• cardiac tissue engineering;
  • cell sheet;
  • vascularization;
  • pluripotent stem cells;
  • bioreactor


  1. Top of page
  3. Cell sheet transplantation for heart diseases
  4. Our Future challenges
  5. Acknowledgments
  6. Literature Cited

Tissue engineering is indispensable for the advancement of regenerative medicine and the development of tissue models. Cell sheet-based method is one the promising strategies for cardiac tissue engineering. To date, cell sheet transplantation using wide variety of cells has been performed for the treatment of various heart diseases. These cell sheet transplantations have shown to ameliorate cardiac dysfunction and improve symptoms of heart failure. Recent progress of the technologies on the layering of cardiac cell sheets accompanied with vascularization and the large scale cultivation system of embryonic stem cell and induced pluripotent stem cell is about to turn the fabrication of thickened human cardiac tissue for transplant and tissue models into reality. Anat Rec, 297:65–72. 2014. © 2013 Wiley Periodicals, Inc.

Despite recent progress in diagnostic methods, drugs, devices, and surgical therapies, heart diseases are major cause of death in the developed world, and the huge and rapidly expanding costs of medical treatments are a major concern. Recently cell-based therapy in accompanied with the progress in stem cell biology has been rapidly applied for diseases of various organs and these regenerative medicine-based therapies are also widely believed to be the novel therapeutic strategy to severe heart diseases. In many cases in cardiac cell therapy for acute myocardial infarction, cells were transplanted to heart through coronary artery using catheters (Menasche, 2011). One of the reasons for their wildly usage for cardiac cell therapy is that these are relatively easy transplantation approaches to apply since the specific techniques for the manipulation of tissues are not necessary. However, it cannot be denied that these strategies have some limitations in terms of a difficulty to regulate the location of transplanted cells and the poor engraftment, which might lead to the marginal outcomes.

One way of overcoming these limitations is to develop bioengineered cardiac tissue grafts using biodegradable scaffolds (Freed et al., 1994). Although some studies reported the bioengineered urinary duct and bladder using the biodegradable scaffolds (Atala et al., 2006; Raya-Rivera et al., 2011), there are few reports describing the usability of tissue-engineered biodegradable scaffolds in the clinical setting so far. There are some limitations on the usage of biodegradable scaffolds such as (1) insufficient control of vascular network formation for the efficient oxygen and nutrient supply and the waste excretion and (2) transplanted cells injury due to significant immunoreactions following the polymer degradation in the scaffold (Yang et al., 2007).

Cell sheet technology using a temperature-responsive culture surface is our original tissue engineering approaches (Fig. 1): “Poly(N-isopropylacrylamide, PIPAAm), a temperature responsive polymer, its copolymer show the hydrophobic state at 37°C and reversibly change to the hydrophilic state bellow 32°C. The temperature responsive culture dishes are covalently grafted with PIPAAm. The surface of these dishes is hydrophobic and cells adhere and proliferate at 37°C. However, by lowering temperature below at 32°C, the surface reversibly change to the hydrophilic state and cells cannot adhere to the surface due to the rapid hydration and the swelling of the grafted PIPAAm, which enables collection of a viable monolayer cell sheet with full preservation of the cell-cell contacts and extracellular matrices. This strategy can therefore be utilized to yield a noninvasive harvest of cultured cells as an intact layer cell sheet containing deposited extracellular matrices that can be collected in a non-enzymatic process by simply reducing the culture temperature to below 32°C for <1 hr” (Matsuura et al., 2013). The enzymatic dissociation is commonly used for collecting cells from culture dishes. However, since these treatments disrupt cell adhesion proteins and extracellular matrices, the reduced cell viability and anoikis are also commonly observed. Conversely, temperature responsive culture surface is capable of harvesting cells noninvasively as intact layer cell sheets containing fully preserved extracellular matrices (Kushida et al., 1999). Since cell-sheets based bioengineered tissues do not contain biodegradable materials and avoid any limitation associated with scaffold degradation, these can be applied for the transplantation. To date, some clinical studies and trials have already started for various types of organ injuries, including cornea diseases based on cornea stem cell deficiency (Nishida et al., 2004), heart failure (Sawa et al., 2012), esophagus after endoscopic cancer resection (Ohki et al., 2012), periodontal diseases (Washio et al., 2010), and osteoarthritis of the knee (Ito et al., 2012).


Figure 1. The schematic illustration of cell sheet-based tissue engineering.

