Binary Colloidal Crystals (BCCs) Drive Spheroid Formation and

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Biological and Medical Applications of Materials and Interfaces

Binary Colloidal Crystals (BCCs) Drive Spheroid Formation and Accelerate Maturation of Human Induced Pluripotent Stem Cells-derived Cardiomyocytes Chang Cui, Jiaxian Wang, Duoduo Qian, Jiayi Huang, Jiao Lin, Peter Kingshott, Peng-Yuan Wang, and Minglong Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b17090 • Publication Date (Web): 07 Jan 2019 Downloaded from http://pubs.acs.org on January 9, 2019

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ACS Applied Materials & Interfaces

Binary Colloidal Crystals (BCCs) Drive Spheroid Formation and Accelerate Maturation of Human Induced Pluripotent Stem Cells-derived Cardiomyocytes Chang Cui, #† Jiaxian Wang, #†△ Duoduo Qian, † Jiayi Huang, † Jiao Lin, ⊥Peter Kingshott, ‡ Peng-Yuan Wang, * ‡⊥Minglong Chen*† †Division

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

210029, China △Department

‡Department

of R&D, HELP Stem Cell Therapeutics, Nanjing 210010, China

of Chemistry and Biotechnology, Swinburne University of Technology, Victoria

3122, Australia ⊥

Center for Human Tissues and Organs Degeneration, Insitute of Biomedicine and

Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China

*Corresponding author: P-Y.W.: [email protected]; M.C.: [email protected] #Equal

first author

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ABSTRACT: The development of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provides significant advances to cell therapy, disease modeling, and drug screening applications. However, the current differentiation protocol is inefficient in mimicking biophysical and biochemical characteristics of cardiac niche. Hence, immature cardiomyocytes are often generated. In this study, hiPSC-CMs were generated on a new family of substrates called monolayer binary colloidal crystals (BCCs). Four BCCs were fabricated with different sizes (2 μm or 5 μm or 0.4 μm or 0.2 μm) and materials (Si or PS or PMMA) abbreviated as 2PS, 5PS, 2PM, and 5PM. BCCs have complex surface micro-/nanotopographies and heterogeneous chemistries which are important modulators in microenvironment in vitro. The results showed that hiPSCs formed adhered spheroids with strong pluripotent markers (Oct4, Nanog, and Sox2) on PM surfaces compared to PS and flat surfaces. After 30-day differentiation, hiPSC-CMs on PM surfaces showed markedly improved myofibril ultrastructures, Ca2+ handling, and electrophysiological properties indicating that more mature hiPSC-CMs were generated. hiPSCCMs generated on 5PM is more similar to adult heart tissue compared to other surfaces in terms of genes (ACTC1, TNNT2, RYR2, SERCA2a, SCN5a, KCNJ2, CACNA1c, ITGB1, GJA1, MYH6, and MYH7) and proteins (ssTnI and cTnI) expressions. We further demonstrated that 5PM surfaces facilitated cadherin switching (from E- to N-) during cardiac differentiation and mature N-cadherin expression, which were positive correlated with the cardiogensis markers (GATA4, MEF2c, and NKX2.5). This study illuminated that a tailored surface nanotopography was beneficial in hiPSCs culture and in situ cardiac differentiation. This one step approach and BCCs can be a next-generation tool for hiPSCs expansion and CM differentiation.

