Cell delivery in regenerative medicine: the cell sheet engineering approach.

Recently, cell-based therapies have developed as a foundation for regenerative medicine. General approaches for cell delivery have thus far involved the use of direct injection of single cell suspensions into the target tissues. Additionally, tissue engineering with the general paradigm of seeding cells into biodegradable scaffolds has also evolved as a method for the reconstruction of various tissues and organs. With success in clinical trials, regenerative therapies using these approaches have therefore garnered significant interest and attention. As a novel alternative, we have developed cell sheet engineering using temperature-responsive culture dishes, which allows for the non-invasive harvest of cultured cells as intact sheets along with their deposited extracellular matrix. Using this approach, cell sheets can be directly transplanted to host tissues without the use of scaffolding or carrier materials, or used to create in vitro tissue constructs via the layering of individual cell sheets. In addition to simple transplantation, cell sheet engineered constructs have also been applied for alternative therapies such as endoscopic transplantation, combinatorial tissue reconstruction, and polysurgery to overcome limitations of regenerative therapies and cell delivery using conventional approaches.     

Myoblast cell sheet transplantation enhances the endogenous regenerative abilities of infant hearts in rats with myocardial infarction

The shortage of heart transplantation donors for infants is severe. Regenerative cell therapy has been expected to offer new methods of treatment, and this study was about regenerative cell therapy for infant hearts. The aims of the present study were to clarify the effects of regenerative cell therapy on the infant heart. The heart impairment model and tissue‐engineered myoblast cell sheets were used for regenerative cell therapy. Infant rats (n = 54) aged 2 weeks and adult rats aged 12 weeks (n = 35) were used. Myocardial infarction (MI) was induced as the heart impairment model and triple‐layer myoblast cell sheets were used for transplantation to MI lesions. Infant rats after MI had better self‐regenerative ability in wall thickness, fibrosis and cardiac function and we observed greater numbers of proliferating cardiomyocytes than in adults. Moreover, infant MI rats treated with myoblast cell sheets showed better results in wall thickness, fibrosis and cardiac function than infant MI rats without myoblast cell sheets, because of the positive effect that myoblast cell sheets had on proliferating cardiomyocytes, increasing vascular networks and accumulating c‐kit‐positive cells. We clarified that regenerative cell therapy enhances the endogenous regenerative ability of infant hearts in rats with MI; moreover, it has a greater therapeutic effect on infant hearts than on adult hearts, because of the ability of infant hearts for cardiomyocyte proliferation. The present paper provides essential new data for clinical therapy in infant patients. Copyright © 2015 John Wiley & Sons, Ltd.     

Scaffold‐free tissue engineering of functional corneal stromal tissue

Blinding corneal scarring is predominately treated with allogeneic graft tissue; however, there is a worldwide shortage of donor tissue leaving millions in need of therapy. Human corneal stromal stem cells (CSSC) have been shown produce corneal tissue when cultured on nanofibre scaffolding, but this tissue cannot be readily separated from the scaffold. In this study, scaffold‐free tissue engineering methods were used to generate biomimetic corneal stromal tissue constructs that can be transplanted in vivo without introducing the additional variables associated with exogenous scaffolding. CSSC were cultured on substrates with aligned microgrooves, which directed parallel cell alignment and matrix organization, similar to the organization of native corneal stromal lamella. CSSC produced sufficient matrix to allow manual separation of a tissue sheet from the grooved substrate. These constructs were cellular and collagenous tissue sheets, approximately 4 μm thick and contained extracellular matrix molecules typical of corneal tissue including collagen types I and V and keratocan. Similar to the native corneal stroma, the engineered corneal tissues contained long parallel collagen fibrils with uniform diameter. After being transplanted into mouse corneal stromal pockets, the engineered corneal stromal tissues became transparent, and the human CSSCs continued to express human corneal stromal matrix molecules. Both in vitro and in vivo, these scaffold‐free engineered constructs emulated stromal lamellae of native corneal stromal tissues. Scaffold‐free engineered corneal stromal constructs represent a novel, potentially autologous, cell‐generated, biomaterial with the potential for treating corneal blindness. Copyright © 2016 John Wiley & Sons, Ltd.     

