- Email: [email protected]
Myocardial tissue engineering for cardiac repair
Author's Accepted Manuscript
Myocardial Tissue Engineering for Cardiac Repair S. Pecha, T. Eschenhagen, H. Reichenspurner
PII: DOI: Reference:
S1053-2498(16)00002-4 http://dx.doi.org/10.1016/j.healun.2015.12.007 HEALUN6139
To appear in:
J Heart Lung Transplant
http://www.jhltonline.org
Cite this article as: S. Pecha, T. Eschenhagen, H. Reichenspurner, Myocardial Tissue Engineering for Cardiac Repair, J Heart Lung Transplant, http://dx.doi.org/10.1016/j. healun.2015.12.007 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 galley proof before it is published in its final citable 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.
State of the Art ReviewMyocardial tissue engineering for cardiac repair
Pecha S.1,3 Eschenhagen T.2,3 Reichenspurner H1,3.
1
2
Department of Cardiovascular Surgery, University Heart Center Hamburg, Germany
Department of Experimental Pharmacology and Toxicology, University Medical Center HamburgEppendorf, Germany 3
DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck
Corresponding author: Dr. Simon Pecha University Heart Center Hamburg Department of Cardiovascular Surgery Martinistr. 52, 20246 Hamburg, Germany Telephone: +49 40 7410 52440 Email: [email protected]
Abstract Number of heart failure patients is increasing in our aging population. Heart transplantation remains the only curative treatment option for end-stage heart failure patients. Due to an organ donor shortage, new organ-independent treatment options are necessary. Different approaches to cardiac repair therapies have been developed and optimized in recent years. One of these promising approaches is myocardial tissue engineering, which describes the creation of three-dimensional engineered heart tissue in-vitro. This review article provides an overview on different approaches to tissue engineering, including essentials to improve tissue quality, choice of ideal cell source and provides an overview on in-vitro and in-vivo studies. Several hurdles that have to be overcome before a clinical application of engineered heart tissue might become a realistic scenario are also acknowledged in this article.
1. Background Incidence and prevalence of heart failure is increasing in an aging population (1). Besides heart transplantation, until now there is no curative treatment option. Due to an organ donorshortage and decreasing number of heart transplantations in Europe in the last years, new organ-independent treatment modalities are necessary. Recent progress in mechanical circulatory support therapy is promising, however still limited by several side effects including infections, bleeding complications, stroke and pump thrombosis especially in patients on long-term support (2). Due to limited endogenous regeneration capacity of the heart in adult mammals, exogenous regenerative strategies have become an attractive treatment option (3) (4) (5). Different approaches for cardiac repair and replacement therapies were described in recent years. Aim of all these methods is to form a contractile heart muscle mass, which mechanically and electrically couples to the host myocardium and supports the contractile function of the injured heart. The technique of direct intramyocardial cell injection
offers advantages in terms of simplicity which is an important issue regarding regulatory affairs and logistics for clinical use (6, 7). However, recent publications have shown limited survival- and retention rate of injected cells (8, 9). Therefore, not only large cell numbers associated with high costs are needed, but there is also a high risk of biodistribution of cells and subsequent teratoma formation. Myocardial tissue engineering, which describes the generation of three-dimensional heart tissues in-vitro, might overcome this problem. However, before clinical application of this technology major hurdles including vascularization of engineered heart tissue or enlargement of grafts have to be overcome.
2. Different approaches to myocardial tissue engineering Cardiomyocytes have an intrinsic capacity to form three-dimensional syncytia. Engineering techniques use this property to generate three-dimensional heart tissue constructs of the desired size and geometry.
Cell sheet technique This technique first described by Shimizu, Okano and colleagues (10) is based on the capacity of cardiomyocytes to form 2D monolayers when cultured in dishes. With the help of thermosensitive culture dish surfaces it is possible to detach intact cellular monolayers when leaving them at room temperature. Stacking of these thin, spontaneously beating 2D monolayers results in 3D myocardial tissue. These scaffold free tissues can then easily be transplanted onto the heart without glue or sutures.
