Published Papers - Noguchi Hirofumi

Differential Ability of Somatic Stem Cells

Koichi Oishi, Hirofumi Noguchi, Hiroshi Yukawa, Shuji Hayashi

CELL TRANSPLANTATION ( SAGE PUBLICATIONS INC )  18 ( 5-6 ) 581 - 589   2009 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Somatic stem cells can be isolated from a variety of sources. Although some studies have suggested that somatic stem cells may represent a cell population that is very similar to embryonic stem (ES) cells, it remains unclear whether somatic stem cells retain the potential to differentiate into any cell type derived from the three germ layers. In this study, we investigated the transdifferentiation potential of somatic stem cells using adipose tissue-derived stem/progenitor cells (ASCs; mesodermal stem cells) and pancreatic stem cells (endodermal stem cells). Previous reports from other groups describe the protocol that has been used to differentiate ASCs or mesenchymal stem cells (MSCs) in bone marrow into insulin-producing cells. Induction 1: ASCs were cultured for 3 days in ultra-low attachment plates under serum-free conditions. Induction 2: ASCs were cultured for 24 h with L-DMEM, and reinduced with serum-free H-DMEM for another 10 h. Unlike previous reports, we did not get ASCs to express any pancreas-specific genes, including insulin-1 or insulin-2. Pancreatic stem cells were induced to differentiate into adipo/osteogenic by the following protocols. Induction protocol 1: ACSs were cultured for 7 days with medium containing indometacin, dexamethasone, hydrocortisone, and insulin for adipogenic differentiation. Induction protocol 2: The cells were cultured for 7 days with medium containing dexamethasone, ascorbate-2-phosphate, and beta-glyecrophosphate for osteogenic differentiation. Although these approaches have been widely used for adipo/osteogenic differentiation from MSCs, adipo/osteogenic differentiation from pancreatic stem cells was not observed. These data suggest that it is not easy for somatic stem cells to transdifferentiate into other germ cell types, at least, under these conditions.

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Comparison of Trypsin Inhibitors in Preservation Solution for Islet Isolation

Hirofumi Noguchi, Michiko Ueda, Shuji Hayashi, Naoya Kobayashi, Teru Okitsu, Yasuhiro Iwanaga, Hideo Nagata, Xiaoling Liu, Hiroki Kamiya, Marlon F. Levy, Shinichi Matsumoto

CELL TRANSPLANTATION ( COGNIZANT COMMUNICATION CORP )  18 ( 5-6 ) 541 - 547   2009 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Islet transplantation has recently emerged as an effective therapy and potential cure for type I diabetes mellitus. Recent reports show that the two-layer method (TLM), which employs oxygenated perfluorochemical (PFC) and University of Wisconsin (UW) solution, is superior to simple cold storage in UW for pancreas preservation in islet transplantation. Moreover, we recently reported that islet yield was significantly higher in the ET-Kyoto solution with ulinastatin (MK)/PFC preservation solution compared with the UW/PFC preservation solution in the porcine model and that the advantages of MK solution are trypsin inhibition and less collagenase inhibition. In this study, we compared ulinastatin with another trypsin inhibitor, Pefabloc, in preservation solution for islet isolation. Islet yield before purification was higher in the MK/PFC group compared with the ET-Kyoto with Pefabloc (PK)/PFC group. The stimulation index was higher for the MK/PFC group than for the PK/PFC group. These data suggest that ET-Kyoto with ulinastatin was the better combination for pancreas preservation than ET-Kyoto with Pefabloc. Based on these data, we now use ET-Kyoto solution with ulinastatin for clinical islet transplantation.

