Severe acute liver failure, even when transient, must be treated by transplantation and lifelong immune suppression. Treatment could be improved by bioartificial liver (BAL) support, but this approach is hindered by a shortage of human hepatocytes. To generate an alternative source of cells for BAL support, we differentiated mouse embryonic stem (ES) cells into hepatocytes by coculture with a combination of human liver nonparenchymal cell lines and fibroblast growth factor-2, human activin-A and hepatocyte growth factor. Functional hepatocytes were isolated using albumin promoter-based cell sorting. ES cell-derived hepatocytes expressed liver-specific genes, secreted albumin and metabolized ammonia, lidocaine and diazepam. Treatment of 90% hepatectomized mice with a subcutaneously implanted BAL seeded with ES cell-derived hepatocytes or primary hepatocytes improved liver function and prolonged survival, whereas treatment with a BAL seeded with control cells did not. After functioning in the BAL, ES cell-derived hepatocytes developed characteristics nearly identical to those of primary hepatocytes.

A human pancreatic beta-cell line that is functionally equivalent to primary beta-cells has not been available. We established a reversibly immortalized human beta-cell clone (NAKT-15) by transfection of primary human beta-cells with a retroviral vector containing simian virus 40 large T-antigen (SV40T) and human telomerase reverse transcriptase (hTERT) cDNAs flanked by paired loxP recombination targets, which allow deletion of SV40T and TERT by Cre recombinase. Reverted NAKT-15 cells expressed beta-cell transcription factors (Isl-1, Pax 6, Nkx 6.1, Pdx-1), prohormone convertases 1/3 and 2, and secretory granule proteins, and secreted insulin in response to glucose, similar to normal human islets. Transplantation of NAKT-15 cells into streptozotocin-induced diabetic severe combined immunodeficiency mice resulted in perfect control of blood glucose within 2 weeks; mice remained normoglycemic for longer than 30 weeks. The establishment of this cell line is one step toward a potential cure of diabetes by transplantation.

Development of an efficient preculture system of islets is ideal. Toward that goal, we constructed a human pancreatic islet-derived fibroblast cell line MNNK-1 for a source as a coculture system for freshly isolated islets to maintain islet functions. Human pancreatic islet cells were nucleofected with a plasmid vector pYK-1 expressing simian virus 40 large T antigen gene (SV40T) and hygromycin resistance gene (HygroR). One of the transduced cell lines, MNNK-1, was established and served as a feeder cell in the coculture for freshly isolated mouse, rat, and pig islets. Morphology, viability, and glucose-responding insulin secretion were analyzed in the coculture system. MNNK-1 cells were morphologically spindle shaped and were negative for pancreatic endocrine markers. MNNK-1 cells were positive for α-smooth muscle actin and collagen type I and produced fibroblast growth factor. Coculture of the mouse, rat, and pig islets with MNNK-1 cells maintained their viability and insulin secretion with glucose responsiveness. A human pancreatic isletderived fibroblast cell line MNNK-1 was established. MNNK-1 cells were a useful means for maintaining morphology and insulin secretion of islets in the coculture system. Key words: Islets; Simian virus 40 large T antigen; Coculture INTRODUCTIONtransplant program through a collaborative relationship with a remote islet isolation center (4,12). The advantage of islet culture before transplantation Since the year 2000, a dramatic turnaround has been made in the field of clinical islet transplantation by the is to decrease total volume of islet mass, to allow adequate assessment of transplantable islets and preparation introduction of the Edmonton Protocol. International multicenter trials of the Edmonton Protocol demonstrate of the patient with immunosuppressive agents. One of the major drawbacks of preculture of islets is a decrease successful replication. The success of the Edmonton protocol has spurred expectations and interests about isin viability and transplantable islet number. Thus, the development of an efficient preculture system of freshly let transplantation, raising people's awareness that islet transplantation is an ideal therapy for patients with type isolated pancreatic islets is one of the important issues to facilitate islet transplantation. 1 diabetes mellitus (33). Current developments in islet transplantation include: 1) islet culture prior to transToward that goal, we have focused on the use of coculture system of islets with pancreatic islet-derived fiplantation (preculture), 2) pancreas shipment using twolayer method, 3) islet infusion using a bag instead of a broblasts, because heterotypic cell interactions between parenchymal cells and nonparenchymal neighbors have syringe (3), 4) achievement of a single donor islet transplant success (15,16) Immunochemical staining for α-SMA and collagen type 1 was carried out using an ENVISION+ Kit/HRP Here we present the effect of MNNK-1 cells on islet functions in the coculture system. Because of the diffi-(DAB) (Dak...

OBJECTIVE-Treatment of diabetic patients by pancreatic islet transplantation often requires the use of islets from two to four donors to produce insulin independence in a single recipient. Following isolation and transplantation, islets are susceptible to apoptosis, which limits their function and probably long-term islet graft survival. RESEARCH DESIGN AND METHODS-To address this issue,we examined the effect of the cell-permeable apoptosis inhibitor pentapeptide Val-Pro-Met-Leu-Lys, V5, on pancreatic islets in a mouse model. RESULTS-V5treatment upregulated expression of anti-apoptotic proteins Bcl-2 and XIAP (X-linked inhibitor of apoptosis protein) by more than 3-and 11-fold and downregulated expression of apoptosis-inducing proteins Bax, Bad, and nuclear factor-B-p65 by 10, 30, and nearly 50%, respectively. Treatment improved the recovered islet mass following collagenase digestion and isolation by 44% and in vitro glucose-responsive insulin secretion nearly fourfold. Following transplantation in streptozotocin-induced diabetic mice, 150 V5-treated islet equivalents functioned as well as 450 control untreated islet equivalents in normalizing blood glucose.CONCLUSIONS-These studies indicate that inhibition of apoptosis by V5 significantly improves islet function following isolation and improves islet graft function following transplantation. Use of this reagent in clinical islet transplantation could have a dramatic impact on the number of patients that might benefit from this therapy and could affect long-term graft survival.

