Cell Transplantation

In developing cell therapy, normal human cells are ideal as a cell source, but considering the serious lack of donor organs, it is unlikely to obtain a large enough amount of human cells. Moreover, even with current culturing techniques, the long-term culturing of normal human cells is difficult. On the other hand, in using xenogenic porcine cells and human tumor tissue-derived cell lines, there is concern that species-specific pathogens can be transmitted (such as infection by porcine endogenous retroviruses), and possible cancer may thus develop in recipients. Therefore, we are making efforts toward establishing reversible immortalized human cell lines that can be economically grown in tissue culture using the techniques of gene transfer in order to solve these problems. I here describe a strategy for establishing human reversibly immortalized cell lines that are intended for practical application in cell therapies. I would like to further contribute toward the realization of tissue engineering in fusional coordination with cell-processing technology by the utilization of such cell line constructing techniques.

Recent advances in islet transplantation using highly purified islets and effective immunosuppression strategies have resulted in substantial improvement in achieving insulin independence in type 1 diabetes patients. However, there are side effects from long-term immunosuppression, and transplant rejection and/or the recurrence of autoimmune attack of the transplanted islets cannot be completely prevented, even with immunosuppressive treatment. Therefore, construction of a safe and functional bioartificial pancreas (BAP) that provides an adequate environment for islet cells may be an important approach to treat diabetic patients. Various types of BAP devices have been developed and examined in animals. In this review, I introduce the previous BAP studies and our approach of BAP development.

The concept of a “cancer stem cell system” that continues to supply cancer component cells has been proposed. It is time to apply stem cell studies, which is a field of expertise in regenerative medicine, to cancer treatment. Cancer treatments that effectively attack cancer stem cells acting as a manufacturing plant for producing differentiated cancer progenies will be designed by revealing the cancer stem cell system. Therefore, in the near future we hope that revolution will occur in cancer therapy to eradicate cancer and prevent the recurrence thereof. In this review we discuss the current situation and problems of cancer stem cell research.

Human pluripotent embryonic stem cells (hESCs) have great promise for research into human developmental biology, development of cell therapies for the treatment of diseases, toxicology, and drug discovery. Traditionally, undifferentiated hESCs are maintained on mouse embryonic fibroblasts (MEFs), which impede the clinical applications of hESCs. Here we have examined the long-term stability of the Japanese hESC line (KhES-1) in feeder-free culture. KhES-1 cells were cultured with MEF conditioned medium (CM) and different doses of basic fibroblast growth factor (bFGF) in six-well-plates of which the surface was coated with Matrigel. KhES-1 cells were maintained for at least 40 passages. In this culture system, the cells maintained stable proliferation rates and steadily expressed Oct-4, Nanog, and alkaline phosphatase. In addition, KhES-1 cells maintained without direct feeder contact formed embryonic bodies with expression of markers from the three germ layers. Here we demonstrated that Japanese human embryonic stem cells KhES-1 were cultured long term in a feeder-free method, while retaining pluripotency in vitro.

Adipose tissue-derived stem/progenitor cells (ASCs) have been reported to differentiate not only into mesodermal cells such as osteoblasts, chondorocytes, and adipocytes, but also to endodermal cells such as hepatocytes and insulin-expressing cells. These stem/progenitor cells are expected to be used for variety of regenerative therapies. This study demonstrates the viability and the adipo/osteogenic potential of cryopreserved ASCs using seven cryopreservation solutions, including 10% DMSO, Cell Freezing Medium-DMSO, Cell Freezing Medium-Glycerol, Cell Banker 1, Cell Banker 1+, Cell Banker 2, and CP-1. ASCs were obtained from mouse subcutaneous adipose tissue. The viability of the cryopreserved ASCs was over 90% with Cell Banker 2 preservation, approximately 90% with Cell Banker 1, Cell Banker 1+, or CP-1 preservation, and less than 80% for 10% DMSO, Cell Freezing Medium-DMSO, or Cell Freezing Medium-Glycerol preservation. No difference in the adipo/osteogenic potential was found between cells with or without cryopreservation in Cell Banker 2. These data suggests that Cell Banker 2 is the most effective cryopreservation solution for ASCs and that cryopreserved as well as noncryopreserved ASCs could be applied for regenerative medicine.