KEYWORDS: hiPSC-CMs, BCC, spheroid, cadherin switching, cell-cell contact


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1. INTRODUCTION Considering human adult cardiomyocytes are post-mitotic, human induced pluripotent stem cellderived cardiomyocytes (hiPSC-CMs) hold great potential for disease modeling1, drug screening2, and cell transplantation3. However, mass production of hiPSC-CMs is still not fully optimized. Differentiated hiPSC-CMs exhibit immature characteristics such as disorganized sarcomere structures4, weak Ca2+ handling properties5, and immature action potentials6. These are key limitations for clinical applications. During embryonic development, stem cells are residing in a complex microenvironment where extracellular matrix (ECM), soluble factors, and mechanical forces exert biochemical and biophysical cues on cells and synergistically influence cell fate7-8. The current iPSCs expansion is costly and inefficient. Also, the current differentiation methods focus on the efficiency of generating CMs from hiPSCs using different chemicals9, failing to provide an appropriate stem cell niche with proper biophysical cues. Therefore, an efficient and productive approach for generation of mature CMs is urgently to be established. It has been recognized that artificial ECM provides biophysical cues that can stimulate cardiac differentiation from stem cells10. Using surface properties of biomaterials to direct stem cell behavior is a more defined, cost-effective, and longer-last approach compared to biochemical cocktails11. Previously, Li’s group12 proposed that morphological changes on grooves and fibers influenced methylation and prompted iPSCs generation. Similar concept has been used for direct reprogramming of fibroblasts into functional CMs using microgrooves10. Besides, Carson et al. reported that the grooved structure can modulate the cellular alignment and further promote the cardiac maturation4. These studies implicated that topographical cues can stimulate cells via


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focal adhesion dependent signal pathway and induce cytoskeletal tension and epigenetic change which is different from chemical cues. Recently, we developed a new family of surfaces called monolayer binary colloidal crystals (BCCs) for cell culture13-15. BCCs are composed of two different particles where the size, material, ratio, or surface chemistry can be varied resulting in that abundant combinations can be made as a high-throughput platform. In addition, different from other approaches, BCCs can be easily fabricated into ordered or disordered structures by changing surface properties and ratios of the particles. We demonstrated that BCCs have been used as a feeder free substrate for generation of hiPSCs from fibroblasts14. Without vitronectin coating, BCCs composed of 2 µm Si particles/110 nm PMMA particles and 5 µm Si particles/400 nm PMMA particles supported generation of hiPSCs and increased the proportion of fully reprogrammed hiPSC colonies compared to control. The result implied that changes of focal adhesions and cytoskeleton of fibroblasts may promote cell reprogramming. In the present study, we further investigated the effects of BCCs on the expansion of hiPSCs and in situ generation of hiPSC-CMs. Four BCCs surfaces abbreviated as 2PS, 5PS, 2PM, and 5PM were selected from our BCCs library based on different surface nanotopographies, roughness, and chemistries. Pluripotency of hiPSCs, cytoskeletal organization, Ca2+ handling, and electrophysiological properties of hiPSC-CMs were analyzed. Our finding showed that a tailored surface provides optimized microenvironment for hiPSCs culture and in situ cardiac differentiation.

2. EXPERIMENTAL SECTION


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BCC fabrication and characterization: Binary colloidal crystal (BCC) monolayers were fabricated according to our previous study14. Briefly, four BCCs, 2SiPM (2 μm silica particles and 0.1 μm poly (methyl methacrylate) (PMMA)), 5SiPM (5 μm silica particles and 0.4 μm PMMA), 2SiPS (2 μm silica particles and 0.2 μm polystyrene), and 5SiPS (5 μm silica particles and 0.4 μm polystyrene) were selected from our library for cell culture. Briefly, BCCs were fabricated by mixing two colloids and depositing them in a confined area on coverslips (Solarbio, YA0352). Furthermore, we also fabricated BCCs on both tissue-culture treated polystyrene well plates. The differences between two substrates were ignorable16-17. Therefore, we only used coverslips as control in this study. The volumes of the two colloidal solutions were calculated to be able to form a monolayer inside the area. After water evaporation, BCCs were heated to stabilize the particle layers. Prior to cell culture, BCCs were sterilized with UV light in biosafety hood, and coated with Matrigel for 1h. Matrigel coated tissue culture plates were used as controls. BCCs were characterized using scanning electron microscopy (FE-SEM; ZEISS SUPRA 40 VP, Carl Zeiss, Germany) and nanoindentation in this study. Other surface properties including surface roughness, wettability and chemistries were characterized by AFM, water contact angle, x-ray photon spectrometer (XPS, AXIS Nova, Kratos Analytical Ltd., Manchester, UK) and published in our previous studies13,

18.

