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SHORT COMMUNICATION Myocardial Tissue Engineering With Cells Derived from Human Induced-Pluripotent Stem Cells and a Native-Like, High-Resolution, 3-Dimensionally Printed Scaffold Ling Gao1, Molly E. Kupfer2, Jangwook P. Jung2, Libang Yang2, Patrick Zhang2, Yong Da Sie3, Quyen Tran3, Visar Ajeti3, Brian T. Freeman2, Vladimir G. Fast1, Paul J. Campagnola3, Brenda M. Ogle2*, Jianyi Zhang1* 1 Department of Biomedical Engineering, School of Medicine, School of Engineering, University of Alabama at Birmingham; 2Department of Biomedical Engineering, University of Minnesota – Twin Cities, and; 3Department of Biomedical Engineering, University of Wisconsin – Madison, Madison. Running title: 3D-Printed Scaffolds for Engineered Myocardium Downloaded from http://circres.ahajournals.org/ by guest on April 29, 2017 B.M.O. and J.Z. are both corresponding authors. Subject Terms: Myocardial Infarction Cell Therapy Stem Cells Addresses correspondence to: Dr. Jianyi Zhang Department of Biomedical Engineering University of Alabama at Birmingham [email protected] Dr. Brenda M. Ogle Department of Biomedical Engineering University of Minnesota – Twin Cities [email protected] In December 2016, the average time from submission to first decision for all original research papers submitted to Circulation Research was 13.4 days. DOI: 10.1161/CIRCRESAHA.116.310277 1 ABSTRACT Rationale: Conventional three-dimensional (3D) printing techniques cannot produce structures of the size at which individual cells interact. Objective: Here, we used multiphoton-excited, 3-dimensional printing (MPE-3DP) to generate a nativelike, extracellular matrix (ECM) scaffold with submicron resolution, and then seeded the scaffold with cardiomyocytes (CMs), smooth-muscle cells (SMCs), and endothelial cells (ECs) that had been differentiated from human induced-pluripotent stem cells (iPSCs) to generate a human, iPSC-derived cardiac muscle patch (hCMP), which was subsequently evaluated in a murine model of myocardial infarction (MI). Downloaded from http://circres.ahajournals.org/ by guest on April 29, 2017 Methods and Results: The scaffold was seeded with ~50,000 human, iPSC-derived CMs, SMCs, and ECs (in a 2:1:1 ratio) to generate the hCMP, which began generating calcium transients and beating synchronously within 1 day of seeding; the speeds of contraction and relaxation and the peak amplitudes of the calcium transients increased significantly over the next 7 days. When tested in mice with surgically induced MI, measurements of cardiac function, infarct size, apoptosis, both vascular and arteriole density, and cell proliferation at week 4 after treatment were significantly better in animals treated with the hCMPs than in animals treated with cell-free scaffolds, and the rate of cell engraftment in hCMP-treated animals was 24.5% at week 1 and 11.2% at week 4. Conclusions: Thus, the novel MPE-3DP technique produces ECM-based scaffolds with exceptional resolution and fidelity, and hCMPs fabricated with these scaffolds may significantly improve recovery from ischemic myocardial injury. Keywords: Heart, myocardial infarction, tissue engineering. Nonstandard Abbreviations and Acronyms: hiPSCs 3D ECM MPLSM MPE-3DP CMs ECs SMCs hCMP CL SMA MI HNA GFP hCD31 BP human induced-pluripotent stem cells three-dimensional myocardial extracellular matrix multi-photon laser scanning microscopy multiphoton-excited three-dimensional printing cardiomyocytes endothelial cells smooth muscle cells hiPSC-derived cardiac muscle patch cycle length -smooth muscle actin myocardial infarction human-specific nuclear antigen green fluorescent protein human specific CD31 bovine pericardium DOI: 10.1161/CIRCRESAHA.116.310277 2 INTRODUCTION Downloaded from http://circres.ahajournals.org/ by guest on April 29, 2017 Although recent large-animal studies have shown that recovery from myocardial injury can be improved by injecting cardiac cells that have been differentiated from human induced-pluripotent stem cells (hiPSCs), the structural support and synchronized contractile activity of a transplanted myocardial tissue equivalent (MTE) could provide additional benefits. Tissue engineers are beginning to improve the effectiveness of MTE engineering for regenerative myocardial therapies1-3 by developing techniques that can enhance the engraftment, improve the MTE maturation4, 5 and long-term functional benefits.6, 7 These efforts may be aided by incorporating some of the more complex features of the myocardial extracellular matrix (ECM) into the tissue’s design. This level of complexity cannot be reproducibly achieved with established manufacturing technologies, but a newer modality, 3D printing, has been successfully used to build structures with defined geometries from heterogeneous materials8 and, consequently, may be used for generating scaffolds that mimic the ECM of native cardiovascular tissues. However, key structural features of the native ECM need to be identified and incorporated (with adequate resolution and reliability) into a scaffold to promote cell function and limit detrimental effects. In addition, from the potential clinical application perspective, a high level of quality control of the scaffold product is required, which can only be achieved by a computer controlled 3D printing technology. The position of crosslinks within a matrix of photoactive biological polymers can be controlled with high resolution. Printers that utilize single-photon excitation coupled to a sequence of photomasks can achieve approximately 30 micron resolution in x, y and approximately 50 micron resolution in z. A more advanced technique, multiphoton excited (MPE) photochemistry, can restrict excitation (and, consequently, the photochemical reaction) in three dimensions via a method that is analogous to multiphoton laser scanning microscopy (MPLSM).9-14 Notably, the resolution of the features (<1 μm) is determined by the MPE point spread function and can therefore approximate the feature size of components of the extracellular matrix.15 The technique can also be combined with rapid prototyping and computeraided design16 to fabricate essentially any 3D structure that can be drawn. For the experiments described in this report, we used our novel technique, multiphoton-excited three-dimensional printing (MPE-3DP), to generate a scaffold with a native-like cardiac ECM architecture from a solution of a photoactive gelatin polymer and then seeded the scaffold with cardiac cells (cardiomyocytes [CMs], endothelial cells [ECs], and smooth muscle cells [SMCs]) that had been differentiated from human, cardiac-lineage, induced-pluripotent stem cells (hciPSCs)17 to generate an hciPSC-derived cardiac muscle patch (hCMP). The hCMP was subsequently characterized via a series of in vitro analyses and then tested in a murine model of ischemic myocardial injury. METHODS A detailed description of the experimental procedures used in this investigation is provided in the online Data Supplement. DOI: 10.1161/CIRCRESAHA.116.310277 3 RESULTS Fabrication of an ECM scaffold based on templates derived from optical image stacks of murine myocardium. Our approach can be summarized in two steps: first, native, murine, adult myocardial tissue was examined to determine the size and distribution of various ECM features, which were incorporated into a 3D template; then, the template was scanned and used to map the positions of crosslinks in a solution of a photoactive polymer (Figure 1A). Importantly, our scanning technique, modulated raster scanning, maps the template directly to the scaffold by monitoring the brightness of each point in the image, and it is accurate to resolutions of less than 1 μm. To our knowledge, this is the first time modulated raster scanning has ever been successfully used to control the fabrication