Biomedical subjects

Masayuki Yamato

Publications and source records attributed to Masayuki Yamato.

At least 19 recordsLinked to original sources

PubMed2006

Expression of Integrin beta3 is correlated to the properties of quiescent hemopoietic stem cells possessing the side population phenotype.

With significant attention paid to the field of tissue-specific stem cells, the identification of stem cell-specific markers is of considerable importance. Previously, the side population (SP) phenotype, with the capacity to efflux the DNA-binding dye Hoechst 33342, has been recognized as a common feature of adult tissue-specific stem cells. In this study, we show that high expression of integrin beta(3) (CD61) is an attribute of SP cells isolated from mouse bone marrow. Additionally, we confirmed that the expression of integrin beta(3) is correlated with properties of quiescent hemopoietic stem cells (HSCs) including the strength of the SP phenotype, cell cycle arrest, expression of HSC markers, and long-term hemopoiesis. Importantly, Lineage(-) (Lin(-))/integrin beta(3)(high) (beta(3)(high)) SP cells have as strong a capacity for long-term hemopoiesis as c-Kit(+)/Sca-1(+)/Lin(-) SP cells, which are regarded as one of the most highly enriched HSC populations. Finally, the integrin beta(3) subunit that is present in SP cells having the properties of HSCs, is associated with integrin alpha(v) (CD51). Therefore, our results demonstrate that high expression of integrin beta(3) is correlated to the properties of quiescent HSCs and suggest that the integrin beta(3) subunit is available as a common surface marker of tissue-specific stem cells.

Animals↗

PubMed2006

Comparison between Holstein cow's milk and Japanese-Saanen goat's milk in fatty acid composition, lipid digestibility and protein profile.

The fatty acid composition, the lipid digestibility and protein profile of Japanese-Saanen goat's milk were characterized. Caprine milk contained substantial quantities of C(4:0) to C(10:0) fatty acids as compared with Holstein cow's milk. The lipids of the former showed significantly higher digestibility in vitro by porcine lipase than those of the latter (P<0.05). As determined by SDS-PAGE, the respective contents of alpha(s1)-casein, one of the major allergens, were 3.9% and 33.7% in caprine and bovine milk.

Animals↗

PubMed2006

Recovery course of full-thickness skin defects with exposed bone: an evaluation by a quantitative examination of new blood vessels.

BACKGROUND: Full-thickness skin defects with exposed bone are often hard to heal. The lack or delayed re-vascularization is considered one of the major causes, and the periosteum is also suggested to have an important role in tissue regeneration. MATERIALS AND METHODS: Full-thickness skin defect wounds with exposed bone were made in the parietal region of Wister rats. The periosteum of the exposed parietal bone was removed in the periosteum-lacking group, but maintained in the control group (periosteum-intact group). The wound was covered by an artificial dermis made of collagen. The wound healing process was histologically compared. Double immunostaining of alpha-smooth muscle actin (SMA) and von Willebrand factor (vWF) was used for re-vascularization examination, and the blood vessel density in the artificial dermis was quantified. RESULTS: The density of the blood vessels in the uninjured parietal tissue was approximately 80 vessels/mm(2). To reach this density, 7 and 21 days were required for the control (periosteum-intact) and the periosteum-lacking groups, respectively. This coincided with complete revascularization, fibroblast migration and the reentry of blood vessels to the upper layer of the wound were observed. CONCLUSION: The described results support the importance of the periosteum in the full-thickness skin defect healing process.

Animals↗

PubMed2006

Development of transplantable genetically modified corneal epithelial cell sheets for gene therapy.

