Functional engineered human cardiac patches prepared from nature's platform improve heart function after acute myocardial infarction

Accepted Manuscript Functional engineered human cardiac patches prepared from nature's platform improve heart function after acute myocardial infarction Qingjie Wang, Hui Yang, Aobing Bai, Wei Jiang, Xiuya Li, Xinhong Wang, Yishen Mao, Chao Lu, Ruizhe Qian, Feng Guo, Tianling Ding, Haiyan Chen, Sifeng Chen, Jianyi Zhang, Chen Liu, Ning Sun PII:

S0142-9612(16)30372-6

DOI:

10.1016/j.biomaterials.2016.07.035

Reference:

JBMT 17639

To appear in:

Biomaterials

Received Date: 31 March 2016 Revised Date:

22 July 2016

Accepted Date: 27 July 2016

Please cite this article as: Wang Q, Yang H, Bai A, Jiang W, Li X, Wang X, Mao Y, Lu C, Qian R, Guo F, Ding T, Chen H, Chen S, Zhang J, Liu C, Sun N, Functional engineered human cardiac patches prepared from nature's platform improve heart function after acute myocardial infarction, Biomaterials (2016), doi: 10.1016/j.biomaterials.2016.07.035. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Functional engineered human cardiac patches prepared from nature's platform improve heart function after acute myocardial infarction Qingjie Wang1,*, Hui Yang1,*, Aobing Bai1, Wei Jiang1, Xiuya Li1, Xinhong Wang1, Yishen Mao1, Chao Lu1, Ruizhe Qian1, Feng Guo2, Tianling Ding5, Haiyan Chen6, Sifeng Chen1, Jianyi Zhang7, Chen Liu6#, Ning Sun1, 3, 4# Department of Physiology and Pathophysiology, 2Department of Cell Biology and Genetics, School of Basic Medical Sciences, 3Research Center on Aging and Medicine, 4State Key Laboratory of Medical Neurobiology, 5Department of Hematology, Huashan Hospital, 6 Department of Cardiac Surgery, Zhongshan Hospital, Fudan University, Shanghai 200032, China 7 Department of Biomedical Engineering, University of Alabama, Birmingham, AL, 35294, USA

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*These authors contributed equally to this work

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#Correspondence should be addressed to Ning Sun ([email protected]) and Chen Liu ([email protected])

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ABSTRACT With the advent of induced pluripotent stem cells and directed differentiation techniques, it is now feasible to derive individual-specific cardiac cells for human heart tissue engineering. Here we report the generation of functional engineered human cardiac patches using human induced pluripotent stem cells-derived cardiac cells and decellularized natural heart ECM as scaffolds. The engineered human cardiac patches can be tailored to any desired size and shape and exhibited normal contractile and electrical physiology in vitro. Further, when patching on the infarct area, these patches improved heart function of rats with acute myocardial infarction in vivo. These engineered human cardiac patches can be of great value for normal and disease-specific heart tissue engineering, drug screening, and meet the demands for individual-specific heart tissues for personalized regenerative therapy of myocardial damages in the future.

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Keywords: Decellularized heart matrix, Engineering human heart tissue, Induced pluripotent stem cells, Cardiomyocytes, Cardiac patch, Myocardial infarction

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INTRODUCTION Cardiovascular disease is now a leading cause of death worldwide [1]. Since postnatal cardiomyocytes exhibit nearly no regenerative capacity, myocardium damages often develop into scar tissues and progressively lead to heart dilatation and failure [2]. Except for heart transplantation, there are no satisfactory treatments for heart failure to date. However, this practice is limited by the scarce number of donor organs as well as the lifelong immunosuppression for patients. Engineered heart tissues (EHTs) represent a promising alternative therapy for myocardium damage. There have been many studies using cardiomyocytes from non-human animals especially from rodents for preparation of bioartificial heart tissues [3]. However, the difference in species and electrophysiological properties of animal cardiomyocytes with human ones making them not an ideal cell source for future clinical applications. With the advent of induced pluripotent stem cells (iPSCs), it is now possible to generate individual-specific human cardiomyocytes for personalized human heart tissue engineering [4-7]. Another important component for heart tissue engineering is the scaffold for cell attachment and growth. A number of scaffolds have been fabricated using synthetic polymers, natural biological materials including collagen and alginate, as well as decellularized heart matrix [8, 9]. In 2008, Ott et al. showed for the first time that the rat heart can be decellularized by perfusion using detergents, which generated natural heart extracellular matrix (ECM) [10]. Compared to the synthetic polymers and biological materials, the decellularized heart matrix represents a better scaffold because it preserved the architecture and ECM protein components of the natural heart. Using similar methods, a recent study by Lu et al. decellularized the mouse whole heart and recellularized it with human pluripotent stem cells-derived multipotent cardiovascular progenitors (MCPs) [11]. This pioneering work demonstrated the possibility of combining human iPSC-derived cardiovascular cells and decellularized heart matrix for preparation of human EHTs. However, the repopulated whole heart was not uniform in cell distribution and still left giant gaps of uncoupled areas. Whether engineered tissues from such recellularized mouse heart are effective in treating myocardial damages remains unknown. It is also worth to note that normal heart tissues are composed of ~40% cardiomyocytes and ~60% cardiac fibroblasts [12]. Using a single population of iPSC-derived MCPs as the “seed” cells to repopulate the whole heart is difficult to control the precise optimal ratio of cardiomyocytes and non-cardiomyocytes for the EHTs. During preparation of this manuscript, Guyette et al., reported recellularization of decellularized human cadaveric heart matrix with hiPSC-derived cardiomyocytes [13]. However, the above mentioned problems were still not solved. In this study, we combined fixed ratio of human iPSCs-derived cardiomyocytes and fibroblasts with pieces of decellularized natural rat heart ECM for preparation of engineered human cardiac patches. This method allowed us to generate human cardiac patches of any desired shape and size with well distributed cells. Further, decellularized natural heart ECM improved maturation of human iPSCs-derived cardiomyocytes. The decellularized natural heart ECM-based human cardiac patches exhibited beating activity and electrophysiology similar with human normal heart muscles and responded well to pharmaceutical agents affecting cardiomyocyte physiology. We further patched such engineered human cardiac patches on the infarct area of the heart of rat MI models. We show that engineered human

