypes have greatly hindered the applications of hiPSC-CMs for cell transplantation and drug screening. Current differentiation methods do not mimic the structural, mechanical, or physiological properties of the cardiogenesis niche. To address this issue, bioengineers have investigated the maturation effects on cardiomyocytes (CMs) with numerous techniques for recreating various characteristics of the native heart in vitro10-12. Indeed, developing a bioengineering approach to achieve efficient differentiation of hiPSCs into mature CMs would be highly beneficial. In a native heart, uncompromised cardiac pump functioning requires the adequate propagation of electrical impulses along the conduction system, which is composed of a group of specialized non-contractile cells that distribute impulses to coordinate the depolarization and contraction of the heart13. Disruptions in this conduction system result in slower or even blocked conduction, which is manifested as desynchronized mechanical actions14. Present differentiation protocols emphasize on generating the working myocardium, such as atrial and ventricular-like CMs, missing the regeneration of essential conduction system. Recently, several groups have used electrical stimulation as an exogenous approach to promote synchronized contraction to advance the function of regenerated CMs11-12. On the other hand, You and others reported that hybrid hydrogel scaffold based on Au nanoparticles increased the endogenous expression of connexin 43 of neonatal rat cardiomyocytes without electrical stimulation15-16. These studies have implicated that biomimetic conductive microenvironment may influence cardiac development by facilitating the electrical communication between cardiomyocytes in vitro.



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Graphene has recently gained considerable interest for tissue engineering applications

due

to

its

high

conductivity,

biocompatibility

and

superior

electromechanical properties17-18. Here, we report the use of graphene as a biocompatible and conductive substrate to differentiate large areas of hiPSC-CMs with comprehensively enhanced maturation. CMs differentiated on graphene sheets exhibited a remarkable degree of contractile protein organization, increased conduction velocity, enhanced Ca2+ handling and electrophysiological properties. RNA sequencing was also performed to investigate the notable changes at the gene expression level. Our findings suggest that graphene as a low-cost, robust and scalable surface material, provides a unique microenvironment and promotes the self-renewal and cardiac differentiation of hiPSCs, which will be useful for fast and high-throughput regenerated CMs with global maturation.

2. MATERIALS AND METHODS 2.1. Fabrication of the graphene substrate. Monolayer graphene was synthesized on a copper foil (25 um thickness) by chemical vapor deposition (CVD) method in a vacuum chamber with a gas mixture of methane and hydrogen (60 and 90 sccm, respectively, total pressure of ~ 250 Pa) at 1045 °C for 10 min. Graphene was transferred onto a coverslip using PMMA-assisted transfer technique. The copper foil was etched using (NH4)2S2O8 at a concentration of 0.5 mol/L, followed by rinsing PMMA/graphene in distilled water for 20 min. Then, PMMA/graphene was transferred to the coverslip, and the sample was left for natural withering. Finally, PMMA was dissolved by immersing the samples in acetone for ~ 12 h. For oxygen plasma modification, the graphene devices were treated by oxygen plasma (10 w, 10 pa) for 20s. The electrical characteristic of modified graphene was performed to test the conductivity. 2.2. Electrical transfer characteristics. Monolayer graphene grown by CVD was transferred onto 300 nm SiO2/Si substrate and patterned using UV lithography and O2 plasma etching (size 270 × 270 µm2). Source and drain electrodes (Ni (5 nm)/Au (50 nm)) were patterned by UV lithography and thermal evaporation. Electrical


