Yuji Haraguchi

Fabrication of functional three-dimensional tissues by stacking cell sheets in vitro

The fabrication of 3D tissues retaining the original functions of tissues/organs in vitro is crucial for optimal tissue engineering and regenerative medicine. The fabrication of 3D tissues also contributes to the establishment of in vitro tissue/organ models for drug screening. Our laboratory has developed a fabrication system for functional 3D tissues by stacking cell sheets of confluent cultured cells detached from a temperature-responsive culture dish. Here we describe the protocols for the fabrication of 3D tissues by cell sheet engineering. Three-dimensional cardiac tissues fabricated by stacking cardiac cell sheets pulsate spontaneously, synchronously and macroscopically. Via this protocol, it is also possible to fabricate other tissues, such as 3D tissue including capillary-like prevascular networks, from endothelial cells sandwiched between layered cell sheets. Cell sheet stacking technology promises to provide in vitro tissue/organ models and more effective therapies for curing tissue/organ failures.

Design of prevascularized three-dimensional cell-dense tissues using a cell sheet stacking manipulation technology.

To survive three-dimensional (3-D) cell-dense thick tissues after transplantation, the improvements of hypoxia, nutrient insufficiency, and accumulation of waste products are required. This study presents a strategy for the initiation of prevascular networks in a 3-D tissue construct by sandwiching endothelial cells between the cell sheets. For obtaining a stable stacked cell sheet construct, a sophisticated 3-D cell sheet manipulation system using temperature-responsive culture dishes and a cell sheet manipulator was developed. When sparsely cultured human umbilical vein endothelial cells (HUVECs) were sandwiched between two myoblast sheets, the inserted HUVECs sprouted and formed network structures in vitro. Additionally, when myoblast sheets and HUVECs were alternately sandwiched, endothelial cell connections through the layers and capillary-like structures were found in a five-layer construct. Moreover, the endothelial networks in the five-layer myoblast sheet construct were observed to connect to the host vessels after transplantation into the subcutaneous tissues of nude rats, resulted in a neovascularization that allow the graft to survive. These results indicated that the prevascularized myoblast sheet constructs could induce functional anastomosis. Consequently, our prevascularizing method using a cell sheet stacking manipulation technology provides a substantial advance for developing various types of three-dimensional tissues and contributes to regenerative medicine.

Creation of human cardiac cell sheets using pluripotent stem cells

Although we previously reported the development of cell-dense thickened cardiac tissue by repeated transplantation-based vascularization of neonatal rat cardiac cell sheets, the cell sources for human cardiac cells sheets and their functions have not been fully elucidated. In this study, we developed a bioreactor to expand and induce cardiac differentiation of human induced pluripotent stem cells (hiPSCs). Bioreactor culture for 14 days produced around 8×10(7) cells/100 ml vessel and about 80% of cells were positive for cardiac troponin T. After cardiac differentiation, cardiomyocytes were cultured on temperature-responsive culture dishes and showed spontaneous and synchronous beating, even after cell sheets were detached from culture dishes. Furthermore, extracellular action potential propagation was observed between cell sheets when two cardiac cell sheets were partially overlaid. These findings suggest that cardiac cell sheets formed by hiPSC-derived cardiomyocytes might have sufficient properties for the creation of thickened cardiac tissue.

Creation of mouse embryonic stem cell-derived cardiac cell sheets

Research on heart tissue engineering is an exciting and promising area. Although we previously developed bioengineered myocardium using cell sheet-based tissue engineering technologies, the issue of appropriate cell sources remained unresolved. In the present study, we created cell sheets of mouse embryonic stem (ES) cell-derived cardiomyocytes after expansion in three-dimensional stirred suspension cultures. Serial treatment of the suspension cultures with noggin and granulocyte colony-stimulating factor significantly increased the number of cardiomyocytes by more than fourfold compared with untreated cultures. After drug selection for ES cells expressing the neomycin-resistance gene under the control of the α-myosin heavy chain promoter, almost all of the cells showed spontaneous beating and expressed several cardiac contractive proteins in a fine striated pattern. When ES-derived cardiomyocytes alone were seeded onto temperature-responsive culture dishes, cell sheets were not created, whereas cocultures with cardiac fibroblasts promoted cell sheet formation. The cardiomyocytes in the cell sheets beat spontaneously and synchronously, and expressed connexin 43 at the edge of adjacent cardiomyocytes. Furthermore, when the extracellular action potential was recorded, unidirectional action potential propagation was observed. The present findings suggest that stirred suspension cultures with appropriate growth factors are capable of producing cardiomyocytes effectively and easily, and that ES-derived cardiac cell sheets may be a promising tool for the development of bioengineered myocardium.

