Published Papers - Noguchi Hirofumi

BETA2/NeuroD protein can be transduced into cells due to an arginine- and lysine-rich sequence

H Noguchi, S Bonner-Weir, FY Wei, M Matsushita, S Matsumoto

DIABETES ( AMER DIABETES ASSOC )  54 ( 10 ) 2859 - 2866   2005.10 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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BETA2/NeuroD, a basic helix-loop-helix transcription factor, is a key regulator of pancreatic islet morphogenesis and insulin gene transcription. Here we report for the first time that the BETA2/NeuroD protein can permeate several cells, including pancreatic islets, due to an arginine- and lysine-rich protein transduction domain sequence in its structure. The BETA2/NeuroD protein was transduced in a dose-dependent manner up to 1 mu mol/l. Transduced BETA2/NeuroD functions similarly to endogenous BETA2/ NeuroD: it binds to the insulin promoter and activates its expression. We also investigated the mechanism of BETA2/ NeuroD protein transduction. The BETA2/NeuroD protein penetrated cells by macropinocytosis and was released from endosomes homogeneously in cytoplasm and nuclei. These data suggest 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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Cell permeable peptide of JNK inhibitor prevents islet apoptosis immediately after isolation and improves islet graft function

H Noguchi, Y Nakai, S Matsumoto, M Kawaguchi, M Ueda, T Okitsu, Y Iwanaga, Y Yonekawa, H Nagata, K Minami, Y Masui, S Futaki, K Tanaka

AMERICAN JOURNAL OF TRANSPLANTATION ( WILEY-BLACKWELL )  5 ( 8 ) 1848 - 1855   2005.08 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Although application of the Edmonton protocol has markedly improved outcomes for pancreatic islet transplantation, the insulin independence rate after islet transplantation from one donor pancreas has proven to remain 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. Pancreas preservation with the two-layer method (TLM) has proven to improve transplant results by protecting isolated islets against apoptosis through the mitochondrial pathway. However, pancreas storage with TLM cannot protect against activation of c-Jun NH(2)-terminal kinase (JNK) in isolated islets. This study investigated whether delivery of a JNK inhibitory peptide (JNKI) via the protein transduction system can prevent apoptosis of islet cells immediately after isolation. For efficient delivery of the (JNKI into isolated islets, we synthesized JNKI as a C-terminal fusion peptide with the 11-arginine protein transduction domain (11R-JNKI). 11R efficiently delivered the JNKI into isolated islets and 11R-JNKI prevented islet apoptosis immediately after isolation and improved islet graft function. These findings suggest that peptide drugs could be useful for the prevention of the impairment of islet cells and lead to improvement in the outcomes for pancreatic islet transplantation.

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Analysis of large-scale nonhuman primate islet isolations

S Matsumoto, Y Iwanaga, T Okitsu, H Noguchi, Y Yonekawa, K Tanaka, DM Strong, JA Reems, LK Gaur

TRANSPLANTATION PROCEEDINGS ( ELSEVIER SCIENCE INC )  37 ( 2 ) 1317 - 1321   2005.03 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Aims. It is important to have clinically relevant large animal models, especially nonhuman primates, to improve the efficacy of islet isolation and transplantation prior to clinical trials. The aim of this study was to improve the efficacy of islet isolation by analyzing large-scale nonhuman primate islet isolations. Methods. Sixty-one islet isolations were evaluated using nonhuman primates. An automated isolation method was scaled down for islet isolation. Islet yields of prepurification, postpurification, and postculture, purity of islets, viability of islets, and functionality with glucose stimulation test were assessed. Initially, we analyzed relationships between endpoints then analyzed additional factors for successful islet isolation. Those factors included donor characteristics, the two-layer method (TLM) of pancreas preservation, trypsin inhibition during digestion, and digestion and collection time. Results. Prepurification islet yields were strongly correlated with postpurification yields and postculture yields. It weakly but significantly correlated with purity, viability, and functionality. The average prepurification yield was 16,267 IE/g with each case divided into either above-average (high-yield group) or below-average groups (low-yield group). In 8 cases, TLM and trypsin inhibition were used and all cases belonged to the high-yield group. There were no significant differences between high- and low-yield groups in terms of donor age, body weight, pancreas weight, and cold ischemic time. The high-yield group had significantly longer digestion times and shorter collection times. Conclusions. TLM, trypsin inhibition, complete digestion, and quick collections were key for successful islet isolation. Analysis of nonhuman primate islet isolation techniques provided useful information, which should help to improve clinical islet transplantation.