The purpose of this study was to establish a method for the fabrication of exogenous gene-transferred, transplantable corneal epithelial cell sheets. Corneo-limbal epithelial cells collected from USA eye bank eyes were transduced with an EGFP-expressing lentiviral vector at differential MOI. Multi-layered corneal epithelial cell sheets were fabricated by co-cultivation of transduced cells and mitomycin C-treated 3T3 feeder layers on temperature-responsive culture dishes. These cultured epithelial cells could be harvested as intact sheets by simply lowering the temperature. The number of EGFP-positive cells was increased as the MOI raised, and at an MOI of 100, nearly 100% of the superficial cells showed strong EGFP expression. Histological analysis revealed that EGFP was expressed in all layers of the cell sheet of which cell source was transduced with the lentiviral vector at an MOI of 100. Immunofluorescence data showed that p63 was also expressed in the basal layer of the same cell sheet. These results suggest that this technique will likely be applicable to ex vivo gene therapies for various corneal disorders.

Cells, Cultured↗

PubMed2006

N-Cadherin is expressed by putative stem/progenitor cells and melanocytes in the human limbal epithelial stem cell niche.

Corneal epithelial stem cells are known to be localized to the basal layer of the limbal epithelium, providing a model system for epithelial stem cell biology; however, the mechanisms regarding the maintenance of these stem cells in their specialized niche remain poorly understood. N-cadherin is a member of the classic cadherin family and has previously been demonstrated to be expressed by hematopoietic stem cells. In the present study, we demonstrate that N-cadherin is expressed by putative stem/progenitor cells, as well as melanocytes, in the human limbal epithelial stem cell niche. In addition, we demonstrate that upon in vitro culture using 3T3 feeder layers, loss of N-cadherin expression occurs with cell proliferation. These results indicate that N-cadherin may be a critical cell-to-cell adhesion molecule between corneal epithelial stem/progenitor cells and their corresponding niche cells in the limbal epithelium.

Adult↗

PubMed2006

Heterotypic cell interactions on a dually patterned surface.

It is worth investigating heterotypic cell-cell interactions by mimicking their in vivo structures and environment. In the present study, physiological cellular response and behavior of hepatocytes and endothelial cells were investigated by controlling their contact periphery in a new co-culture system. Rat primary hepatocytes and bovine endothelial cells were co-cultured on a dually patterned surface. Hepatic physiological functions such as albumin secretion and ammonium metabolism were enhanced by increasing heterotypic cell-cell interactions in a patterned co-culture. Furthermore, enhanced hepatic functions through heterotypic interactions are effective within a limited area apart from endothelial cells as evidenced by immunofluorescence staining of hepatic intracellular albumin, indicating that heterotypic interactions act in a paracrine manner. Thus, heterotypic cell communications that play indispensable roles in increasing hepatic physiological functions should be obtained with an increasing periphery of two-cell domains. These findings are important for the reconstruction of complex tissues such as liver and pancreas.

Animals↗

PubMed2006

The effect of micropores in the surface of temperature-responsive culture inserts on the fabrication of transplantable canine oral mucosal epithelial cell sheets.

Primary canine oral mucosal epithelial cells were cultured on temperature-responsive dishes and cell culture inserts to fabricate transplantable epithelial cell sheets. When 3T3 feeder layers and fetal bovine serum were eliminated from dish culture, the harvested cell sheets became significantly more fragile. In contrast, when epithelial cells were cultured on inserts having submicron-scale pores, cell sheet fragility was eliminated. Keratin expression profiles showed no differences among the harvested cell sheets, but the expression of p63, a putative stem/progenitor marker, was strongly dependent on the presence of 3T3 feeder layers and serum. These results suggest that the maintenance of stem/progenitor cells is influenced by the apical/basal supply of nutrients as well as culture supplements.

Animals↗

PubMed2006

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.

Acrylic Resins↗

PubMed2006

Bio-functionalized thermoresponsive interfaces facilitating cell adhesion and proliferation.