For SEM, BCCs were coated with 5 nm gold before

imaging. For nanoindentation, three spots were measured on each surface and two samples for each BCC (n = 6). Human iPSCs culture: The hiPSC cell line NC5 (Help Stem Cell Innovations, NC2001) was expanded on Matrigel (Corning, 354277) coated surfaces. The seeding density of the hiPSCs was


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1×106 per well (10 cm2) on each surface. Cells were maintained with mTeSR medium (Stemcell, 05850) in a 5% CO2 and 37°C environment. Cardiac differentiation of hiPSCs: hiPSCs were subjected to directed cardiac differentiation as described before9. In brief, medium was changed to RPMI 1640 (Gibco, 1744361) with B-27 (Gibco, A1895601), when hiPSCs reached 80% confluence. For the early stages of differentiation, the cells were also exposed to the GSK3-β inhibitor CHIR 99021 (6 μM, Selleck, S2924) followed by the Wnt antagonist IWR-1 (5 μM, Sigma-Aldrich, 10161). Contracting cells were noted from day 8 and were fed every alternate day with RPMI 1640 supplemented with B27 supplement (Gibco, 17504-044). During day 15-20, medium was changed to purification medium, which consists of glucose-free DMEM (Gibco, 11966025) supplemented with 4 mM lactic acid and sterile 1M Na-HEPES. After 30 days of in vitro differentiation, the cells were trypsinized and replated on gelatin-coated coverslips (Solarbio, YA0352) for further experiments. Immunofluorescence staining: Cells were fixed with 4% paraformaldehyde (PFA) in DPBS for 20 min at RT, permeabilized with 0.1% Triton-X 100 for 10 min. Cells were then incubated with the following primary antibodies overnight at 4°C: rabbit anti-Oct4 (CST, 2750S), mouse antiSSEA-4 (R&D, MAB1435) for pluripotency staining of hiPSCs; rabbit anti-α-tubulin (abcam, ab18251) and mouse anti-α-actinin (Sigma, A7811) for structural staining of hiPSC-CMs; rabbit anti-N-Cadherin (abcam, ab76057) and mouse anti-E-Cadherin (abcam, ab1416) for cell adhesion staining. The secondary antibodies were donkey anti-rabbit IgG (Alexa Fluor® 488, abcam, ab150073) and goat anti-mouse IgG (Alexa Fluor® 555, abcam, ab150118). Nuclei were visualized with DAPI (Beyotime, C1006). Images were captured using a Zeiss fluorescence microscope (Zeiss, Axio Vert A1). Measurements of the cardiomyocyte size were performed


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with ImageJ software (National Institutes of Health, 1.8.0_77) as previously described16. The aspect ratio was defined as long axes/short axes. The circularity index was defined as 4π area/perimeter2. Flow cytometry: Signal cardiomyocytes were obtained with trypsin and fixed in 4% PFA for 20 minutes. To analysis intracellular proteins, cells were permeabilized with 0.1% Triton-X 100 for 10 min. The following primary antibodies were applied: rat anti-SSEA3 (Alexa Fluor® 488, R&D, FAB1434G), rabbit anti-cardiac troponin I (Alexa Fluor® 488, abcam, ab196384), rabbit anti-MYL7 (PE, Miltenyi, 130-117-546) and mouse anti-MYL2 (Alexa Fluor® 488, Novus, NBP1-30249G). The stained cells were counted using a BD FACS Calibur. The following data analysis was performed using FlowJo software. Measurements of Calcium transient: Dissociated hiPSC-CMs were reseeded on gelatin-coated 12-well plates. Cells were treated with 5 μM Fluo-3, AM (Life Technologies, F1242) and 0.02% Pluronic (Life Technologies, 3000MP) and then washed with Tyrode’s solution. Images of Ca2+ were recorded with an sCMOS (Tucam, 2300KPA). Recordings of spontaneous calcium transients were analyzed with ImageJ. Patch clamp: Whole-cell patch clamp was used to record the action potentials (APs) on the Axopatch 200B amplifier (Axon). AP was examined using the following intracellular solution: 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 Tyrode’s solution composition was as follows: 140 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 10 mM glucose and 10 mM HEPES (pH 7.3). The quantitative analysis of action potential was performed as previously described19.