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cardiac patches effectively helped reduce the infarct size and improved the heart function after acute MI. Our data indicated that, using decellularized natural heart ECM and human iPSCs-derived cardiac cells, it is feasible to generate individual-specific human cardiac patches of different sizes and shapes. These human cardiac patches can be of great value for drug screening and study of inherited heart diseases, as well as meeting the demands for individual-specific (size and shape) EHTs for personalized regenerative therapy of myocardial infarctions.

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MATERIALS AND METHODS Animals Sprague-Dawley (SD) rats were obtained from the Laboratory Animal Care Facility of Shanghai Medical College at Fudan University. All the protocols in this study were approved by the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Publication No. 85-23, Revised) and was carried out under the supervision of the Fudan University Institutional Animal Care and Use Committee. All rats received cyclosporine (10 mg/kg/day, supplemented with food) to suppress immune rejection after xeno-transplantation.

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Decellularization and characterization of rat cadaveric hearts For preparation of decellularized natural heart extracellular matrix (DC-ECM), we used 10-12-weeks-old SD rat cadaveric heart and decellularized it with methods modified from the previously published protocol [11]. Briefly, hearts were obtained immediately after euthanasia of adult rats and kept frozen at -80˚C. Before decellularization, the hearts were thawed in deionized water at room temperature. A blunted 20-gauge needle was cannulated into the ascending aorta to allow retrograde coronary perfusion. Sterile deionized water was perfused for 15-30 min at 2.0 ml/min, followed by perfusion with sterile phosphate-buffered saline (PBS). The heart was then perfused with 1% sodium dodecyl sulphate (SDS) for two hours, 1% Triton X-100 with 0.5% EDTA (PH 8.0) for another 30 min at room temperature, followed by washing with antibiotic-containing deionized water and PBS (100 U/ml penicillin (Life Technologies, USA), 100 µg/ml streptomycin (Life Technologies) and 1.25 µg/ml amphotericin B (Sigma Aldrich, USA)) for two hours.

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Derivation, culture and characterization of hiPSCs The protocol for derivation of human skin fibroblasts in this study was approved by the Institutional Ethical Review Board of Fudan University for Human Subjects. Human iPSC lines were generated as previously described [14] using the CytoTune®-iPS 2.0 Sendai Reprogramming Kits (Life Technology) following manufacturer's instruction. The cells were assayed for the expression of pluripotency markers by immunofluorescence and alkaline phosphatase staining. Derived hiPSCs were maintained on tissue culture dishes coated with Matrigel (growth factor reduced; BD Biosciences) using mTeSR-1 medium (Stemcell Technology) for subsequent analyses and differentiation. Teratoma formation To form teratomas, approximately 2 million undifferentiated human skin fibroblast derived-hiPSCs were harvested, mixed with Matrigel, and injected subcutaneously to

ACCEPTED MANUSCRIPT immunodeficient mice. After 6–8 weeks, teratomas were dissected, fixed with 10% formaldehyde in PBS, embedded in paraffin wax, sectioned and stained with H&E.

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Differentiation of hiPSCs Differentiation into cardiac lineage was carried out with the protocol described by Sean P Palecek et al. [15]. The hiPSCs were initially cultured in mTeSR1 medium on Matrigel-coated plates until they were ~90% confluent. Medium was changed to RPMI/B-27 without insulin, consisting of RPMI 1640 (Corning) and B-27 minus insulin (Life technologies). On day 0-1, medium was supplemented with 12µm CHIR-99021 (Selleck) in RPMI/B-27 without insulin. After 24 hours, IWR-1 (5µm, Sigma) was added into the fresh RPMI/B-27. On day 5-6, medium was changed to RPMI/B-27 minus insulin. On day 7 of differentiation and every 2 day thereafter, aspirated the medium and added RPMI/B-27 medium. Cultures were maintained in a 37˚C, 5% CO2 environment. Contracting cells should be observed from day 7 post differentiation. Spontaneous differentiation of hiPSCs were carried out for obtaining CD90+ cells. Briefly, hiPSCs were digested from culture plates and floating embryiod bodies (EBs) were formed using ultralow attachment plates (Corning). EBs were then cultured with DMEM high glucose (GIBCO) supplemented with 20% FBS under a 37˚C, 5% CO2 environment for 14 days with medium change every 2 days. The spontaneously differentiated EBs were then dissociated and subjected to fluorescence activated cell sorting (FACS) using anti-CD90 antibodies (Supplementary Table 1).