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Cell sheet transplantation for heart diseases

  1. Top of page
  3. Cell sheet transplantation for heart diseases
  4. Our Future challenges
  5. Acknowledgments
  6. Literature Cited

A wide variety of cell sources have been transplanted to many heart diseases through intracoronary infusion and intramyocardium injection approaches. However, the transplanted cells tend to disappear soon after transplantation when using these delivery methods. When cardiomyocytes were transplanted via direct cell injection, very small number of cells were engrafted due to very small amounts of remained extracellular matrices, insufficient primary oxygen and nutrient supply (Zhang et al., 2001). In contrast, more transplanted cells were seen to persist when using cell sheet transplantation compared to intra-myocardium transplantation (Matsuura et al., 2009; Sekine et al., 2011). Integrin-mediated signaling pathway is well known to regulate cell survival. After the cells were transplanted as cell sheets, a lot of β1 integrin positive cells were observed, which led to less cardiomyocyte apoptosis, and in contrast with direct intra myocardium injection (Sekine et al., 2011). Collectively, cell sheet-based delivery method is more suitable for engraftment and prolonged effects.

Various types of cells have been transplanted as cell sheets into damaged hearts and skeletal myoblast cell sheet transplantation is the most established strategy so far. “On the basis of findings that skeletal myoblasts can be prepared from host tissues for autologous transplantation, they are ischemia-resistant, and are able to differentiate into cells of nonmyocytes lineage, the efficacy of the transplantation were widely evident in the various types of damaged heart including a rat model of myocardial infarction (Memon et al., 2005; Sekiya et al., 2009; Miyagawa et al., 2010), a hamster model of dilated cardiomyopathy (Kondoh et al., 2006), and a canine model of dilated cardiomyopathy (Hata et al., 2006), suggest that skeletal myoblasts are considered to have a therapeutic potential.” (Matsuura et al., 2013). Consistent with the animal experiment data described above, autologous myoblast cell sheet transplantation significantly improved the cardiac function of the patient who suffered from severe heart failure due to dilated cardiomyopathy and “his clinical condition improved markedly, leaving him without any arrhythmia and able to discontinue using a left ventricular assist system and avoid cardiac transplantation” (Sawa et al., 2012). These findings suggest that skeletal myoblasts with the appropriate delivery methods such as cell sheet transplantation might greatly contribute to the cardiac function and QOL of heart failure patients.

Aside from skeletal myoblasts, cell sheets using “mesenchymal stem cells from adipose tissue (Miyahara et al., 2006) and menstrual blood (Hida et al., 2008), adipocytes (Imanishi et al., 2011), cardiomyocytes from neonatal rats (Miyagawa et al., 2005), cardiomyocytes co-cultured with endothelial cells (Sekine et al., 2008), dermal fibroblasts co-cultured with endothelial progenitor cells (Kobayashi et al., 2008), Sca-1(+) cardiac progenitor cells from adult murine hearts (Matsuura et al., 2009) and ES/iPS-derived cardiomyocytes have been applied to cell sheet-based transplantation to hearts damaged by myocardial infarction and cardiomyopathy in the animal experiments.” (Matsuura, J Control Release, 2013). Although some studies have reported different degrees of cardiomyogenesis, paracrine effects such as angiogenesis and cardioprotection mediated by transplanted cells-secreted cytokines and growth factors are the main mechanism for the improvement of cardiac function. Skeletal myoblasts induced angiogenesis and attenuated the excess fibrous tissue formation after myocardial infarction mainly through the production of VEGF and HGF. Adiponectin-mediated attenuation of inflammatory responses after myocardial infarction might be the main mechanisms on the improvement of cardiac function following adipocyte sheet transplantation (Imanishi et al., 2011). VCAM-1 secreted from cardiac progenitor cells greatly contributed to endothelial cell migration, prevent cardiomyocytes damage from oxidative stress and promote the engraftment of cells after transplantation through α4β1 integrin in the heart (Matsuura et al., 2009).