Adipose tissue-derived stem cells (ASCs) are expected to have clinical applications as well as other stem cells, because ASCs can be obtained safely from adult donors and used in autologous therapies without concern about rejection and the need for immunosuppression. However, the use of gene transfer with Sendai virus (SeV) vectors, which can efficiently introduce foreign genes without toxicity into several cells, with ASCs has not yet been investigated. This study documents on the use of SeV vectors for gene transfer to ASCs. The dose-dependent GFP expression of ASCs transfected with SeV vectors after 48 h of culture at 37°C was first evaluated. Next, the cellular toxicity of ASCs transfected with SeV vectors was verified. In addition, SeV vectors were compared with adenovirus (AdV) vectors. Finally, the time-dependent GFP expression of ASCs transfected with SeV vectors was evaluated. The results showed that transfection of ASCs with SeV vectors results in more efficient expression of transgene (GFP expression) in the ASCs than with AdV vectors after 48 h of culture at 37°C. Moreover, while the transfection of ASCs with AdV vectors at high MOIs was cytotoxic (a lot of transfected cells died) that of ASCs with SeV vectors at high MOIs was not necessarily cytotoxic. In addition, the preservation of multilineage ASCs transfected with SeV was observed. In conclusion, this is the first report describing the successful use of SeV-mediated gene transfer in ASCs, and the results indicate that SeV may thus provide advantages with respect to safety issues in gene therapy.

The term “immunoisolation” refers to the encapsulation of a graft in a selectively permeable membrane. Encapsulation of cellular grafts may provide a way to protect the graft from immune attack without the need for immunosuppressive agents. Although numerous types of artificial materials have been used for encapsulating membranes, their incomplete biocompatibility causes foreign body reaction against the membranes. A new technique has been developed, called cell sheet engineering using temperature-responsive culture dishes, that allows the use of living cells as an immunoisolating membrane in this study. Using this method, the cultured cells can be easily harvested in the shape of a sheet by a simple change of the temperature without the use of proteolytic enzymes. A cell sheet can be created with three-dimensional structure by making multiple cell sheet layers. In this study, a new technique of macroencapsulation (bioartificial organs) has been developed using chondrocyte sheets. Among the various candidate cells, pancreatic islet cells were selected for a bioartificial organ in this study. A chondrocyte sheeting immunodelusive immunoisolated bioartificial pancreas (CSI-BAP) was manufactured by means of cell sheet engineering. An auricular cartilage, which is a histologically elastic cartilage from dogs (beagle), was used as a source of immunoisolating membrane. CSI-BAP was made by multilayering the chondrocyte sheets, and the donor's islets were located between each sheet. Islets were isolated and prepared from the dog (ALLO-model) and Brown Norway (BN) rat (XENO-model). The CSI-BAP was cultured for 83 days and the cultured medium was collected every 24 h to measure the insulin concentrations. The CSI-BAP was examined histologically using hematoxyhin and eosin (H&E), and azan dye staining. In addition, immunohistochemical staining was performed to demonstrate the insulin production of CSI-BAP. Insulin secretion of CSI-BAP on day 16 was reduced to 21.4% of the insulin secretion level of day 10, which was the start point of measurement. Although a gradual reduction was observed, insulin secretion was maintained for 3 months. The CSI-BAP was capable of secreting insulin to the culture medium during the observation period. Histological evaluations demonstrated the good viability of the islets, and immunohistochemistry showed the positive staining of insulin. This novel technology may be used for other kinds of endocrine cells or hepatocytes, which may become the models for immunoisolated bioartificial organs in the near future.