Apelin peptides block the entry of human immunodeficiency virus (HIV)

The orphan G protein‐coupled receptor APJ has been shown to be a coreceptor for human and simian immunodeficiency virus (HIV and SIV) strains. We have determined that some HIV and SIV strains use APJ as a coreceptor to infect the brain‐derived NP‐2/CD4 cells. Because apelin is an endogenous ligand for the APJ receptor, we examined the inhibitory effects of apelin peptides on HIV infection, and found that the apelin peptides inhibit the entry of some HIV‐1 and HIV‐2 into the NP‐2/CD4 cells expressing APJ. The inhibitory efficiency has been found to be in the order of apelin‐36>apelin‐17>apelin‐13>apelin‐12.

Concise Review: Cell Therapy and Tissue Engineering for Cardiovascular Disease

Cardiovascular disease is a major cause of morbidity and mortality, especially in developed countries. Various therapies for cardiovascular disease are investigated actively and are performed clinically. Recently, cell‐based regenerative medicine using several cell sources has appeared as an alternative therapy for curing cardiovascular diseases. Scaffold‐based or cell sheet‐based tissue engineering is focused as a new generational cell‐based regenerative therapy, and the clinical trials have also been started. Cell‐based regenerative therapies have an enormous potential for treating cardiovascular disease. This review summarizes the recent research of cell sources and cell‐based‐regenerative therapies for cardiovascular diseases.

Inhibition of HIV-1 infection by zinc group metal compounds

Thirty-seven metal compounds were examined for inhibitory activities against infection with human immunodeficiency virus type 1 (HIV-1). Zinc group metal compounds, namely, zinc acetate, zinc chloride, zinc nitrate, cadmium acetate and mercury chloride, showed anti-HIV-1 activities. Cadmium and mercury compounds at 1-10 microg/ml and zinc compounds at 100 microg/ml strongly inhibited HIV-1 infection, although the cadmium, mercury and zinc compounds had severe cytotoxities at 100, 100 and 1000 microg/ml, respectively. They inhibited transcription of HIV-1 RNA and HIV-1 production at concentrations at which they did not affect the growth of HIV-1-producing cells. They had little effect on syncytium formation resulting from cocultivation of uninfected with HIV-1-producing cells. Nor did they affect HIV-1 DNA synthesis following HIV-1 infection. The metal compounds may owe their anti-HIV-1 effects to inhibition of HIV-1 DNA to RNA transcription, rather than inhibition of the adsorption, penetration or reverse transcription step of HIV-1 infection.

Scaffold-free tissue engineering using cell sheet technology

Cell sheet technology is a tissue engineering methodology requiring no scaffolds. Confluent cultured cells can be harvested as an intact cell sheet using a temperature-responsive polymer, poly(N-isoproplyacrylamide) (PIPAAm), grafted cell culture surface, offering noninvasive control of cell attachment and detachment only by changing the temperature across 32 °C, without any protease treatments. Avoiding protease treatment preserves complete cell–cell junctions, cell surface proteins, and the extracellular matrix in the cell sheet. Therefore, functional three-dimensional (3D) tissue can be easily fabricated by layering cell sheets without the use of scaffolds. Cell sheet technology has been applied in regenerative medicine for several tissues, and a number of clinical trials have already started. In addition, to fabricate more complex and functional 3D tissue, cell micro-patterning technology can be combined with cell sheet technology, and this interdisciplinary technology could produce interesting results. In this review, recent advances in temperature-responsive culture surfaces, cell sheet and cell micro-patterning technologies are summarized and discussed. In addition, the application of these technologies to regenerative medicine and the re-construction of various tissues including heterogeneous tissues are also discussed.

Regenerative Therapies Using Cell Sheet-Based Tissue Engineering for Cardiac Disease

At present, cardiac diseases are a major cause of morbidity and mortality in the world. Recently, a cell-based regenerative medicine has appeared as one of the most potential and promising therapies for improving cardiac diseases. As a new generational cell-based regenerative therapy, tissue engineering is focused. Our laboratory has originally developed cell sheet-based scaffold-free tissue engineering. Three-dimensional myocardial tissue fabricated by stacking cardiomyocyte sheets, which are tightly interconnected to each other through gap junctions, beats simultaneously and macroscopically and shows the characteristic structures of native heart tissue. Cell sheet-based therapy cures the damaged heart function of animal models and is clinically applied. Cell sheet-based tissue engineering has a promising and enormous potential in myocardial tissue regenerative medicine and will cure many patients suffering from severe cardiac disease. This paper summarizes cell sheet-based tissue engineering and its satisfactory therapeutic effects on cardiac disease.

Cell sheet transplantation for heart tissue repair

Cell transplantation is attracting considerable attention as the next-generation therapy for treatment of cardiovascular diseases. We have developed cell sheet engineering as a type of scaffold-less tissue engineering for application in myocardial tissue engineering and the repair of injured heart tissue by cell transplantation. Various types of cell sheet transplantation have improved cardiac function in animal models and clinical settings. Furthermore, cell-based tissue engineering with human induced pluripotent stem cell technology is about to create thick vascularized cardiac tissue for cardiac grafts and heart tissue models. In this review, we summarize the current cardiac cell therapies for treating heart failure with cell sheet technology and cell sheet-based tissue engineering.