Cell Transplantation

Embryonic stem (ES) cells are expected to be a potential donor source for neural transplantation. We have obtained motoneuron-enriched neural progenitor cells by culturing mouse ES cells with retinoic acid (RA). The cells also expressed mRNA of a neurotrophic factor, neurotrophin-3 (NT-3). The left motor cortex area of mice was damaged by cryogenic brain injury, and the neural cells were transplanted underneath the injured motor cortex, neighboring to the paraventricular region. We found that the cells expressing neuronal phenotypes not only remained close to the implantation site, but also exhibited substantial migration penetrating into the damaged lesion, in a seemingly directed manner up to cortical region. We found that some of the neural cells differentiated into Islet1-positive motoneurons. It seems likely that the ability of the ES cell-derived neural progenitor cells to respond in vivo to guidance cues and signals that can direct their migration and differentiation may contribute to functional recovery of the recipient mice. We found that an “island of the mature neuronal cells” of recipient origin emerged in the damaged motor cortex. This may be associated with the neuroprotective effects of the ES cell-derived neural cells. The ES cells differentiated into CD31+ vasculoendothelial cells with the RA treatment in vitro. Furthermore, the grafted cells may provide sufficient neurotrophic factors such as NT-3 for neuroprotection and regeneration. The grafted neural cells that migrated into residual cortex and differentiated into neurons had purposefully elongated axons that were stained with anti-neurofilament middle chain (NFM) antibody. Our study suggests that motoneurons can be induced from ES cells, and ES cells become virtually an unlimited source of cells for experimental and clinical neural cell transplantation.

Hepatocyte transplantation and artificial organ hepatic support require a number of functionally mature hepatocytes. However, their growth activity and functional behaviors are much smaller in culture after isolation from the liver. We examined whether continuously differentiating hepatocytes from multipotent hepatic stem cells that were isolated by using flow cytometry and propagated clonally in culture could be a source of clinical application. They actually gave rise to cells that were functionally equal to mature hepatocytes found in the adult liver, which secreted albumin into culture medium and metabolized harmful ammonium into urea. These data suggest that stem cell-derived hepatocytes are a useful cell source for developing therapeutic strategies, such as cell transplantation, gene therapy, and artificial liver organ to treat various liver disorders.

Chondrocytes in articular cartilage synthesize collagen type II and large sulfated proteoglycans, whereas the same cells cultured in monolayer (2D) dedifferentiate into fibroblastic cells and express collagen type I and small proteoglycans. On the other hand, a pellet culture system was developed as a method for preventing the phenotypic modulation of chondrocytes and promoting the redifferentiation of dedifferentiated ones. Because the pellet culture system forms only one cell aggregate each tube by a centrifugator, the pellet could not be applied to produce a tissue-engineered cartilage. Therefore, we tried to form chondrocyte aggregates by a rotational culture, expecting to form a large number of aggregates at once. In order to increase cell–cell interactions and decrease chondrocyte–material interaction, dishes with low retention of protein adsorption and cell adhesiveness were used. In addition, rotational shaking of the dish including cells was attempted to increase the cell–cell interaction. The shaking speed was set at 80 rpm, so the cells would be distributed in the center of the dish to augment the frequency of cell–cell contact. Under these conditions, bovine articular chondrocytes started aggregating in a few hours. At 24–36 h of rotational culture, aggregates with smooth surfaces were observed. Parameters such as increase of culture time and addition of TGF-β controlled diameters of the aggregates. There were many fusiform cells at the periphery of the aggregates, where the cells tended to form a multilayered zone in cross sections. In addition, lacune-like structure, which was almost the same as pellet culture, was observed. It was found that the internal structure of the aggregates was similar to that of pellets reported previously. Therefore, the aggregates formed by a rotational culture could become an essential component to make tissue-engineered artificial cartilage.

We devised a novel nerve prosthesis composed of an elastomeric gelatinous tube and multifilament gelatinous fibers, both of which were prepared from styrene-derivatized gelatin, which allows in situ formation of a bioactive substance-incorporated gel. An in vitro study showed that the axonal regeneration potential of a photocured gelatin layer impregnated with laminin, fibronectin, and NGF was almost comparable with that of coated Matrigel. A nerve conduit and fibers prepared from photoreactive gelatin was subjected to visible-light irradiation with rotation in the presence of camphorquinone as a photoinitiator using a custom-designed apparatus. A sample of transparent gelatinous conduit with an inner diameter of 1.2 mm and a wall thickness of 0.6 mm and gelatin fibers ranging from 10 to 100 μm in diameter were produced. The photocured elastomeric gelatinous tube was flexible and had structural integrity that allowed mechanical handling without breaking. A novel nerve guidance prosthesis composed of tubes packed with fibers was assembled. This photofabrication technology may enable the design of a tailor-made shape and rapid morphogenesis and functional recovery of damaged nerve tissue.