Bio-functionalized thermoresponsive culture interfaces co-immobilized with cell adhesive peptide, RGDS, and cell growth factor, insulin (INS), are investigated to promote initial cell adhesion and cell growth for further cell sheet engineering applications. These bio-functionalized interfaces were prepared by electron beam-induced copolymerization of N-isopropylacrylamide (IPAAm) with its carboxyl-derivatized analog, 2-carboxyisopropylacrylamide (CIPAAm), and grafting onto tissue culture polystyrene dishes, followed by immobilization of RGDS and/or INS to CIPAAm carboxyls. Adhesion and proliferation of bovine carotid artery endothelial cells (ECs) were examined on the RGDS-INS co-immobilized thermoresponsive interfaces. Immobilized RGDS facilitated initial EC adhesion on the surfaces and INS modification was demonstrated to induce EC proliferation, respectively. More pronounced EC growth was indicated by co-immobilization of appropriate amount of RGDS and INS. This may be due to synergistic effect of direct co-stimulation of adhered ECs by surface-immobilized RGDS and INS molecules. ECs grown on the RGDS-INS co-immobilized thermoresponsive interfaces can also be recovered spontaneously as viable tissue monolayers by solely reducing culture temperature. RGDS-INS co-immobilized thermoresponsive interfaces strongly supported initial EC adhesion and growth than unmodified thermoresponsive surfaces even under serum-free culture. Addition of soluble growth factors to serum-free culture medium effectively induced EC proliferation to confluency. Co-immobilization of cell adhesion peptides and growth factors on thermoresponsive surfaces should be effective for rapid preparation of intact cell sheets and their utilization to regenerative medicine.

Acrylamides↗

PubMed2006

Electrical coupling of cardiomyocyte sheets occurs rapidly via functional gap junction formation.

Previously, we have successfully created pulsatile myocardial tissue grafts using our novel technology, "cell sheet engineering", that layers cell sheets fabricated on temperature-responsive culture dishes to form three-dimensional (3-D) structures. Electrical coupling is established between layered neonatal rat cardiomyocyte sheets, resulting in the synchronized beating of 3-D myocardial tissues. However, the mechanism by which these layered cardiomyocyte sheets communicate electrically is not well-understood. In this study, we used a multiple-electrode extracellular recording system and demonstrated that bilayer cardiomyocyte sheets coupled electrically with slight delays 34+/-2 min (mean+/-SEM) after layering. These delays gradually decreased and the electrical actions of layered cell sheets were completely coupled 46+/-3 min (mean+/-SEM) after initial layering. Immunohistological analysis showed that connexin43, a gap junction (GJ)-related protein, existed not only at cell-to-cell interfaces but also on the free cell membrane in the cardiomyocyte sheet. Additionally, neither connexin40 nor connexin45, but only connexin43 was detected between bilayer cardiomyocyte sheets within 30 min after layering. Dye transfer assay demonstrated that the exchange of small molecules via GJs occurred within 30 min. The cell sheet manipulation technique using the temperature-responsive culture dishes has substantial advances and the exciting potential in the fields of cell and tissue physiology, as well as tissue engineering.

Action Potentials↗

PubMed2006

Transplantable retinal pigment epithelial cell sheets for tissue engineering.

The native retinal pigment epithelium (RPE) exists as a monolayer structure and is critically involved in the maintenance of photoreceptors. Damage or destruction of the RPE due to a variety of diseases therefore often results in loss of vision. With regenerative purposes in mind, we have examined various culture conditions such as the initial cell density and the addition of various supplements in an effort to produce transplantable RPE cell sheets that can be harvested without defects. We demonstrate that the cell density in cultured RPE sheets increased linearly with the number of seeded cells and that RPE sheets were harvested without defects and limited contraction due to cytoskeletal reorganization, when TGF-beta2 was added to the growth medium. Results from histological analysis and the measurement of trans-epithelial resistance also demonstrates that these RPE cell sheets exist as monolayer structure, similar to the native RPE, with intact cell-to-cell junctions. Therefore, these methods provide significant insight into the fabrication of transplantable RPE cell sheets that can be applied to RPE regenerative therapies to restore lost vision.

Cell Count↗

PubMed2006

Polysurgery of cell sheet grafts overcomes diffusion limits to produce thick, vascularized myocardial tissues.