Decellularized heart tissue The principle of this technique has first been described by Taylor and colleagues (7). It aims at reconstituting a natural heart tissue by decellularizing a whole heart or parts of it and reseeding it with cardiac cells. Decellularization can be done for example by Langendorffperfusion with Triton X-100 and sodium dodecyl substrate (SDS), a procedure, which eradicates almost all cells from the tissue but leaves connective tissue and blood vessels behind. The remaining matrix is then perfused with cardiac cells (11). Weymann et al. have shown, that also decellularization of human-sized porcine hearts is possible. Those decellularized hearts can be used as a whole heart tissue-engineering platform (12, 13). However, adequate recellularization and contractile properties of those hearts are still in a very early stage and recellularisation itself shows many unsolved questions. Hydrogel technique This approach developed by Eschenhagen, Elson and colleagues in 1994 (14), was the first cardiac tissue engineering technique. It uses liquid hydrogels like collagen I (15) (14) (16), fibrin (17) or matrigel (18) as matrices. These hydrogels are combined with cardiac cells and put into casting molds with inserted anchoring constructs. The casting mold helps to build the myocardial tissue of desired form and size, while the anchoring constructs allow the tissue to fix and to develop forces between two anchoring points. The liquid hydrogels entrap the cells within the three-dimensional environment and stimulate the natural ability of the cells to form intercellular connections and to build functional and electrical syncytia. The anchoring points are necessary, because good quality of engineered heart tissue (EHT) is only achieved when the constructs grow under continuous mechanical load.
Prefabricated matrices Another tissue engineering approach is to seed prefabricated solid matrices with cardiomyocytes. Several materials including collagen, alginate, gelatin sponges, polyglycolic acid and poly (glycerol sebatic) acid matrices (19) (20) have been used for this method. An advantage of this technique is the possibility to create any desired geometry and size of prefabricated matrix, which then can be seeded with cells.
3. Cell source For several years, embryonic chicken- or neonatal mouse- or rat cardiomyocytes have been the only available cell sources for myocardial tissue engineering. Recent advantages in cell biology allow for the generation of cardiomyocytes from human pluripotent stem cells (embryonic and induced pluripotent stem cells, hESC, hiPSC) at high efficiency (21, 22). Although recent publications demonstrate possible limitations of hiPSC-based strategies for clinical applications (23, 24), they also offer advantages over hESC-approaches. Human iPSC lack the ethical concerns associated with hESC and even if some immune responses were noted in a mouse model (17), hiPSC may offer the opportunity for an autologous approach without the need for long-term immunosuppression. It is currently not clear, though whether time constraints and regulatory hurdles associated with an autologous approach (e.g. each stem cell product will have to undergo extensive safety testing) finally favor the use of an allogeneic approach with well-defined, HLA-matched stem cell banks.
4. Essentials in myocardial tissue engineering Non-cardiomyocytes Cardiac muscle tissue consists of cardiomyocytes and non-myocytes like fibroblasts, endothelial cells and smooth muscle cells. There is strong evidence that non-myocytes contribute to the growth of cardiomyocytes in culture and that they are essential for generation of EHT. It has been shown that EHT made from unpurified heart muscle cell mix developed 3-fold higher forces compared to EHT from pure cardiomyocytes (25). Furthermore, pure hPSC derived cardiomyocytes did not form three-dimensional EHTs but required the addition of cardiac fibroblasts to generate three-dimensional heart tissue (26). Furthermore, the addition of stromal and endothelial cells to hESC-derived cardiomyocytes improved structure and function of the tissue (27, 28). On the other hand, fibrin-based EHTs can readily be made from almost 100% pure hiPSC-cardiomyocytes (own unpublished observation), questioning the generality of this conclusion. Strain In several hydrogel-based EHT studies, it has been shown that mechanical strain is essential for cardiomyocyte alignment and maturation of tissue. The simplest way of mechanical strain is static tension of an EHT between two fixed anchoring points (14, 16). However, improved cardiac tissue function and structure has been observed in EHTs that were cultured in a manner that they can beat against two flexible anchoring points (17). This auxotonic contraction imitates the physiological conditions of the heart in vivo. Vascularization EHT constructs from native heart cell populations form primitive vascular networks with clear lumina, even without the addition of endothelial cells (16, 25, 27). It is unlikely that the vascular structures serve more than a paracrine function in the absence of perfusion, but they
might contribute to in-vivo vascularization after EHT transplantation. The impact of an invitro vascularization on EHT maturation needs to be determined in future studies.