of a tissue-engineered scaffold and, consequently, our results are particularly relevant for applications that require the fibrillar and mesh-like structures present in cardiac tissue.18 Downloaded from http://circres.ahajournals.org/ by guest on April 29, 2017 We chose to base our template on the distribution of fibronectin in murine myocardium (Figure 1B and 1C). Fibronectin is uniformly distributed around each CM, so it can be used to determine the dimensions of each individual cell compartment to form a grid (here termed, Adult Simulate). The scaffold was generated from a solution of gelatin methacrylate, which can be crosslinked into complex structures with high efficiency, allows creation of complex structures with thickness of approximately 100 m (Online Video I, II and III), is biologically inert when both crosslinked and degraded, and the denatured collagen exposes cell binding sites (including Arg-Gly-Asp, RGD), which should readily adhere to the seeded cells and support biochemical signaling via focal adhesions.19 Analysis of the native myocardium suggested that CMs reside in channels that are approximately 15 μm by 100 μm which, when incorporated in the hCMP scaffold (Figure 1D and 1E), yielded a robust structure with high reproducibility and exceptional fidelity in both coverage area (≥95%) and intensity variation (≥85%). Integration of hciPSC-derived cardiac cells in hCMPs. The hciPSCs were reprogrammed from human cardiac fibroblasts and differentiated into hciPSCCMs, hciPSC-SMCs, and hciPSC-ECs; then, the differentiated cells were characterized via the expression of lineage-specific markers (Online Figure I), and the hCMP was formed by seeding ~50,000 cells (in a 2:1:1 ratio of hciPSC-CMs, hciPSC-SMCs, and hciPSC-ECs, respectively) into the fabricated scaffold, where they quickly became assimilated and occupied most of the free space (Figure 1D). The hciPSC-CMs began generating calcium transients (Figure 2A) and beat synchronously across the entire hCMP within one day of seeding (Online Video IV and V). Over the next seven days, the speeds of contraction and relaxation, the peak amplitude of the calcium transients (Figure 2B and 2C), and the expression of several genes required for contractile function (cTnT, cTnI, and MHC) and for generating calcium transients (SERCA2RYR2, CASQ2) increased significantly (Online Figure II). Contractile and calcium-transient gene expression was also greater in the hCMPs than in monolayers grown from equivalent populations of hiPSC-derived cardiac cells on day 7. Optical mapping of transmembrane potentials (Figure 2D and 2E) revealed macroscopically continuous action potential propagation in hCMPs, showing an excellent functional electrophysiological communications between cells. Conduction velocity in hCMPs increased linearly with the increase of pacing cycle length (CL), reaching 18.8±0.8 cm/s at 800 ms pacing CL (Figure 2F). Action potential durations, APD50 and APD80, were about 214±10.4 and 270±12.7 ms at 800 ms pacing CL, separately, and also showed significant correlation with pacing rate (Figure 2G). Autofluorescence images obtained via 2-photon microscopy on day 7 (Figure 2H) indicated that the cells had aligned with the channels of the fabricated scaffold (Figure 2I) to form multinucleate cells with an aspect ratio (i.e., cell length:width) of approximately 5.5:1 (Figure 2J), which is close to the characteristic ratio observed in native CMs (7:1). Analysis of hCMPs stained for expression of the CM protein cTnI, -smooth muscle actin DOI: 10.1161/CIRCRESAHA.116.310277 4 ( SMA), and the endothelial marker CD31 (Figure 2K) indicated that the original 2:1:1 ratio of hciPSCCMs, -SMCs, and -ECs was largely retained (Figure 2L), with CMs comprising ~45% of the remaining cells. The 7-day survival rate of hciPSC-CMs in the hCMP was similar to that observed when cells were maintained on Matrigel-coated plates and significantly greater than the rate achieved by culturing the cells with bovine pericardium or polyethylene glycol (Figure 2M). In vivo evaluations of hCMP transplantation in a murine model of myocardial infarction. Downloaded from http://circres.ahajournals.org/ by guest on April 29, 2017 In vivo assessments of the hCMPs were performed in a murine model of myocardial infarction (MI). MI was surgically induced as described previously,20 then, animals in the MI+hCMP group were treated with two hCMPs, animals in the MI+Scaffold group were treated with two patches of scaffold material without cells, and animals in the MI group received neither the hCMP nor the cell-free scaffold. The hCMPs and scaffolds were positioned over the site of infarction (Figure 3A) and held in place by covering them with a piece of decellularized bovine pericardium that was sutured to the heart to avoid movement of hCMPs off the heart. The decellularized bovine pericardium above the hCMP has beneficial effect of preventing the adhesion between the heart/hCMP and the chest. The Sham group underwent all surgical procedures for MI induction except the ligation step and recovered without either experimental treatment. Quantitative PCR measurements for expression of the human Y chromosome indicated that 24.5±2.6% of the transplanted cells remained engrafted in the hearts of MI+hCMP animals for at least one week after transplantation (Figure 3B). By week 4, the engraftment rate had declined to 11.2±2.3%, which is still much higher than the rate reported in previous studies of cell-based myocardial therapy.21, 22 In order to assess the engraftment rate by counting the grafted human cells as evidenced by human-specific nuclear antigen (HNA) positivity, we also used histologic assessment of consecutive sections of the heart that spans the entire area covered by the engrafted patch, and counting the grafted human cells as evidenced by HNA positivity.17, 23 The engraftment rate of the 7 hearts studied using the histology method is 13.6 ± 2.2%, which is in agreement with the quantitative PCR (11.2 ± 2.3%). The ratio of the hciPSC-tri lineage cardiac cells at week 1 and 4 was also examined. The engrafted hciPSC-CMs were identified by the expression of both green fluorescent protein (GFP) and cTnI, engrafted hiPSC-ECs were identified by expression of human specific CD31 (hCD31), and engrafted hiPSC-SMCs were identified by the expression of both GFP and SMA. At week 1 post transplantation the ratio of hciPSC-CMs, hciPSC-SMCs, and hciPSC-ECs was 1.42: 0.93: 1; at week 4 the ratio was 0.67: 0.86: 1 for hciPSC-CMs, hciPSC-SMCs, and hciPSC-ECs, respectively. Hematoxylin and Eosin (HE) staining (Figure 3C), and immunofluorescence analysis of HNA, GFP, cTnI, SMA, and hCD31 expression (Figure 3C and 3D) showed evidence of all three transplanted cell lineages in the treated region Cardiac function was evaluated on day 28 after injury via echocardiographic assessments (Figure 4A) of left-ventricular ejection fraction and fractional shortening; both parameters were significantly greater for animals in the MI+hCMP group than in MI+Scaffold or MI animals (Figure 4B and 4C), while measurements in the MI+Scaffold and MI groups were similar. Infarcts were also significantly smaller, while the thickness of the infarcted region of the myocardial wall was significantly greater, in MI+hCMP hearts than in MI or MI+Scaffold hearts (Figure 4D-4F), and analyses of TUNEL-stained sections (Online Figure IIIA and IIIB) and sections stained for the presence of CD31 and SMA (Online Figure IIIC-IIIE) indicated that apoptotic cells were significantly less common, while vascular structures (including arterioles) were significantly more common, at the border of the infarcted region in hCMP-treated animals than in the corresponding regions of hearts from animals in the MI+Scaffold or MI groups. hCMP treatment was also associated with significant increases in