Although widely used, DMSO is toxic for pancreatic islets. We combined hydroxyethyl starch (HES) with DMSO to simplify the procedure of freezing and thawing, and to decrease the toxicity of DMSO. A preclinical study was performed using islets from beagle dogs. After storage for 4 weeks, the islets were thawed and examined. The islet structure was well maintained after thawing. Although the number of the islets decreased to 71.2 ± 20.1%, the function of the islets was evaluated by static incubation after thawing and showed a 1.80 ± 0.78 stimulation index. We have introduced this technique for the cryopreservation of human islets from non-heart-beating donors. Twelve cases of human islet cryopreservation were performed. The sample tube of each human cryopreservation was thawed to evaluate the morphology, contamination, and endocrine function. Although fragmentation was observed in five samples (41.6%), the other seven (58.4%) showed a normal structure when evaluated by microscopic and electron microscopic study. The stimulation index (SI) of static incubation deteriorated from 3.37 ± 3.02 to 1.34 ± 0.28 after thawing. We divided the thawed islets into two groups: group 1 (n = 8), SI >1.2; group 2 (n = 4), SI <1.2. The group 1 islets showed a higher rate of normal structure (87%) than did group 2 (25%). Moreover, the SI before cryopreservation was 4.01 ± 3.57 in group 1, which was higher than the SI of 2.11 ± 0.72 in group 2. Based on the good results from the preclinical study using a large-animal model, this method was introduced for clinical application. Even from the pancreata of non-heart-beating donors, a successful islet cryopreservation was achieved. However, the isolated islets with poor function should not be cryopreserved for transplantation.

For islet transplantation, it is important to obtain an available islet mass adequate for diabetes reversal from a single donor pancreas. A recent report demonstrated that the use of M-Kyoto solution instead of UW solution improved islet yields in the two-layer method for pancreas preservation. The present study investigated whether the ductal injection of a large volume of preservation solution (UW and M-Kyoto solution) before pancreas storage improves islet yields. Islet yield both before and after purification was significantly higher in the ductal injection (+) group compared with the ductal injection (–) group. TUNEL-positive cells in the ductal injection (+) group were significantly decreased in comparison to the ductal injection (–) group. The ductal injection of preservation solution increased the ATP level in the pancreas tissue and reduced trypsin activity during the digestion step. Annexin V and PI assays showed that the ductal injection prevents islet apoptosis. In a transplant model, the ductal injection improved islet graft function. These findings suggest that the ductal injection of preservation solution, especially the M-Kyoto solution, leads to improved outcomes for pancreatic islet transplantation. Based on these data, this technique is now used for clinical islet transplantation from non-heart-beating donor pancreata or living donor pancreas.

The inert fluid perfluorocarbon (PFC) has been used since about 1960 in liquid respiration and artificial blood for mammals. PFC has been used to successfully resuscitate tardigrades that had been dried and exposed to a high barometric pressure of 6,000 atmospheres. Next, scientists attempted to experimentally preserve organs that had been removed from animals, dried, and immersed in PFC. Since 1998 preservation and resuscitation experiments have been conducted with mammalian hearts using 2,015 rats and 70 pigs. Among those experiments, the maximum time after desiccation until successful resuscitation was 26 days for a rat heart and 37 days for a pig heart. However, these results could not be reproduced. Finally, in 2005, this laboratory demonstrated that a rat heart removed under 2 atmospheres pressure and a CO₂ partial pressure of 400 hPa, followed by desiccation for 24 h, could be revived and heterotypically transplanted. Moreover, these results were reproducible. The preservation time can be extended to 72 h if, after immersing isolated rat hearts in PFC, they are dried by air exposure under a CO₂ partial pressure of 100 hPa. The present report documents the resuscitation of this heart after 72 h of preservation followed by heterotrophic transplantation.

Although islet transplantation is a promising therapeutic option for the treatment of type 1 diabetes, the shortage of suitable donor tissues remains a major obstacle. Pancreatic stem/progenitor cells residing within the ductal epithelium have been used to generate human islet-like clusters, but there is no efficient strategy for facilitating differentiation of progenitor cells into insulin-producing cells. A previous study reported that exogenous PDX-1 protein can be transduced into pancreatic stem/progenitor cells and induce differentiation of the cells into insulin-producing cells without requiring gene transfer technology. This study provides genetic and biochemical evidence that cell membrane heparan sulfate proteoglycans are required for extracellular PDX-1 internalization. Heparin, one of the soluble glycosaminoglycans (GAGs), inhibited PDX-1 internalization, while chondroitin sulfate A, B, and C caused only very limited inhibition. Cell treatment with heparinase-III demonstrated impaired PDX-1 internalization, while treatment with chondroitinase ABC, or with chondroitinase AC, was completely ineffective in inhibiting PDX-1 internalization. Different mutant cell lines originating from CHO K1 cells and defective in GAG biosynthesis were also examined. PDX-1 internalization was significantly reduced in both pgs A-745 mutant cells, which are defective in a enzyme that initiates GAG synthesis, and pgs B-618 cells, which produce about 15% of the amount of GAGs synthesized by wild-type cells. These data indicate that cell-surface heparan sulfate proteoglycans are required for PDX-1 internalization and that PDX-1 protein transduction could be a valuable strategy for inducing insulin expression in pancreatic stem/progenitor cells without requiring gene transfer technology.