Since embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) have the unlimited growth ability and the cardiomyocyte differentiation potency, the usage of ESC and iPSC is the only way to collect enough number and quality of cardiomyocytes for regenerative medicine and disease models so far. Therefore when cardiomyocytes from these stem cells were transplanted to injured hearts, transplanted cardiomyocytes were expected to engraft and function as the direct contractile elements with host myocardium in a coordinate manner. When ESC-derived cardiomyocytes were transplanted to infarcted hearts, these cardiomyocytes have been reported to show the contraction accompanied with the contraction of host myocardium in intact and injured areas (Shiba et al., 2012), suggesting that ESC-derived cardiomyocytes might have the enough properties to function as contractile elements after transplantation. Cardiomyocytes are known to not only function as contractile elements, but also secrete angiogenic growth factors such as VEGF (Levy et al., 1995), in particular hypoxic condition. Since it is well known that transplanted cells are exposed to hypoxic condition immediately following transplantation, angiogenic potential of cells in autocrine/paracrine fashion might be important to promote their engraftment and subsequent function. Recent reports have suggested that cardiac cell sheets derived from ESC-derived cells including cardiomyocytes, endothelial cells and mural cells (Masumoto et al., 2012), and iPSC-derived cardiomyocytes (Kawamura et al., 2012) promoted angiogenesis in the infarcted myocardium, which led to the improvement of cardiac function. It should be noted that cardiomyocytes were the main source for several angiogenic growth factors expression in cell sheets and the co-existence of endothelial cells enhanced these expression in cardiomyocytes (Masumoto et al., 2012). We previously reported that prevascularization of cardiac cell sheets composed of cardiomyocytes, endothelial cells, and fibroblasts from neonatal rat hearts might be critical for cardiomyocyte engraftment and transplantation-mediated improvement of cardiac function of infarcted hearts (Sekine et al., 2008). Therefore transplantation of cardiomyocytes as cardiac tissue, but not suspension might be appropriate for angiogenesis-mediated cardiomyocytes engraftment in the myocardium.

Myocardial Tissue Engineering by Cell Sheet Layering

When cell sheets are detached from the cell culture surface, extracellular matrices and adhesion proteins are fully preserved, which enable to prompt establishment of tissue by layering cell sheets. In accompanied with the evidences on the cell sheet properties such as the lack of an inflammatory response otherwise associated with the degradation of biopolymers, the cell sheet technology is particularly well-suited for generating dense, thicker tissues, such as those required for myocardial tissue engineering.

Monolayered cardiac cell sheet derived from neonatal rat hearts showed the spontaneous beating (Shimizu et al., 2001), and two cell sheets showed the synchronous beating after the layering (Shimizu et al., 2002). The gap junction proteins such as connexin 43 were also preserved even after cell detaching from the cell culture surface, the electrical communication between cell sheets was established within 1 hr (Haraguchi et al., 2006). These findings indicate that layering monolayered cardiac cell sheet might enable to fabricate the three-dimensional tissue with the electrically communicative properties. When layered cardiac cell sheets were transplanted onto the dorsal subcutaneous tissues of nude rats, the transplanted tissue was microvascularized in a couple of days, and the spontaneous beating of the grafts observed even after 1 yr (Shimizu et al., 2006). Collectively, cardiac grafts that were fabricated in vitro were engrafted in vivo and the appropriate vascularization enabled to maintain their function for a long time.

Although cell sheet layering is the useful method for fabricating three-dimensional tissues, the layering procedure might be troublesome in some cases. For example, the little bit longer culture period in the lower temperature for cell detaching occasionally makes cell sheets shrinkage. We have recently developed the method, cell sheet stamp method, to stack cell sheets for fabricating of functional three-dimensional tissues in vitro using the hydrogel-coated plunger (Sasagawa et al., 2010; Haraguchi et al., 2012). The repeated layering of cells sheet using this stamp method are capable of fabricating three-dimensional tissue easily and these technologies are thought to be useful for developing the automated three-dimensional tissue fabricating machines.

Vascularization of 3D-Layered Cardiac Graft

Cell sheet layering methods enable to fabricate three-dimensional tissue. However, Vascularization is thought to be the indispensable technology for fabricating more thickened tissue. According to our previous study (Shimizu et al., 2006), 80 μm (triple layers) is the thickness limitation in a single transplantation. However, since the transplanted layered cardiac grafts on the subcutaneous tissue were sufficiently vascularized, the graft might function as the vascular bed for the second grafts. On the basis of these hypothesis, we previously reported that triple layered cardiac tissue transplanted onto the subcutaneous cardiac tissue 24 hr after the first transplantation were fully vascularized, and 10-times transplantation of triple layered cardiac tissue every 24 hr enabled to fabricate cardiac tissue whose thickness was around 1 mm accompanied with microvasculature (30 layered) in the subcutaneous tissue (Shimizu et al., 2006). Furthermore when these fabricated grafts were ectopic transplanted to the neck with blood vessel anastomosis, the blood flow within the grafts were observed in a few second and subsequently the grafts showed the spontaneous pulsation.