the number of cells that expressed the proliferation marker Ki67 (Online Figure IV). Thus, the transplanted hCMPs appeared to improve recovery from myocardial injury by, at least in part, reducing apoptosis, promoting angiogenesis, and increasing cell proliferation. DOI: 10.1161/CIRCRESAHA.116.310277 5 DISCUSSION Downloaded from http://circres.ahajournals.org/ by guest on April 29, 2017 The fate specification of progenitor cells in the developing heart is critically dependent on the spatial and temporal factors of the ECM where the progenitor cells reside. In human pluripotent stem cell differentiation to endothelial cells, the rate and quality of derived endothelial cells are critically influenced by the temporal factors in 3D ECM. 24 The myocardial ECM provides substrates for cell adhesion, sequesters soluble factors, and serves as a conduit for mechanical signaling. Our recent efforts to characterize the ECM of the developing heart have provided us with an extensive body of knowledge regarding the nanoscale distribution of the ECM,25 and by combining this knowledge with MPE-3DP, we were able to generate a scaffold that is structurally native-like.16 The technique couples MPE with modulated raster scanning to induce crosslinks in a solution of a photoactive gelatin polymer, thereby creating an ECM scaffold with exceptionally high resolution. Notably, construction was guided by a template composed of features that had been identified in the ECM of a native adult murine heart, which suggests that the technique may be able to replicate the unique architecture of the myocardial ECM in each individual. Furthermore, when hCMPs were generated by seeding the scaffolds with hciPSC-derived cardiac cells and transplanted into infarcted mouse hearts, the treatment was associated with significant improvements in cardiac function, infarct size, apoptosis, vascular density, and cell proliferation. Thus, the hCMPs produced for this report may represent an important step toward the clinical use of 3D-printing technology. Our novel technique is the first to achieve a resolution of 1 μm or less and, consequently, can reliably control the thickness of the walls that separate adjacent channels. Channel-wall thickness may be a particularly important parameter for myocardial tissues, because contractions in adjacent fibers must be synchronized by signaling mechanisms that traverse the wall. Notably, the seeded cells quickly settled into the engineered channels of the scaffolds generated for this report, and synchronized beating was observed as early as one day after cell seeding, which suggests that individual cells interacted with the features of the scaffold, and that inter-channel coupling mechanisms were quickly established. The functional improvement associated with hCMP transplantation in the murine MI model suggests that this goal may have been at least partially achieved. Thus, in future investigations we will use optical mapping technology to determine whether transplanted hCMPs are electromechanically coupled to native myocardial tissues.26 In conclusion, we have developed a method by which the principles of 3D printing and photochemistry can be combined to generate an ECM scaffold with unprecedented resolution from a template based on the architecture of native myocardial ECM. When hCMPs were generated by seeding the scaffolds with hciPSC-derived cardiac cells, the scaffold promoted cell viability and electromechanical coupling in vitro, and the hCMPs were associated with high levels of cell engraftment, as well as significant improvements in cardiac function, infarct size, apoptosis, vascularity, and cell proliferation in a murine MI model. Subsequent investigations will focus on methods for creating hCMPs of sufficient size for largeanimal studies and means to improve effectiveness of the hCMPs by incorporating mixtures of ECM proteins into the scaffold. SOURCES OF FUNDING This work was supported by the following funding sources: National Science Foundation, Award CBET1445650; Lillehei Heart Institute, UMN, High Risk High Reward; Institute for Engineering and Medicine, UMN, Pilot Grant, and NIH RO1 HL 99507, HL114120, HL 131017, UO1 134764. DISCLOSURES None. DOI: 10.1161/CIRCRESAHA.116.310277 6 REFERENCES 1. 2. 3. 4. 5. 6. 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Biomaterials. 2010;31:5536-44. DOI: 10.1161/CIRCRESAHA.116.310277 7 Downloaded from http://circres.ahajournals.org/ by guest on April 29, 2017 20. Nakamura Y, Yasuda T, Weisel RD and Li RK. Enhanced cell transplantation: preventing apoptosis increases cell survival and ventricular function. Am J Physiol Heart Circ Physiol. 2006;291:H939-47. 21. Nguyen PK, Riegler J and Wu JC. Stem cell imaging: from bench to bedside. Cell Stem Cell. 2014;14:431-44. 22. Xiong Q, Ye L, Zhang P, Lepley M, Swingen C, Zhang L, Kaufman DS and Zhang J. Bioenergetic and functional consequences of cellular therapy: activation of endogenous cardiovascular progenitor cells. Circ Res. 2012;111:455-68. 23. Nakamura Y, Wang X, Xu C, Asakura A, Yoshiyama M, From AH and Zhang J. Xenotransplantation of Long Term Cultured Swine Bone Marrow-Derived Mesenchymal Stem Cells. Stem Cells. 2006. 24. Zhang S, Dutton JR, Su L, Zhang J and Ye L. The influence of a spatiotemporal 3D environment on endothelial cell differentiation of human induced pluripotent stem cells. Biomaterials. 2014;35:378693. 25. Hanson KP, Jung JP, Tran QA, Hsu SP, Iida R, Ajeti V, Campagnola PJ, Eliceiri KW, Squirrell JM, Lyons GE and Ogle BM. Spatial and temporal analysis of extracellular matrix proteins in the developing murine heart: a blueprint for regeneration. Tissue Eng Part A. 2013;19:1132-43. 26. Matiukas A, Pertsov AM, Kothari P, Cram A and Tolkacheva EG. Optical mapping of electrical heterogeneities in the heart during global ischemia. Conf Proc IEEE Eng Med Biol Soc. 2009;2009:6321-4. DOI: 10.1161/CIRCRESAHA.116.310277 8 FIGURE LEGENDS Figure 1. hCMP fabrication via 3D-MPE. (A) The ECM and associated crosslinking solution are passed through the optical interrogation path while the laser power and dwell time are modulated to deposit ECM at each x, y location in each z plane. The submicron-scale features produced in the ECM scaffold are displayed in the inset (scale bar = 1 μm). Three-dimensional structures can be generated by combining multiple layers with the same or different ECM pattern. (B) Sections from the heart of an adult mouse were immunofluorescently stained for the presence of fibronectin and scanned via MPE (scale bar = 200 μm); then, (C) the distribution of fibronectin in the native tissue was simulated in a template. The simulated channels (green, 100 μm × 15 μm) are shown overlaying the fibronectin pattern of the native tissue (red) in the inset (scale bar = 100 μm). (D-E) The simulated template was used to determine the position of crosslinks in a solution of gelatin methacrylate, thereby producing a native-like ECM scaffold (D); then, the scaffold was seeded with hiPSC-derived CMs, ECs, and SMCs to generate the hCMPs (E). The complete hCMP is shown in the larger image (scale bar = 400 μm), while the individual channels and incorporated cells are visible in the inset (scale bar = 50 μm). Downloaded from http://circres.ahajournals.org/ by guest on April 29, 2017 Figure 2. In vitro assessments of the hCMP. (A) Calcium transients were recorded in hCMPs on day 1, 3, and 7 after cell seeding, and used to calculate the (B) peak amplitude (F/F0; n ≥ 50 cells per time point). (C) Videos of the beating hCMPs were taken on day 1, 3, and 7 and evaluated with motion vector analysis software to calculate the speeds of contraction and relaxation (n=4 hCMPs per time point). (D-G) Action potential propagation in hCMP was measured on day 7 (n=4). (D) Representative isochronal map of activation spread and (E) selected optical Vm traces