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Flow cytometry Sheets of cells were dissociated as previously reported [16]. Briefly, cells were digested with Collagenase I (1 mg/ml, Sigma) with DNase I (40 Unit/ml, Calbiochem) in PBS for 20 min, and followed by 0.25% trypsin/EDTA treatment for 5 min at 37˚C. For sorting CD90+ cells from spontaneously differentiated hiPSCs, cells were washed and stained with the primary anti-CD90 antibodies at 37˚C for 30 min, washed, and then sorted with a FACSAria (BD Biosciences). For analysis of cTnT positive cardiomyocytes from differentiated hiPSCs, cells were fixed and permeabilized with fixation and permeabilization solution (BD Biosciences) for 30 min, and stained with the primary anti-cardiac troponin T (cTnT) antibodies or isotype control antibodies (Supplementary Table 1) for 30 min, washed, and stained with appropriate secondary antibodies. After staining, the cells were washed, resuspended, and evaluated with a FACSCalibur (BD Biosciences) and CELLQuest software. Generation of 3D cardiac patch The decellularized heart ECM was cut into pieces of desired shape and size using a surgical scissor under sterile condition. Individual native heart ECM pieces were put in wells of either 96-well or 48-well plates as a sheet, with the endocardial side facing up. The mixture of hiPSC-derived cardiac cells (75% hiPSC-derived cardiomyocytes and 25% hiPSC-derived CD90+ cells) were then seeded onto the ECM sheet at 104 cells/mm2. Resulting cardiac patches were cultured in DMEM supplemented with 5% FBS and antibiotics. In order to inhibit proliferation of non-myocytes and preserve initial purity of hiPSC-CMs, the media was switched to RPMI/B27 the next day and changed every 2 days.

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Quantitative real time PCR Total RNA was extracted using the Trizol reagent (Life technologies) according to the manufacturer's instructions. RNA (2ng) was reverse-transcribed into cDNA by the AMV Reverse Transcription System (Promega). Quantitative RT-RCR was performed with the SYBR Green QPCR system (Bioscience) with GAPDH as an internal control. The nucleotide primer sequences can be found in the Supplementary Table 2.

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DNA quantification Approximately 10 mg of cadaveric heart and decellularized tissue were subjected to DNA extraction using EasyPure Genomic DNA kit (Transgen Biotech) according to manufacturer's instructions. The DNA extracts were next used for DNA quantification using Epoch microplate reading (BioTek).

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Western Blotting The cardiac patches and ECM were homogenized, and lysed in RIPA buffer supplemented with protease inhibitor cocktail (Sigma Aldrich). Protein was electrophoresed and separated by 10% SDS-PAGE and transferred to polyvinlidene difluoride membranes (Millipore). The membrane was incubated with antibodies of human CX43, α-actinin and CD31, then corresponding secondary antibodies. Blots were visualized using an enhanced chemiluminescence (Applygen).

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Scanning electron microscopy (SEM) Samples were fixed with 1.25% (vol/vol) glutaraldehyde for 2 h at 4˚C, followed by washing with PBS (three times; 15 min each), and then post-fixed with 1% osmium tetroxide for 2 h. After osmium tetroxide post-fixation, the tissue were washed again three times with PBS. Then the samples were dehydrated by graded ethanol series (50%, 70%, 90% and 100%) and air-dried. Samples were sputter-coated with gold and visualized using a Hitachi SU8010 field emission gun SEM (Hitachi, Japan).

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Transmission electron microscopy (TEM) Samples were fixed with 2.5% (vol/vol) glutaraldehyde for 1 h at room temperature, followed with three washes with PBS (15 min each) and post-fixed with 1% osmium tetroxide for 2-3 h. The samples were washed in PBS, dehydrated in graded ethanol (50%, 70%, 90% and 100%) and then in propylene oxide for 10 min. Next samples were embedded, sectioned at about 70-nm thickness and stained with lead citrate. Micrographs were captured using a PHILIPS CM-120 transmission electron microscope (PHILIPS, Holland). Electrophysiological assessment of the engineered human cardiac patches The electrophysiological properties of engineered cardiac patches were examined using the microelectrode array (MEA) data acquisition system MEA-2100 (Multi Channel Systems) [17, 18]. Contracting engineered human cardiac patches were plated on gelatin coated MEA probes. Local activation time at each of the 60 electrodes was determined and used for the generation of color-coded activation maps as reported [19]. Data analysis was performed

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Induction of myocardial infarction in SD rats and treatment Myocardial infarction (MI) was induced in male SD rats (250 to 300 g) by left anterior descending coronary artery (LAD) ligation under 2.5% isoflurane anesthesia as previously described [20]. The animals were randomly divided into four groups. Animals in the "patch" group were treated by transplanting the engineered human cardiac patch (Patch, n=9). Animals in the ECM group were treated with the natural heart ECM alone (ECM, n=9), and both the ECM and the cells were withheld from animals in the MI group (MI, n=7). In the sham group, animals were exposed to identical open-chest surgery but without LAD ligation or any treatment (Sham, n=8). The LV contractile performance was examined by echocardiography on a Vevo770 Imaging System (VisualSonics, Canada) equipped with a 30MHz transducer at 1, 2, 3, and 4 weeks after injury and transplantation. Short-axis views were obtained and both conventional 2-dimensional images and M-Mode images at the level of midpapillary muscle as described previously in details [21]. Measurements of left ventricular internal systolic dimension (LVIDs) and left ventricular internal diastolic dimension (LVIDd) in M-mode were performed. LV ejection fractions (EF) and fractional shortening (FS) were calculated according to equations: EF= (LVIDd3- LVIDs3)/ LVIDd3×100%; and FS= (LVIDd- LVIDs)/ LVIDd×100%.

