Cell Transplantation

The number of diabetic patients in the world is increasing in recent years and the prevention of diabetes mellitus is therefore one of the urgent medical issues. Exogenous insulin is used for the control of blood glucose in diabetic patients; however, hypoglycemic episodes are unavoidable. Over the last several decades, islet transplantation has been developed as a promising method to achieve strict control of blood glucose and a potential cure for type 1 diabetes. However, due to the shortage of donor pancreata, alternative sources of islets have been sought through the generation of beta cells from stem cells, use of porcine islets, and beta cell expansion with growth factors. However, differentiation and expansion of embryonic and pancreatic stem cells and expansion of differentiated beta cells in vitro is limited. Expansion of primary beta cells by growth factors is also hampered by the senescence of the cells. Thus, we focused on establishing a human pancreatic beta cell line that is functionally equivalent to primary beta cells and can yield large amounts of cells for transplantation. Using Cre/loxP-based reversible immortalization, we constructed a reversibly immortalized pancreatic beta cell clone (NAKT-15). The cells may overcome the limitation of primary pancreatic beta cells for transplantation to control type 1 diabetes. In order to avoid the use of immunosuppressive agents, we are currently engaged in developing an implantable bag-type bioartificial pancreas. In this article, I discuss the hurdles of the current therapy for diabetes and introduce the possible future treatment of diabetes.

Since Berry and Friend developed methods to isolate hepatocytes from the liver by a collagenase digestion technique in 1969, studies in laboratory animals have demonstrated that hepatocyte transplantation could potentially be used for the treatment of liver failure and inborn errors of liver-based metabolism. Healthy human hepatocytes are an ideal source for hepatocyte transplantation; however, their relative scarcity is one of the major drawbacks, further compounded by the competing demands of liver transplantation. Notably, most of the hepatocytes are isolated from discarded livers that are not suitable for organ transplantation for a variety of reasons, including excessive fat content. Importantly, the hepatocyte isolation procedure itself exerts major stress on hepatocytes by the disruption of cell-to-cell and cell-to-matrix contacts, resulting in hepatocytic apoptosis. Prevention of apoptosis would maximize yield of healthy cells and maintain hepatocyte differentiated function in culture. In this review, we describe methods to prevent apoptosis by utilizing both antiapoptotic molecules and matrices. We also introduce a new type of liver tissue engineering, hepatocyte sheet transplantation, which utilizes unwoven cloth having a cellular adhesive property.

Human embryonic stem (hES) cells have the ability to differentiate into a variety of different cell lineages and potentially provide a source of differentiated cells for many therapeutic uses. Here we investigated an efficient method of hepatic differentiation from hES cells. A human ES cell line, KhES-1, was used and maintained by a nonfeeder method. KhES-1 cells were cultured for 5 days in the presence of human activin A (50 ng/ml) and then treated with a deleted variant of hepatocyte growth factor (dHGF) at 0, 100, or 500 ng/ml for 7 days. The resultant cells were biologically analyzed. The expression of the endodermal genes SOX17 and FOXA2 increased in KhES-1 cells after activin A treatment. In contrast, Oct4, a self-renewal undifferentiated marker, decreased in a time-dependent manner in KhES-1 cells. Following a 7-day treatment of the resultant cells with dHGF, especially at 500 ng/ml, KhES-1 cells showed an expression of the hepatic makers albumin, AFP, and CK18. Transitional electron microscopy showed well-developed glycogen rosettes and a gap junction in KhES-1 cells treated with 500 ng/ml of dHGF. We developed an efficient method to differentiate KhES-1 cells into hepatocyte-like cells in vitro using 50 ng/ml of activin A and 500 ng/ml of dHGF.