recorded during pacing with cycle length (CL) of 800 and 300 ms. AT, activation time; Dependence of (F) conduction velocity (CV) and (G) action potential duration (APD50 and APD80) on pacing CL. (H) hCMPs were stained with DAPI on day 7; then, autofluorescence images were obtained via 2-photon microscopy (scale bar = 20 μm) and used to calculate the cells’ (I) angle of alignment relative to the long axis of the engineered channel and (J) aspect ratio. (K) On day 7, ECs, SMCs, and CMs were identified in the hCMPs via immunofluorescence staining for the presence of CD31, -smooth-muscle actin (SMA), and cardiac troponin I (cTnI), respectively; then (L) the proportion of each cell type was calculated (5 fields per hCMP and 4 hCMPs). (M) Known quantities of hiPSC-CMs were seeded into Matrigel, polyethylene glycol (PEG), decellularized bovine pericardium, or the engineered scaffold and cultured for 7 days; then, the cells were immunofluorescently stained for cTnI expression, and cell survival was quantified as the ratio of the number of cTnI+ cells observed to the number of seeded cells. **P<0.01. Figure 3. hCMP engraft and survive after transplantation into the hearts of mice with MI. Myocardial infarction was surgically induced in mice. Animals in the MI+hCMP group were treated with two hCMPs (0.1 million cells total). (A) The representative image showed two transplanted hCMPs on the mouse heart. (B) The engraftment rate was determined in animals from the MI+hCMP group at week 1 and 4 after injury via quantitative PCR measurements of the human Y chromosome (n=4 hearts per time point). (C) The representative HE staining image and human specific nuclear antigen (HNA) immunostaining image showed engrafted hCMP on the epicardial surface of the heart at week 4 after transplantation. BP=bovine pericardium. (D) Sections taken from the region of patch in MI+hCMP animals at week 4 were immufluorescently stained for the presence of HNA, cTnI, green fluorescent protein (GFP), SMA, and the human isoform of the endothelial marker CD31 (hCD31); nuclei were counterstained with DAPI (scale bar = 50 m). (i-ii) Engrafted hciPSC-CMs were identified by the expression of both HNA and cTnI (i) or both GFP and cTnI (ii); (iii) engrafted hiPSC-SMCs were identified by the expression of both GFP and SMA; (iv) engrafted hiPSC-ECs were identified by the expression of hCD31. DOI: 10.1161/CIRCRESAHA.116.310277 9 Figure 4. hCMP transplantation improves cardiac function and reduces infarct size after MI. (A-C) Cardiac function was evaluated at week 1 and 4 via (A) echocardiographic assessments of (B) leftventicular ejection fraction and (C) fractional shortening. (D-F) Sections of hearts from animals in different groups were (D) Masson’s trichrome–stained for histological assessments of (E) infarct size and (F) infarct wall thickness. Infarct size was calculated as a percentage of the circumflexion length of the left ventricular free wall, and infarct wall thickness was calculated as a percentage of the thickness of the septal wall. *p<0.05. BP=bovine pericardium. Downloaded from http://circres.ahajournals.org/ by guest on April 29, 2017 DOI: 10.1161/CIRCRESAHA.116.310277 10 NOVELTY AND SIGNIFICANCE What Is Known? Cellular therapy for myocardial repair has shown some benefit in preclinical and clinical settings, but limited cell retention and associated lack of robust electromechanical coupling remain the major problems. Greater benefits may be achieved if the transplanted cardiac cells are provided with structural support such that synchronized contractile activity of the graft can be integrated to recipient heart. One approach to generate a structural support is 3D printing, which has recently advanced to include the printing of biological materials such cells and extracellular matrix proteins (ECM). Downloaded from http://circres.ahajournals.org/ by guest on April 29, 2017 What New Information Does This Article Contribute? The article describes the implementation of multiphoton-excited three-dimensional printing (MPE3DP) to generate a scaffold with native-like cardiac ECM architecture. The scaffold can effectively harbor stem cell-derived cardiac cell types and can be handled for effective transfer to the damaged heart. The cell-seeded scaffold (termed human cardiac muscle patch, hCMP) beats synchronously in a culture dish prior to transplant and with extended culture time beats with increased speed and calcium handling suggesting cardiomyocyte maturation. In vivo, measurements of cardiac function and infarct size were significantly better in infarcted hearts treated with the hCMPs than in hearts treated with cell-free scaffolds. The mechanisms for therapeutic improvement include improved cell engraftment, increased cellular proliferation and vascular supply with decreased apoptosis. We demonstrate multiphoton-excited, 3D printing to generate a human cardiac muscle patch (hCMP) that could be effectively populated with cardiac cell types because it mimics the structural dimensions and protein composition of the native myocardium. In a mouse model of myocardial infarction, hCMP transplantation results in higher levels of cell engraftment, cell proliferation and myocardial vascular density, which are accompanied by a significant reduction of infarct size and improvement of left ventricular function. This work pushes the technical limits of 3D printing for cardiac tissue equivalents such that cells and extracellular matrix proteins could be manufactured precisely as designed. DOI: 10.1161/CIRCRESAHA.116.310277 11 Figure-1 A Adult simulate E hCMP Downloaded from http://circres.ahajournals.org/ by guest on April 29, 2017 D Printed scaffold C Adult B Downloaded from http://circres.ahajournals.org/ by guest on April 29, 2017 100 m 100 m Figure-3 A D hCMP-1 i HNA DAPI cTnI HNA DAPI cTnI hCMP-2 B Engraftment rate (%) DAPI HNA cTnI HNA DAPI cTnI 32 24 Downloaded from http://circres.ahajournals.org/ by guest on April 29, 2017 16 8 0 Week 1 Week 4 ii GFP cTnI GFP cTnI DAPI C hCMP iii GFP SMA GFP SMA cTnI DAPI BP iv hCD31 iV HNA DAPI hCMP BP DAPI hCD31 cTnI DAPI Figure-4 A MI Ejection fraction (%) Sham (5) MI (7) MI+Scaffold (7) MI+hCMP (8) 100 80 MI+hCMP C Sham (5) MI (7) MI+Scaffold (7) MI+hCMP (8) * * 60 40 20 0 Week 1 Week 4 60 45 * * 30 15 0 Week 1 BP D MI E MI (7) MI+Scaffold (7) MI+hCMP (7) 75 60 45 30 15 0 * * MI+Scaffold F Week 4 hCMP BP MI+hCMP MI (7) MI+Scaffold (7) MI+hCMP (7) Percentage of infarct thickness (%) Sham Percentage of Infarct size (%) Downloaded from http://circres.ahajournals.org/ by guest on April 29, 2017 B MI+Scaffold Fractional shorting (%) Sham 100 80 60 40 20 0 * * Downloaded from http://circres.ahajournals.org/ by guest on April 29, 2017 Myocardial Tissue Engineering With Cells Derived from Human Induced-Pluripotent Stem Cells and a Native-Like, High-Resolution, 3-Dimensionally Printed Scaffold Ling Gao, Molly Kupfer, Jangwook Jung, Libang Yang, Patrick Zhang, Yong Sie, Quyen Tran, Visar Ajeti, Brian Freeman, Vladimir Fast, Paul Campagnola, Brenda Ogle and Jianyi Zhang Circ Res. published online January 9, 2017; Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2017 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. Online ISSN: 1524-4571 The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circres.ahajournals.org/content/early/2017/01/09/CIRCRESAHA.116.310277 Data Supplement (unedited) at: http://circres.ahajournals.org/content/suppl/2017/01/09/CIRCRESAHA.116.310277.DC1 Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Circulation Research is online at: http://circres.ahajournals.org//subscriptions/ CIRCRES/2016/310277 R2 Supplemental Material Myocardial tissue engineering with cardiac cells derived from human induced-pluripotent stem cells and a native-like, high-resolution, 3-dimensionally printed scaffold Ling Gao1, Molly E. Kupfer2, Jangwook