ET-Kyoto solution (ET-K) is an extracellular-type organ preservation solution containing the cytoprotective disaccharide, trehalose. A previous study reported the supplement of dibutyryl cyclic adenosine monophosphate (db-cAMP) in conventional ET-K to attenuate lung ischemia-reperfusion injury. In this study, the efficacy of this modified ET-K for liver preservation was investigated by comparison with University of Wisconsin solution (UW). ET-K was supplemented with db-cAMP (2 mmol/L). Lewis rats were randomly assigned to two groups, and liver grafts were flushed and stored at 4°C for 24 h with ET-K or UW before syngeneic liver transplantation. The graft function and histological changes at 4 h posttransplant as well as 7-day survival were evaluated. Recipient rat survival rate was significantly higher in the ET-K group than in the UW group. Preservation in ET-K resulted in a significant reduction in serum parenchymal transaminase level and promotion of bile production in comparison with UW. The serum hyaluronic acid level, an indicator of sinusoidal endothelial cell injury, was significantly lower after ET-K preservation than that in UW. Histologically, at 4 h after transplantation, the liver grafts preserved in UW solution demonstrated a greater degree of injury than those in ET-K, which appeared to be apoptosis, rather than necrosis. The continuity of the sinusoidal lining was better preserved in ET-K than in UW. In conclusion, ET-K supplemented with db-cAMP is superior to UW in rat liver preservation. This modified ET-K might therefore be a novel candidate for the procurement and preservation of multiple organs.

Islet transplantation is associated with an elevated rate of early graft failure. The isolation process leads to structural and functional abnormalities. The reestablishment of the cell–matrix relationship is important to modulate the survival and function of islets. Thus, we evaluated the effect of human fibronectin (hFN) and self-assembling peptide nanofiber (SAPNF) in the ability to support islet function in vitro and after transplantation into streptozotocin (STZ)-induced diabetic severe combined immunodeficiency (SCID) mice. Human isolated islets were cultured with hFN or SAPNF for 7 days. Their ability to maintain insulin production/glucose responsiveness over time was evaluated. Islets embedded in hFN, SAPNF, or alone were transplanted into STZ-induced diabetic SCID mice. Islet grafts were removed after 14 days to evaluate insulin content, insulin expression, and apoptosis. SAPNF-entrapped islets maintained satisfactory morphology/viability and capability of glucose-dependent insulin secretion for over 7 days, whereas islets cultured in hFN underwent widespread deterioration. In vivo grafts containing human islets in SAPNF showed remarkably higher insulin content and expression when compared with human islets in hFn or alone. RT-PCR revealed lower caspase-3 expression in SAPNF islets grafts. These studies indicate that the reestablishment of the cell–matrix interactions by a synthetic matrix in the immediate postisolation period is a useful tool to maintain islet functions in vitro and in vivo.

The evaluation of engraftment is important to assess the success of islet transplantation, but it is complex because islet transplantation usually requires two or more donors to achieve euglycemia. Islet transplantation from NHBDs was evaluated using new assessment forms for the secretory unit of islet in transplantation (SUIT) and engrafted islet rate (EIR) indexes. Insulin independence was obtained when the SUIT index was more than 28, which might indicate that 28% of the β-cell mass of a normal subject is required for insulin independence. Because the average EIR for a single transplantation is about 30, the percentage of engrafted islets following one transplantation is about 30%, assuming that a normal subject has 1 million islet equivalents. Although few cultured islet transplants have been performed, the increase of the SUIT and EIR indexes in patients who received cultured islets was significantly lower than in patients who received fresh islets, suggesting that fresh islets may be more effective than cultured islets. The SUIT and EIR indexes are thus considered to be useful values for evaluating islet transplantation, especially for single islet transplantation.