Hypoxia resistance Neonatal rat cardiomyocytes have a high degree of hypoxia resistance. This is clearly an advantage for the generation of EHT from these cells. The immaturity of the cells with their high capacity for anaerobic glycolytic metabolism might be an advantage for cell survival during in-vitro culture and immediately after in vivo transplantation of the EHTs, when there is no vascularization of the graft. In a recent publication, measurements of oxygen concentration in large fibrin EHTs has shown 2% and less in the middle of the construct (29). Maturation EHTs show a more mature, native heart muscle-like phenotype than 2D cultured cardiomyocytes. A high degree of sarcomeric organisation, formation of M-bands, rod-shaped formation, and a relatively normal ratio of nuclei, sarcomeres and mitochondria characterize 3D EHTs. Furthermore, 3D EHTs have quite normal action potential shape and duration and physiological responses like Frank-Starling mechanism, force-frequency relationship and a positive inotropic reaction to calcium and isoprenaline (14, 17, 30). However, EHT tissues do not achieve a fully mature phenotype of native heart muscle tissue. For example, in 3D EHTs the ratio of the adult α-myosin heavy chain isoform to the fetal ß-isoform was 7:1, while it was >100:1 in the adult rat heart (31). Furthermore, the contractile forces of EHTs are less than those of isolated adult heart preparations. In future, further improvements of cardiac maturation will be necessary. Possible factors influencing the degree of maturation are culture conditions, (e.g. media composition, nutrient and oxygen supply), time of in vitro culture and conditions of contraction during maturation (auxotonic stretch).
5. Engineered heart tissue for cardiac repair To regenerate failing hearts, different approaches including cell injection and implantation of in-vitro generated three-dimensional cardiac tissue have been described. The simplicity of cell injections may be outweighed by low cell retention rate, which raises safety issues when injecting cells with potential teratogenic potential like hESC or hiPSC (8). Therefore, strict quality control needs to be implemented before cell-injection technique is approaching clinical use. The EHT approach with its tissue patches transplanted onto the heart might have a much higher cell retention rate, although a direct head-to head comparison with cell injection therapy has not been performed yet. Besides the implantation of tissue patches onto failing hearts, there are some efforts to generate whole biological ventricular assist devices. Although, a quite challenging approach and to date only used in small animal models, it might be a future possibility to overcome biventricular heart failure (32, 33). However, it has to be admitted that due to several unsolved problems like vascularization, or enlargement of graft size, no clinical trials have been performed yet, and it still seems to be a long way until a clinical application of engineered heart tissue might become a realistic scenario. The possibility to stimulate endogenous repair process of the failing heart by injection of biological scaffolds like alginate has been proven in animal experiments and was now used in a first prospective randomized human clinical trial (AUGMENT HF). Patients receiving alginate patches had an improvement in peak VO2 max, NYHA class as well as 6 minute walk test after 6 months. However, a worse outcome in terms of major adverse events (78% vs. 45% of patients), including death (15% vs. 8%) was observed in patients receiving alginate patches compared to control group treated with optimal medical therapy only.
In-vivo studies Over the years, several in-vivo EHT implantation studies, performed in small animal models, have been published with partly promising results. Most studies have been conducted with stacked cell-sheets (34, 35), hydrogel-based EHTs (36) (28, 37) or scaffold-free cell syncytia (27). EHTs from neonatal rat cardiomyocytes survived on host hearts after transplantation for an extended period of time and formed several 100 µm thick muscle strands. This was initially surprising, because there is no vascularization immediately after transplantation. Reasons for this might be the immature phenotype of neonatal rat cardiomyocytes (and most likely of ESCs), which makes them resistant to hypoxia. Furthermore, in contrast to native myocardial tissue, the hydrogel-based and scaffold free EHTs are relatively loose packed with cells, which allows for enhanced diffusion capacity until the relatively quick process of host vessel ingrowth takes place. However, the period until true vascularization by host vessel perfusion is a critical point and might be one of the major circumstances contributing to cell death after EHT implantation. In a study by Shimizu et al. it has been shown that implantation of more than three cell sheets (80 µm) resulted in widespread cell death. However when performing repeated implantations of three layers of cell sheets with a time interval of 1- to 2 days between the implants, building of thick cardiac tissue was possible (38). Although this approach probably cannot be translated into clinics, it gives a hint how fast host vessel ingrowth can occur.
Several attempts have been conducted to realize an in-vitro vascularization and perfusion of the EHT before transplantation. In one of the most promising approaches by Tee et al (39) loops of arterio-venous rat epigastric arteries were isolated and casted together with neonatal rat cardiomyocytes and matrigel in a mold. This construct resulted in a well-vascularized tissue, with a defined arterial and venous pedicle that can be anastomosed to host vasculature. Further approach to install in-vitro vascularization of EHTs was the creation of micro-
channels, generated by enzymatically dissolving alginate fibers or collagen based microchannels. Those micro-channels were populated by endothelial cells and, when perfused with culture medium, contributed to enhanced tissue maturation (29) (40). However, further studies comparing pre-vascularized constructs with conventional EHTs need to be conducted.