Recently, the field of tissue engineering has progressed rapidly, but poor vascularization remains a major obstacle in bioengineering cell-dense tissues, limiting the viable size of constructs due to hypoxia, nutrient insufficiency, and waste accumulation. Therefore, new technologies for fabricating functional tissues with a well-organized vasculature are required. In the present study, neonatal rat cardiomyocytes were harvested as intact sheets from temperature-responsive culture dishes and stacked into cell-dense myocardial tissues. However, the thickness limit for layered cell sheets in subcutaneous tissue was approximately 80 microm (3 layers). To overcome this limitation, repeated transplantation of triple-layer grafts was performed at 1, 2, or 3 day intervals. The two overlaid grafts completely synchronized and the whole tissues survived without necrosis in the 1 or 2 day interval cases. Multistep transplantation also created approximately 1 mm thick myocardium with a well-organized microvascular network. Furthermore, functional multilayer grafts fabricated over a surgically connectable artery and vein revealed complete graft perfusion via the vessels and ectopic transplantation of the grafts was successfully performed using direct vessel anastomoses. These cultured cell sheet integration methods overcome long-standing barriers to producing thick, vascularized tissues, revealing a possible solution for the clinical repair of various damaged organs, including the impaired myocardium.

Animals↗

PubMed2006

An actuated pump on-chip powered by cultured cardiomyocytes.

Cellular functions are frequently exploited as processing components for integrated chemical systems such as biochemical reactors and bioassay systems. Here, we have created a new cell-based microsystem exploiting the intrinsic pulsatile mechanical functions of cardiomyocytes to build a cellular micropump on-chip using cardiomyocyte sheets as prototype bio-microactuators. We first demonstrate cell-based control of fluid motion in a model microchannel without check valves and evaluate the potential performance of the bio-actuation. For this purpose, a poly(dimethylsiloxane) (PDMS) microchip with a microchannel equipped with a diaphragm and a push-bar structure capable of harnessing collective cell fluid mechanical forces was coupled to a cultured pulsating cardiomyocyte sheet, activating cell-based fluid movement in the microchannel by actuating the diaphragm. Cell oscillation frequency and correlated fluid displacement in this system depended on temperature. When culture temperature was increased, collective cell contraction frequency remained cooperative and synchronous but increased, while displacement was slightly reduced. We then demonstrated directional fluid pumping within microchannels using cantilever-type micro-check valves made of polyimide. A directional flow rate of nL min(-1) was produced. This cell micropump system could be further developed as a self-actuated and efficient mechanochemical transducer requiring no external energy sources for various purposes in the future.

Animals↗

PubMed2006

Bioengineered cardiac cell sheet grafts have intrinsic angiogenic potential.

Previously, we have demonstrated the long-term survival of myocardial cell sheet constructs in vivo, with microvascular network formation throughout the engineered tissues. The understanding and control of these vascularization processes are a key factor for creating thicker functional tissues. Here, we show that cardiac cell sheets express angiogenesis-related genes and form endothelial cell networks in culture. After non-invasive harvest and stacking of cell sheets using temperature-responsive culture dishes, these endothelial cell networks are maintained and result in neovascularization upon in vivo transplantation. Interestingly, we also discovered that all of the graft vessels are derived from the grafts themselves and these vessels migrate to connect with the host vasculature. Finally, blood vessel formation within the grafts can be controlled by changing the ratio of endothelial cells. In conclusion, myocardial tissue grafts engineered with cell sheet technology have their own inherent potential for the in vivo neovascularization that can be regulated in vitro.

Animals↗

PubMed2006

Demonstration of a PDMS-based bio-microactuator using cultured cardiomyocytes to drive polymer micropillars.