Because of a worldwide shortage of renal grafts, kidneys procured from donors after cardiac death (DCD) have recently become an important source of renal transplants. However, DCD kidneys often have complications with delayed graft function (DGF) and recipients require hemodialysis (HD) in the early period after kidney transplantation (KTx). This study evaluated serum NGAL as a potential specific parameter to predict early functional recovery of transplanted DCD kidneys. The average serum neutrophil gelatinase-associated lipocalin (NGAL) level in normal samples was 53 ± 30 ng/ml, while that in patients with chronic renal failure requiring HD was markedly raised at 963 ± 33 ng/ml. In patients undergoing a living-related KTx from a living donor (n = 11), serum NGAL level decreased rapidly after KTx, and only in two cases, with serum NGAL levels over 400 ng/ml on postoperative day 1 (POD1), was HD required due to DGF. In contrast, all patients undergoing a KTx from a DCD (n = 5) required HD due to DGF. Even in these cases, serum NGAL levels decreased rapidly several days after a KTx prior to the recovery of urine output and preceding the decrease in serum creatinine level. The pattern of decline in serum NGAL was biphasic, the decrease after the second peak indicating a functional recovery within the next several days. These data suggest that monitoring of serum NGAL levels may allow us to predict graft recovery and the need for HD after a KTx from a DCD.

Several studies have shown that erythropoietin (EPO) can protect the kidneys from ischemia-reperfusion injury and can raise the hemoglobin (Hb) concentration. Recently, the EPO molecule modified by carbamylation (CEPO) has been identified and was demonstrated to be able to protect several organs without increasing the Hb concentration. We hypothesized that treatment with CEPO would protect the kidneys, partly due to the increased peritubular capillaries. The therapeutic effect of CEPO was evaluated using an endothelial tube formation assay in vitro, and a rat ischemia-reperfusion injury model in vivo. EPO treatment showed the tendency of increased tube formation, while CEPO treatment induced more capillary-like formation than EPO. Ischemia-reperfusion-induced kidneys exhibited characteristic nuclei of apoptosis in tubular epithelial cells with decreased peritubular capillaries, while EPO treatment inhibited tubular apoptosis with preserved endothelial cells. Moreover, CEPO-treated kidneys showed minimal tubular apoptosis with increased peritubular capillary endothelial cells. In conclusion, we identified a new therapeutic approach using CEPO to protect kidneys from ischemia-reperfusion injury by promoting angiogenesis.

The replacement of a necrotic tubular epithelium with functional tubular epithelial cells is required for recovery from acute renal failure (ARF). A rat renal progenitor-like (rKS56) cell line was recently established derived from the S3 segment of renal proximal tubules. The therapeutic efficacy of rKS56 cells was examined in a rat model of cisplatin-induced ARF. rKS56-lacZ cells expressing β-galactosidase were injected into SD rats either at the subcapsule of the left kidney (rKS-SC) or via the left renal artery (rKS-IA) 2 days after the injection of cisplatin. Bluo-gal(+) rKS56-lacZ cells were observed in the subcapsule in the rKS-SC group on day 5, and were further increased in number on day 9, accompanied by partial distribution in the corticomedullary junction, but not in the rKS-IA group. A portion of Bluo-gal(+) cells coexpressed Ki-67, aquaporin-1, hepatocyte growth factor (HGF), and c-Met. rKS-SC treatment significantly improved the tubular injury scores, ameliorated tubular cell apoptosis, and induced cell proliferation. The renal function also significantly improved in the rKS-SC group on day 5. These results demonstrate that locally implanted rKS56 cells could differentiate into tubular epithelial cells, thereby accelerating the recovery from tubular injury, most likely by producing tubular trophic factors. These results suggest the therapeutic potential of this novel approach for patients with end-stage renal failure.

Anionic constituents in the peritubular capillary basement membranes and the glomerular endothelial cells have been demonstrated to function as a size- and charge-selective barrier. Cationic colloidal iron staining of human biopsy specimens revealed a glycocalyx on the surface of the glomerular basement membrane (GBM), peritubular capillary (PTC) endothelial cells, and brush border of the tubular epithelial cells of normal kidney. However, the glycocalyx was abolished in the PTC wall of C4d-positive acute humoral rejected kidney, and in the GBM as well as the PTC wall of a chronic, allograft, nephropathy kidney. In addition, cyclosporine eliminated the glycocalyx in the PTC wall, while treatment with heparin inhibited the elimination of the PTC glycocalyx. In conclusion, the glycocalyx on the surface of the GBM and PTC is an important component in the endothelial cell barrier.