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Comparison of Sendai Virus-Mediated Gene Transfer Efficiency to Adhesive and Floating Adipose Tissue-Derived Stem Cells

Hiroshi Yukawa, Hirofumi Noguchi, Koichi Oishi, Makoto Inoue, Mamoru Hasegawa, Michinari Hamaguchi, Nobuyuki Hamajima, Shuji Hayashi

CELL TRANSPLANTATION ( COGNIZANT COMMUNICATION CORP )  18 ( 5-6 ) 601 - 609   2009 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Sendai virus (SeV) vectors have potential clinical applications because they can efficiently introduce foreign genes without toxicity into various organs. A recent study reported the green fluorescent protein (GFP) gene transfer to adipose tissue-derived stem cells (ASCs) with SeV vectors results in more efficient expression of GFP than AdV and identified the preservation of the multilineage potential of ASCs transfected with SeV vectors. This study assessed the gene transfer efficiency to floating ASCs with SeV vectors. Although a slight cytotoxicity was observed, the efficiency of gene transfer to cells in the floating state was much higher at all times and all concentrations at MOIs of 2, 10, and 20 than in the adhesion state. Moreover, ASCs transfected with SeV vectors in floating state have the same potential for their differentiation into specific tissues, such as adipocytes and osteocytes, as untransfected ASCs. These data suggest that SeV transfection to ASCs in the floating state could therefore be useful for gene transfer technology.

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Continuous, but not occasional, oral ethanol intake reduces the success of intraportal transplanted islets of Langerhans: An experimental study

X. Liu, S. Matsumoto, H. Noguchi, Y. Yonekawa, Y. Iwanaga, T. Okitsu, H. Nagata, S. Miyakawa, C. Song, A. Jackson, B. Naziruddin, M. F. Levy

TRANSPLANTATION PROCEEDINGS ( ELSEVIER SCIENCE INC )  40 ( 2 ) 441 - 443   2008.03 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Background. Islet transplantation is gradually gaining acceptance for the treatment of type 1 diabetes mellitus. One of the unknown questions is alcohol intake; we have prohibited alcohol intake after islet transplantation although there is no solid evidence to support this. Materials and Methods. In this study, we employed a mouse model to determine the effect of oral ethanol intake on transplanted islets. Either 500 or 150 islets were infused selectively into the right liver lobe of chemically induced diabetic mice. After transplantation, mice were orally administered either water (as a control) or various concentrations of ethanol for 14 consecutive days occasionally (once per day) or continuously (all intake was alcohol). Blood glucose levels were monitored and oral glucose tolerance tests (OGTT) performed. Results. After 500 islets had been transplanted, all mice, were cured from diabetes, but the continuous alcohol intake group showed significantly prolonged time to diabetes reversal and significantly lower glucose clearance rates by OGTT compared with the control group. After 150 islet transplantations, the diabetes cure rate in the continuous alcohol intake group was significantly lower than the control group (continuous alcohol vs control: 3/8 vs 11/12, P < .05). However, the occasional alcohol intake group showed no difference from the control group, even with as few as 150 islets transplanted per mouse. Conclusion. The present results demonstrated that continuous but not occasional alcohol intake reduced the success of intraportal islet transplantation.

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Effect of JNK inhibitor during islet isolation and transplantation

H. Noguchi, S. Matsumoto, N. Kobayashi, S. Hayashi, Y. Iwanaga, H. Nagata, A. Jackson, B. Naziruddin, T. Okitsu, M. F. Levy

TRANSPLANTATION PROCEEDINGS ( ELSEVIER SCIENCE INC )  40 ( 2 ) 379 - 381   2008.03 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Although islet transplantation has been remarkably improved by the Edmonton protocol, the insulin independence rate after islet transplantation from one donor pancreas has remained low. The c-Jun NH2-terminal kinases (JNKs) are classic stress-activated protein kinases; many cellular stresses have been shown to stimulate JNK activation. JNK in the pancreas is activated during brain death, pancreas procurement, and organ preservation, and its activity is progressively increased during the isolation procedure. Moreover, JNK activity in the transplanted liver after islet transplantation increases markedly within 24 hours. In this study, we show the effect of a JNK inhibitor during islet isolation and transplantation. Use of the JNK inhibitor in pancreas preservation, islet culture, and/or islet transplantation prevents islet cell apoptosis and improves islet graft function. These findings suggest that inhibition of JNK could prevent the impairment of islet cells and improve outcomes after pancreatic islet transplantation.