Stem cell‐based therapy in gastroenterology and hepatology

Protagonists of a new scientific era, stem cells are promising tools on which regenerative medicine relies for the treatment of human pathologies. Stem cells can be obtained from various sources, including embryos, fetal tissues, umbilical cord blood, and also terminally differentiated organs. Once forced to expand and differentiate into functional progenies, stem cells may become suitable for cell replacement and tissue engineering. The manipulation and/or stimulation of adult stem cells seems to be particularly promising, as it could improve the endogenous regenerative potential without risks of rejection and overcome the ethical and political issues related to embryonic stem cell research. Stem cells are already leaving the bench and reaching the bedside, despite an incomplete knowledge of the genetic control program driving their fate and plasticity. In gastroenterology and hepatology, the first attempts to translate stem cell basic research into novel therapeutic strategies have been made for the treatment of several disorders, such as inflammatory bowel diseases, diabetes mellitus, celiachy and acute or chronic hepatopaties. Nonetheless, critical aspects need to be further addressed, including the long‐term safety, tolerability and efficacy of cell‐based treatments, as well as their carcinogenic potential. Aim of this review is to summarize the state‐of‐the‐arts on gastrointestinal and hepatic stem cells and on stem cell‐based therapies in gastroenterology and hepatology, highlighting both the benefits and the potential risks of these new tools for the treatment and prevention of human diseases.     

Fibrin,the preferred scaffold for cell transplantation after myocardial infarction? An old molecule with a new life

Fibrin is a topical haemostat, sealant and tissue glue, which consists of concentrated fibrinogen and thrombin. It has broad medical and research uses. Recently, several studies have shown that engineered patches comprising mixtures of biological or synthetic materials and progenitor cells showed therapeutic promise for regenerating damaged tissues. In that context, fibrin maintains cell adherence at the site of injury, where cells are required for tissue repair, and offers a nurturing environment that protects implanted cells without interfering with their expected benefit. Here we review the past, present and future uses of fibrin, with a focus on its use as a scaffold material for cardiac repair. Fibrin patches filled with regenerative cells can be placed over the scarring myocardium; this methodology avoids many of the drawbacks of conventional cell‐infusion systems. Advantages of using fibrin also include extraction from the patient's blood, an easy readjustment and implantation procedure, increase in viability and early proliferation of delivered cells, and benefits even with the patch alone. In line with this, we discuss the numerous preclinical studies that have used fibrin–cell patches, the practical issues inherent in their generation, and the necessary process of scaling‐up from animal models to patients. In the light of the data presented, fibrin stands out as a valuable biomaterial for delivering cells to damaged tissue and for promoting beneficial effects. However, before the fibrin scaffold can be translated from bench to bedside, many issues must be explored further, including suboptimal survival and limited migration of the implanted cells to underlying ischaemic myocardium. Copyright © 2016 John Wiley & Sons, Ltd.     

Middle ear mucosal regeneration with three‐dimensionally tissue‐engineered autologous middle ear cell sheets in rabbit model

The likelihood of recurrent retraction and adhesion of newly formed tympanic membrane is high when middle ear mucosa is extensively lost during cholesteatoma and adhesive otitis media surgery. If rapid postoperative regeneration of the mucosa on the exposed bone surface can be achieved, prevention of recurrent eardrum adhesion and cholesteatoma formation, for which there has been no definitive treatment, can be expected. Suture‐less transplantation of tissue‐engineered mucosal cell sheets was examined immediately after the operation of otitis media surgery in order to quickly regenerate middle ear mucosa lost during surgery in a rabbit model. Transplantable middle ear mucosal cell sheets with a three‐dimensional tissue architecture very similar to native middle ear mucosa were fabricated from middle ear mucosal tissue fragments obtained in an autologous manner from middle ear bulla on temperature‐responsive culture surfaces. Immediately after the mucosa was resected from middle ear bone bulla inner cavity, mucosal cell sheets were grafted at the resected site. Both bone hyperplasia and granulation tissue formation were inhibited and early mucosal regeneration was observed in the cell sheet‐grafted group, compared with the control group in which only mucosal removal was carried out and the bone surface exposed. This result indicates that tissue engineered mucosal cell sheets would be useful to minimize complications after the surgical operation on otitis media and future clinical application is expected. Copyright © 2013 John Wiley & Sons, Ltd.     