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Histology and immunofluorescence staining Rat cadaveric hearts, decellularized hearts and engineered cardiac patches were fixed, paraffin-embedded and sectioned, followed with hematoxylin and eosin (H&E) staining. Moreover, fixed hearts were embedded into optimal cutting temperature compound (OCT, Sakura Finetek, Japan), frozen, and sectioned into 8-µm-thick sections. The slides were permeabilized with 0.5% Triton X-100 for 15 min, and blocked with normal goat serum for 30 min. The slides were then incubated with appropriate primary antibodies at 4˚C overnight. Last, the samples were washed three times and stained with appropriate secondary antibodies and DAPI. Before imaging, all slides were covered with cover glass in hardening mounting medium. Tissue images were recorded with the fluorescence microscope Leica DMi8 (Leica). The antibodies are described in the Supplementary Table 1. The ischemic hearts were obtained and fixed 4 weeks after MI. The heart tissue below the ligation site were sectioned into 5 slices sampled at 2-mm intervals from the apex. Masson's trichrome staining and H&E staining were performed for histological analysis. Infarct size was measured as previously reported [22]. The ventricular wall thickness was measured across the infarcted region of the heart wall from the closest section plane. For immunohistochemistry analyses, cryosections were used. Antibodies against von Willebrand factor (vWF) and α-smooth muscle actin (α-SMA) were stained for vascular density measurement as described previously [23]. Statistical analysis Experimental data was reported as mean±SEM. To compare between the different of two groups, two-tailed Student's t test was used. Statistical differences among more than two

ACCEPTED MANUSCRIPT groups were analyzed with one-way analysis of variance (ANOVA) tests. When P value is less than 0.05, significant differences were determined.

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RESULTS Decellularization of rat heart and characterization of the natural heart ECM The overall schema of making the human cardiac patches using decellularized natural heart matrix and human iPSCs-derived cells is shown in Fig. 1A. Decellularization of the cadaveric rat heart was performed according to the methods described in the Methods and Materials section. Continuous perfusion of the rat heart with detergent solution removed the cellular components and led to a transparent whole heart matrix (Fig. 1B). For the decellularized heart tissues, H&E and DAPI staining showed no visible cell nuclei or double-stranded DNA within the decellularized heart matrices, whereas the native heart tissues showed very dense cellularity (Fig. 1C). DNA content of the decellularized ECM was nearly removed from the native hearts (Fig. 1D), confirming the removal of cellular contents. ECM proteins, such as fibrinogen, laminin, and collagen type III, remained to be present within the decellularized heart matrices and were detected by immunofluorescence staining (Fig. 1E). Scanning electron microscopy (SEM) also showed that the texture and orientation of the ECM of the decellularized heart was preserved, whereas cardiac cells were removed (Fig. 1F). These results indicated a complete removal of cells from the heart tissues and well preservation of the heart ECM, which is consistent to previous reports of generating decellularized rat and mouse heart matrices [10, 11].

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Generation of human cardiomyocytes from hiPSCs We next generated human iPSCs from skin fibroblasts of a 18 years old male and a 30 years old male individual as previously described [24]. Three weeks after reprogramming, colonies with human embryonic stem cell (hESC)-like morphology were picked (Supplementary Fig. 1A), expanded and established as individual iPSC lines. The expanded iPSCs expressed the pluripotency markers Nanog, Oct4, Sox2, and SSEA4, exhibited strong alkaline phosphatase activities (Supplementary Fig. 1B), and formed teratoma after transplantation into immunodeficient mice (Supplementary Fig. 1C). These results confirmed the pluripotent nature of the established hiPSC lines. We next differentiated the hiPSCs into the cardiovascular lineage using a method slightly modified from previous reports [11]. The detailed protocol used in this study is shown in Fig. 2A. Beating cells were observed on day 8 after induction of differentiation (Fig. 2B, and Supplementary video 1). Our modified differentiation protocol consistently yielded an efficiency above 90% for cardiac Troponin T (cTnT) positive cardiomyocytes generation from our established hiPSC lines (Fig. 2C). These beating cells stained positive for cardiomyocyte-specific markers sarcomeric α-actinin (α-actinin), cardiac troponin T cTnT , myosin light chain 2V (MLC2V), and the gap-junction protein Connexin 43 (CX43, between adjacent cells) (Fig. 2D). On the other hand, it is established that nonmyocytes are also critical components for heart tissue formation and function [25]. A combination of hiPSC-derived 75% cardiomyocytes and 25% CD90+ nonmyocytes is optimal for EHTs, yielding enhanced structural and functional properties [26]. We thus used the same combination of hiPSC-derived beating cells (75%) and CD90+ cells as the founding cells for

ACCEPTED MANUSCRIPT following cardiac patch preparations. CD90+ cells were obtained by FACS using dissociated day 14 post spontaneous differentiation of hiPSCs (Fig. 2C).