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Analysis of donor- and isolation-related variables from non-heart-beating donors (NHBDs) using the Kyoto islet isolation method

Xiaoling Liu, Shinichi Matsumoto, Teru Okitsu, Yasuhiro Iwanaga, Hirofumi Noguchi, Yukihide Yonekawa, Hideo Nagata, Hiroki Kamiya, Michiko Ueda, Nobuyo Hatanaka, Shuichi Miyakawa, Naoya Kobayashi, Chunfang Song

CELL TRANSPLANTATION ( COGNIZANT COMMUNICATION CORP )  17 ( 6 ) 649 - 656   2008 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Recently, we demonstrated that islet transplantation from non-heart-beating donors (NHBDs) using the Kyoto islet isolation method (KIIM) successfully reversed patients' diabetes state. In this study, we evaluated the effects of donor- and isolation-related variables on islet isolation results from NHBDs by KIIM. Twentyone islet preparations from the pancreata of NHBDs were isolated by KIIM. Islet preparations that met transplantation criteria and achieved improved patient diabetes control after transplantation were defined as successful isolations. Potential risk factors deemed to affect islet isolation results, such as age, gender, body mass index, hospital stay, donors' blood biochemical tests, a modified pancreata procurement method, and isolation and purification procedure-related variables, were analyzed. Seventeen out of 21 islet isolations (81%) were successful isolations. Postpurification islet yield was 447,639 +/- 39,902 islet equivalents (IE) in the successful isolation group and 108,007 +/- 31,532 IE in the failure group. Donor age was significantly younger in the success group (41.9 +/- 4.0 years old in the success group vs. 57.5 +/- 2.2 years old in the failure group, p = 0.003). Chronic pancreatitis significantly decreased islet yields (p = 0.006). Phase 1 time was significantly shorter (p = 0.010) and undigested tissue volume was significantly smaller (p = 0.020) in the success group. Purity was in positive correlation to postpurification islet yield, while donor age was in reverse correlation to postpurification islet yield. KIIM enables us to perform islet transplantation from NHBDs; however, the decision to use pancreata from older donors or those with chronic pancreatitis requires careful consideration.

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Cell surface heparan sulfate proteoglycans mediate the internalization of PDX-1 protein

Michiko Ueda, Shinichi Matsumoto, Shuji Hayashi, Naoya Kobayashi, Hirofumi Noguchi

CELL TRANSPLANTATION ( COGNIZANT COMMUNICATION CORP )  17 ( 1-2 ) 91 - 97   2008 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Although islet transplantation is a promising therapeutic option for the treatment of type I 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-Ill 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 requiting gene transfer technology.

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Ductal injection of preservation solution increases islet yields in islet isolation and improves islet graft function

Hirofumi Noguchi, Michiko Ueda, Shuji Hayashi, Naoya Kobayashi, Teru Okitsu, Yasuhiro Iwanaga, Hideo Nagata, Yusuke Nakai, Shinichi Matsumoto

CELL TRANSPLANTATION ( COGNIZANT COMMUNICATION CORP )  17 ( 1-2 ) 69 - 81   2008 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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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.

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Cryopreservation of mouse adipose tissue-derived stem/progenitor cells

Koichi Oishi, Hirofumi Noguchi, Hiroshi Yukawa, Takamichi Miyazaki, Ryuji Kato, Yasuo Kitagawa, Minoru Ueda, Shuji Hayashi

CELL TRANSPLANTATION ( SAGE PUBLICATIONS INC )  17 ( 1-2 ) 35 - 41   2008 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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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.