Development of a protocol for maintaining viability while shipping organoid‐derived retinal tissue

Retinal organoid technology enables generation of an inexhaustible supply of three‐dimensional retinal tissue from human pluripotent stem cells (hPSCs) for regenerative medicine applications. The high similarity of organoid‐derived retinal tissue and transplantable human fetal retina provides an opportunity for evaluating and modeling retinal tissue replacement strategies in relevant animal models in the effort to develop a functional retinal patch to restore vision in patients with profound blindness caused by retinal degeneration. Because of the complexity of this very promising approach requiring specialized stem cell and grafting techniques, the tasks of retinal tissue derivation and transplantation are frequently split between geographically distant teams. Delivery of delicate and perishable neural tissue such as retina to the surgical sites requires a reliable shipping protocol and also controlled temperature conditions with damage‐reporting mechanisms in place to prevent transplantation of tissue damaged in transit into expensive animal models. We have developed a robust overnight tissue shipping protocol providing reliable temperature control, live monitoring of the shipment conditions and physical location of the package, and damage reporting at the time of delivery. This allows for shipping of viable (transplantation‐competent) hPSC‐derived retinal tissue over large distances, thus enabling stem cell and surgical teams from different parts of the country to work together and maximize successful engraftment of organoid‐derived retinal tissue. Although this protocol was developed for preclinical in vivo studies in animal models, it is potentially translatable for clinical transplantation in the future and will contribute to developing clinical protocols for restoring vision in patients with retinal degeneration.     

Three‐dimensional functional human myocardial tissues fabricated from induced pluripotent stem cells

The most radical treatment currently available for severe heart failure is heart transplantation; however, the number of donor hearts is limited. A better approach is to make human cardiac tissues. We developed an original cell sheet‐based tissue‐engineering technology to fabricate human cardiac tissue by layering myocardial cell sheets. Human induced pluripotent stem (iPS) cells were differentiated into cardiomyocytes to fabricate cardiomyocyte sheets. Initially, three‐layer human iPS cardiomyocyte (hiPSCM) sheets were transplanted on subcutaneous tissues of nude rats. Next, to fabricate thicker tissue, three‐layer sheets were transplanted on one day, then additional three‐layer sheets were transplanted onto them the following day, after the first sheets were vascularized. On day 3, the final three‐layer sheets were again transplanted, creating a nine‐layer graft (multi‐step transplantation procedure). In the last step, six‐layer sheets were transplanted on fat tissues of the inguinal portion, which were subsequently resected together with the femoral arteries and veins to make transplantable grafts with connectable vessels. They were then transplanted ectopically to the neck portion of other rats by anastomosing vessels with the host's jugular arteries and veins. Transplanted three‐layer hiPSCMs were beating and, histologically, showed a cardiac muscle‐like structure with vascular systems. Moreover, transplanted hiPSCMs proliferated and matured in vivo. Significantly thicker tissues were fabricated by a multi‐step transplantation procedure. The ectopically transplanted graft survived and continued to beat. We succeeded in fabricating functional human cardiac tissue with cell sheet technology. Transplanting this cardiac tissue may become a new treatment option for severe heart failure. Copyright © 2015 John Wiley & Sons, Ltd.     