To support cardiac function of the failing heart, implanted EHT needs to be electrically coupled to the host myocardium. This has been proven in studies of neonatal rat cardiomyocyte EHTs- and cell sheets (35, 37). However, there is only meager direct histological proof of cardiomyocyte coupling and its mechanism is yet undefined. Probably few cell-cell connections are sufficient to provide electrical coupling, or the improved conduction of remote cells within the infarction area due to paracrine effects might play a role. Further explanation would be the electric conduction via cardiac fibroblasts, which has been shown in another context (41).
Improvement of cardiac function of failing hearts is the central goal of cardiac repair. In a study by Zimmermann et al. implantation of multiloop-EHTs in rats showed improvement of systolic and diastolic function 4 weeks after myocardial infarction. EHTs initially contained 12.5 million cells, of which 4 million cells survived the in-vitro period. An uncertain number of cells survived the transplantation, however the mean thickness of newly formed myocardium was 440 micrometer. In a control group, non-cardiomyocyte constructs did not improve left ventricular function (37). However, further studies with much smaller 3D EHT constructs or non-cardiomyocyte patches have reported similar therapeutic effects. This suggests, that other effects, like increased mechanical wall stability, angiogenesis or paracrine effects might contribute to the observed therapeutic benefits.
Engineered heart tissue from hPSC With recent advantages in cell biology and the availability of high numbers of hPSC derived cardiomyocytes, first three-dimensional tissue engineered constructs, (e.g. cell sheets, hydrogel-based EHTs and scaffold-based 3D constructs) have been generated from hiPSC and hESC derived cardiomyocytes (28, 42-44). Although tissue quality is still not as good as that from neonatal rat cardiomyocyte EHTs, improving protocols with cocultures, addition of growth factors and mechanical and pacing conditioning improves tissue quality (45). First promising animal studies have been performed with cell sheets and hydrogel-based hiPSC EHTs. In a preliminary study, implantation of fibrin-based hiPSC-cardiomyocyte EHTs, resulted in a guinea pig myocardial injury model in significant improvement of left ventricular cardiac function (46). Similar results were reported for implantation of hiPSC derived cardiomyocyte cell-sheet in a porcine cardiomyopathy model (47). Further animal studies are needed to confirm these first promising results, and to go further steps towards a clinical use of hPSC-based EHT to repair injured hearts.
6. Problems to be solved on the way to clinical application Advantages in the field of myocardial tissue engineering have been promising in recent years, however prior to clinical application several major hurdles have to be overcome and it will still be a long way until the use of engineered heart tissue might become a realistic scenario for clinical use. The recent advantages in cell biology, with the possibility to generate unlimited numbers of cardiomyocytes from hiPSCs or hESCs have been a relevant progress in terms of cell availability. However, refinement of differentiation protocols is necessary to minimize variabilities between cell lines and to receive reproducible differentiation efficacy of cardiomyocytes. Use of pluripotent stem cells always raises safety issues in terms of teratoma formation. Especially the application of iPSC additionally carries the risk for chromosome instability and
induction of mutations leading to malignancy. Therefore, strict testing of the cell products is essential prior to clinical application. Antibody-related selection protocols can probably help to overcome this limitation (48). Furthermore, in terms of cardiac repair, graft size and thickness are very important issues to support the failing hearts and to improve cardiac function. Actually, the biological diffusion capacity limits the graft thickness (50-200 µm), and reliable concepts for in-vitro as well as in-vivo vascularization and perfusion are necessary to enlarge the thickness of the three-dimensional grafts. For clinical use, up scaling of graft size is a major limitation, which has not been solved to date. Different experimental approaches for the generation of pre-vascularized grafts have been described (39), however the ideal method has yet to be defined. In terms of immunological issues, the use of hiPSC derived cardiomyocytes at least theoretically allows for an autologous approach without need for immunosuppression. However, this autologous solution is time-consuming and would at least need 6 to 9 months for generation of a patient-specific EHT patch. Besides the time concerns, logistic and economic hurdles would have to be handled, as each iPSC cell line is from a regulatory board view, a new product and needs to undergo extensive toxicity testing prior to clinical application. A more realistic scenario might be a bank of different iPSC or ESC lines, which can be transplanted human leucocyte antigen (HLA) matched, requiring only minimal immunosuppressive regimen.