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characteristics of the field effect transistor (FET) device were measured using a Keithley 2612 analyzer in an ambient environment. 2.3. Raman analysis. Raman spectroscopy was conducted using a LabRAM HR800 system with an excitation wavelength of 514.5 nm. The laser power used was below 0.5 mW to avoid laser-induced heating or damage. To obtain the Raman images, an x-y stage was used to move the sample with a step size of 2 µm, and a Raman spectrum was recorded at every point. 2.4. Scanning electron microscopy (SEM). Cells cultured on the graphene and coverslips were fixed with 4% paraformaldehyde (PFA) for 15 min and then washed 3 times with PBS. The samples were dehydrated at room temperature, and then the morphologies of cells on coverslips or graphene sheets were investigated by SEM (FEI Inspect F50). 2.5. Human pluripotent stem cell culture and cardiac differentiation. KB3 (homemade) and NC5 (Help Stem Cell Innovations, NC2001) hiPSC lines were seeded on both graphene substrate and coverslips after Matrigel (Corning, 354277) coating. Human iPSCs were cultured with daily renewal of mTeSR™ (Stemcell, 05850) medium. In brief, the cardiac differentiation medium consisted of RPMI 1640 (Gibco, 1744361) and B-27 (Gibco, A1895601), with extra CHIR-99021 (6 µM, Selleckchem, S2924) on Day 0-1 and IWR-1 (5 µM, Sigma, 10161) on day 3-4 was applied as previously described6. Cells were maintained in 5% CO2/37°C environment. 2.6. Immunofluorescent staining. hiPSC-CMs were fixed in 4% PFA for 20 min and permeabilized with 0.1% Triton-X 100 for 5 min as previously described. The cells were incubated with the following primary antibodies overnight at 4°C: mouse anti-TRA-1-60 (1:200, R&D, MAB4770), mouse anti-TRA-1-81 (1:200, CST, 4745S), mouse anti-SSEA-4 (1:200, R&D, MAB1435), rabbit anti-Oct4 (1:200, CST, 2750S), rabbit anti-Nanog (1:200, CST, 4903S), rat anti-human/mouse SSEA-3 (Alexa Fluor®488, 1:200, BioLegend, 330306), rabbit anti-α-actinin (1:200, CST, 6487S), mouse anti-cardiac troponin T (1:200, Abcam, ab8295), rabbit anti-Cx43 (1:200,



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Abcam, ab11370) and rabbit anti-MLC2v (1:200, Abcam, ab79935). Then cells were washed with PBS and incubated with Alexa-conjugated secondary antibodies, including donkey anti-mouse IgG (1:500, Abcam, ab150106), goat anti-rabbit IgG (1:500, Abcam, ab150077), and goat anti-mouse IgG (1:500, Abcam, ab150113). Nuclei were stained with DAPI (Life Technologies, P36931). Images were captured using a Zeiss fluorescence microscope (Axio Imager A2, Zeiss) with ZEISS Microscope Software ZEN. For confocal microscopy analysis, cells were visualized using a fluorescence confocal microscope (Nikon ECLIPSE TI). ImageJ (National Institutes of Health, 1.8.0_77) software was used to determine major and minor axis lengths (to provide a measure of aspect ratio), cell area, cell perimeter and sarcomere length as described before10. Cardiomyocytes were defined as rod shape cells once their aspect ratio was over 1.511. 2.7. Transmission electron microscopy (TEM). Single hiPSC-CMs dissociated from graphene or coverslips were fixed with 2.5% glutaraldehyde overnight at 4°C. Samples were then washed with PBS and stained in 2% uranyl acetate. Finally, sections were observed with a JEM-1010 microscope at 75 kV. The morphology of cardiomyocytes was analyzed with ImageJ. 2.8. Flow cytometry analysis. Single hiPSC-CMs were obtained from graphene or coverslips with trypsin. Then cells were fixed with 4% PFA for 15 min and washed with PBS. For intracellular epitopes, cells were permeabilized in permeabilization buffer (0.1% Triton X-100 in PBS) for 5 min at room temperature. Single cell suspensions were then incubated with primary antibodies: anti-cTnT-FITC (10 µl/test, Miltenyi Biotec, 130-106-687) and anti-MLC2v-PE (10 µl/test, Miltenyi Biotec, 130-106-133) in PBS for 30 min. REA Control-FITC (10 µl/test, Miltenyi Biotec, 130-104-611) and REA Control-PE (10 µl/test, Miltenyi Biotec, 130-104-613) were used as isotype controls19. Fluorescence-activated cell sorting was performed using a flow cytometer (BD FACS Calibur). The percentages of cells positive for cTnT and MLC2v were analyzed with FlowJo software (FlowJo, LLC, v10.0.7).