It is quite important the communication of endothelial cells between the grafts and the vascular bed for fabricating three-dimensional tissue. When cardiac cell sheets composed of cardiomyocytes, fibroblast, and endothelial cells from neonatal rat hearts were transplanted to the subcutaneous tissue, the mature vessels made up of endothelial cells in cell sheets and the host tissue-derived cells were observed (Sekiya et al., 2006). Furthermore, endothelial cells in cardiac cell sheets greatly contributed to robust microvascular network formation within the graft area when in transplantation to infarcted hearts, which led the better graft engraftment (Sekine et al., 2008). Conversely, transplantation of cardiac cell sheets without endothelial cells showed the marginal microvascular network and improvement of cardiac function.

Recently, we have succeeded to fabricate the three-dimensional cardiac tissue using two types of vascular bed. One is the ex vivo vascular bed using the isolated tissue with a connectable artery and vein (Sekine et al., 2013), another is the in vitro vascular bed using collagen-based microchannels (Sakaguchi et al., 2013). In this concept, repeated layering of cardiac cell sheets at the adequate time interval might enable to vascularize cell sheets enough for fabricating thickened tissue. Triple-layered cardiac cell sheets containing endothelial cells were overlaid on the isolated femoral muscle as vascular beds, and the layered cardiac cell sheets were perfused with the culture medium from the vascular beds using a bioreactor system. “Endothelial cells in cell sheets connected to capillaries in the vascular bed and formed tubular lumen, creating in vitro perfusable blood vessels in the cardiac cell sheets” (Sekine et al., 2013). Since it takes 3 days to create perfusable blood vessels in cardiac cell sheets in this condition, 12-layered cardiac tissues were fabricated in 12 days. Another approach is the layering of cell sheets on the collagen-based microchannel vascular bed in accompanied with the medium perfusion (Sakaguchi et al., 2013). “Endothelial cells in cardiac cell sheets migrated to vascularize in the collagen gel and connected with the microchannels, and finally medium flowed into the cell sheets through the microchannels” (Sakaguchi et al., 2013). Since it takes 5 days to create perfusable blood vessel in the cardiac cell sheets in this condition, 12-layered cardiac tissue was fabricated in 20 days.

Cardiac Tissue Engineering Using ESC/iPSC-Derived Cells

The cell sources for cardiac tissue engineering remained the problems. Although many reports have suggested the cardiomyocyte differentiation potency of somatic stem cells, their potential seems to be below the needs for clinical application in terms of the number and function of cardiomyocytes. Pluripotent stem cells including ESC and iPSC have the big advantage in terms of their indefinite proliferative and high efficient cardiomyocyte differentiation potential. Furthermore, iPSC technologies have opened the possibilities for fabricating the autologous cardiac tissue for regenerative medicine and drug screening (Yoshida and Yamanaka, 2011).

Recently, we have succeeded to fabricate cardiac cell sheet using ESC-derived cardiomyocytes (Matsuura et al., 2011). Mouse ESC proliferated and differentiated with the embryoid body (EB) formation in a stirred suspension culture system. By using mouse ESC expressing cardiomyocytes specific drug resistant gene, cardiomyocytes were purified after the drug treatment. Consistent with evidence that vertebrate heart tissue is mainly composed of fibroblast and cardiomyocytes, cell sheets were fabricated when purified cardiomyocytes were cocultured with fibroblasts, but without coculture.

Not only cardiomyocyte number, but also their viability after the cardiac differentiation of pluripotent stem cells are critical aspects for fabricating functional thickened cardiac tissue, as cardiomyocytes in cell sheets are thought to function as the contractile elements and angiogenic growth factor secretory elements after transplantation. The culture environment is indispensable for collecting enough quantities of viable cardiomyocytes (Matsuura et al., 2012a). When mouse ESC were densely cultivated in a perfusion bioreactor system that was capable of maintaining the culture conditions such as pH and dissolved oxygen concentration in a normal range, cardiomyocyte yield was increased. Furthermore, a lot of cardiomyocytes were survived in cell sheets after the differentiation in this type of culture environment.