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Comparison of M-Kyoto solution and Histidine-Tryptophan-Ketoglutarate solution with a trypsin inhibitor for pancreas preservation in islet transplantation

Hirofumi Noguchi, Michiko Ueda, Shuji Hayashi, Naoya Kobayashi, Hideo Nagata, Yasuhiro Iwanaga, Teru Okitsu, Shinichi Matsumoto

TRANSPLANTATION ( LIPPINCOTT WILLIAMS & WILKINS )  84 ( 5 ) 655 - 658   2007.09 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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The use of University of Wisconsin (UW) preservation solution in islet transplantation has some disadvantages, including inhibition of collagenase activity for pancreatic digestion. Histidine-tryptophan-ketoglutarate (HTK) solution has demonstrated an efficacy similar to UW solution for organ preservation in clinical pancreas transplantation. Recently, we reported that islet yield from porcine pancreata was significantly greater when they were preserved using M-Kyoto solution compared with UW solution. Here, we compared HTK solution with ulinastatin (M-HTK) and M-Kyoto solution for islet yield. In porcine islet isolation, islet yield after purification was significantly greater in the M-Kyoto/perfluorochemical (PFC) group compared with the M-HTK/PFC group. The M-Kyoto/PFC group had a significantly lower ADP/ATP ratio compared with the M-HTK/PFC group, suggesting that different islet yields might be due to the differences as energy sources of the solutions used. In conclusion, M-Kyoto/PFC solution is better for pancreas preservation before islet isolation than M-HTK/PFC solution.

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Cell-permeable pentapeptide V5 inhibits apoptosis and enhances insulin secretion, allowing experimental single-donor islet transplantation in mice

Jorge D. Rivas-Carrillo, Alejandro Soto-Gutierrez, Nalu Navarro-Alvarez, Hirofumi Noguchi, Teru Okitsu, Yong Chen, Takeshi Yuasa, Kimiaki Tanaka, Michiki Narushima, Atsushi Miki, Haruo Misawa, Yasuhiko Tabata, Hee-Sook Jun, Shinichi Matsumoto, Ira J. Fox, Noriaki Tanaka, Naoya Kobayashi

DIABETES ( AMER DIABETES ASSOC )  56 ( 5 ) 1259 - 1267   2007.05 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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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 - V5 treatment 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-kappa 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 apo ptosis 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.

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Activation of c-jun NH2-terminal kinase during islet isolation

Hirofumi Noguchi

ENDOCRINE JOURNAL ( JAPAN ENDOCRINE SOC )  54 ( 2 ) 169 - 176   2007.04 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Pancreatic islet transplantation has been remarkably improved by the Edmonton protocol; however, it is not easy to achieve insulin independence after islet transplantation from one donor pancreas. The islet isolation procedure itself destroys cellular and noncellular components of the pancreas that probably play a role in supporting islet survival. Further islet transplantation exposes cells to a variety of stressful stimuli such as proinflammatory cytokines. The reduction in islet mass immediately after isolation and transplantation implicates P cell death by apoptosis and the prerecruitment of intracellular death signalling pathways. The c-Jun NH2-terminal kinases (JNKs) are classic stress-activated protein kinases and many cellular stresses have been shown to stimulate JNK activation. JNK in the pancreas is activated during brain death, pancreas procurement, and organ preservation, and its activity is progressively increased during the isolation procedure. Moreover, JNK activity in the transplanted liver after islet transplantation increases markedly within 24 hrs. Use of the JNK inhibitor in pancreas preservation, islet culture, and/or islet transplantation prevents islet apoptosis and improves islet graft function. These findings suggest that the control of JNK activation is important for pancreatic islet transplantation.