Natural cellular functions are increasingly exploited for integrated chemical systems such as biochemical reactors and biosensors. We propose to utilize the intrinsic mechanical function of cardiomyocytes, converting chemical energy into mechanical energy. In this report, we demonstrate the working principle of our proposed poly(dimethylsiloxane) (PDMS) based cardiomyocyte bio-microactuator using fabricated PDMS micropillars driven to repetitive motion by attached pulsating cardiomyocytes. Sheets of PDMS embedded with an array of micropillars were fabricated and modified for cardiomyocyte attachment in culture. Primary neonatal rat cardiomyocytes were cultured on the array, attaching to the micropillars and substratum successfully, and exhibiting their typical spontaneous, pulsatile phenotype. Micropillars beat with the coupled cells spontaneously without any triggers. The beat frequency was 1.4 Hz at 37 degrees C and the displacement of the top of the pillar that beat most strongly in our observation was 2.8+/-0.2 microm. From this result, contractile forces of cultured cardiomyocytes were estimated to exceed 3.5 microN. The estimated force is far greater than that of a previously described hydrogel-based cardiomyocyte bio-microactuator (K. Morishima et al., in Micro Total Analysis Systems 2003, ed. M. A. Northrup et al., The Transducers Research Foundation, San Diego, CA, vol. 2, pp. 1125-1128). PDMS compatibility as a base material for bio-microactuator design using cultured cardiomyocytes was verified. This PDMS-based cell microactuator worked for about one week without exchange of the culture medium, and this system could be developed for various purposes in the future as self-actuated and efficient mechanochemical transducers without external energy source requirements.

Algorithms↗

PubMed2006

Fabrication of transplantable human oral mucosal epithelial cell sheets using temperature-responsive culture inserts without feeder layer cells.

To exclude bacteria- or animal-derived factors from cultured fabrication of transplantable epithelial cell sheets, primary human oral mucosal epithelial cells were seeded on temperature-responsive culture inserts having submicron-scale pores. Supplying culture medium containing human autologous serum to both apical and basal sides of human epithelial cells allows these cells to grow to confluence. These proliferating cells created stratified epithelial layers even when 3T3 feeder layers and fetal bovine serum were eliminated from culture. Normal keratin expression profiles were obtained with these cells, and basal and midlayer cells expressed p63, a putative stem/progenitor marker. These results suggest that temperature-responsive culture inserts can be useful in clinical settings that require the exclusion of xenogeneic factors.

Animals↗

PubMed2006

Maskless liquid-crystal-display projection photolithography for improved design flexibility of cellular micropatterns.

We previously developed an all-in-one photopolymerization device by modifying a commercially available liquid crystal display projector (LCDP) for the preparation of micropatterned surfaces and microfluidic channels without the need for expensive photomasks. In the present study, we demonstrate a simple and reliable method for rapid prototyping of cell micropatterning with high resolution using the modified LCDP device. Fabrication of complicated and flexible patterns was achieved using this device with positive-type photoresist in a two-step process. First, micropatterns on the silanized coverslips were fabricated from positive photoresist. Second, acrylamide monomer solution containing polymerization initiator was dropped onto the micropatterned positive photoresist and copolymerized on the silanized coverslips in situ by thermally initiated radical polymerization. After the reaction, the remnant micropatterned photoresist is easily dissolved, resulting in a polyacrylamide-silane micropattern on the coverslip. The resultant polyacrylamide layer is highly hydrophilic and repels both protein adsorption and cell adhesion. Cells seeded on the micropatterned surfaces therefore attach and spread only on unpolymerized silanized glass surfaces, conforming to the pattern design. This technique is therefore useful for inexpensive, rapid prototyping of surface micropatterns using polymer materials.

Acrylic Resins↗

PubMed2006

Corneal epithelial stem cell delivery using cell sheet engineering: not lost in transplantation.

Cell-based therapies have now generated significant interest as novel drug delivery systems, with various adult cell types used in treating a wide range of diseases. To overcome the limits that restrict treatments for corneal surface dysfunction, corneal epithelial stem cells expanded ex vivo have been applied as an alternative approach. While previous studies used various carrier substrates, we present a novel method using cell sheet engineering with temperature-responsive culture dishes to create carrier-free corneal epithelial stem cell sheets that can be transplanted without sutures. Results from clinical trials reveal successful transplantation with the recovery of lost visual acuity in all cases. Cell sheet engineering, therefore, presents a novel method for the delivery of corneal epithelial stem cells, and can also be applied for other approaches of cellular therapeutics.

Corneal Transplantation↗