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Quantitative RT-PCR (qRT-PCR): RNA was isolated with Trizol (Invitrogen) following the manufacturer’s instructions. cDNA was produced using iScript cDNA Synthesis Kit (170-8891, Bio-RAD). Real-time PCR was conducted using SYBR green PCR kit (170-8882AP, Bio-RAD) and performed on a 7900HT Real-Time PCR System (Life Technologies). All PCR reactions were in quadruplicate and normalized to β-actin or GAPDH which were considered as the housekeeping genes. Primers are shown in Table S1, Supporting Information. Study procedures were approved by the Bioethics Committee of the First Affiliated Hospital of Nanjing Medical University (2014-SR-090). Human adult heart tissues collected during the surgical procedures were snap-frozen in liquid nitrogen and followed by protein analysis and qRT-PCR. Western Blot: Western blot was conducted as previously described by our group19. The primary antibodies used in the present study were as follows: GAPDH (1:1000, Cell Signaling, 2118S), N-Cadherin (1:1000, abcam, ab76057), E-Cadherin (1:1000, abcam, ab1416), cTnI (1:1000, abcam, ab47003), and ssTnI (1:1000, Abcam, ab8293). Secondary antibodies employed in this work included anti-rabbit IgG antibody (1:5000, Cell Signaling, 7074P2) or anti-mouse IgG antibody (1:5000, Cell Signaling, 7076). Statistical Analysis: SPSS Statistics 19.0 was used for statistical analysis. Normally distributed data were shown as the means ± standard deviation (SD) estimated by one-way analysis of variance (ANOVA) followed by Dunnet test. Skew distributed data were expressed as the median (interquartile range), estimated by Nemenyi test. A p-value less than 0.05 was considered statistically significant.

3. RESULTS AND DISCUSSION


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Surface characterization of BCCs Monolayer binary colloidal crystals (BCCs) is a new type of substrates for stem cell expansion and manipulation13-14. In the present study, four BCCs surfaces composed of large silica (2 or 5 micron) and small polymer particles (either PS or PMMA in 100 or 400 nm) abbreviated as 2PS, 5PS, 2PM, and 5PM were selected (Figure 1). Surface characterizations of these BCCs have been done previously13 where different surface topographies, roughness, and wettability can be seen using scanning electron microscopy (SEM), atomic force microscopy (AFM), and water contact angle goniometer. Human iPSCs cultured on BCCs Modification of cell culture substrates has been involved in generation of cardiomyocytes20. We recently reported that graphene coated substrates could enhance the globe maturation of differentiated cardiomyocytes via its electroconductive property16. Our previous results have shown that optimized BCCs facilitated the generation of hiPSCs by increasing the proportion of fully reprogrammed hiPSC colonies14. This result suggested that the complex surface properties of BCCs may be able to mimic the in vivo microenvironment or alter the in vitro niche such as protein adsorption. Although BCCs have shown the potential in cell reprogramming, their effect on longer term iPSCs culture and differentiation was unknown. Our group previously showed that without protein coating (i.e. gelatin, vitronectin, and Matrigel), hiPSCs and hESCs (i.e. H9 cells) cannot attach on any surfaces including BCCs21. Herein, human iPSCs (NC5) were maintained on Matrigel coated surface and in mTeSR medium using standard protocol16. hiPSCs were seeded on Matrigel-coated BCCs and coverslip controls. After 2 days, hiPSCs grew into monolayers on 2PS, 5PS, and control surfaces. However, hiPSCs formed stereoscopic clusters on 2PM and 5PM surfaces after 2 days (Figure 2a). On 5PM