Combination of platelet‐rich plasma within periodontal ligament stem cell sheets enhances cell differentiation and matrix production

The longstanding goal of periodontal therapy is to regenerate periodontal tissues. Although platelet‐rich plasma (PRP) has been gaining increasing popularity for use in the orofacial region, whether PRP is useful for periodontal regeneration is still unknown. The purpose of this study was to determine whether a mixture of periodontal ligament stem cell (PDLSC) sheets and PRP promoted bone regeneration, one of the most important measurement indices of periodontal tissue regenerative capability in vitro and in vivo. In this study, we evaluated the effects of different doses of PRP on the differentiation of human PDLSCs. Then cell sheet formation, extracellular matrix deposition and osteogenic gene expression in response to different doses of PRP treatment during sheet grafting was investigated. Furthermore, we implanted PDLSC sheets treated with 1% PRP subcutaneously into immunocompromised mice to evaluate their bone‐regenerative capability. The results revealed that 1% PRP significantly enhanced the osteogenic differentiation of PDLSCs. Based on the production of extracellular matrix proteins, the results of scanning electron microscopy and the expression of the osteogenic genes ALP, Runx2, Col‐1 and OCN, the provision of 1% PRP for PDLSC sheets was the most effective PRP administration mode for cell sheet formation. The results of in vivo transplantation showed that 1% PRP‐mediated PDLSC sheets exhibited better periodontal tissue regenerative capability than those obtained without PRP intervention. These data suggest that a suitable concentration of PRP stimulation may enhance extracellular matrix production and positively affect cell behaviour in PDLSC sheets. Copyright © 2014 John Wiley & Sons, Ltd.     

Autologous human nasal epithelial cell sheet using temperature‐responsive culture insert for transplantation after middle ear surgery

Postoperative mucosal regeneration of the middle ear cavity and the mastoid cavity is of great importance after middle ear surgery. However, the epithelialization of the mucosa in the middle ear is retarded because chronic inflammation without epithelialization aggravates gas exchange and clinical function. These environmental conditions in the middle ear lead to postoperative retraction and adhesion of the newly‐formed tympanic membrane. Therefore, if the mucosa on the exposed middle ear bone surface can be rapidly regenerated after surgery, the surgical treatments for cholesteatoma and adhesive middle ear disease can potentially be improved. In this study, we successfully generated a cell sheet designed for the postoperative treatment of cholesteatoma. We used nasal cells to create an artificial middle ear mucosal cell sheet with a three‐dimensional (3D) configuration similar to that of the middle ear mucosa. The sheets consisted of multi‐layered mucosal epithelia and lower connective tissue and were similar to normal middle ear mucosa. This result indicates that tissue‐engineered mucosal cell sheets would be useful to minimize complications after surgical operations in the middle ear and future clinical applications are expected. Copyright © 2015 John Wiley & Sons, Ltd.     

Functional life‐long maintenance of engineered liver tissue in mice following transplantation under the kidney capsule

The ability to engineer biologically active cells and tissue matrices with long‐term functional maintenance has been a principal focus for investigators in the field of hepatocyte transplantation and liver tissue engineering. The present study was designed to determine the efficacy and temporal persistence of functional engineered liver tissue following transplantation under the kidney capsule of a normal mouse. Hepatocytes were isolated from human α‐1 antitrypsin (hA1AT) transgenic mouse livers. Hepatocytes were subsequently transplanted under the kidney capsule space in combination with extracellular matrix components (Matrigel) for engineering liver tissues. The primary outcome of interest was to assess the level of engineering liver tissue function over the experimental period, which was 450 days. Long‐term survival by the engineered liver tissue was confirmed by measuring the serum level of hA1AT in the recipient mice throughout the experimental period. In addition, administration of chemical compounds at day 450 resulted in the ability of the engineered liver tissue to metabolize exogenously circulating compounds and induce drug‐metabolizing enzyme production. Moreover, we were able to document that the engineered tissues could retain their native regenerative potential similar to that of naïve livers. Overall, these results demonstrated that liver tissues could be engineered at a heterologous site while stably maintaining its functionality for nearly the life span of a normal mouse. Copyright © 2009 John Wiley & Sons, Ltd.     