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Activation of c-Jun NH2-terminal kinase (JNK) pathway during islet transplantation and prevention of islet graft loss by intraportal injection of JNK inhibitor

H. Noguchi, Y. Nakai, M. Ueda, Y. Masui, S. Futaki, N. Kobayashi, S. Hayashi, S. Matsumoto

DIABETOLOGIA ( SPRINGER )  50 ( 3 ) 612 - 619   2007.03 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Aims/hypothesis Although application of the Edmonton protocol has markedly improved the outcome for pancreatic islet transplantation, the insulin independence rate after islet transplantation from one donor pancreas has remained low. During the isolation process and subsequent clinical transplantation, islets are subjected to severe adverse conditions that impair survival and ultimately contribute to graft failure. The aim of this study was to map the c-Jun NH2-terminal kinase (JNK) pathway that mediates islet loss during islet transplantation and to clarify whether intraportal injection with JNK inhibitor during islet transplantation can prevent islet graft loss. Methods We measured JNK activity in the liver, fat and muscle of diabetic mice and in the liver immediately after islet transplantation. We examined the effect of intraportal injection of JNK inhibitory peptide at islet transplantation. Results JNK activity became progressively higher at least until 24 h after transplantation. The cell-permeable peptide of JNK inhibitor was delivered not only in the liver but also in other insulin target organs, preventing JNK activation in the liver at least until 24 h after transplantation and reducing JNK activity in these insulin target organs. Moreover, the peptide inhibitor prevented islet graft loss immediately after transplantation and improved islet transplant outcome. Conclusions/interpretation These findings suggest that control of the JNK pathway is extremely important in islet transplantation and that intraportal injection of JNK inhibitor during islet transplantation (addition of JNK inhibitor to transplant media) could prevent the impairment of islet cells, leading to improved outcome for pancreatic islet transplantation.

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BETA2/NeuroD protein transduction requires cell surface heparan sulfate proteoglycans

Hirofumi Noguchi, Michiko Ueda, Shinichi Matsumoto, Naoya Kobayashi, Shuji Hayashi

HUMAN GENE THERAPY ( MARY ANN LIEBERT INC )  18 ( 1 ) 10 - 17   2007.01 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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BETA2/NeuroD protein is important for regulating insulin gene transcription and for the terminal differentiation of islet cells, including insulin- and glucagon-producing cells. We reported that BETA2/NeuroD protein can permeate several cell types, including pancreatic islets, because of an arginine- and lysine-rich protein transduction domain (PTD) sequence in its structure. Here we provide genetic and biochemical evidence that cell membrane heparan sulfate proteoglycans are involved in extracellular BETA2/NeuroD internalization. We tested whether soluble glycosaminoglycans (GAGS) could inhibit BETA2/NeuroD internalization. Heparin almost completely prevented BETA2/NeuroD entry, whereas chondroitin sulfate A, B, and C caused only limited inhibition. Moreover, treatment with heparinase III impaired BETA2/NeuroD internalization, whereas treatment with chondroitinase ABC, or with chondroitinase AC, was completely ineffective in inhibiting BETA2/NeuroD internalization. We also examined various mutant cell lines originating from CHOK1 cells and defective in GAG biosynthesis. The observation using mutant cell lines supports the notion that the selective sulfation of heparan sulfate is an important determinant for NeuroD/heparan sulfate recognition. These data indicate that cell surface heparan sulfate proteoglycans are required for BETA2/NeuroD internalization and that BETA2/NeuroD protein transduction could be a safe and valuable strategy for enhancing insulin gene transcription without requiring gene transfer technology.

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Construction and transplantation of an engineered hepatic tissue using a polyaminourethane-coated nonwoven polyteltrafluoroethylene fabric

Alejandro Soto-Gutierrez, Nalu Navarro-Alvarez, Jorge D. Rivas-Carrillo, Kimiaki Tanaka, Yong Chen, Haruo Misawa, Teru Okitsu, Hirofumi Noguchi, Noriaki Tanaka, Naoya Kobayashi