The present work clearly demonstrates the usefulness of V5 for maintaining the functions of monkey hepatocytes in tissue culture.

Our patient presented with advanced ovarian angiosarcoma and bone metastases and still achieved 7 years of progression-free survival after treatment with a gemcitabine-based regimen. Recently, MYC amplification was proposed to occur in a proportion of primary 31 and radiation-induced angiosarcomas 32 . Enhanced expression of c-Myc is an important mediator leading to disease development 33 . We therefore report our case and the results of retrospective MYC gene amplification and c-Myc protein expression analyses, discuss the relevance of those factors in terms of therapy and prognosis, and review the related literature. CASE DESCRIPTIONA 29-year-old woman was admitted to the emergency room with abdominal pain and fever. Abdominal palpation revealed rebound tenderness. Transvaginal ultrasonography revealed a cystic mass of approximately 9 cm in the right pelvis, within which several solid masses existed. The masses were recognized as blood clots or other artefacts unrelated to the tumour component. A small amount of ascites in the Douglas pouch was also observed.Laboratory data revealed a white blood cell count of 9320×10 3 /μL, with 85.6% neutrophils, and 2.30 mg/dL serum C-reactive protein. On the following day, serum C-reactive protein increased to 11.22 mg/dL, indicating a level of inflammation.Clinically, a rupture or torsion of the right ovarian tumour with acute peritonitis was suspected. Intravenous administration of ceftriaxone sodium hydrate 2 g daily was initiated and continued for 3 days.Magnetic resonance imaging revealed a cystic right ovarian tumour whose cystic portion contained a fluid or blood component [ Figure 1(A,B)]. Computed tomography revealed low-density areas in several bones, suggestive of osteolytic bone metastases [ Figure 2(A)]. We also examined the tumour markers cancer antigen 125, carbohydrate antigen 19-9, and carcinoembryonic antigen, whose ABSTRACT Angiosarcoma is a rare and aggressive type of sarcoma, and primary angiosarcoma of the ovary is extremely rare. We report the case of a 29-year-old woman who was diagnosed with ovarian angiosarcoma and possible bone metastases. We treated this patient with a gemcitabine-based regimen as postoperative adjuvant chemotherapy, after which she achieved at least 7 years of progression-free survival, an extremely long duration given the aggressive features of this tumour. We retrospectively performed immunohistochemical analyses and fluorescence in situ hybridization to make a pathology diagnosis and to investigate the tumour features. MYC amplification and c-Myc protein overexpression were positively detected. It might be possible to correlate the effectiveness of the gemcitabinebased chemotherapeutic regimen with MYC gene amplification and c-Myc protein overexpression.

Normal human islet cells are an ideal source for pancreas-targeted cell therapies, but the availability of human donor pancreata for islet isolation is severely limited. To effectively utilize such scarce donor organs for cell therapies, it is crucial to develop an excellent isolation, effective cryopreservation, and efficient gene transfer techniques for the transportation of isolated cells. In the present study, we investigate the effect of University of Wisconsin (UW) solution and ascorbic acid-2 glucoside (AA2G) on the cryopreservation of human islets. We also evaluate the gene transfer efficiency of a lentiviral vector expressing the E. coli LacZ gene, Lt-NLS/LacZ, in human islets. Human islets were isolated with a standard digestion method at the University of Alberta. Isolated islets were transported to Japan for 40 h and then subjected to cryopreservation experiments. The following preservation solutions were tested: UW solution with 100 micro g/mL of AA2G, UW solution, 100% fetal bovine serum (FBS), and CMRL supplemented with 10% FBS. Following three months of cryopreservation, the islets were thawed and analyzed for viability, glucose-sensitive insulin secretion, proinsulin gene expression profile, and in vivo engraftment. The islets were also subjected to monolayer formation with 804G-cell-line-derived extracellular matrix (ECM), followed by Lt-NLS/LacZ transduction. The viability, morphology, glucose-sensitive insulin secretion, proinsulin gene expression, and monolayer formation efficiency of the thawed cryopreserved islets are significantly better maintained by the use of UW solution. When AA2G (100 microg/mL) is combined with UW, such parameters are further improved. The adequate engraftment of UW + AA2G-cryopreserved human islets is achieved in the liver of nude mice. Efficient Lt-NLS/LacZ transduction is identified in monolayered islets cryopreserved with UW solution with AA2G. The present work demonstrates that the combination of UW solution with AA2G (100 microg/mL) would be a useful cryopreservation means for human islets. Human islets monolayer-cultured with 804G-derived ECM are efficiently transduced with a lentiviral vector Lt-NLS/LacZ.

The present work clearly demonstrates the usefulness of dHGF (100 ng/ml) for maintaining the differentiated functions of human hepatocytes in tissue culture.