Fabrication of tissue‐engineered cell sheets by automated cell culture equipment

Most cells for regenerative medicine are currently cultured manually. In order to promote the widespread use of regenerative medicine, it will be necessary to develop automated culture techniques so that cells can be produced in greater quantities at lower cost and with more stable quality. In the field of regenerative medicine technology, cell sheet therapy is an effective tissue engineering technique whereby cells can be grafted by attaching them to a target site. We have developed automated cell culture equipment to promote the use of this cell sheet regenerative treatment. This equipment features a fully closed culture vessel and circuit system that avoids contamination with bacteria and the like from the external environment, and it was designed to allow 10 cell sheets to be simultaneously cultured in parallel. We used this equipment to fabricate 50 sheets of human oral mucosal epithelial cells in five automated culture tests in this trial. By analyzing these sheets, we confirmed that 49 of the 50 sheets satisfied the quality standards of clinical research. To compare the characteristics of automatically fabricated cell sheets with those of manually fabricated cell sheets, we performed histological analyses using immunostaining and transmission electron microscopy. The results confirmed that cell sheets fabricated with the automated cell culture are differentiated in the same way as cultures fabricated manually.     

Carrier‐free epithelial cell sheets prepared by enzymatic degradation of collagen gel

Limbal stem‐cell deficiency by ocular trauma or disease causes corneal opacification and vision loss. Conventional tissue engineering using biodegradable scaffolds has met with limited success. In this study, we developed a novel method for preparing carrier‐free epithelial cell sheets, which have potential for use in repairing defects of the ocular surface. Stratified corneal epithelial cell sheets were prepared in culture dishes coated with biodegradable type I collagen. Haematoxylin and eosin staining, electron microscopy and immunohistochemistry were performed to characterize the cell sheets. Then, carrier‐free epithelial sheets were successfully engineered using specific collagenase to degrade the collagen gel. Cell sheets of four to six cell layers after culture for 14 days were similar to natural rabbit corneal epithelia, as shown by pathological examination. Microvillus, tight cell–cell junctions and desmosome junctions were observed via electron microscopy. K3 and basement membrane components, such as type IV collagen and laminin, were expressed in the cells sheets and integrin β1 was maintained in basal cells. This novel method of using collagenase to degrade collagen gel is both simple and effective in preparing intact carrier‐free epithelial cell sheets. Such sheets have great potential for application during in vivo corneal regeneration. Copyright © 2010 John Wiley & Sons, Ltd.     

Micropatterned three‐dimensional hydrogel system to study human endothelial–mesenchymal stem cell interactions

The creation of vascularized engineered tissues of clinically relevant size is a major challenge of tissue engineering. While it is known that endothelial and mural vascular cells are integral to the formation of stable blood vessels, the specific cell types and optimal conditions for engineered vascular networks are poorly understood. To this end, we investigated the vasculogenic potential of human mesenchymal stem cell (MSC) populations derived from three different sources: (a) bone marrow aspirates; (b) perivascular cells from the umbilical cord vein; and (c) perivascular cells from the umbilical cord artery. Cell populations were isolated and identified as MSCs according to their phenotypes and differentiation potential. Human umbilical vein endothelial cells (HUVECs) were used as a standard for endothelial cells. A novel co‐culture system was developed to study cell–cell interactions in a spatially controlled three‐dimensional (3D) fibrin hydrogel model. Using microfluidic patterning, it was possible to localize hydrogel‐encapsulated HUVECs and MSCs within separate channels spaced at 500, 1000 or 2000 µm. All three MSC populations had similar expression profiles of mesenchymal cell markers and similar capacity for osteogenic and adipogenic differentiation. However, bone marrow‐derived MSCs (but not umbilical vein or artery derived MSCs) showed strong distance‐dependent migration toward HUVECs and supported the formation of stable vascular networks resembling capillary‐like vasculature. The presented approach provides a simple and robust model to study the cell–cell communication of relevance to engineering vascularized tissues. Copyright © 2009 John Wiley & Sons, Ltd.     