P. Jung2, Libang Yang2, Patrick Zhang2, Yong Da Sie3,Quyen Tran3, Visar Ajeti3, Brian T. Freeman2, Vladimir G. Fast1, Paul J. Campagnola3, Brenda M. Ogle2*, Jianyi Zhang1* 1 Department of Biomedical Engineering, School of Medicine, School of Engineering, University of Alabama at Birmingham; 2 Department of Biomedical Engineering, University of Minnesota – Twin Cities, Minneapolis, MN 555455 USA; 3 Department of Biomedical Engineering, University of Wisconsin – Madison, Madison, WI 53706 USA Short title: Gao, 3D-printed scaffolds for engineered myocardium *Addresses for correspondence Dr. Jianyi Zhang, Department of Biomedical Engineering, School of Medicine, School of Engineering, University of Alabama at Birmingham, Birmingham, AL 35233 205-934-8421, E-mail: [email protected] Dr. Brenda M. Ogle, Department of Biomedical Engineering, College of Science and Engineering, University of Minnesota – Twin Cities, Minneapolis, MN, 55455, 612-624-5948, E-mail: [email protected] 1 CIRCRES/2016/310277 R2 SUPPLEMENTARY METHODS Creation of the ECM scaffold via 3D-MPE printing Methacrylated gelatin (100 mg/mL) was mixed with sodium 4-[2-(4-morpholino)benzoyl-2-dimethylamino]-butylbenzenesulfonate (MBS) (1 mM) at 1% v/v, and crosslinking was performed on a glass slide, which served as the non-specific background. Two-photon excitation of the MBS photoactivator was induced with a 100 fs Ti:sapphire laser (Mira, Coherent, Santa Clara, CA) at 780 nm. A 10x, 0.5 NA objective lens was used, and the average power at the focus was kept constant (~100 mW at 78 MHz repetition rate). Upon UV absorption, MBS degrades to benzoyl and an α-aminoalkyl radical, which then attack residues containing aromatic groups and free amines1; then, the radical protein links to a second protein molecule, generating a covalent bond. Our custom-built multiphoton instrument, which has been described in detail previously, 2 can produce scaffolds with more complexity and in a greater variety of sizes than can be achieved with a commercial laser-scanning microscope. The Ti:sapphire laser is coupled to a upright microscope stand (Axioskop 2, Zeiss, Thornewood, NY), and scanning is performed by combining a laser-scanning galvos (Cambridge Technolgoies, Bedford, MA) with a motorized stage (x-y-z, Ludl Electronic Products Ltd, Hawthorne, NY). The apparatus is controlled with LabVIEW software, and data is acquired with a field-programmable gate array (FPGA) board (Virtex-II PCI-7831R, National Instruments, Austin, TX).2 The laser power entering the optical train is regulated with a 10 kHz electro-optic modulator (EOM) (Conoptics, Danbuty, CT), and the laser is rapidly shuttered with a second, higher-speed EOM (maximum 100 MHz; Conoptics). Parameters such as power, scanning area, the scan rate of the galvanometer, and repetition of the scanning pattern (i.e., the number of scans per layer) are set via a graphical user interface (GUI), while the TPEF of the entrapped residual photoactivator 2 CIRCRES/2016/310277 R2 serves as the online diagnostic signal for crosslinking and is read by the FPGA. The microscope is also equipped with phase-contrast and two-photon fluorescence imaging capabilities for characterizing the scaffold (e.g. immunofluorescence and quality control), and the minimum size of the features in the crosslinked protein structure corresponds to the two-photon excited point spread function (PSF). For example, when using 0.75 NA and 780 nm two-photon excitation, the lateral and axial resolution are about 600 nm and 1.8 μm, respectively2, 3 MPE polymerization can also produce features with sub-micron resolution, because of chemical nonlinearity in the free radical kinetics,3 but this does not occur during the crosslinking of proteins. The FPGA was incorporated into the system to exploit the parallelism of command executions (80 MHz clock rate) and to avoid bottlenecks in communication between the CPU and the hardware, which occurs through four first in, first out (FIFO) channels. Two FIFO channels relay information from the main LabVIEW program to the FPGA for control of the galvanometer mirrors and fast EOM shutter, while the other two record information from the PMT to create an image of the fabrication; thus, communication between the CPU and hardware occurs in near real-time. The source code of the instrument control software is freely available at: http://campagnola.molbio.wisc.edu/. The scanning approach (i.e., “modulated raster scanning”) combines the advantages of both raster scanning and vector scanning.4 The galvanometers are raster scanned at maximum speed (~40 kHz), while the laser is shuttered at an even greater rate (10 MHz-40kHz) with the fast EOM. This approach is more accurate than modulating the scanning speed and/or step size of the galvanometers, because it is run over the whole field of view at constant speed and with the same pulse energy and repetition rate. The average power entering the fast-shuttering EOM is set by the slower EOM, which allows the entire scanning process to be performed at constant peak power and, consequently, 3 CIRCRES/2016/310277 R2 the fractional “on-time” within each pixel defines the integrated exposure dose, which is a linear process that directly correlates to the resulting protein concentration. Thus, we achieve a linear map between the intensity in the original image data and the fabricated structure. Fabrication was based on 8-bit, 3D image files (.bmp or .tif) that were acquired with sampling that satisfied the Nyquist criterion, which is crucial for ensuring that the tissue is accurately represented and that the resolution (i.e., the sizes of the features) matches that of the original image, as well as for optimizing the structural integrity of the fabricated scaffold in 3D. Fabrication proceeded through the image stack via a Z-step pattern, and upon completion, the fidelity of the structure to the image stack was measured by using FIJI software to compare, pixel-by-pixel, the gray-scale intensity of the original (or processed) image to the two-photon excited fluorescence image of the fabricated construct. Generation and characterization of hciPSC-derived cardiac cells Recent reports from our laboratory suggest that the cells derived from induced-pluripotent stem cells (iPSCs) may be more effective for treatment of myocardial injury, and that the Ca2+ handling profile of iPSC-derived CMs is more cardiac-like, if the iPSCs are reprogrammed from cardiac, rather than dermal, fibroblasts.5, 6 Thus, the CMs, SMCs, and ECs used in this study were generated from human, cardiac-lineage, iPSCs (hciPSCs), which had been reprogrammed from male, human, cardiac fibroblasts by transfecting the cells with lentiviruses coding for OCT4, SOX2, KLF4, and C-MYC.5 After reprogramming, the hciPSCs were engineered to constitutively express green fluorescent protein (GFP), cultured in Matrigel-coated plate with human iPSC growth medium, and regularly passaged every 6-7 days. 4 CIRCRES/2016/310277 R2 hciPSCs were differentiated into ECs and SMCs as described previously.7, 8 Briefly, the undifferentiated cells were treated with a GSK-3β inhibitor and ascorbic acid to induce mesoderm differentiation. Five days later, cells that expressed CD34 (i.e., hciPSC-derived vascular progenitor cells [hciPSC-VPCs]) were collected via magnetic nanoparticle selection; then, the hciPSC-VCs were differentiated into ECs by culturing them on fibronectin-coated flasks with EC-developmental medium (EGM2-MV; Lonza, Basel, Switzerland) or into SMCs by culturing them on collagen IV-coated flasks with SMC-developmental medium (SmGM-2; Lonza) containing PDGF-BB and TGF-β. ECs were purified to >95% via flow-cytometry selection for both CD31 and CD144 expression and then characterized via the expression of CD31, CD144, and von Willebrand factor-8 (vWF-8). SMCs were characterized via the expression of smooth-muscle actin (SMA), smooth-muscle 22 alpha (SM22), and calponin. hciPSCs were differentiated into CMs as previously reported. 