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Generation and evaluation of the engineered human cardiac patches We next combined 75% hiPSC-derived cardiomyocytes and 25% hiPSC-derived CD90+ cells with pieces of decellularized natural rat heart ECM to generate human individual-specific cardiac patches. Individual pieces of heart matrix were put in wells of either 96-well or 48-well plates as a sheet, with the endocardial side facing up. The mixture of cells were then added to the matrix sheet at 104 cells per mm2 and maintained under standard cell culture conditions for more than two weeks until they formed a compact tissue-like structure (Fig. 3A and Supplementary Fig. 2). The newly formed cardiac patches usually started exhibiting beating activity from day 5-10 after cell-matrix co-culture (Supplementary video 2). Histological analysis by H&E staining showed that the cells attached and grew well on the decellularized natural heart matrix sheets, although not mature and as aligned as the adult heart tissues (Fig. 3B). Double immunofluorescence staining of the ECM protein laminin and the cardiomyocyte-specific marker cTnT showed co-localization of the cardiomyocytes and matrix, further indicating a well attachment and growth of cells on the natural heart matrix (Fig. 3C). These newly formed cardiac patches contained human cardiomyocytes, smooth muscle cells and endothelial cells (Fig. 3D, and Supplementary Fig. B-C)) and formed junction structures between cardiomyocytes as indicated by expression of connexin-43 (CX43), which is the predominant cardiac gap-junction protein (Fig. 3D, and Supplementary Fig. 3A). These results demonstrated that the engineered human cardiac patches were formed by a very well combination of cells and the natural heart ECM and were composed of cellular components similar to normal heart tissues.

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The natural heart ECM enhanced maturation of human iPSC-derived cardiac cells in vitro. Previous studies demonstrated that ECM has critical influences on vertebrate heart formation and function [11, 27, 28]. To see whether the natural heart ECM is beneficial for the growth and maturation of hiPSC-derived cardiac cells, we examined the relative expression of several cardiac-specific genes and key cardiac maturation genes (for the list of primers see Supplementary Table 1) in our engineered human cardiac patches, and compared with those of cell aggregates formed by hiPSC-derived cardiomyocytes plus CD90+ cells only (without matrix). Both cardiac patches and matched cell aggregates were cultured in the same medium till day 28. As shown in Fig. 4A, compared to the cell aggregates only, genes required for cardiac and contractile function (cTnT and MYH6) were significantly upregulated in the engineered human cardiac patches. Markers of cardiomyocyte maturation (ANF and BNP) were also examined. Expression levels of atrial natriuretic factor (ANF) and B-type natriuretic peptide (BNP), which are involved in normal and diseased heart physiology, were significantly increased in the engineered human cardiac patches compared to those in cell aggregates. The expression of genes encoding sarcomeric protein MYL2 (MLC2v) and MYL7 (MLC2a) were also upregulated in the engineered human cardiac patches. Moreover, the ratios of MYH7/MYH6 and MYL2/MYL7, which indicates the

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maturation of cardiac tissues, were found to be significantly increased in the engineered human cardiac patches. To further assess the maturation state of the cardiac cells and tissue structures, we investigated the ultra-structure of the engineered cardiac patches and cell aggregates only under transmission electron microscopy (TEM) (Fig. 4B). We found wider Z-lines within the engineered cardiac patches than those of the cell aggregates only, suggesting a more mature sarcomeric structure within the engineered cardiac patches. These results further indicated that the natural heart ECM enhanced maturation of hiPSC-derived cardiac cells in vitro.

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The engineered human cardiac patches exhibited normal electrical properties and responded well to pharmaceutical agents in vitro We consistently observed that the engineered human cardiac patches exhibited spontaneous contractions in vitro and thus could be useful in testing different pharmaceutical reagents that targeting heart tissues. To this end, we next studied the electrophysiological properties of the engineered human cardiac patches using multi-electrode array (MEA) system. The engineered human cardiac patches at day 28 of culture were seeded on the MEA probes (Fig. 5A). By recording electrical activities from electrodes underlying these contractile patches, we were able to obtain detailed activation maps that reflecting initiation of contractions and electrical conductions within these engineered human cardiac patches. These maps revealed that the activation wavefront propagated from the pacemaker area (red) to the latest (dark blue) activation sites (Fig. 5B). We next tested the response of the engineered human cardiac patches to different pharmaceutical agents known to affect cardiomyocyte physiology. As shown in Fig. 5C, administration of epinephrine (0.1 µg/ml), an adrenergic receptor agonist, to our engineered human cardiac patches led to a positive chronotropic response, which was partially reversed by the β-adrenergic receptor blocker motoprolol (0.25 µg/mL). Adding nifedipine, a potent dihydropyridine L-type Ca2+-channel blocker, reduced beating rate and extended field potential durations which reflect the QT intervals in electrocardiogram (Fig. 5D). In addition, the engineered human cardiac patches responded well to E4031 (1 µg/mL), a selective blocker of hERG K+ channel, led to a decrease of the beating rate and prolonged field potential duration (Fig. 5E). Taken altogether, these results demonstrated that the human cardiac patches engineered with our strategy expressed the molecular units and ion channel systems required for normal heart electrophysiology and displayed electrical functional properties very similar to normal heart tissues. These engineered human cardiac patches could be used as an efficient tool for cardiac drug testing in vitro. Patching the engineered human cardiac patch on infarct heart improves cardiac function after acute MI To examine whether our engineered human cardiac patches is useful in regenerative therapy for myocardial damage in vivo, we transplanted the patches into SD rats with left anterior descendant coronary (LAD) ligation-induced acute myocardial infarction. The engineered human cardiac patches were patched on top of the infarct area of the rat hearts right after LAD ligation using a fibrin patch-based method [29] with the cellular side facing toward the infarct of the heart (Fig. 6, A and B).