TRANSPLANTATION ( LIPPINCOTT WILLIAMS & WILKINS )  83 ( 2 ) 129 - 137   2007.01 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Background. Acute liver failure (ALF) is a serious condition that has a high mortality rate. Construction of an efficient culture and transplantation engineering system of hepatic tissue is an important approach to treat patients suffering from ALF to provide short-term hepatic support until the damaged liver spontaneously recovers or a donor liver becomes available for transplantation. Here, we evaluate the construction and transplantation of an engineered hepatic tissue (EHT) using primary isolated hepatocytcs cultured onto polyaminourethane (PAU)-coated, nonwoven polytetrafluoroethylene (PTFE) fabric. Methods. The isolated hepatocytes cultured onto PAU-coated PTFE fabric were able to adhere and spread over the individual fibers of the net and formed hepatic clusters after 3 days, such clusters revealed Gap junctions and well-developed bile canaliculi. Results. When PAU-coated PTFE was utilized, ammonia-, and diazepam- metabolizing capacities and albumin production ability were significantly increased compared with collagen control. To test the function of this hepatic tissue in vivo, we transplanted a nonwoven PAU-coated PTFE fabric inoculated with one million hepatocytes on the surface of the spleen of Balb/c mice suffering from ALF induced by 90% hepatectomy, and found that this FIAT prolonged the survival of liver failure-induced mice without adverse effects. Ultrastructure analyses showed good attachment of the cells on the surface of PTFE fabric and strong albumin expression seven days after the newly formed hepatic tissue was transplanted. Conclusion. We have here demonstrated the efficient construction and transplantation of hepatic tissue using primary hepatocytes and PAU-coated PTFE fabric.

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A new mouse model for intraportal islet transplantation with limited hepatic lobe as a graft site

Yukihide Yonekawa, Teru Okitsu, Kenjiro Wake, Yasuhiro Iwanaga, Hirofumi Noguchi, Hideo Nagata, Xiaoling Liu, Naoya Kobayashi, Shinichi Matsumoto

TRANSPLANTATION ( LIPPINCOTT WILLIAMS & WILKINS )  82 ( 5 ) 712 - 715   2006.09 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Intraportal site is the standard for grafting in clinical islet transplantation. In the mouse model, the whole liver has been used as the grafting site to mimic clinical islet transplantation. However, this model lacks the potency to directly assess the contribution of the islet graft to diabetes control. Only demonstrating the immediate recurrence of diabetes in a surviving recipient after the removal of the islet graft can validate this assessment. In this study, we develop a mouse model of intraportal islet transplantation equipped with the potency of this assessment by injecting islets selectively into the right hepatic lobe under temporal clamp of the left portal vein. The mouse of this model survives after the right hepatectomy by which the islet graft is removed. This model can be applied to investigate both the specific graft-recipient interaction in the liver and the islet graft contribution to the control of diabetes.

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A single transplantation of the islets can produce glycemic stability and reduction of basal insulin requirement

Mariko Sassa, Kazuhito Fukuda, Shimpei Fujimoto, Kentaro Toyoda, Yoshihito Fujita, Shinichi Matsumoto, Teru Okitsu, Yasuhiro Iwanaga, Hirofumi Noguchi, Hideo Nagatac, Yukihide Yonekawa, Takeshi Ohara, Masahiko Okamoto, Koichi Tanaka, Yutaka Seino, Nobuya Inagaki, Yuichiro Yamada

DIABETES RESEARCH AND CLINICAL PRACTICE ( ELSEVIER IRELAND LTD )  73 ( 3 ) 235 - 240   2006.09 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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We investigated glycemic stability and insulin requirement 1 month after a single transplantation of the islets from non-heart-beating donors or a living donor. Overall blood glucose levels decreased immediately after transplantation. The M-value and mean amplitude of glycemic excursions (MAGE) decreased significantly from 53.0 (range, 8.9-91.0) to 4.2 (0.6-8.8, P < 0.05) and from 8.5 mM (4.8-11.7) to 3.3 mM (2.0-4.5, P < 0.05), respectively. The values after transplantation were lower than the first quartile of 102 type 2 diabetic control patients. The estimated HbA(1c) level decreased significantly from 7.9% (5.7-10.9) to 5.4% (4.7-5.9, P < 0.05). The supplement of basal insulin decreased 43% from 0.31 units/kg/day (0.16-0.37) to 0.18 units/kg/day (0-0.22, P < 0.05). while that of stimulated insulin did not decrease significantly, from 0.28 units/kg/day (0.13-0.51) to 0.21 units/kg/day (0-0.41). Thus, only one islet transplantation can be sufficient to attain metabolic stability, probably by effective supply of basal insulin secretion, sufficient to avoid life-threatening severe hypoglycemia and prevent or delay the progress of secondary complications of diabetes by decreasing the HbA(1c) level. (c) 2006 Elsevier Ireland Ltd. All rights reserved.