Influence of culture conditions and extracellular matrix alignment on human mesenchymal stem cells invasion into decellularized engineered tissues

The variables that influence the in vitro recellularization potential of decellularized engineered tissues, such as cell culture conditions and scaffold alignment, have yet to be explored. The goal of this work was to explore the influence of insulin and ascorbic acid and extracellular matrix (ECM) alignment on the recellularization of decellularized engineered tissue by human mesenchymal stem cells (hMSCs). Aligned and non‐aligned tissues were created by specifying the geometry and associated mechanical constraints to fibroblast‐mediated fibrin gel contraction and remodelling using circular and C‐shaped moulds. Decellularized tissues (matrices) of the same alignment were created by decellularization with detergents. Ascorbic acid promoted the invasion of hMSCs into the matrices due to a stimulated increase in motility and proliferation. Invasion correlated with hyaluronic acid secretion, α‐smooth muscle actin expression and decreased matrix thickness. Furthermore, hMSCs invasion into aligned and non‐aligned matrices was not different, although there was a difference in cell orientation. Finally, we show that hMSCs on the matrix surface appear to differentiate toward a smooth muscle cell or myofibroblast phenotype with ascorbic acid treatment. These results inform the strategy of recellularizing decellularized engineered tissue with hMSCs. © 2015 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd.     

Transplantation of three‐dimensional artificial human vascular tissues fabricated using an extracellular matrix nanofilm‐based cell‐accumulation technique

We have established a novel three‐dimensional (3D) tissue‐constructing technique, referred to as the ‘cell‐accumulation method’, which is based on the self‐assembly of cultured human cells. In this technique, cells are coated with fibronectin and gelatin to construct extracellular matrix (ECM) nanofilms and cultured to form multi‐layers in vitro. By using this method, we have successfully fabricated artificial tissues with vascular networks constructed by co‐cultivation of human umbilical vein‐derived vascular endothelial cells between multi‐layers of normal human dermal fibroblasts. In this study, to assess these engineered vascular tissues as therapeutic implants, we transplanted the 3D human tissues with microvascular networks, fabricated based on the cell‐accumulation method, onto the back skin of nude mice. After the transplantation, we found vascular networks with perfusion of blood in the transplanted graft. At the boundary between host and implanted tissue, connectivity between murine and human vessels was found. Transmission electron microscopy of the implanted artificial vascular tubules demonstrated the ultrastructural features of blood capillaries. Moreover, maturation of the vascular tissues after transplantation was shown by the presence of pericyte‐like cells and abundant collagen fibrils in the ECM surrounding the vasculature. These results demonstrated that artificial human vascular tissues constructed by our method were engrafted and matured in animal skin. In addition, the implanted artificial human vascular networks were connected with the host circulatory system by anastomosis. This method is an attractive technique for engineering prevascularized artificial tissues for transplantation. Copyright © 2015 John Wiley & Sons, Ltd.     

Fetal dermal fibroblasts exhibit enhanced growth and collagen production in two‐ and three‐dimensional culture in comparison to adult fibroblasts

The high morbidity of tendon injuries and the poor outcomes observed following repair or replacement have stimulated interest in regenerative approaches to treatment and, in particular, the use of cell‐based analogues as alternatives to autologous and allogeneic graft repair. Given the known regenerative properties of fetal tissues, the objective of this study was to assess the biological and mechanical properties of tissue‐engineered three‐dimensional (3D) composites seeded with fetal skin cells. Dermal fibroblasts were isolated from pregnant rats and their fetuses and characterized in monolayer culture and on 3D resorbable polyester scaffolds. To determine the differences between fetal and adult fibroblasts, DNA, total protein and types I and III collagen production were measured. In addition, morphology and mechanical properties of the 3D constructs were examined. In monolayer culture, fetal fibroblasts produced significantly more types I and III collagen and displayed serum‐independent growth, while adult fibroblasts elaborated less collagen and exhibited reduced cell spreading and attachment under low‐serum conditions. In 3D culture, fetal constructs appeared more developed based on gross examination, with significantly more total DNA, total protein and normalized type I collagen production compared to adult specimens. Finally, after 35 days, fetal fibroblast‐seeded constructs possessed superior mechanical properties compared to adult samples. Taken together, these findings indicate that fetal dermal fibroblasts may be an effective source of cells for fabricating tissue equivalents to regenerate injured tendons. Copyright © 2009 John Wiley & Sons, Ltd.     