9 Briefly, the undifferentiated cells were expanded on a Matrigel ®-coated dish for 4 days; then, differentiation was induced on day 0 by culturing the cells with a GSK-3β inhibitor in RPMI basal medium plus B27 without insulin (B27–). The cells were recovered 24 hours later, cultured in RPMI basal medium plus B27– for 2 days, and then cultured in RPMI basal medium plus B27– and a Wnt signaling inhibitor; beating cells usually appeared about 8 days after differentiation was initiated. The hciPSC-CMs were purified to >95% via metabolic selection10 and characterized via the expression of cardiac troponin I (cTnI), cardiac troponin T (cTnT), α-sarcomeric actin (αSA), Connexin 43, and ventricular myosin light chain-2 (MLC-2v). Creation of the human iPSC-derived cardiac muscle patchs (hCMPs) and control patches The hCMPs (2 mm × 2 mm, 100 μm thick) were created by combining the 5 CIRCRES/2016/310277 R2 3D-MPE–fabricated scaffold with a 2:1:1 ratio of hciPSC-CMs, hciPSC-ECs, and hciPSC-SMCs. A total of ~50,000 cells (i.e., 12,500 cells/mm 2) were seeded onto the surface of the scaffold; then, the hCMPs were cultured in Dulbecco minimum essential medium (DMEM) with 10% fetal calf serum for 1 week before in vitro analyses were performed or for 1 day before in vivo transplantation. For the in-vitro experiments, control patches were created by combining the same density and ratio of hciPSC-CMs, -ECs, and -SMCs with a chemoselectively cross-linked polyethylene glycol (PEG) gel11 or with decellularized bovine pericardium; the control patches were also cultured in DMEM with 10% fetal calf serum for 1 week. Measurement of calcium transients hCMPs were loaded with 5 µM Fluo-4 AM (Life Technologies, USA) and incubated at 37C and 5% CO2 for 30 min; then, the intercellular AM esters were de-esterified by incubating the hCMPs with Tyrode’s salt solution at 37C and 5% CO2 for another 30 min. Intracellular calcium transients were recorded with a Zeiss Cell Observer Spinning Disk Confocal microscope equipped with a 20X (NA = 0.5) objective and an environmental chamber (37C, 5% CO2) over 30 to 120 s. Movies were analyzed with FIJI software to measure the fluorescence intensities for 2 to 8 regions of interest (F) and for 3 to 8 background regions (F0) per acquisition. In vitro analysis of contractility Videos of the beating hCMPs were taken at 24 fps; then, the speed of contraction and relaxation, as well as the peak beat rate, of the hCMP was determined with motion vector analysis software developed by Huebsch, et al.12 Optical mapping of activation hCMPs were transferred to a perfusion chamber mounted on an inverted microscope 6 CIRCRES/2016/310277 R2 and perfused with Hank's balanced salt solution at 37oC. Preparations were stimulated with 2-ms rectangular pulses delivered from a bipolar electrode. Cells were stained with Vm-sensitive dye RH-237 (5 μM) for 5 min. To eliminate motion artifact from optical recordings, cell contractions were inhibited by supplementing staining and perfusion solutions with 10 μM of blebbistatin. Dye fluorescence was excited using an Hg/Xe arc lamp and a 560/55-nm excitation filter and measured at >650 nm using a 16x16 photodiode array at spatial resolution of 110 µm per diode, as previously described.13 Optical signals were digitally filtered to increase the signal-to-noise ratio. Activation times were measured at the 50% level of action potential amplitude and used to construct the isochronal maps of activation spread. Conduction velocity was calculated at each recording site from local activation times and averaged across the whole map using custom-written data analysis software. Action potential durations were measured at 50% and 80% levels of repolarization (APD50 and APD80, respectively) as intervals between repolarization and activation times. Cardiac monolayer culture Cell suspensions containing a 2:1:1 ratio of hciPSC-CMs, -ECs, and -SMCs were seeded on a gelatin coated plate at a density of 1.2x105 cells/cm2 and cultured in DMEM with 10% fetal calf serum; the culture media was changed every 2 days. Quantitative RT-PCR Analysis Total RNA was extracted by using Qiashredder and RNeasy mini kits (Qiagen, USA) as directed by the manufacturer's instructions; then, the RNA (1-2 μg per 20 μL reaction) was reverse transcribed with SuperScript™ II Reverse Transcriptase (Thermo Scientific, USA) and appropriate primers (Supplemental Table 1). Quantitative PCR was performed with Maxima SYBR Green Master Mix (Thermo Scientific) on a Realplex2 Real-Time 7 CIRCRES/2016/310277 R2 PCR system (Eppendorf, USA). Measurements were calculated via the 2−∆Ct method and normalized to glyceraldehyde phosphate dehydrogenase RNA levels. Murine model of myocardial infarction and hCMP administration All experimental procedures that involved animals were approved by the Institutional Animal Care and Use Committee of the University of Minnesota, performed in accordance with the Animal Use Guidelines of the University of Minnesota, and consistent with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publication No 85-23). Twelve-week-old immunodeficient NOD-scid/γc–/– mice (Jackson Laboratory) were anesthetized with an intraperitoneal injection of sodium pentobarbital (35 mg/kg), intubated, and ventilated with a small animal respirator (Harvard Apparatus); then, a left thoracotomy was performed to expose the heart, and the left-anterior descending coronary artery was permanently ligated with an 8.0 surgical silk suture. Fifteen minutes after ligation, animals in the MI+hCMP group were treated with two hCMPs, and animals in the MI+Scaffold group were treated with two 3D-MPE–printed scaffolds that lacked any cells. Both treatments were applied to the epicardial surface over the infarcted area—the hCMP was oriented approximately parallel to the alignment of the surface myocardium and held in place by covering them with a piece of decellularized bovine pericardium that was sutured to the heart to avoid movement of hCMPs off the heart and prevent adhesions between the heart and the rib cage. Both the hCMP and the cell-free scaffold were withheld from animals in the MI group, and animals in the Sham group underwent all surgical procedures for MI induction except the ligation step and received neither of the experimental treatments. The chest was closed in layers and the animals were allowed to recover. Echocardiographic assessments of cardiac function Echocardiographic measurements were obtained via the method recommended by the 8 CIRCRES/2016/310277 R2 American Society of Echocardiography and performed on a Vevo770 Imaging System equipped with an RMV 707B transducer (15-45MH; VisualSonics Inc, Canada); both conventional 2-dimensional images and M-Mode images of the heart in a parasternal short axis view were acquired. Left ventricular (LV) internal diameters at end-diastole (LVIDed) and end-systole (LVIDes) were determined from 8 consecutive images, and LV ejection fractions (EF) and fractional shortening (FS) were calculated according to the following equations: EF=(LVIDed3−LVIDes3)/LVIDed3×100%;FS=(LVIDed−LVIDes)/LVIDed×100%. Immunohistochemical evaluations The hCMPs were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.2% Triton X-100 for 15 min, and then blocked with 5% donkey serum for 30 min. Primary antibodies (rabbit anti-cTnI [Abcam, USA], mouse anti-cTnT [Thermo Scientific, USA], mouse anti-SMA [Sigma-Aldrich, USA], and goat anti-CD31 [Santa Cruz, USA]) were diluted in the blocking solution, and the hCMPs were incubated at 4°C; on the following day, the hCMPs were incubated with secondary antibodies for 1 h and then stained with DAPI. The alignment of cells in the hCMP channels was evaluated via