Conflict of Interest: There is no conflict of interest. No funding was received.
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Myocardial Tissue Engineering for Cardiac Repair S. Pecha, T. Eschenhagen, H. Reichenspurner
PII: DOI: Reference:
S1053-2498(16)00002-4 http://dx.doi.org/10.1016/j.healun.2015.12.007 HEALUN6139
To appear in:
J Heart Lung Transplant
http://www.jhltonline.org
Cite this article as: S. Pecha, T. Eschenhagen, H. Reichenspurner, Myocardial Tissue Engineering for Cardiac Repair, J Heart Lung Transplant, http://dx.doi.org/10.1016/j. healun.2015.12.007 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 galley proof before it is published in its final citable 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.
State of the Art ReviewMyocardial tissue engineering for cardiac repair
Pecha S.1,3 Eschenhagen T.2,3 Reichenspurner H1,3.
1
2
Department of Cardiovascular Surgery, University Heart Center Hamburg, Germany
Department of Experimental Pharmacology and Toxicology, University Medical Center HamburgEppendorf, Germany 3
DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck
Corresponding author: Dr. Simon Pecha University Heart Center Hamburg Department of Cardiovascular Surgery Martinistr. 52, 20246 Hamburg, Germany Telephone: +49 40 7410 52440 Email: [email protected]
Abstract Number of heart failure patients is increasing in our aging population. Heart transplantation remains the only curative treatment option for end-stage heart failure patients. Due to an organ donor shortage, new organ-independent treatment options are necessary. Different approaches to cardiac repair therapies have been developed and optimized in recent years. One of these promising approaches is myocardial tissue engineering, which describes the creation of three-dimensional engineered heart tissue in-vitro. This review article provides an overview on different approaches to tissue engineering, including essentials to improve tissue quality, choice of ideal cell source and provides an overview on in-vitro and in-vivo studies. Several hurdles that have to be overcome before a clinical application of engineered heart tissue might become a realistic scenario are also acknowledged in this article.
1. Background Incidence and prevalence of heart failure is increasing in an aging population (1). Besides heart transplantation, until now there is no curative treatment option. Due to an organ donorshortage and decreasing number of heart transplantations in Europe in the last years, new organ-independent treatment modalities are necessary. Recent progress in mechanical circulatory support therapy is promising, however still limited by several side effects including infections, bleeding complications, stroke and pump thrombosis especially in patients on long-term support (2). Due to limited endogenous regeneration capacity of the heart in adult mammals, exogenous regenerative strategies have become an attractive treatment option (3) (4) (5). Different approaches for cardiac repair and replacement therapies were described in recent years. Aim of all these methods is to form a contractile heart muscle mass, which mechanically and electrically couples to the host myocardium and supports the contractile function of the injured heart. The technique of direct intramyocardial cell injection
offers advantages in terms of simplicity which is an important issue regarding regulatory affairs and logistics for clinical use (6, 7). However, recent publications have shown limited survival- and retention rate of injected cells (8, 9). Therefore, not only large cell numbers associated with high costs are needed, but there is also a high risk of biodistribution of cells and subsequent teratoma formation. Myocardial tissue engineering, which describes the generation of three-dimensional heart tissues in-vitro, might overcome this problem. However, before clinical application of this technology major hurdles including vascularization of engineered heart tissue or enlargement of grafts have to be overcome.
2. Different approaches to myocardial tissue engineering Cardiomyocytes have an intrinsic capacity to form three-dimensional syncytia. Engineering techniques use this property to generate three-dimensional heart tissue constructs of the desired size and geometry.
Cell sheet technique This technique first described by Shimizu, Okano and colleagues (10) is based on the capacity of cardiomyocytes to form 2D monolayers when cultured in dishes. With the help of thermosensitive culture dish surfaces it is possible to detach intact cellular monolayers when leaving them at room temperature. Stacking of these thin, spontaneously beating 2D monolayers results in 3D myocardial tissue. These scaffold free tissues can then easily be transplanted onto the heart without glue or sutures.