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Echocardiography demonstrated that, compared to the ECM only group (MI heart treated with the natural heart ECM alone, n=9) and the MI only group (MI heart without treatment, n=7), left-ventricular ejection fraction (LVEF) was significantly recovered in animals of the Patch group (treated with the engineered human cardiac patches, n=9) (Fig. 6C). Fractional shortening (FS) that reflects regional myocardial contractility was also significantly increased in the Patch group compared with that in the ECM only group and the MI only group (Fig. 6D). Consistent with improvements in LVEF and FS, left ventricular systolic inner diameter (LVIDs) and left ventricular diastolic inner diameter (LVIDd) were smaller in the patch group than those in the ECM only group and the MI only group (Fig. 6, E and F; Supplementary Fig. 4). All these results indicated that transplantation of our engineered human cardiac patches improved the LV function of the heart with MI and thus had a beneficial effect for the treatment of acute MI. To better understand the mechanisms underlying the beneficial effects of the engineered human cardiac patches transplantation in treating rat acute MI, we performed histological analyses of heart samples from each group at week 4 by examining thin sections of gross specimen via Masson’s trichrome and H&E staining (Fig. 7A and B). Histological analysis showed that, after LAD occlusion, a transmural MI was present in heart samples of all the three groups (patch, ECM only, and MI only group). The average infarct size in the Patch group was significantly smaller compared with that of the other two groups at week 4 after MI (Fig. 7C). In addition, patch-treated rats showed a pronounced wall thickening compared with other two groups (Fig. 7D). Previous reports have shown that transplantation of hiPSC-derived cardiac cells promoted neovascularization after injury [7]. We therefore assessed whether transplantation of our engineered human cardiac patches promoted neovascularization in the peri-infarct area of hearts after LAD ligation. The immunofluorescence analyses of vWF and SMA expression in the peri-infarct area showed that vascular density was significantly higher in the patch group than in other two groups (Fig. 8, A and B). Histological analyses of heart samples from each group at week 4 showed that engineered human cardiac patches tightly attached to the MI area, integrated with the rat cardiac muscle in texture, with no obvious boundary observed (Supplementary Fig. 5). No significant cell death were observed, suggesting most of the cellular components of the patch survived for 4 weeks. Further, staining with the human-specific nuclear antigen (HNA) also showed that hiPSC-derived cells in the engrafted patches were still alive at the transplantation site at week 4 (Fig. 9). Moreover, we didn't observe the formation of teratoma after the engineered human cardiac patches transplantation (Supplementary Fig. 5). These data suggested that the mechanisms for the engineered human cardiac patches improving heart function after acute MI could be via promoting angiogenesis in the peri-infarct area by the prolonged survival of the cells in the transplanted cardiac patches. DISCUSSION Using pieces of decellularized natural heart ECM as the scaffold plus a thin layer of fixed ratio of hiPSC-derived cardiomyocytes (75%) and CD90+ nonmyocytes (25%) as the seeding cells, we have successfully made functional engineered human cardiac patches in this study. There are several advantages to make such cardiac patches using our method. First, the decellularized heart ECM preserved the natural ECM components and thus was optimal for

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the attachment and growth of cardiac cells. Indeed, our data showed that the hiPSC-derived cardiac cells attached and grew very well on the natural ECM. Further, the natural heart ECM provided a supportive microenvironment for the hiPSC-derived cardiac cells and improved their maturation during prolonged culture. Second, the natural heart ECM can be cut by surgical scissors to generate pieces of any desired sizes and shapes, thus avoiding making sophisticated molds of fixed shapes and sizes for EHTs. Third, seeding the hiPSC-derived cardiac cells on a layer of natural heart ECM piece led to a more homogeneous distribution of the hiPSC-derived cardiac cells on the scaffold. Our engineered human cardiac patches all exhibited a uniform contractile and electrical activities. We feel that this is better than the method of recellularizing the whole decellularized heart by perfusion of seeding cells, which usually leaves giant gaps of uncoupled areas. Fourth, the hiPSC-derived cells were seeded on the endocardial side of the natural heart ECM, leaving the epicardial side intact. We believe this provided a better pattern of cardiac patch formation, because only one side of the patch contain cells. When this patch is transplanted/patched on the heart, the epicardial part of the natural heart ECM will act as a cover for and protect the cellular contents of the patch from exposing to the outside. There are also several disadvantages for our engineered human cardiac patches to be overcome in the future. The hiPSC-derived cardiac cells are not mature and as aligned as the adult heart tissue. This is because the current best cardiac differentiation protocol of hiPSCs still generates cardiomyocytes similar to human fetal cardiomyocytes [30]. The maturation of the hiPSC-derived cardiomyocytes still needed to be improved. Also, alignment of the cardiomyocytes in the patch could be enhanced by constant stretching of the newly made cardiac patches with tailor-made appliances in the future. Furthermore, although we followed a recent report and used a ratio of hiPSC-derived 75% cardiomyocytes and 25% CD90+ nonmyocytes as the seeding cells for the patch formation [26], it may not represent the optimal combination achieving the best treatment effect in vivo. The optimal ratio of hiPSC-derived cardiomyocytes and different fibroblasts for the best treatment effect of myocardial damage remain to be determined. However, the reason for us to choose hiPSC-derived CD90+ cells as supporting fibroblasts was that they are from the same individual so as to further minimize the immunogenicity of the engineered human cardiac patches for future clinical use. Moreover, CD90+ nonmyocytes are also critical components for heart tissue formation and function. They express relative high levels of adhesion proteins which promote cell-cell contact and may facilitate maturation signaling for the heart tissues [31, 32]. In addition, although the current cardiovascular differentiation protocol for human pluripotent stem cells can reach above 90% purity for cardiomyocytes generation, there is still residual non-cardiomyocytes in the population. New techniques needed to be developed for obtaining highly pure cardiomyocytes either by identifying new satisfactory surface markers or improving differentiation protocols. In this study, we observed some endothelial cells and smooth muscle cells in the engineered human cardiac patches. These cells may came from the residual nonmyocytes from the differentiated hiPSCs or from the CD90+ cells. Indeed, previous study reported that CD90 was expressed in endothelial cells and smooth muscle cells [33]. Our engineered human cardiac patches exhibited normal beating properties and electrophysiological activities in the dish, and responded well to various pharmaceutical