Simple suspension culture system of human iPS cells maintaining their pluripotency for cardiac cell sheet engineering

In this study, a simple three‐dimensional (3D) suspension culture method for the expansion and cardiac differentiation of human induced pluripotent stem cells (hiPSCs) is reported. The culture methods were easily adapted from two‐dimensional (2D) to 3D culture without any additional manipulations. When hiPSCs were directly applied to 3D culture from 2D in a single‐cell suspension, only a few aggregated cells were observed. However, after 3 days, culture of the small hiPSC aggregates in a spinner flask at the optimal agitation rate created aggregates which were capable of cell passages from the single‐cell suspension. Cell numbers increased to approximately 10‐fold after 12 days of culture. The undifferentiated state of expanded hiPSCs was confirmed by flow cytometry, immunocytochemistry and quantitative RT–PCR, and the hiPSCs differentiated into three germ layers. When the hiPSCs were subsequently cultured in a flask using cardiac differentiation medium, expression of cardiac cell‐specific genes and beating cardiomyocytes were observed. Furthermore, the culture of hiPSCs on Matrigel‐coated dishes with serum‐free medium containing activin A, BMP4 and FGF‐2 enabled it to generate robust spontaneous beating cardiomyocytes and these cells expressed several cardiac cell‐related genes, including HCN4, MLC‐2a and MLC‐2v. This suggests that the expanded hiPSCs might maintain the potential to differentiate into several types of cardiomyocytes, including pacemakers. Moreover, when cardiac cell sheets were fabricated using differentiated cardiomyocytes, they beat spontaneously and synchronously, indicating electrically communicative tissue. This simple culture system might enable the generation of sufficient amounts of beating cardiomyocytes for use in cardiac regenerative medicine and tissue engineering. Copyright © 2013 John Wiley & Sons, Ltd.     

Transplantation of cancerous cell sheets effectively generates tumour‐bearing model mice

Tumour‐bearing mice were created by transplanting cancerous cell sheets onto the subcutaneous tissue of the dorsal region, using luciferase gene‐transfected mammary gland adenocarcinoma cells, 4T1‐luc2, to investigate the tumourigenicity of the cell sheet relative to a conventional injection of cell suspension. Contiguous breast cancerous cell sheets were harvested from temperature‐responsive culture dishes by reducing the temperature from 37 °C to 20 °C; the sheets were then transplanted onto the dorsal side of the mouse subcutaneous tissue, using a chitin‐based supporting membrane. Cell suspensions obtained by trypsin digestion were subcutaneously injected into the dorsal region of mice. The tumour growth of the transplanted cancer cells was evaluated by the tumour volume and by the bioluminescence from luciferase‐gene transfected cancer cells, using an in vivo imaging system. The cell sheet method improved the 4 T1‐luc2 engraftment efficiency in living mouse tissues at the initial stage by 13‐fold compared with that from injecting cell suspensions. On day 14 after the transplantation, the tumour formation at the transplanted area of cell sheet‐transplanted mice also accelerated, and the mean tumour volume became 1116 mm³, which was 10 times larger than that in cell suspension‐transplanted mice. The cell sheets engrafted on the recipient tissues efficiently due to the preserved extracellular matrix on their basal sides, such that cancer cells were supplied with sufficient oxygen and nutrients from the host tissues to develop tumour tissues. Therefore, cancerous cell sheet‐based transplantation is a promising method for efficiently creating cancer‐bearing mice. Copyright © 2013 John Wiley & Sons, Ltd.