Decellularized heart tissue The principle of this technique has first been described by Taylor and colleagues (7). It aims at reconstituting a natural heart tissue by decellularizing a whole heart or parts of it and reseeding it with cardiac cells. Decellularization can be done for example by Langendorffperfusion with Triton X-100 and sodium dodecyl substrate (SDS), a procedure, which eradicates almost all cells from the tissue but leaves connective tissue and blood vessels behind. The remaining matrix is then perfused with cardiac cells (11). Weymann et al. have shown, that also decellularization of human-sized porcine hearts is possible. Those decellularized hearts can be used as a whole heart tissue-engineering platform (12, 13). However, adequate recellularization and contractile properties of those hearts are still in a very early stage and recellularisation itself shows many unsolved questions. Hydrogel technique This approach developed by Eschenhagen, Elson and colleagues in 1994 (14), was the first cardiac tissue engineering technique. It uses liquid hydrogels like collagen I (15) (14) (16), fibrin (17) or matrigel (18) as matrices. These hydrogels are combined with cardiac cells and put into casting molds with inserted anchoring constructs. The casting mold helps to build the myocardial tissue of desired form and size, while the anchoring constructs allow the tissue to fix and to develop forces between two anchoring points. The liquid hydrogels entrap the cells within the three-dimensional environment and stimulate the natural ability of the cells to form intercellular connections and to build functional and electrical syncytia. The anchoring points are necessary, because good quality of engineered heart tissue (EHT) is only achieved when the constructs grow under continuous mechanical load.
Prefabricated matrices Another tissue engineering approach is to seed prefabricated solid matrices with cardiomyocytes. Several materials including collagen, alginate, gelatin sponges, polyglycolic acid and poly (glycerol sebatic) acid matrices (19) (20) have been used for this method. An advantage of this technique is the possibility to create any desired geometry and size of prefabricated matrix, which then can be seeded with cells.
3. Cell source For several years, embryonic chicken- or neonatal mouse- or rat cardiomyocytes have been the only available cell sources for myocardial tissue engineering. Recent advantages in cell biology allow for the generation of cardiomyocytes from human pluripotent stem cells (embryonic and induced pluripotent stem cells, hESC, hiPSC) at high efficiency (21, 22). Although recent publications demonstrate possible limitations of hiPSC-based strategies for clinical applications (23, 24), they also offer advantages over hESC-approaches. Human iPSC lack the ethical concerns associated with hESC and even if some immune responses were noted in a mouse model (17), hiPSC may offer the opportunity for an autologous approach without the need for long-term immunosuppression. It is currently not clear, though whether time constraints and regulatory hurdles associated with an autologous approach (e.g. each stem cell product will have to undergo extensive safety testing) finally favor the use of an allogeneic approach with well-defined, HLA-matched stem cell banks.
4. Essentials in myocardial tissue engineering Non-cardiomyocytes Cardiac muscle tissue consists of cardiomyocytes and non-myocytes like fibroblasts, endothelial cells and smooth muscle cells. There is strong evidence that non-myocytes contribute to the growth of cardiomyocytes in culture and that they are essential for generation of EHT. It has been shown that EHT made from unpurified heart muscle cell mix developed 3-fold higher forces compared to EHT from pure cardiomyocytes (25). Furthermore, pure hPSC derived cardiomyocytes did not form three-dimensional EHTs but required the addition of cardiac fibroblasts to generate three-dimensional heart tissue (26). Furthermore, the addition of stromal and endothelial cells to hESC-derived cardiomyocytes improved structure and function of the tissue (27, 28). On the other hand, fibrin-based EHTs can readily be made from almost 100% pure hiPSC-cardiomyocytes (own unpublished observation), questioning the generality of this conclusion. Strain In several hydrogel-based EHT studies, it has been shown that mechanical strain is essential for cardiomyocyte alignment and maturation of tissue. The simplest way of mechanical strain is static tension of an EHT between two fixed anchoring points (14, 16). However, improved cardiac tissue function and structure has been observed in EHTs that were cultured in a manner that they can beat against two flexible anchoring points (17). This auxotonic contraction imitates the physiological conditions of the heart in vivo. Vascularization EHT constructs from native heart cell populations form primitive vascular networks with clear lumina, even without the addition of endothelial cells (16, 25, 27). It is unlikely that the vascular structures serve more than a paracrine function in the absence of perfusion, but they
might contribute to in-vivo vascularization after EHT transplantation. The impact of an invitro vascularization on EHT maturation needs to be determined in future studies.