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For quantification of multinucleation, cells were stained with propidium iodide (Beyotime Biotechnology, C1052) as described before20. Briefly, cardiomyocytes obtained from graphene or coverslips were dissociated with trypsin and fixed with ice-cold 75% ethanol for 12 h. Cell suspensions were centrifuged at 300 g for 5 min before stained by 50 µl/ml PI and 20 µl/ml RNase A at 37˚C for 30 min. Finally, cells were detected with a flow cytometer (BD FACS Calibur). 2.9. Optical mapping. To access the conduction velocities of hiPSC-CMs, optical mapping was carried out21. 4 µM voltage-sensitive dye (Di-4, Life Technologies, D1199) was used to load hiPSC-CMs for 20 min at room temperature. After loading, cells were washed three times in PBS. Trans-membrane potentials were recorded in Tyrode’s solution using an Axio Vert.A1 Zeiss microscope armed with an sCMOS detector (Zyla 4.2 PLUS, Andor) at a sampling rate of 100 Hz. Isochronal map was generated using MATLAB (MathWorks, R2014a). Data were analyzed using ImageJ. 2.10. Measurements of calcium transients. Intracellular calcium transients were analyzed by loading hiPSC-CMs with 5 µM Fluo-3, AM (F1242, Life Technologies) and 0.02% Pluronic (P3000MP, Life Technologies) according to the manufacturer’s protocol. Cells were then washed in indicator-free medium to remove any dye that nonspecifically associated with the cell surface and incubated for 30 min to allow complete de-esterification of intracellular AM esters. Electrical stimulation or 50 mmol/L caffeine was applied to evoke calcium transients. Images were recorded using a Zyla 4.2 PLUS sCMOS camera. Recordings were analyzed with ImageJ and OriginPro-8. 2.11. Patch-clamp recordings. Patch-clamp experiments were carried out using a Axopatch 200B amplifier (Axon) as previously described22. The action potential of hiPSC-CMs was recorded in current-clamp mode by whole-cell patch-clamp technique at 37°C. Pipette solution consisted of: 120 mM K-aspartate, 25 mM KCl, 5 mM Mg2ATP, 1.8 mM CaCl2, 5 mM HEPES, 10 mM EGTA and 10 mM Glucose (pH 7.3). The external bath solution contained 140 mM NaCl, 5 mM KCl, 1.8 mM CaCl2,



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1 mM MgCl2, 10 mM Glucose and 10 mM HEPES (pH 7.3). Action potential recordings were performed with current pulse at 1 Hz steady-state pacing. For ICa recording, hiPSC-CMs were recorded with an internal solution designed to buffer intracellular Ca2+: 130 mM CsCl, 20 mM TEA-Cl, 1.8 mM MgCl2, 5 mM Na2ATP, 10 mM Glucoase, 5 mM EGTA and 10 mM HEPES (pH 7.3) as previously described. Cells were bathed in a standard solution containing: 120 mM Choline chloride, 20 mM CsCl, 1 mM MgCl2, 1.8 mM CaCl2, 10 mM HEPES and 10 Glucose (pH 7.3). ICa was elicited with a voltage ramp from -80 to +60 mV at 20 mV/s. ICa, L was recorded from a holding potential of -40mV using the same solution. To assess IK currents, hiPSC-CMs were bathed in a solution containing: 135 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 0.33 mM NaH2PO4 and 10 Glucose (pH 7.3). Patch pipettes were filled with internal solution containing: 45 mM KCl, 85 mM K-aspartate, 5 mM Na-pyruvate, 5 mM Mg2ATP, 10 mM EGTA and 10 Glucose (pH 7.3). All experiments were carried out at 37°C and data were analyzed with Clampfit 8.0 (Axon Instrument). 2.12. Western Blot. Proteins were extracted and analyzed from hiPSC-CMs as previously described23. Cell extracts were resolved by SDS/PAGE (Beyotime, P0012A), transferred to nitrocellulose membrane before incubation with the following primary antibodies overnight at 4°C: ssTnI (1:1000, Abcam, ab8293), cTnI (1:1000, Abcam, ab47003), cTnC (1:1000, Abcam, ab8285) and Cx43 (1:1000, Abcam, ab11370). Then, samples were washed three times before incubation with secondary antibodies. Western blot bands were acquired with a Molecular Imager ChemiDocTM XRS+ Imaging System (Bio-Rad). Quantification was carried out with Image LabTM Software (Bio-Rad). 2.13. Quantitative RT-PCR. Total RNA was isolated from hiPSC-CMs according to the manufacturer’s protocol (DP430, Tiangen). A total of 50 ng RNA was reverse-transcribed to cDNA using iScript cDNA Synthesis Kit (170-8891, Bio-RAD). Quantitative PCR using SYBR Green (170-8882AP, Bio-RAD) was performed