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A newly developed bioartificial pancreas successfully controls blood glucose in totally pancreatectomized diabetic pigs

Hideaki Ikeda, Naoya Kobayashi, Yoshihito Tanaka, Shuhei Nakaji, Chen Yong, Teru Okitsu, Mizuko Oshita, Shinichi Matsumoto, Hirofumi Noguchi, Michiki Narushima, Kimiaki Tanaka, Atsushi Miki, Jorge David Rivas-Carrillo, Alejandro Soto-Gutierrez, Nalu Navarro-Alvarez, Koichi Tanaka, Hee-Sook Jun, Noriaki Tanaka, Ji-Won Yoon

TISSUE ENGINEERING ( MARY ANN LIEBERT INC )  12 ( 7 ) 1799 - 1809   2006.07 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Construction of a safe and functional bioartificial pancreas (BAP) that provides an adequate environment for islet cells may be an important approach to treating diabetic patients. Various types of BAP devices have been developed, but most of them involve extravascular implantation of islets in microcapsules or diffusion chambers. These devices have poor diffusive exchange between the islets and blood, and often rupture. To overcome these problems, we developed a new type of BAP composed of polyethylene-vinyl alcohol (EVAL) hollow fibers that are permeable to glucose and insulin and a poly-amino-urethane-coated, non-woven polytetrafluoroethylene (PTFE) fabric that allows cell adhesion. Porcine islets attached to the surface of the PTFE fabric, but not to the surface of the EVAL hollow fibers, allowing nutrient and oxygen exchange between blood flowing inside the fibers and cells outside. We inoculated this BAP with porcine islets and connected it to the circulation of totally pancreatectomized diabetic pigs. We found that blood glucose levels were reduced to a normal range and general health was improved, resulting in longer survival times. In addition, regulation of insulin secretion from the BAP was properly controlled in response to glucose both in vitro and in vivo. These results indicate that our newly developed BAP may be a potential therapy for the treatment of diabetes in humans.

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Amelioration of diabetes in mice after single-donor islet transplantation using the controlled release of gelatinized FGF-2

Jorge David Rivas-Carrillo, Nalu Navarro-Alvarez, Alejandro Soto-Gutierrez, Teru Okitsu, Yong Chen, Yasuhiko Tabata, Haruo Misawa, Hirofumi Noguchi, Shinichi Matsumoto, Noriaki Tanaka, Naoya Kobayashi

CELL TRANSPLANTATION ( COGNIZANT COMMUNICATION CORP )  15 ( 10 ) 939 - 944   2006 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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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.

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A human beta-cell line for transplantation therapy to control type 1 diabetes

M Narushima, N Kobayashi, T Okitsu, Y Tanaka, SA Li, Y Chen, A Miki, K Tanaka, S Nakaji, K Takei, AS Gutierrez, JD Rivas-Carrillo, N Navarro-Alvarez, HS Jun, KA Westerman, H Noguchi, JRT Lakey, P Leboulch, N Tanaka, JW Yoon

NATURE BIOTECHNOLOGY ( NATURE PUBLISHING GROUP )  23 ( 10 ) 1274 - 1282   2005.10 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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A human pancreatic b-cell line that is functionally equivalent to primary b-cells has not been available. We established a reversibly immortalized human b-cell clone ( NAKT-15) by transfection of primary human b-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 b-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.

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