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surfaces, hiPSC clusters were slightly larger with denser cell number compared to 2PM surfaces. After 4 days, pluripotency of hiPSCs was confirmed using immunofluorescence staining, i.e. OCT4 and SSEA-4 (Figure 2b). hiPSCs expressed OCT4 and SSEA-4 on all surfaces suggesting that BCCs exhibited excellent biocompatibility without interfering iPSCs culture and expansion. Accordingly, FACs analysis of SSEA-3 proved that pluripotent, healthy hiPSC colonies could be formed among five surfaces (Figure 2b). The difference is that 3D spheroids were formed on PM group while 2D-like colonies were formed on PS group and control. Morphological analysis of hiPSCs colonies showed that colonies on PM surfaces had a lower aspect ratio and higher circularity compared to the control (n = 17-18, Figure 2c and 2d). The morphology of hiPSCs colonies has been reported that is a critical factor of their pluripotency22. The pluripotency of hiPSCs was further confirmed using quantitative real time PCR (qRT-PCR) on OCT4, NANOG and SOX2. The result showed that higher gene expressions were found on PM group especially 5PM surface compared to the PS groups and control (n = 4, Figure 2e). In our previous study13, the wettability on 2PM and 2PS are similar (43-53 degree), 5PM is about 60 degree, and 5PS is the most hydrophobic (~ 83 degree). The roughness of 2PM and 2PS is similar (Ra = 90-100 nm), 5PM is about double (~ 180 nm), and 5PS is about triple (~ 270 nm). In addition, the main difference between PM and PS groups is surface chemistry. Therefore, the cause of 3D-like spheroid formation of hiPSCs was due to a collective effect of these surface factors but not one of them. The complex surface properties may also influence the adsorption of Matrigel or media proteins, and then result in different cell morphology. Controlling cell spreading is one of the key to modulate cell fate including maintaining pluripotency and/or improving differentiation and cell function23-24. It is more defined to manipulate cell spreading and morphology using surface nanotopographies compared to surface


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chemistry. We previously reported that nanogrooves can prolong the in vitro beating of mice cardiomyocytes because more organized cytoskeletal structure was developed25. It was also reported that mESCs aggregated into 3D-like structure on patterned substrates including grooves, hexagons, and pillars with higher levels of pluripotent gene expressions compared to control22. In the current study, we demonstrated that optimized PM surfaces allowed hiPSCs to form 3D-like spheroids and maintained their pluripotency. Cardiac differentiation on BCCs We next induced the cardiac differentiation of hiPSCs in situ using small molecules9 (Figure 3a). Cells were counted for each substrate before and after the differentiation procedure (i.e. day 0 and day 30), which were defined as input and output amount respectively. There were no significant differences in output amount between BCC groups and controls except 2PS, and the output cells is about double of the input cells (Figure S1, Supporting Information). Spontaneous beating cardiomyocytes were observed on all surfaces after 8 days. At the very beginning, the beating areas from control and PS groups were weak and even undetectable (2PS group), whereas cardiomyocytes from PM groups beat strongly (Day8, Video S1-5, Supporting Information). The morphology of hiPSC-CMs was still 2D-like cell sheets on control and PS groups while these formed 3D-like spheroids on PM groups (Figure 3b). hiPSC-CMs was digested and replated to analysis the maturation after 30 days in culture. Fluorescence-activated cell sorting (FACS) analysis of cardiac troponin I (cTnI) showed high differentiation efficiency (~95%) of hiPSCs on all surfaces after purification (Figure S2, Supporting Information). It has been reported that MLC2v positive means ventricular-like cardiomyocytes, while MLC2v positive/MLC2a negative means mature ventricular-like cardiomyocytes26-27. In chamber specific analysis, a similar proportion of MLC2v-positive hiPSC-CMs (~95%) was found on all surfaces


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(Figure 3c-d) suggesting that the majority of hiPSC-CMs were ventricular-like cardiomyocytes. However, the ratio of mature ventricular cardiomyocytes, defined as MLC2v+/MLC2a- cells, was higher on PM groups (~80%) than PS groups and control (

Binary Colloidal Crystals (BCCs) Drive Spheroid Formation and

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