We developed a bioartificial liver (BAL) containing human hepatoblastoma cell line, HepG2, with the addition of ammonia removal activity by transfecting a glutamine synthetase (GS) gene and estimated the efficacy using pigs with ischemic liver failure. GS-HepG2 cells showed 15% ammonia removal activity of porcine hepatocytes, while unmodified HepG2 had no such activity. The established GS-HepG2 cells were grown in a circulatory flow bioreactor to 3.5–4.1 × 109 cells. Survival time of the animals treated with GS-HepG2 BAL was significantly prolonged compared to the cell-free control (14.52 ± 5.24 h vs. 8.53 ± 2.52 h) and the group treated with the BAL consisting of unmodified wild-type HepG2 (9.58 ± 4.52 h). Comparison showed the cell-containing BAL groups to have significantly fewer incidences of increased brain pressure. Thus, the GS-HepG2 BAL treatment resulted in a significant improvement of survival time and pathological parameters in pigs with ischemic liver failure.

Green tea polyphenols have recently attracted medical attention as bioactive agents with anticancer, antimicrobial, and antiviral effects. We discovered their new usage as preservative agents for tissue transplants. We preserved rat aortas in a DMEM solution containing polyphenols extracted from green tea leaves. The preserved aortas retained original structures and mechanical strength, and were devoid of any undesirable cell secretions for over a month under physiological conditions. In addition, aortas from Lewis rats preserved for a month and transplanted to allogenic ACI rats completely avoided rejection by the host, suggesting that the polyphenols have immunosuppressive actions on the aortic tissues. From these results, we conclude that polyphenol treatment of aortic tissue transplant can maintain its viability for extended periods of time either before or after transplantation, and the method can be applicable to other transplantation situations.

Successful immunosuppressive therapy is critical for liver transplantation. However, a considerable number of patients show clinical resistance to the therapy and experience rejection episodes, or alternatively exhibits serious adverse effects of drugs. We examined the in vitro response of peripheral blood mononuclear cells (PBMCs) to immunosuppressive drugs in cirrhosis patients awaiting liver transplantation. We evaluated the suppressive efficacy of prednisolone, methylprednisolone, cyclosporine, and tacrolimus on the in vitro blastogenesis of PBMCs obtained from 22 cirrhosis patients and 31 healthy subjects. In vitro drug concentrations giving 50% inhibition of PBMC blastogenesis (IC₅₀s) were calculated. Two out of these 22 patients received liver transplantation from living donors, and their clinical courses were surveyed until 5 weeks after operation. The median IC₅₀ values for prednisolone, cyclosporine, and tacrolimus against blastogenesis of PBMCs from cirrhosis patients were significantly lower than those of PBMCs from healthy subjects (p < 0.01). However, large individual differences were observed in the IC₅₀ values of the immunosuppressive drugs examined, especially in the cirrhosis patients. One recipient exhibiting high PBMC sensitivity to tacrolimus (IC₅₀ = 0.001 ng/ml) showed good clinical course without rejection until 5 weeks after liver transplantation. The other recipient exhibiting relatively low PBMC sensitivity to taclolimus (IC₅₀ = 0.30) showed allograft rejection at 1 week after operation. We concluded from these observations that PBMCs of cirrhosis patients are vulnerable to the immunosuppressive effects of prednisolone and calcineurin inhibitors. However, large individual variations in the IC₅₀ values suggest that patients exhibiting relatively lower sensitivity to these drugs may have risks of rejection, whereas highly sensitive patients are possibly able to reduce the dose of immunosuppressive drugs to avoid serious drug-adverse effects, after liver transplantation.

Development of a subcutaneously implantable bioartificial pancreas (BAP) with immunoisolatory function could have a great impact on the treatment of diabetes mellitus. We have developed an implantable BAP device with an ethylene vinyl alcohol (EVAL) membrane. In the present study, we used basic fibroblast growth factors (bFGF), which was incorporated in a carrier for sustained release, in order to induce neovascularization when the device was implanted subcutaneously. To maintain the vasculature thus formed, a cell infusion port was attached to the BAP device, through which the device was filled with human liver vascular endothelial cell line TMNK-1, and the vasculature could be adequately maintained. Mice were divided into the following three groups. In group 1, a bFGF-free BAP device was implanted subcutaneously. In group 2, a sustained-release bFGF-impregnated BAP device was implanted. In group 3, a sustained-release bFGF-impregnated BAP device was implanted, and 3 × 106 TMNK-1 cells were infused into the implanted device every week. Neovascularization induced in the subcutaneous tissue around the implanted BAP device was macroscopically examined and histologically evaluated. In addition, the tissue blood flow was measured using a laser blood flow meter. In mice in group 3, neovascularization was significantly induced and maintained until week 8 postimplantation. It was confirmed by scanning electron microscopy that infused TMNK-1 cells adhered to the inner polyethylene surface of the device. It was demonstrated that the use of bFGF and vascular endothelial TMNK-1 cells induced and maintained adequate vasculature and tissue blood flow surrounding the implantable bag-type BAP device. We believe that the present study will contribute to BAP development for the treatment of diabetes.