The shortage of organ donors has impeded the development of human hepatocyte transplantation. Immortalized hepatocytes could provide an unlimited supply of transplantable cells. To determine whether immortalized hepatocytes could provide global metabolic support in end-stage liver disease, rat hepatocyte clones were developed by transduction with the gene encoding the Simian virus 40 T antigen (SVT) using the human artificial minichromosome (HAC). The SVLT sequence was excised by FRT recombination. Following HAC infusion, the transduced hepatocytes express SVT, blasticidine resistance (BS), and the PGK promoter TK gene. Forty-six cell clones were obtained and at least partially characterized, as previously described, for albumin, α-1-antitrypsin, glucose-6-phosphatase (G6Pase), dipeptidylpeptidase 4 (Dpp4), γ-glutamyltransferase 1 (Ggt), SVT, and β-actin expression using RT-PCR. Clones were also assessed for albumin secretion into the culture medium using ELISA. All of the cell line secreted approximately 10 mg/dl of albumin, which is equivalent to the amount secreted by primary hepatocytes. In further experiments, this cell line will be used for transplantable cells or artificial organ using HAC. These results represent an important step toward the development of immortalized hepatocytes.

To complete a successful liver transplantation (LTx) from non-heart-beating donors (NHBD), it is necessary to both improve the energy status in liver grafts and to reduce the exposure to free radicals. This study investigated the effects of short perfusion with oxygenated buffer on the grafts prior to cold preservation. In addition, the effects of the antioxidant, biliverdin, for reduction of free radicals was investigated. Male Wistar rats were used. Livers were retrieved, preserved in UW solution, and perfused for 60 min with oxygenated Krebs-Henseleit solution. Rats were allocated to six groups as follows (n = 5): i) control group—no warm ischemia (WI) and cold preservation, ii) HBD group—no WI with cold preservation for 6 h; iii) NHBD group—with 30 min of WI and cold preservation, iv) NM group—with WI including nafamostat mesilate infusion before cardiac arrest and cold preservation; v) PRE group—with WI, 30-min pre-cold preservation perfusion with oxygenated buffer after cardiac arrest, and cold preservation, vi) BV group—with the same treatment as the PRE group plus the addition of biliverdin to the pre-cold preservation perfusion. The portal flow volume, bile production, AST, and TNF-α in perfusate, energy charge (EC), and ATP level in the tissue, and histological findings were investigated. The portal flow volume in the NM, PRE, and BV groups were higher than in the NHBD group. The bile production in the PRE and BV groups were also higher than in the NHBD group. The EC and ATP level of the BV group after reperfusion were higher than those of the NHBD group. Pre-cold preservation perfusion and addition of biliverdin to perfusate improved viability of grafts from NHBD. The results indicate that the preservation of the energy status and microcirculation of the graft is important for successful LTx from NHBD.

The scattered cell clusters that can differentiate into hepatocytes or biliary epithelial cells have been isolated from primary cultures of adult porcine livers. We have generated 11 clonal cell lines from this system and identified liver progenitor cells (LPCs) among the clonal lines. These clonal lines expressed c-kit, HNF-1, HNF-6, and/or CK19 mRNA. An immunocytochemical study of the clonal lines indicated that clonal line CL-11 expressed liver epithelial cell markers CK14, vimentin, CK18, and BD-1. The expression of albumin and α1-antitrypsin (α1-AT) mRNA was only upregulated in CL-11 among the clonal lines when they were grown as aggregates. Under these conditions, CL-11 also exhibited ammonia metabolic activity and several indicators that suggest hepatocytic differentiation, including the upregulation of liver-specific genes such as dipeptidyl peptidase IV, CYP1A1, and CYP3A4 mRNA, and the downregulation of biliary cell markers such as γ-glutamyltrans-peptidase (GGT), CK19, and HNF6 mRNA. After culturing CL-11 in Matrigel, the expression of GGT and HNF6 mRNA was upregulated. These results indicate that CL-11 has dual potential: the ability to differentiate as hepatocytes or as bile duct cells. The isolation of scattered cells could provide a simple method to generate LPC lines from adult livers.