Hypoxia resistance Neonatal rat cardiomyocytes have a high degree of hypoxia resistance. This is clearly an advantage for the generation of EHT from these cells. The immaturity of the cells with their high capacity for anaerobic glycolytic metabolism might be an advantage for cell survival during in-vitro culture and immediately after in vivo transplantation of the EHTs, when there is no vascularization of the graft. In a recent publication, measurements of oxygen concentration in large fibrin EHTs has shown 2% and less in the middle of the construct (29). Maturation EHTs show a more mature, native heart muscle-like phenotype than 2D cultured cardiomyocytes. A high degree of sarcomeric organisation, formation of M-bands, rod-shaped formation, and a relatively normal ratio of nuclei, sarcomeres and mitochondria characterize 3D EHTs. Furthermore, 3D EHTs have quite normal action potential shape and duration and physiological responses like Frank-Starling mechanism, force-frequency relationship and a positive inotropic reaction to calcium and isoprenaline (14, 17, 30). However, EHT tissues do not achieve a fully mature phenotype of native heart muscle tissue. For example, in 3D EHTs the ratio of the adult α-myosin heavy chain isoform to the fetal ß-isoform was 7:1, while it was >100:1 in the adult rat heart (31). Furthermore, the contractile forces of EHTs are less than those of isolated adult heart preparations. In future, further improvements of cardiac maturation will be necessary. Possible factors influencing the degree of maturation are culture conditions, (e.g. media composition, nutrient and oxygen supply), time of in vitro culture and conditions of contraction during maturation (auxotonic stretch).
5. Engineered heart tissue for cardiac repair To regenerate failing hearts, different approaches including cell injection and implantation of in-vitro generated three-dimensional cardiac tissue have been described. The simplicity of cell injections may be outweighed by low cell retention rate, which raises safety issues when injecting cells with potential teratogenic potential like hESC or hiPSC (8). Therefore, strict quality control needs to be implemented before cell-injection technique is approaching clinical use. The EHT approach with its tissue patches transplanted onto the heart might have a much higher cell retention rate, although a direct head-to head comparison with cell injection therapy has not been performed yet. Besides the implantation of tissue patches onto failing hearts, there are some efforts to generate whole biological ventricular assist devices. Although, a quite challenging approach and to date only used in small animal models, it might be a future possibility to overcome biventricular heart failure (32, 33). However, it has to be admitted that due to several unsolved problems like vascularization, or enlargement of graft size, no clinical trials have been performed yet, and it still seems to be a long way until a clinical application of engineered heart tissue might become a realistic scenario. The possibility to stimulate endogenous repair process of the failing heart by injection of biological scaffolds like alginate has been proven in animal experiments and was now used in a first prospective randomized human clinical trial (AUGMENT HF). Patients receiving alginate patches had an improvement in peak VO2 max, NYHA class as well as 6 minute walk test after 6 months. However, a worse outcome in terms of major adverse events (78% vs. 45% of patients), including death (15% vs. 8%) was observed in patients receiving alginate patches compared to control group treated with optimal medical therapy only.
In-vivo studies Over the years, several in-vivo EHT implantation studies, performed in small animal models, have been published with partly promising results. Most studies have been conducted with stacked cell-sheets (34, 35), hydrogel-based EHTs (36) (28, 37) or scaffold-free cell syncytia (27). EHTs from neonatal rat cardiomyocytes survived on host hearts after transplantation for an extended period of time and formed several 100 µm thick muscle strands. This was initially surprising, because there is no vascularization immediately after transplantation. Reasons for this might be the immature phenotype of neonatal rat cardiomyocytes (and most likely of ESCs), which makes them resistant to hypoxia. Furthermore, in contrast to native myocardial tissue, the hydrogel-based and scaffold free EHTs are relatively loose packed with cells, which allows for enhanced diffusion capacity until the relatively quick process of host vessel ingrowth takes place. However, the period until true vascularization by host vessel perfusion is a critical point and might be one of the major circumstances contributing to cell death after EHT implantation. In a study by Shimizu et al. it has been shown that implantation of more than three cell sheets (80 µm) resulted in widespread cell death. However when performing repeated implantations of three layers of cell sheets with a time interval of 1- to 2 days between the implants, building of thick cardiac tissue was possible (38). Although this approach probably cannot be translated into clinics, it gives a hint how fast host vessel ingrowth can occur.
Several attempts have been conducted to realize an in-vitro vascularization and perfusion of the EHT before transplantation. In one of the most promising approaches by Tee et al (39) loops of arterio-venous rat epigastric arteries were isolated and casted together with neonatal rat cardiomyocytes and matrigel in a mold. This construct resulted in a well-vascularized tissue, with a defined arterial and venous pedicle that can be anastomosed to host vasculature. Further approach to install in-vitro vascularization of EHTs was the creation of micro-
channels, generated by enzymatically dissolving alginate fibers or collagen based microchannels. Those micro-channels were populated by endothelial cells and, when perfused with culture medium, contributed to enhanced tissue maturation (29) (40). However, further studies comparing pre-vascularized constructs with conventional EHTs need to be conducted.