Artificial bones have often used for bone regeneration due to their strength, but they cannot provide an adequate environment for cell penetration and settlement. We therefore attempted to explore various materials that may allow the cells to penetrate and engraft in bone defects. PuraMatrix™ is a self-assembling peptide scaffold that produces a nanoscale environment allowing both cellular penetration and engraftment. The objective of this study was to investigate the effect of PuraMatrix™ on bone regeneration in a mouse bone defect model of the calvaria. Matrigel™ was used as a control. The expression of bone-related genes (alkaline phosphatase, Runx2, and Osterix) in the PuraMatrix™-injected bone defects was stronger than that in the Matrigel™-injected defects. Soft X-ray radiographs revealed that bony bridges were clearly observed in the defects treated with PuraMatrix™, but not in the Matrigel™-treated defects. Notably, PuraMatrix™ treatment induced mature bone tissue while showing cortical bone medullary cavities. The area of newly formed bones at the site of the bone defects was 1.38-fold larger for PuraMatrix™ than Matrigel™. The strength of the regenerated bone was 1.72-fold higher for PuraMatrix™ (146.0 g) than for Matrigel™ (84.7 g). The present study demonstrated that PuraMatrix™ injection favorably induced functional bone regeneration.

Factors affecting cell viability, plating efficiency, and survival of hepatocytes after cryopreservation have been investigated. We focused especially on the effect of including trehalose and related oligosaccharides in the cryopreservation fluid. This was supplemented with either glucose, trehalose, maltotriose, or other sugars, in addition to dimethyl sulfoxide (10%) and first tested with primary rat hepatocytes cooled in a controlled rate freezer. After thawing, viability by trypan blue exclusion of cells frozen in oligosaccharide-supplemented medium was significantly higher than for those cryopreserved without oligosaccharides. Use of oligosaccharides with higher molecular weights resulted in greatest improvement in viability. Moreover, attachment and survival rates in plastic dishes were approximately 1.2–1.8-fold greater after freezing in the presence of di-, tri-, and tetrasaccharides. Human hepatocytes isolated from untransplantable liver showed the same tendency regarding viability, but cell adherence was not similarly improved by the addition of oligosaccharides. Possible reasons for these differences may be prior cell damage during extended cold ischemia of the human liver, donor age, or cell degradation caused by progression of fatty liver in humans, and/or species differences.

Freshly isolated porcine hepatocytes are a very attractive cell source in the cell-based therapies to treat liver failure because of unlimited availability. However, due to the loss of hepatocyte functions in vitro, there is a need to develop a functional culture system to keep the cells metabolically active. Here we compared the effect of a self-assembling peptide nanofiber (SAPNF) as an extracellular matrix (ECM) with collagen type I on hepatocyte metabolic and secretion activities following hepatocyte isolation. Isolated porcine hepatocytes were cultured in SAPNF and collagen type I. Morphological assessment at different time points was performed by using SEM and phase contrast microscope. Metabolic and secretion activities were comparatively performed in the groups, by means of ammonia, lidocaine, and diazepam as well as albumin. Hepatocytes cultured on SAPNF revealed a three-dimensional spheroidal formation, thus maintaining cell differentiation status during 2 weeks of culture. On the other hand, hepatocytes in collagen revealed a spread shape, and by day 14 no hepatocyte-like cells were observed, but cells with long shape were present, thus revealing a degree of dedifferentiation in collagen culture. Hepatocytes in SAPNF were capable of drug-metabolizing activities and albumin secretion in higher ratio than those cultured on collagen. The present work clearly demonstrates the usefulness of SAPNF for maintaining differentiated functions of porcine hepatocytes in culture.