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(7900HT, Applied Biosystems). GAPDH was selected as the housekeeping gene for qRT-PCR. Primers are shown in Table S1. 2.14. RNA isolation and sequencing. hiPSC-CMs were lysed in Trizol (Life Technologies), followed by DNase I treatment for 30 min (Ambion, DNA-freeTM Kit). Total RNA was extracted and 3µg were used to generate a Polyadenylated RNA-seq libraries using Illumina TruSeq Stranded Total RNA HT Sample Prep Kit. Sequencing was performed using an Illumina HiSeq 2500, at CAS-MPG Partner Institute for Computational Biology Omics Core, Shanghai, China. Total and mapped read numbers were listed in Table S2. 2.15. Statistical analysis. All analyses were performed using SPSS Statistics 19.0 (IBM). Normally distributed data are presented as mean ± SD, estimated by Student’s t test. Skew distributed data are presented as median (interquartile range) and compared using Mann-Whitney U test. One-way ANOVA analysis followed by LSD and S-N-K tests were performed to analyze quantitative RT-PCR results. A p-value < 0.05 was considered significant.

3. RESULTS 3.1 Preparation of graphene sheets. Graphene sheets were prepared as previously described by our group (Figure 1a)24. To further confirm the ability of graphene to mediate electrical conduction, the electrical transfer characteristics were investigated. The electrical transport properties of a graphene field effect transistor device measured at room temperature is shown in Figure 1b. The Dirac point locates at ~ 18 V, suggests that graphene is lightly p-doped. Quantification of such transfer curve

µ= illustrated a hole mobility of ~ 4170 cm2v-1s-1 ( L is the length and W is width of device,

L dI ∗ ds W ∗ (ε 0ε r / d ) ∗Vds dV g

, where

ε 0 and ε r are the dielectric of air and

SiO2 respectively, d is the thickness of SiO2 layer, Vds is 10 mV). Results (Figure 1c)



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demonstrated that graphene substrates exerted excellent electron mobility and electrical conductivity. 3.2 Biological influences of graphene substrate on hiPSCs. Two hiPSC lines (KB3 and NC5) were cultured and differentiated into CMs with small molecules treatment on graphene sheets (Figure 2a). Paralleled cultivation on coverslips was performed as controls6. Raman spectra of hiPSCs/hiPSC-CMs grown on graphene substrate were all dominated by characteristic G band (~ 1580 cm-1) and 2D band (~ 2700 cm-1)25, indicative of the presence and integrity of graphene (Figure 2b, 2c; Figure S1). To assess the biological influences of graphene on hiPSCs, we first carried out the SEM scan. Results illustrated similar cellular and colony sizes 24-hour after cell seeding (Figure 2d, 2e; Figure S1), indicating an excellent biocompatibility. Moreover, hiPSCs maintained pluripotency on graphene sheets as shown by immunofluorescent staining of typical pluripotency markers, including TRA-1-60, TRA-1-81, OCT4, NANOG, SSEA-3, SSEA-4 (Figure 2f; Figure S2). 3.3 Graphene substrate improved cardiomyocyte phenotype. Previous studies have reported that biomaterials influence stem cell fates by ‘giving’ cues to stem cells via their physical properties10. We next investigated the effect of graphene substrate on cardiac differentiation. A small molecule-based monolayer differentiation protocol was adapted for cardiac regeneration6. Spontaneously contracting CMs were observed under light microscopy at days 7-9 after initiation. After 3 to 4 weeks in culture, hiPSC-CMs were digested and immunostaining illustrated that cells differentiated on both coverslips and graphene sheets robustly expressed cardiac Troponin T and cardiac contractile proteins α-actinin (Figure 3a, 3f), followed by FACS analysis confirming similar differentiation efficiency (~ 95%) and similar proportion of ventricular-like cardiomyocytes (~ 80%) between two groups (Figure S3). Although adult human CMs are rod-shaped with a length of approximately 100 µm, hiPSC-CMs are round cells that display fetal-like features during early cardiac development26. We then performed morphologic quantification under fluorescence microscope. Result indicated ~ 60% of CMs differentiated on graphene sheets displayed a rod-like


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phenotype, while only ~ 38% of CMs from coverslips displayed a rod-like shape (Rod-like shape cells Ratio: 38.00 ± 2.16% vs. 60.67 ± 1.70%, p

Graphene Sheet-Induced Global Maturation of Cardiomyocytes

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