To support cardiac function of the failing heart, implanted EHT needs to be electrically coupled to the host myocardium. This has been proven in studies of neonatal rat cardiomyocyte EHTs- and cell sheets (35, 37). However, there is only meager direct histological proof of cardiomyocyte coupling and its mechanism is yet undefined. Probably few cell-cell connections are sufficient to provide electrical coupling, or the improved conduction of remote cells within the infarction area due to paracrine effects might play a role. Further explanation would be the electric conduction via cardiac fibroblasts, which has been shown in another context (41).
Improvement of cardiac function of failing hearts is the central goal of cardiac repair. In a study by Zimmermann et al. implantation of multiloop-EHTs in rats showed improvement of systolic and diastolic function 4 weeks after myocardial infarction. EHTs initially contained 12.5 million cells, of which 4 million cells survived the in-vitro period. An uncertain number of cells survived the transplantation, however the mean thickness of newly formed myocardium was 440 micrometer. In a control group, non-cardiomyocyte constructs did not improve left ventricular function (37). However, further studies with much smaller 3D EHT constructs or non-cardiomyocyte patches have reported similar therapeutic effects. This suggests, that other effects, like increased mechanical wall stability, angiogenesis or paracrine effects might contribute to the observed therapeutic benefits.
Engineered heart tissue from hPSC With recent advantages in cell biology and the availability of high numbers of hPSC derived cardiomyocytes, first three-dimensional tissue engineered constructs, (e.g. cell sheets, hydrogel-based EHTs and scaffold-based 3D constructs) have been generated from hiPSC and hESC derived cardiomyocytes (28, 42-44). Although tissue quality is still not as good as that from neonatal rat cardiomyocyte EHTs, improving protocols with cocultures, addition of growth factors and mechanical and pacing conditioning improves tissue quality (45). First promising animal studies have been performed with cell sheets and hydrogel-based hiPSC EHTs. In a preliminary study, implantation of fibrin-based hiPSC-cardiomyocyte EHTs, resulted in a guinea pig myocardial injury model in significant improvement of left ventricular cardiac function (46). Similar results were reported for implantation of hiPSC derived cardiomyocyte cell-sheet in a porcine cardiomyopathy model (47). Further animal studies are needed to confirm these first promising results, and to go further steps towards a clinical use of hPSC-based EHT to repair injured hearts.
6. Problems to be solved on the way to clinical application Advantages in the field of myocardial tissue engineering have been promising in recent years, however prior to clinical application several major hurdles have to be overcome and it will still be a long way until the use of engineered heart tissue might become a realistic scenario for clinical use. The recent advantages in cell biology, with the possibility to generate unlimited numbers of cardiomyocytes from hiPSCs or hESCs have been a relevant progress in terms of cell availability. However, refinement of differentiation protocols is necessary to minimize variabilities between cell lines and to receive reproducible differentiation efficacy of cardiomyocytes. Use of pluripotent stem cells always raises safety issues in terms of teratoma formation. Especially the application of iPSC additionally carries the risk for chromosome instability and
induction of mutations leading to malignancy. Therefore, strict testing of the cell products is essential prior to clinical application. Antibody-related selection protocols can probably help to overcome this limitation (48). Furthermore, in terms of cardiac repair, graft size and thickness are very important issues to support the failing hearts and to improve cardiac function. Actually, the biological diffusion capacity limits the graft thickness (50-200 µm), and reliable concepts for in-vitro as well as in-vivo vascularization and perfusion are necessary to enlarge the thickness of the three-dimensional grafts. For clinical use, up scaling of graft size is a major limitation, which has not been solved to date. Different experimental approaches for the generation of pre-vascularized grafts have been described (39), however the ideal method has yet to be defined. In terms of immunological issues, the use of hiPSC derived cardiomyocytes at least theoretically allows for an autologous approach without need for immunosuppression. However, this autologous solution is time-consuming and would at least need 6 to 9 months for generation of a patient-specific EHT patch. Besides the time concerns, logistic and economic hurdles would have to be handled, as each iPSC cell line is from a regulatory board view, a new product and needs to undergo extensive toxicity testing prior to clinical application. A more realistic scenario might be a bank of different iPSC or ESC lines, which can be transplanted human leucocyte antigen (HLA) matched, requiring only minimal immunosuppressive regimen.
Conflict of Interest: There is no conflict of interest. No funding was received.
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