β-Cell replacement therapy via islet transplantation is a promising possibility for the optimal treatment of type 1 diabetes; however, such an approach is severely limited by the shortage of donor organs. This problem could be overcome if it were possible to generate transplantable islets from stem cells. We showed previously that adult β-cells might originate from duct or duct-associated cells. Ductal progenitor cells in the pancreas would become particularly useful for therapies that target β-cell replacement in diabetic patients, because duct cell types are abundantly available in the pancreas of these patients and in donor organs. In this study, we examined which embryonic transcription factors in adult mouse and human duct cells could efficiently induce their differentiation into insulin-expressing cells. Infection with the adenovirus expressing PDX-1, Ngn3, NeuroD, or Pax4 induced the insulin gene expression. NeuroD was the most effective inducer of insulin expression in primary duct cells. Surprisingly, adenovirus Pax4 strongly induced Ngn3 expression, while Pax4 is considered the downstream target of Ngn3. These data suggest that the overexpression of transcription factors, especially NeuroD, facilitates pancreatic stem/progenitor cell differentiation into insulin-producing cells.

Fibroblast growth factor (FGF)-2 has been recognized to be a key element involved in angiogenesis and a putative factor involved in stem cell-mediated islet regeneration. However, the usefulness of FGF-2 in an islet transplantation setting has not yet been explored. We therefore evaluated the effect of FGF-2 on both islet culture and islet transplantation. Isolated islets were cultured in the presence of 100 ng/ml FGF-2 for a week and then the glucose-responding insulin secretion and insulin contents were measured. Gelatinized FGF-2 (100 ng), which allowed the controlled release of FGF-2, was used for islet transplantation of streptozotocin-induced diabetic mice. Islets (150 IEQ), obtained from a single donor, mixed with gelatinized FGF-2, were transplanted into the subrenal capsule of the mice and the animals were observed for 30 days. Revascularization around the islet grafts was examined. The blood glucose levels were measured and the intraperitoneal glucose tolerance test (IPGTT) was performed. The supplementation of FGF-2 maintained proper insulin secretion and insulin contents in an in vitro culture. The use of gelatinized FGF-2 facilitated revascularization and favorable islet engraftment, thus resulting in an amelioration of the blood glucose levels in diabetic mice. The utilization of FGF-2 showed increased contents of insulin in the islet grafts and revealed a similar pattern as that of normal healthy mice in IPGTT. In contrast, the transplantation of islets without FGF-2 supplementation showed poor revascularization and failed to control the blood glucose levels in the diabetic mice.

Functional demands on a bioartificial liver support (BAL) device are not limited to biosynthetic activities, but must also encompass metabolic removal of potentially toxic substances. For most BALs, however, the concept and design are exclusively directed to biosynthetic support. To add the ability to metabolize and remove toxic substances, we designed a double-compartment cell culture apparatus (DCCA). Two compartments are separated from each other by a compact epithelial cell sheet spread over a synthetic microporous membrane. When a renal proximal convoluted tubular cell line that had been transduced with the human multidrug-resistant (MDR) gene, PCTL-MDR, was introduced into one of the compartments (hereafter referred to as the “inner” compartment) of the DCCA, a compact cellular monolayer was formed on the membrane. Ammonium ions passed across the membrane, but glucose and its metabolite lactate could not, indicating that the DCCA allowed selective transportation of cellular metabolites. In addition to PCTL-MDR, HepG2, a cell line of hepatic-origin, transduced with CYP3A4 (designated GS-3A4-HepG2), was seeded on the opposite side of the membrane, and the metabolism and transportation of lidocaine were studied. The lidocaine metabolite, monoethylglycinexylidide, was detected in the inner compartment across the PCTL-MDR cell layered membrane, indicating that metabolism and the selective transportation of metabolites between the two compartments occurred by cooperation of renal and hepatic cells. These results suggest that this type of DCCA represents a novel BAL that possesses biotransporting activities, as well as biosynthetic and metabolic activities.