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agents targeting heart physiology. It is thus possible to use them as a tool for drug testing. Since the hiPSC-derived cardiac cells exhibited a more mature phenotype in the engineered human cardiac patches than that of the hiPSC-derived cardiomyocytes only, it could be better to use them for drug testing in the future. Using hiPSCs derived from disease-specific patients, it is also possible to generate disease-specific cardiac tissues using the methods in this study. This will provide a new angle for modeling and studying specific heart diseases. Finally, our engineered human cardiac patches showed beneficial effects in treating acute MI, which is similar with cardiac patches made by different methods in other studies [7, 29, 34-36]. Our cardiac patches improved left ventricular function after patching on top of the infarct area of rat with acute MI after LAD ligation. After 4 weeks post-implantation, the patch remained viable and stayed on top of the host myocardium. Mover, it seemed that the cells survived much better and longer in our engineered human cardiac patches after transplantation than those by direct myocardial injection of suspended cells, since it is well acknowledged that cells usually die and disappear several weeks after direct injection [37]. The beneficial treating effect of the engineered human cardiac patches for MI in this study may closely associated with prolonged survival of cells after transplantation. The treating effect of our engineered human cardiac patches for large animal models awaits further studies. Furthermore, the treatment of the acute MI model may not be highly representative of the future human therapies, since it would be relatively rare to have interventions performed immediately after infarction. The treatment effects of the engineered human cardiac patches on sub-acute or chronic MI model should be tested in the future. In summary, it is feasible and relatively easy to generate individual-, size-, and shape-specific human cardiac patches using our methods. These engineered human cardiac patches can be of great value to meet individual-specific demands of cardiac repair, as well as for drug screening and study of inherited heart diseases.

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CONCLUSIONS It is feasible to generate individual-specific human cardiac patches of different sizes and shapes using decellularized natural heart ECM and human iPSCs-derived cardiac cells. These human cardiac patches exhibited normal contractile and electrical physiology in vitro and improved heart function of rats with acute myocardial infarction in vivo when patching on the infarct area. The engineered human cardiac patches can be of great value for drug screening and study of inherited heart diseases, as well as meeting the demands for individual-specific (personalized cardiac cells, size and shape) EHTs for personalized regenerative therapy of myocardial damages. ACKNOWLEDGEMENT This work was supported by the National Natural Science Foundation of China (NSFC No. 81322003, No.31571527) (N.S.); the Recruitment Program of Global Experts of the Organization Department of the Central Committee of the CPC (N.S.); the Science and Technology Commission of Shanghai Municipality (No. 13JC1401704) (N.S.); National Key scientific research projects 2014CBA02003; and the Research Center on Aging and Medicine of Fudan University research fund No.13dz2260700.

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FIGURE LEGENDS Figure 1. Decellularization of rat hearts. A, Schematic diagram of the work flow of engineering the artificial cardiac patches. hFbs, human fibroblasts; hCMs, hiPSC-derived cardiomyocytes. B, Representative images of the process of decellularization of rat hearts. (1)

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before decellularization; (2) after perfusion with deionized water; (3)after perfusion with PBS; (4) after perfusion with enzymatic solutions; (5) after perfusion with 1% SDS solution; (6) after perfusion with 1% Triton X-100 solution. C, H&E and DAPI staining of sections from the cadaveric native rat heart (left) and decellularized heart (right). No nuclear staining was observed in decellularized hearts. Scale bars, 100 µm. D, DNA content of native cardaveric rat hearts and decellularized hearts. Error bars mean SEM of 3 independent experiments. **p < 0.01. E, Immunofluorescence staining of major ECM proteins (laminin (upper), fibronectin (middle), and collagen III (lower)) of cadaveric native rat hearts and decellularized hearts.. F, Scanning electron micrographs of cadaveric and decellularized hearts shows the collagen and elastin fibers are preserved in the decellularized heart ECM, but cells throughout all tissue layers were removed. Red arrows indicate the vascular structural.