multiphoton microscopy. ImageJ software was used to fit an ellipse to the outline of each cell, which was visible because of cellular autofluorescence, and the ellipse was used to determine the cell’s aspect ratio and angle relative to horizontal; then, the angle of orientation for each cell was normalized to the average longitudinal angle of the channel walls. Murine hearts were cut into halves from the middle of the infarct. One of the halves was frozen at the optimal cutting temperature for cryo-sectioning, and the other half was stored in 10% formalin for paraffin-embedded sectioning. Embedded tissues were cut into 7-μm sections; then, the sections were fixed with 4% paraformaldehyde for 20 min at room temperature, permeabilized in 0.1% Triton X-100 at 4ºC for 10 min, and blocked 9 CIRCRES/2016/310277 R2 with UltraV block (Thermo Scientific, USA) for 7 min. Primary antibodies (mouse anti-human specifc nuclear antigen, HNA [Emdmillipore, USA], goat anti-GFP [Abcam, USA], goat anti-CD31 [Santa Cruz, USA], mouse anti-human specific CD31 [hCD31, Dako, USA], mouse anti-SMA [Sigma-Aldrich, USA], rabbit anti-cTnI [Abcam, USA], mouse anti-cTnT [Abcam, USA], rabbit anti-Ki67 [Abcam, USA]) were added to the UltraV block buffer, and the sections were incubated at 4ºC. On the following day, secondary antibodies conjugated with fluorescent markers (Jackson ImmunoResearch Lab, USA) were added; then, the sections were incubated for 1 h at room temperature, stained with DAPI, washed, and examined under a confocal microscope (Zeiss 710, USA). Engrafted hciPSC-cardiovascular cells were identified by the expression of HNA or GFP, engrafted hciPSC-CMs were identified by the expression of both GFP and cTnI, or both HNA and cTnI, engrafted hiPSC-ECs were identified by hCD31 expression, and engrafted hiPSC-SMCs were identified by the expression of both GFP and SMA. Vascular density was evaluated by counting the number of vascular structures that were positive for either CD31 or SMA expression; then, the images used for the CD31 and SMA evaluations were superimposed, and arteriole density was evaluated by counting the number of structures that expressed both CD31 and SMA. Cells and vessels were counted in 5 fields per section, 5 sections per animal. Engraftment rate assessment Engraftment rates were evaluated via quantitative PCR (qPCR) assessments of the human Y-chromosome as previous reported.14 Briefly, whole hearts of female mice were collected and digested overnight at 56 oC with proteinase K; then, the total DNA was isolated from the digested buffer with a QIAGEN DNA isolation kit. The number of cells in each mouse heart was determined by comparing the number of cycles required for each 10 CIRCRES/2016/310277 R2 sample to a standard curve calculated from the DNA of known quantities of undifferentiated hciPSCs, and the engraftment rate was calculated as the number of cells in each animal divided by the number of cells administered (1x105). Analyses were performed with the SYBR Green kit (Thermo Scientific, USA) on an Eppendorf Realplex2 PCR system (Eppendorf, USA) with the following primers: sense, ATCAGCCTAGCCTGTCTT-CAGCAA; anti-sense, TTCACGACCAACAGCACAGCAATG. To assess the engraftment rate by counting the grafted human cells as evidenced by HNA positivity, we also used histologic assessment of consecutives sections of the heart that spans the entire area covered by the engrafted patch, and counting the grafted human cells as evidenced by HNA positivity. 5, 15 Hearts were frozen or paraffin-embedded and transversely cut into 7-μm sections from base to apex. Total cell nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Engrafted hciPSCs-derived cardiovascular cells were identified by counting the human specific nuclear antigen (HNA)-positive cells in every 15th serial section of the heart and then multiplying by 15 to obtain the total number of engrafted hciPSC-cardiovascular cells per heart. The engraftment rate of transplanted hciPSC-cardiovascular cells was calculated by dividing the total number of engrafted cells by the number of cells administered (1x105) and multiplying by 100. Infarct size and thickness measurements Hearts were frozen or paraffin-embedded and cut into 7-μm sections; then, the sections were stained with an Accustain Trichrome Stains (Masson) kit (Sigma-Aldrich, USA). The infarct size was measured from Masson’s trichrome-stained sections as the percentage of the surface area of the left ventricular anterior wall occupied by scar, and infarct thickness was calculated as the ratio of the thicknesses of the fibrous region to the thickness of the septal wall. For each heart, the infarct size was averaged from three areas of the heart: just below the ligation suture, midway between the ligation suture and 11 CIRCRES/2016/310277 R2 the apex, and a section close to the apex. Apoptosis Sections cut from embedded hearts were TUNEL stained with an In-situ Cell Death Detection Kit (Roche Applied Science, Germany) and viewed at 40x magnification as previously reported.5 The number of TUNEL-positive cells and the total number of cells were counted in 5 to 6 slides per animal and 5 fields per slide. Statistical Analyses Data are presented as mean ± standard error. Statistical significance (P<0.05) between two experimental groups was determined via the Student’s two-tailed t-test, assuming unequal variance. Comparisons among more than two experimental groups were evaluated via one-way ANOVA followed by Tukey post-hoc analysis. All statistical analyses were performed with SPSS software (version 13.0, SPSS, USA). 12 CIRCRES/2016/310277 R2 SUPPLEMENTAL REFERENCES 1. Kojima K, Masato I, Morishita H and Hayashi N. A novel water-soluble photoinitiator for the acrylic photopolymerization type resist system. Chem Mater. 1998;10:3429-3433. 2. 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Zhang X, Shen MR, Xu ZD, Hu Z, Chen C, Chi YL, Kong ZD, Li ZF, Li XT, Guo SL, Xiong SH and Zhang CS. Cardiomyocyte differentiation induced in cardiac progenitor cells by cardiac fibroblast-conditioned medium. Exp Biol Med (Maywood). 2014;239:628-37. 7. Lian X, Bao X, Al-Ahmad A, Liu J, Wu Y, Dong W, Dunn KK, Shusta EV and Palecek SP. Efficient differentiation of human pluripotent stem cells to endothelial progenitors via small-molecule activation of WNT signaling. Stem Cell Reports. 2014;3:804-16. 8. Bao X, Lian X, Dunn KK, Shi M, Han T, Qian T, Bhute VJ, Canfield SG and Palecek SP. Chemically-defined albumin-free differentiation of human pluripotent stem cells 13 CIRCRES/2016/310277 R2 to endothelial progenitor cells. Stem Cell Res. 2015;15:122-9. 9. Lian X, Zhang J, Azarin SM, Zhu K, Hazeltine LB, Bao X, Hsiao C, Kamp TJ and Palecek SP. Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/beta-catenin signaling under fully defined conditions. Nat Protoc. 2013;8:162-75. 10. Tohyama S, Hattori F, Sano M, Hishiki T, Nagahata Y, Matsuura T, Hashimoto H, Suzuki T, Yamashita H, Satoh Y, Egashira T, Seki T, Muraoka N, Yamakawa H, Ohgino Y, Tanaka T, Yoichi M, Yuasa S, Murata M, Suematsu M and Fukuda K. Distinct metabolic flow enables large-scale purification of mouse and human pluripotent stem cell-derived cardiomyocytes. Cell Stem Cell. 2013;12:127-37. 11. Jung JP, Sprangers AJ, Byce JR, Su J, Squirrell JM, Messersmith PB, Eliceiri KW and Ogle BM. ECM-incorporated hydrogels cross-linked via native chemical ligation to engineer stem cell microenvironments. Biomacromolecules. 2013;14:3102-11. 12. Huebsch N, Loskill P, Mandegar MA, Marks NC, Sheehan AS, Ma Z, Mathur A, Nguyen TN, Yoo JC, Judge LM, Spencer CI, Chukka AC, Russell CR, So PL, Conklin BR and Healy KE. Automated Video-Based Analysis of Contractility and Calcium Flux in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes Cultured over Different Spatial Scales. Tissue Eng Part C Methods. 2015;21:467-79. 