Since it is well known that human pluripotent stem cells are sensitive to some stresses such as shear stress compared with mouse pluripotent stem cells, the development of large scale cultivation system for human pluripotent stem cells remains a big challenge. Recently, we have developed the three-dimensional suspension culture bioreactor system with the low-shear stress agitation impeller and an optical dissolved oxygen sensor, and succeeded to induce cardiovascular differentiation of human iPSC with high efficacy (Matsuura et al., 2012b; Fig. 2). In the bioreactor system, robust human iPSC-derived EB were treated with BMP4, ActivinA, bFGF, VEGF, and a Wnt inhibitor in the hypoxic condition. The expression of NKX2.5, HCN4, TNNT2, and MYL7 was upregulated from day 8 and that of MYL2 was upregulated from day 11 (Fig. 2A). At 12 days after cardiac induction using 100 mL bioreactor, over 50 million of cardiomyocytes were collected in a single run. After the enzymatic dissociation of these differentiated cells and cultured for 5 days, cell sheets were fabricated. As shown in Fig. 2B, large majority of cells in cell sheets were cardiomyocytes and small number of fibroblasts surrounded cardiomyocytes. Since connexin 43 was abundantly expressed in cardiomyocytes (Fig. 2C), the electrical excitation was smoothly propagated to the whole area and layering cell sheets. Herein, the development of large cultivation system for human iPSC-derived cardiomyocytes production might contribute to the human cardiac tissue engineering.


Figure 2. Human iPS cell-derived cardiac cell sheets. A: The relative mRNA expression of cardiac genes during differentiation in the bioreactor-based culture (undifferentiated hiPSC = 1.0, n = 2 − 3). The relative mRNA expression level was calculated using a standard curve of β-actin mRNA levels. The primer sequences are available upon request. B: High content image analyses of cell components in cell sheets. Cells were stained with the antibodies for cardiac troponin T (TnT, green), SM22 (red), and Nkx2.5 (light blue). Nuclei were stained with Hoechst. Bars, 200 µm. The percentage of cells expressing each protein was calculated and shown the graph. C: The gap junction protein expression between cardiomyocytes in cell sheets. Cells were stained with the antibodies for TnT (green) and connexin 43 (red). Nuclei were stained with Hoechst. Bars, 200 µm. Data were presented as the means ± standard deviation.

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Our Future challenges

  1. Top of page
  3. Cell sheet transplantation for heart diseases
  4. Our Future challenges
  5. Acknowledgments
  6. Literature Cited

Our future concept of cardiac tissue engineering using cell sheet technology is shown in Fig. 3. One direction is regenerative medicine. The three-dimensional suspension culture system has enabled us to collect enough number of viable cardiomyocytes, which should accelerate the development of technologies for cell sheet-based transplantation to severe heart failure. Furthermore, the development of technologies for fabricating perfusable three-dimensional tissue will enable us to use the regenerated tissue fabricated in vitro for the replacement therapy to the severe organ injury.


Figure 3. The schematic illustration of the future goal of the cell sheet-based cardiac tissue engineering.

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Another direction is tissue models for the drug screening and the research on the underlying mechanisms of the diseases. The studies of iPSC-derived cardiomyocytes from subjects with genetic disorders have been strenuously performed all over the world for elucidating the underlying molecular mechanisms of disease onset and the subsequent drug screening (Carvajal-Vergara et al., 2010; Moretti et al., 2010; Itzhaki et al., 2011; Sun et al., 2012; Kim et al., 2013). Although evaluation at single cell level might be enough for genetic disorder-related ion channel diseases, such as long QT syndrome, they may not be useful for cardiomyopathy, as the progression of cardiomyopathy through cardiomyocyte hypertrophy, apoptosis and fibrosis is thought to be the result of the mutual interaction between cardiomyocytes and surrounding cells, such as fibroblasts (Tian and Morrisey, 2012). Furthermore, cardiac dysfunction in many patients with cardiomyopathy is not always clinically evident soon after birth, and thus continuous volume and hemodynamic stress might also promote disease progression. In that respect, the development of the pulsatile human cardiac tube will be useful for tissue models that can provide us with new insight into understanding the underlying mechanisms of various types of cardiomyopathies.


  1. Top of page
  3. Cell sheet transplantation for heart diseases
  4. Our Future challenges
  5. Acknowledgments
  6. Literature Cited

This work was funded by a grant from the Japan Society for the Promotion of Science (JSPS) through the “Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program),” initiated by the Council for Science and Technology Policy (CSTP) and also supported by an open research grant from The Japan Research Promotion Society for Cardiovascular Disease.

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  1. Top of page
  3. Cell sheet transplantation for heart diseases
  4. Our Future challenges
  5. Acknowledgments
  6. Literature Cited
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