We created a hepatic failure pig model that was suitable for the assessment of cell therapies, such as hepatocyte transplantation and bioartificial livers, using a laparoscopic surgical technique. In our model, all of three hepatic arteries were resected, 5, 7.5, or 10 ml of carbon tetrachloride (CCL₄) was injected into the liver through the portal vein, and subsequently the portal vein was mechanically occluded for 30 min. After the portal occlusion was released, a liver biopsy was performed, and then the surgery was completed. Blood samples were regularly taken during the surgery in order to perform biochemical examinations. All of five pigs in which 5 ml of CCL₄ was infused recovered spontaneously and survived; in contrast, all of five pigs that received 10 ml CCL₄ died within 1.5 h after surgery. The pigs in which 7.5 ml CCL₄ was administered developed liver failure and survived for 6.4 h on average (±1.4 SD). Induction of liver failure with the use of 7.5 ml CCL₄ and 30-min hepatic ischemia fulfilled five of the six criteria that were proposed by Terblanche and Hickman: reversibility, reproducibility, death from liver failure, a therapeutic window, and a large-animal model. We believe that our model is the first report on creation of a reliable model for liver failure in pigs to assess the efficacy of liver-targeted cell therapies.

Natural immunological tolerance can be induced in certain types of allogeneic liver transplantation in rats. To screen for genes associated with the induction of tolerance, suppression subtractive hybridization was performed in the rat liver transplantation model between a DA donor and PVG recipient combination where spontaneous immunological tolerance is known to occur without any immunosuppressive treatment. As a result, 112 genes were cloned from a DA liver graft that survived for 20 days in the fully allogeneic PVG recipient. After confirmation of the expression intensity using an in-house manufactured DNA array with cDNAs from the DA graft, 36 genes were classified in the highly expressed group and 26 moderately expressed group. In the first group, there were 8 immunoglobulin-related genes and 6 MHC class II-related genes, suggesting the existence of an underlying rejection response. Among those genes, an antiapoptotic gene in the p38 MAP kinase pathway, heme oxygenase gene (HO-1), and a ras cascade gene, IQ motif containing GTPase activating protein 1 (Iqgap1), retained biological significance. The results suggested that the molecular response to a liver graft tends to be antiapoptotic and to terminate the rejection response. Unfortunately, there was no gene identified that qualified as a putative immunosuppressive protein, liver suppressor factor-1 (LSF-1). The panel of genes identified in the present work will be a useful panel of candidate genes to investigate the induction of spontaneous tolerance.

Green tea polyphenols have been recently reported to promote the preservation of tissues, such as blood vessels, corneas, nerves, islet cells, articular cartilage, and myocardium, at room temperature. These findings indicate the possibility of a new method of tissue banking without freezing. A main active ingredient of green tea, epigallocatechin-3-gallate (EGCG), is a polyphenol that possesses antioxidant, antimicrobial, antiproliferative, and free radical scavenging effects. This study examined the effects of EGCG regarding skin preservation. Skin sample biopsy specimens measuring 1 × 1 cm from GFP rats were held in sterile containers with 50 ml preserving solution at 4°C and 37°C for up to about 8 weeks. Periodically, some of the preserved skin specimens were directly examined histologically and others were transplanted into nude mice. Histological examinations of skin preserved at 4°C revealed a degeneration of the epidermal and dermal layers from 5 weeks in all groups. In the groups preserved at 37°C, degeneration and flakiness of the epidermal layer were demonstrated starting at 2 weeks preservation regardless of addition of EGCG. After 2–7 weeks of preservation the rat skin grafted to nude mice in the EGCG groups stored at 4°C showed successful engraftment. However, grafts preserved at 4°C without EGCG and at 37°C did not demonstrate GFP-positive keratinocyte or fibroblasts. In conclusion, the present findings suggest the future clinical usefulness of EGCG for skin preservation without freezing; however, the mechanism by which EGCG promotes skin preservation still remains unclear.