It is difficult to a produce highly functional bioartificial liver (BAL) using only hepatocytes, because it is believed that liver-specific three-dimensional structure is necessary to maintain high function for BAL. But it is difficult to construct a culture system with liver-specific three-dimensional structure in vitro. To realize a highly functional culture system with liver-specific three-dimensional structure, we developed a culture system using liver slices that keep liver-specific architecture, such as liver lobule and hepatic microvascular system. Liver slices were embedded in agarose gel to maintain them under a moist and three-dimensional environment. We examined the viability and function of liver slices by using various shapes of agarose gel. Liver slices were cultured 1) under stationary condition (control), 2) directly embedded in gel, and 3) embedded in cylindrical gel for good drainage of medium and ventilation of air. The viability and function of the incubated liver slices were evaluated by LDH leakage, histomorphology, and immunohistochemistry. At 10 days, the morphological condition and function of liver slices embedded in cylindrical gel were maintained better than liver slices directly embedded in gel or in the stationary condition. We suggest that high functionality and morphological condition of liver slices could be maintained by embedding in cylindrical gel. In the future, it is possible that this method could be used to develop a highly functional bioartificial liver.

Because human hepatic stellate cells (HSCs) perform a crucial role in the progress of hepatic fibrosis, it is of great value to establish an immortalized human cell line that exhibits HSC characteristics and grows well in tissue cultures for the development of antifibrotic therapies. Thus, we engineered an immortalized human hepatic stellate cell (HSC) line TWNT-4 by retrovirally inducing human telomerase reverse transcriptase (hTERT) into LI 90 cells established from a human liver mesenchymal tumor. Parental LI 90 entered replicative senescence, whereas TWNT-4 showed telomerase activity and proliferated for more than population doubling level (PDL) 200 without any crisis. TWNT-4 expressed platelet-derived growth factor-β receptor (PDGF-βR), α-smooth muscle actin (α-SMA), and type I collagen (α1) and was considered to be an activated form of HSCs. Treatment of TWNT-4 cells with either 100 U/ml of IFN-γ or 1 ng/ml of rapamycin (Rapa) for 14 days led to lower expression of type I collagen (α1) at RNA and protein levels. Exposure of TWNT-4 cells to both of IFN-γ (10 U/ml) and Rapa (0.1 ng/ml) for 14 days effectively decreased the expression of type I collagen (α1), PDGF-βR, and α-SMA expression and suppressed TGF-β1 secretion of TWNT-4 cells. We successfully induced apoptosis by transducing TNF-related apoptosis-inducing ligand (TRAIL) into TWNT-4 cells using adenovirus vectors Ad/GT-TRAIL and Ad/PGK-GV-17. These findings suggested that immortalized activated HSC line TWNT-4 would be a useful means to develop antifibrotic therapies.

Platelets, which contain many growth factors such as platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β), are being used in clinical applications as platelet-rich plasma (PRP). Only a few studies, however, have been conducted on the growth factors present in PRP and on the clinical applications using the drug delivery system (DDS). For the purpose of clinical application, we first modified the PRP preparation method and assessed the amounts of growth factors contained in the human platelet concentrates. Furthermore, we assessed fibrin glue as a DDS of platelet concentrates. Platelet precipitations were made by twice centrifuging human whole blood. The precipitated platelet was resuspended to yield the platelet concentrates. The growth factor concentrations were measured. Fibrin glue sheets containing this platelet concentrate were implanted in rabbit pinna and samples were obtained for immunostaining (anti-PDGF antibody) to assess the use of PRP over time using the fibrin glue as the DDS. The mean concentration of growth factors present in the platelet concentrates was three times or greater than that of conventional PRP. Furthermore, the results indicated that when the platelet concentrate was used with fibrin glue as a carrier, the contents were released over a period of about 1 week. This raises the possibility that this system may be useful in clinical applications.

Regeneration of connective tissue attachment is the ultimate goal of periodontal therapy. It has been suggested that periodontal ligament cells possess the potential to create new connective tissue attachment. However, as cells from gingiva and alveolar bone occupy the root surface during initial wound healing, population by periodontal ligament cells is limited in vivo. We have been developing a new periodontal regeneration technique using in vitro tissue culture of periodontal ligament remaining on a periodontally involved root. The purpose of this study was to examine the periodontal healing after transplantation of teeth with reduced periodontal ligament that had been cultured in vitro. Twenty-five incisors from four beagles were used. After the teeth were extracted, the periodontal ligament and cementum were removed from coronal part of the roots and the roots were planed. The periodontal ligament of the apical part was retained. Fourteen teeth of the experimental group were transplanted following culture for 6 weeks. Eleven teeth of the control group were similarly prepared and immediately transplanted without tissue culture. Four weeks after transplantation, the specimens were prepared for histological analysis. Downgrowth of junctional epithelium on the root of experimental group was significantly less than control. Most of the root planed surfaces of experimental group were covered with periodontal ligament fibers oriented parallel or inclined to the root surfaces and limited new cementum formation was observed near the apical end of the planed root. There was no significant difference between groups in observations on the root surface with remaining periodontal ligament. From the above results, it was concluded that periodontal tissue culture of teeth with root planed surface and remaining periodontal ligament could reduce the extent of epithelium downgrowth and increase connective tissue adhesion on the root planed surface, as well as minimize damage to remaining periodontal ligament, after transplantation of teeth.