13. Sowell B and Fast VG. Ionic mechanism of shock-induced arrhythmias: role of intracellular calcium. Heart rhythm : the official journal of the Heart Rhythm Society. 2012;9:96-104. 14. Ye L, Chang YH, Xiong Q, Zhang P, Zhang L, Somasundaram P, Lepley M, Swingen C, Su L, Wendel JS, Guo J, Jang A, Rosenbush D, Greder L, Dutton JR, Zhang J, Kamp TJ, Kaufman DS, Ge Y and Zhang J. Cardiac repair in a porcine model of acute myocardial infarction with human induced pluripotent stem cell-derived cardiovascular cells. Cell stem cell. 2014;15:750-61. 15. Nakamura Y, Wang X, Xu C, Asakura A, Yoshiyama M, From AH and Zhang J. 14 CIRCRES/2016/310277 R2 Xenotransplantation of Long Term Cultured Mesenchymal Stem Cells. Stem Cells. 2006. 15 Swine Bone Marrow-Derived CIRCRES/2016/310277 R2 ONLINE FIGURE LEGENDS Online Figure I. Immunofluorescent analysis of lineage-specific marker expression in differentiated hiPSC-derived cardiac cells. The lineage of the differentiated hciPSC-CMs was confirmed via the expression of cardiac troponin I (cTnI), cardiac troponin T (cTnT), ventricular myosin light chain 2 (MLC-2v), α-sarcomeric actin (αSA), and the gap-junction protein connexin-43 (Con-43); the lineage of the differentiated hciPSC-SMCs was confirmed via the expression of -smooth muscle actin (SMA), SM22, and calponin; and the lineage of the differentiated hciPSC-ECs was confirmed via the expression of CD31, CD144, and von Villebrand factor 8 (vWF-8). Nuclei were counterstained with DAPI (scale bar = 50μm). Online Figure II. Expression of contractile- and calcium-transient–related genes in hCMPs and in monolayers of hiPSC-derived cardiac cells. Quantitative PCR measurements of the expression of genes involved in (A) contractile activity (cTnT, cTnI, atrial myosin light chain 2 [MLC2a], MLC2v, α myosin heavy chain [αMHC], βMHC), and (B) generating calcium transients (sarcoplasmic reticulum Ca2+-ATPase 2SERCA2, phospholamban [PLB], sodium/calcium exchanger 1 [NCX1], ryanodine receptor 2 [RYR2], calsequestrin 2 [CASQ2]) were performed in hCMPs and in monolayers composed of equivalent populations of cells on day 1 and day 7 after manufacture. Results were normalized to measurements obtained in monolayers on day 1. *P<0.05, **P<0.01; n=3-4 hCMPs or monolayers per time point with 3 replicates per measurement. Online Figure III. hCMP transplantation reduces apoptosis and increases angiogenesis after MI. (A) Apoptotic cells were identified in sections from the border-zone of ischemia in the hearts of animals from the MI (top row), MI+Scaffold (middle row), and MI+hCMP (bottom row) groups via TUNEL staining; CMs were 16 CIRCRES/2016/310277 R2 visualized via immunofluorescent staining for the presence of cTnI and nuclei were counterstained with DAPI. (B) Apoptosis was quantified as the percentage of cells that were positive for TUNEL staining. (C) Sections from the border-zone of ischemia in the hearts of MI (top row), MI+Scaffold (middle row), and MI+hCMP (bottom row) animals were immunofluorescently stained for the presence of the CD31, SMA, and cTnI, and nuclei were counterstained with DAPI; then (D) vascular density was determined by quantifying the expression of CD31, and (E) arteriole density was determined by quantifying the co-expression of CD31 and SMA. **P<0.01, n = 5-6 in each group; scale bar = 50 m. Online Figure IV. hCMP transplantation promotes cell proliferation after MI. (A) Proliferating cells were identified in sections from the border-zone of infarct in hearts on day 28 after MI by staining for expression of the proliferation marker Ki67 (green). (B) Proliferation was quantified as the number of Ki67+ cells per high-power field (HPF). *P<0.05, **P<0.01, n=5-6 in each group. Scale bar = 100m. 17 CIRCRES/2016/310277 R2 ONLINE VIDEO LEGENDS Online Video I. MPE-3DP to fabricate adult simulate grid layer by layer. The video shows layer-by-layer fabrication of a scaffold of dimensions 400 x 400 x 100 microns. This is the same structure as was used for the in vitro experiments. For in vivo studies, structures like the one shown here were tiled together to generate millimeter scale structures. Fabrication of each layer required approximately 10 seconds. The fluorescent contrast arises from the two-photon excitation of Rose Bengal entrapped in the matrix. Online Video II. 360 view of 3D rendering of adult simulate grid. The video shows a 3D projection of the series of fabricated layers shown in the S1 video, with a 360 view of the fabricated structure. Online Video III. Animation depicting the MPE-3DP process. First, a solution containing the polymer or protein of interest and associate photocrosslinking agent is added to the reservoir. The reservoir sits upon a slide on the stage of a multiphoton microscopy rig. The pulsed laser is actuated and raster scanned across the x-y plane of a given field of view. The dwell time of the laser at a given focal volume is dictated by the intensity of the digital template at a given x-y position. The longer the dwell time, the more crosslinking occurs and therefore a more stable structural feature is formed. The scan time is on the scale of microseconds for each focal volume, milliseconds for each x-y line and seconds for the entire raster (i.e., x-y plane). The animation shows a significantly slowed visual depiction of proteins of focal volumes undergoing polymerization on three different z planes. Not shown in the video is the digital template, which dictates the final form of the structure. As noted at the end of the animation, the final structure can be manipulated for use in vitro and in vivo. Online Video IV. Spontaneous and synchronous contractile activity in a hCMP on 18 CIRCRES/2016/310277 R2 day 1 after fabrication (magnification: 100 x). Online Video V. Spontaneous and synchronous contractile activity in a hCMP on day 3 after fabrication (magnification: 100 x). 19 CIRCRES/2016/310277 R2 Online Table I. Primers for quantitative RT-PCR Standard Gene Gene product Forward name Reverse Size bp GAPDH GAPDH TCGACAGTCAGCCGCATCTTCTTT ACCAAATCCGTTGACTCCGACCTT 94 TNNI3 cTNI CCTCACTGACCCTCCAAACG 104 TNNT2 cTNT TTCACCAAAGATCTGCTCCTCGCT TTATTACTGGTGTGGAGTGGGTGTGG 165 MYH6 ɑMHC CTCCGTGAAGGGATAACCAGG TTCACAGTCACCGTCTTCCC 248 MYH7 ßMHC ACCAACCTGTCCAAGTTCCG TCATTCAAGCCCTTCGTGCC 131 MYL2 MLC 2v ACATCATCACCCACGGAGAAGAGA ATTGGAACATGGCCTCTGGATGGA 164 MYL7 MLC 2a GGAGTTCAAAGAAGCCTTCAGC AAAGAGCGTGAGGAAGACGG 178 ACTN2 -actinin 2 CTTCTACCACGCTTTTGCGG CCATTCCAAAAGCTCACTCGC 136 ATP2A2 SERCA 2 TCACCTGTGAGAATTGACTGG AGAAAGAGTGTGCAGCGGAT 149 CASQ2 calsequestrin 2 GTTGCCCGGGACAATACTGA CTGTGACATTCACCACCCCA 142 PLN Phospholamban ACAGCTGCCAAGGCTACCTA GCTTTTGACGTGCTTGTTGA 191 SLC8A1 NCX 1 CTGGAATTCGAGCTCTCCAC ACATCTGGAGCTCGAGGAAA 108 RYR2 RYR 2 TTGGAAGTGGACTCCAAGAAA CGAAGACGAGATCCAGTTCC 141 20 GAGGTTCCCTAGCCGCATC Online Figure I hiPSC-CMs hiPSC-SMCs hiPSC-ECs cTnI DAPI aSMA DAPI CD31 DAPI cTnT MLC-2v DAPI SM22 DAPI CD144 DAPI Con-43 aSA DAPI Calponin DAPI vWF-8 DAPI Online Figure II A ** * 4 3 * * Monolayer, day 1 hCMP, day 1 Monolayer, day 7 hCMP, day 7 * * 2 1 0 cTnT cTnI MLC2a MLC2v aMHC aActinin2 ** ** B 4 bMHC * * 10 3 * * 2 8 6 4 1 2 SERCA2a PLB NCX1 RYR2 0 CASQ2 Monolayer, day 1 hCMP, day 1 Monolayer, day 7 hCMP, day 7 Online Figure III A B DAPI cTnI MI TUNEL cTnI DAPI cTnI MI MI+Scaffold MI+hCMP TUNEL DAPI cTnI TUNEL DAPI cTnI Percentage (TUNEL+ cells /Total cells) TUNEL cTnI ** 48 ** 36 24 12 0 MI+Scaffold TUNEL cTnI DAPI cTnI TUNEL DAPI cTnI MI+hCMP C CD31 B aSMA CD31 aSMA DAPI cTnI Vascular density (mm-2) D ** 1600 MI MI+Scaffold MI+hCMP ** 1200 MI CD31 aSMA CD31 aSMA DAPI cTnI 800 400 0 MI+Scaffold CD31 MI+hCMP aSMA CD31 aSMA DAPI cTnI Arteriole density (mm-2) E ** 600 450 300 150 0 MI MI+Scaffold MI+hCMP ** Online Figure IV A Ki67 DAPI Ki67 DAPI MI MI+Scaffold B MI MI+Scaffold MI+hCMP ** Ki67+ cells /HPF 20 15 10 5 0 * Ki67 DAPI MI+hCMP