Macrophages play a pivotal role in the development of newly formed vascular networks, in addition to their normal immunological functions. This research focuses on peritoneal macrophages as a novel source in cell implantation therapy for ischemic diseases. In this study, production of angiogenic growth factors by peritoneal macrophages and its in vivo effect of neovascularization were evaluated. Mononuclear cells from the peritoneal cavity (P-MNCs) enriched with macrophages were isolated and stimulated with hypoxia and interleukin-1β (IL-1β) to mimic an ischemic tissue environment in vitro. Expression of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) of mRNA in P-MNCs was apparently enhanced by hypoxic stimulation, and the production of VEGF protein was also augmented by hypoxia and IL-1β. A rat ischemic hind limb model was created and P-MNCs (8 × 106/limb) were injected into the ischemic muscles. The blood flow, which was assessed using the colored microsphere method, showed that the percentage blood flow was significantly increased by P-MNCs injection 4 weeks after surgery (48.3 ± 16.8% in noninjected ischemic limb vs. 84.3 ± 13.0% in the P-MNCs-injected limb). A histological analysis revealed that the number of capillaries detected by alkaline phosphatase staining was increased in the P-MNCs group 4 weeks after injection. Furthermore, the number of α-smooth muscle actin-positive vessels also showed a significant increase following P-MNC injection. The injected P-MNCs labeled with fluorescence were detected in the interstitial space of ischemic muscles, and VEGF protein expression of the implanted cells was confirmed by immunohistochemistry. These results indicate that peritoneal macrophages stimulate capillary formation and arteriogenesis in the ischemic limbs, possibly through the production of angiogenic growth factors. These findings suggest that the physiological angiogenic property of peritoneal macrophages could therefore be utilized for neovascularization in cell implantation therapy.

Ischemia followed by reperfusion leads to severe organ injury and dysfunction. Inflammation is considered to be the most important cause of graft dysfunction in kidney transplantation subjected to ischemia. The mechanism that triggers inflammation and renal injury after ischemia remains to be elucidated; however, cellular stress may induce apoptosis during the first hours and days after transplantation, which might play a crucial role in early graft dysfunction. Bcl-2 is known to inhibit apoptosis induced by the etiological factors promoting ischemia and reperfusion injury. Accordingly, we hypothesized that an augmentation of the antiapoptotic factor Bcl-2 may thus protect tubular epithelial cells by inhibiting apoptosis, thereby ameliorating the subsequent tubulointerstitial injury. We examined the effects of Bcl-2 overexpression on ischemia-reperfusion (I/R) injury using Bcl-2 transgenic mice (Bcl-2 TG) and their wild-type littermates (WT). To investigate the effects of I/R injury, the left renal artery and vein were clamped for 45 min, followed by reperfusion for 0–96 h. Bcl-2 TG exhibited decreased active caspase protein in the tubular cells, which led to a reduction in TUNEL-positive apoptotic cells. Consequently, interstitial fibrosis and phenotypic changes were ameliorated in Bcl-2 TG. In conclusion, Bcl-2 augmentation protected renal tubular epithelial cells from I/R, and subsequent interstitial injury by inhibiting tubular apoptosis.

Multiple roles have been already recognized for CCN2 in cartilage development and regeneration. However, the effects of CCN2 on bone regeneration remain to be elucidated. In this study, the utility of CCN2 on bone regeneration was examined in vitro and in vivo in combination with hydroxyapatite (HAp) as a scaffold. Human bone marrow stromal cells (hBMSCs) were isolated from human iliac bone marrow aspirates of healthy donors and expanded, and the effects of CCN2 on their proliferation and migration were examined in vitro. The proliferation of hBMSCs on a plastic or HAp plate was significantly enhanced by CCN2. Moreover, the migration of hBMSCs also dramatically increased by CCN2. Interestingly, a C-terminal signal modular fragment of CCN2 (CT-module) also enhanced the cell proliferation and migration as efficiently as the full-length CCN2. Next, in order to estimate the effect of CCN2 on the migration and survival of hBMSCs and bone formation inside the HAp scaffold in vivo, two experiments were performed. First, the porous HAp carrier was cultured with hBMSCs for a week, and the cell–scaffold hybrid was transplanted with or without CCN2 subcutaneously into immunocompromised mice. CCN2 accelerated the hBMSC-like cell migration and survival inside the porous HAp within 4 weeks after transplantation. Second, the porous HAp carrier with or without CCN2 was directly implanted into bone defects within a rabbit mandible, and bone regeneration inside was evaluated. As a result, CCN2 efficiently induced the cell invasion and bone formation inside the porous HAp scaffold. These findings suggest that CCN2 and its CT-module fragment could be useful for regeneration and reconstruction of large-scale bone defects.