The transplantation of a bioartificial pancreas has been regarded as a potential method for successful islet transplantation without any immunosuppressive agents. The subcutaneous site is a very attractive site for transplantation of a bioartificial pancreas because of its advantage of an easy operation site. Our group has been reporting that transplantation of a bioartificial pancreas to the subcutaneous site can reverse hyperglycemia in diabetic recipients. Regarding shapes of a bioartificial pancreas, it is believed that a bag form has an advantage because it is easy to prepare a large quantity. Our group previously reported successful transplantation of a bioartificial pancreas in bag form, a mesh-reinforced polyvinyl alcohol bag (MRPB), implanted in the peritoneal cavity. We also reported that the effect of subcutaneous islet transplantation can be greatly improved with prevascularization treatment. In the present study, we attempted to combine MRPB to our protocol of subcutaneous prevascularization. The main problem of this trial is that the procedure of MRPB implantation injures the prevascularized blood vessel networks. To solve this problem, we made a slight alternation in our protocol, and designed new devices on the basis of MRPB. The new devices, possessing the ability to induce neovascularization, were prepared by collagen coating on the surface of MRPB and were implanted with/without different doses of FGF-2 impregnated in gelatin microspheres. When using 5 μg of FGF-2, more blood vessels were observed on the surface of type I/IV collagen-coated MRPB compared with the original MRPB and type I collagen-coated MRPB. Quite a few blood vessels were observed either around the injection site of 50 μg of FGF-2 impregnated in gelatin microspheres alone or around the implantation site of FGF-2-free gelatin microspheres and type I collagen-coated MRPB or type I/IV collagen-coated MRPB. Here we demonstrated that the combination of both FGF-2 impregnated in gelatin microspheres and collagen-coated MRPB could give an effective system of neovascularization suitable for subcutaneous implantation of a bioartificial pancreas.

In pancreatic islet transplantation, revascularization is crucial for the graft's survival and function. In this study, the endothelium of isolated islets and revascularization and function of islet isografts in diabetic rat were investigated. Islets were isolated from Lewis rats by collagenase digestion method and were examined using immunohistochemistry (CD31 stain) on days 0, 1, 3, and 7 after isolation. The number of CD31-positive cells in these isolated islets was counted (mean ± SD%). Isografts (freshly isolated islets: group A, and islets cultured for 7 days: group B) transplanted in the renal subcapsule of streptozotocin-induced diabetic Lewis rats were examined using immunohistochemistry (CD31 stain) on days 3, 5, and 7 after transplantation. Intravenous glucose tolerance tests (IVGTT) were performed on days 3 and 7 after transplantation. The number of CD31-positive cells in the isolated islets on days 0, 1, 3, and 7 after isolation were: 17.3 ± 4.1%, 8.2 ± 0.7%, 2.1 ± 0.8%, and 0.8 ± 0.5%, respectively (p < 0.05). On day 5 after transplantation, CD31-positive cells were not detected in group A and B grafts, but were detected in both groups in periphery of the islets. On day 7, CD31-positive microvessels were present throughout the entire graft. IVGTT values in groups A and B on days 3 and 7 after transplantation did not show significant differences. In renal subcapsular isografts in diabetic rats, revascularization into islet grafts occurs from the surrounding host tissue 5 days after transplantation, but has no influence on the response to glucose during this period.

Whole pancreas or β-cell transplantation has opened the way for the treatment of advanced stage of diabetes mellitus. However, it is always limited by the scarcity of transplantation materials. The amniotic membrane is part of the fetal membrane and is composed of amniotic epithelium (HAE) and mesenchymal (HAM) cells that are derived from the inner cell mass in the blastocyst. Thus, HAE and HAM cells may have the potential to differentiate into various organs. The aim of our study was to assess the possibility of HAE cells differentiating into insulin-producing cells. In vitro, HAE cells stimulated with nicotinamide induced insulin mRNA in the culture cells. In vivo, HAE cells were capable of normalizing the blood glucose level of diabetic mice after several weeks of implantation into streptozotocin-induced diabetic mice. The distribution of human cells and human insulin secretion in mouse tissue studied by immunohistochemistry for anti-human-specific β-2-microglobulin and anti-human-specific insulin shows the same location in mouse tissue. These studies suggest that HAE cells have the potential to differentiate into β-cells in vivo, and hence that HAE cells have therapeutic potential for the treatment of type I diabetes mellitus.