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Figure 2. Differentiation of the hiPSCs into cardiomyocytes. A, The differentiation protocol for the hiPSCs to cardiomyocytes used in this study. B, Bright-field images of the typical morphology of day 5 hiPSCs (left) and day 12 hiPSC-CMs (right) post differentiation are shown at 100 times magnification. C, The efficiencies of differentiation of hiPSCs to cTnT+ cardiomyocytes (upper panel) were usually >90%. hiPSC-derived CD90+ nonmyocytes were sorted by FACS (lower panel). D, The hiPSC-derived cardiomyocytes expressed typical striated cardiomyocyte markers α-actinin, cTnT, the ventricle-specific protein myosin light chain 2v (MLC2v), and the gap-junction protein connexin-43 (CX43, green, identified by white arrows). Nuclei were counterstained with DAPI. Scale bars, 50 µm.

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Figure 3. Generation and histological analysis of the engineered human cardiac patches. A, Schematic diagram showing the preparation of the engineered human cardiac patches. hiPSC-derived cardiomyocytes plus CD90+ cells are combined and seeded on the pieces of decellularized native rat heart ECM for extended culture. B, H&E staining of sections of constructed cardiac patches. The hiPSC-derived cardiac cells were well distributed and attached on the native ECM. Scale bars, 100 µm. C, A representative section of the engineered cardiac patches co-immunostained by anti-cTnT and anti-Laminin antibodies. Scale bars, 100um. D, Western blot analysis of ECM and engineered cardiac patches (ECPs). Note the expression of connexin43 (Cx43), CD31 and α-smooth muscle actin (α-SMA) in the engineered human cardiac patches. Figure 4. The decellularized heart ECM improved maturation of hiPSC-derived cardiomyocytes. A, Quantitative PCR analyses of the transcriptional expression of cardiac-associated genes, including cardiomyocyte-specific genes (cTnT, MYH6, and MYH7), cardiac-related transcription factor (NKX2.5), and cardiomyocyte maturation markers (ANF, BNP, MYH7/MYH6, MYL2, MYL7, and MYL2/MYL7), normalized to GAPDH expression. Data are shown as the mean ± SEM of three independent experiments. *P<0.05, **P<0.01. B, Ultra-structural analysis of the engineered human cardiac patches and the hiPSC-derived cardiac cell aggregates by TEM. The width of myofibrillar Z-lines was measured and compared. ECPs, engineered human cardiac patches; Z, Z-line. All error bars show SEM of three independent experiments. *P<0.05.

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Figure 5. The engineered human cardiac patches exhibited normal electrophysiology and responded to cardiac pharmaceutical reagents. A and B, A representative phase-contrast micrograph of the engineered cardiac patch cultured on the MEA probe (A) and the resulting activation map (B). C-E, Contraction rate recording of the engineered human cardiac patches in response to epinephrine and metoprolol (C), nifedipine (D) and E4031 (E) treatment. *P<0.05, **P<0.01 compared with controls; ##P<0.01 compared with epinephrine alone. Data were mean±SEM of three independent experiments and analyzed by t test.

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Figure 6. The engineered human cardiac patches improved cardiac function after patching on the infarct heart after acute MI. A, Schematic view showing patching the engineered human cardiac patch on the heart by the fibrin patch–based method. B, Actual view of the engineered human cardiac patch patched on the heart. C-D, The global left ventricular function were evaluated weekly by echocardiography for LV ejection fractions (C) and fractional shortening (D) at week 1, 2, 3 and 4 after MI injury and treatment across the MI (n=7), ECM (n=9), and Patch groups (n=9). Significant improvement in LV function was achieved in rats that received the engineered human cardiac patches. E, The left ventricular systolic inner diameter (LVIDs) and F, the left ventricular diastolic inner diameter (LVIDd) were significantly smaller in the Patch group than those of the MI and ECM groups. Data were shown as mean±SEM, *P<0.05, **P<0.01 versus MI; ##P<0.01, #P<0.05 versus the ECM group.

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Figure 7. Transplantation of the engineered human cardiac patches alleviated left ventricular remolding in rat MI model. Representative Masson’s trichrome staining (A) and H&E staining (B) graphs of dissected hearts in the MI, ECM and Patch group 4 weeks after MI and treatment. C, Animals treated with the engineered human cardiac patches showed a significantly reduced infarct area at 4 weeks after MI (MI: 40.58%, n=7; MI+ECM: 38.09%, n=9; MI+Patch: 25.87%, n=9). D, Quantification of left ventricular wall thickness. Data represents mean±SEM. *P<0.05, **P<0.01 versus the MI group; ##P<0.01, #P<0.05 versus the ECM group.

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Figure 8. The engineered human cardiac patches increased vascular density surrounding the infarct area. A-D, Double immnunostaining of von Willebrand factor (vWF) and α-smooth muscle actin (α-SMA) at 4 weeks after treatment for the border zone of the infarct in the hearts of animals from the MI (A), ECM (B) and Patch (D) groups. Scale bars, 50µm. E, Vascular density was quantified by calculating the number of the vWF-positive vascular structures. **P<0.01 versus MI; ##P<0.01 versus ECM group. Figure 9. Immunostaining of engineered human cardiac patches at the transplant site 4 weeks after transplantation. Upper panel: representative immunostaining for HNA (human cell nuclei, red) and DAPI of human cardiac patches at the transplant site. Bar, 500 µm. Lower panel: higher magnification of the boxed area in the upper panel.

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