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Effective islet isolation method with extremely high islet yields from adult pigs

Y Yonekawa, S Matsumoto, T Okitsu, T Arata, Y Iwanaga, H Noguchi, H Nagata, JJ O'Neil, K Tanaka

CELL TRANSPLANTATION ( COGNIZANT COMMUNICATION CORP )  14 ( 10 ) 757 - 762   2005 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Achieving good islet isolation is one of the most important factors for successful islet transplantation. Porcine pancreas is suitable for islet isolation research due to its anatomical and physiological similarities to human pancreas. In this study, we evaluated a new porcine islet isolation method designed to maximize islet yield and compared it with our previous open pan method and the standard method using a Ricordi chamber (Ricordi method). We performed 15 porcine islet isolations, five each with the new method, the open pan method, and the Ricordi method. The new method features several important improvements. Pancreata remain uncut and are kept intact during collagenase intraductal injection, a large filtration chamber to handle whole pancreata, low concentration of collagenase (Liberase(TM) HI) for digestion, and large plastic containers for large-scale islet purification. All isolated islets were assessed for yield, purity, viability and in vitro function. Islets isolated with this new method were transplanted under the kidney capsules of SCID mice with chemically induced diabetes for in vivo functional assessment (n = 8). With the new method. we obtained on average more than 1,000,000 islet equivalents (IE) (1,236,266 213,486 IE) (mean SE) before purification and 800,000 IE (879,815 222,729 IE) after purification from one adult pig. Islet yield per pancreas was significantly higher compared with our previous open pan method (30,666 +/- 11,532 IE, p < 0.01) and the Ricordi method (317.073 +/- 86.093 IE, p < 0.05). All mice, transplanted with 1000 islets from the new method, returned to normoglycemia within 4 days after transplantation. Our new method makes it possible to obtain extremely high porcine islet yield with good function. It should produce useful information for human islet isolation and transplantation, and might be applied to single donor clinical xenogeneic transplantation.

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A new cell-permeable peptide allows successful allogeneic islet transplantation in mice

H Noguchi, M Matsushita, T Okitsu, A Moriwaki, K Tomizawa, SY Kang, ST Li, N Kobayashi, S Matsumoto, K Tanaka, N Tanaka, H Matsui

NATURE MEDICINE ( NATURE PUBLISHING GROUP )  10 ( 3 ) 305 - 309   2004.03 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Calcineurin inhibitors such as cyclosporine A and FK506 have been used for transplant therapy and treatment of autoimmune diseases. However, the inhibition of calcineurin outside the immune system has a number of side effects, including hyperglycemia. In the search for safer drugs, we developed a cell-permeable inhibitor of NFAT (nuclear factor of activated T cells) using the polyarginine peptide delivery system(1,2). This peptide provided immunosuppression for fully mismatched islet allografts in mice. In addition, it did not affect insulin secretion, whereas FK506 caused a dose-dependent decrease in insulin secretion. Cell-permeable peptides can thus provide a new strategy for drug development and may eventually be useful clinically.

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Esophageal cancer associated with right aortic arch: Report of two cases

H Noguchi, Y Naomoto, M Haisa, T Yamatsuji, K Shigemitsu, T Ohkawa, T Nobuhisa, R Ono, M Gunduz, N Tanaka

DISEASES OF THE ESOPHAGUS ( BLACKWELL PUBLISHING ASIA )  16 ( 4 ) 332 - 334   2003 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Esophageal carcinoma associated with a right aortic arch is very rare. In such cases, the dissection of right paratracheal lymph nodes is difficult. Herein, we report two cases of thoracic esophageal carcinoma with right aortic arch, for which the left door open method was used to provide a good surgical view. Postoperative chemotherapy and radiotherapy were used for both cases and no evidence of recurrence or metastasis has been noted in the 24-month postoperative period.

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Controlled expansion of human endothelial cell Populations by cre-loxP-based reversible immortalization

H Noguchi, N Kobayashi, KA Westerman, M Sakaguchi, T Okitsu, T Totsugawa, T Watanabe, T Matsumura, T Fujiwara, T Ueda, M Miyazaki, N Tanaka, P Leboulch

HUMAN GENE THERAPY ( MARY ANN LIEBERT INC PUBL )  13 ( 2 ) 321 - 334   2002.01 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Endothelial cells (ECs) play multiple physiological functions and are central to many pathological processes. Various biological studies as well as cell and gene therapy applications would benefit substantially from a procedure that would result in the expansion in culture of large numbers of highly differentiated human ECs. Here, we report the amplification in vitro of human EC populations, which occurred during the first phase of reversible immortalization resulting from the retroviral transfer of an oncogene that was subsequently excised by Cre-loxP-mediated site-specific recombination. Human umbilical vein endothelial cells (HUVECs) and human liver sinusoidal endothelial cells (HLSECs) were transduced with a retroviral vector that expresses the simian virus 40 large T (SV40T) gene flanked by positive and negative selectable markers and a pair of loxP recombination targets. Transduced HUVECs and HLSECs yielded clones with greatly extended life spans, referred to as HNNT-1 and HNNT-2 cells, respectively. HNNT-1 and HNNT-2 cells showed morphological characteristics of ECs and were maintained in culture up to population doubling level (PDL) 80 for HNNT-1 and PDL 65 for HNNT-2 cells. HNNT-1 and HNNT-2 cells were not tumorigenic when transplanted into severe combined immunodeficiency mice and were sensitive to ganciclovir as well as G418. Both cell clones expressed EC markers, which include factor VIII, VEGF receptors (Flt-1 and KDR/Flk-1), and CD34, and endocytosed acetylated low-density lipoproteins. Formation of capillary-like structures in a Matrigel assay was observed with HNNT-1 and HNNT-2 cells until at least PDL 50. Complete elimination of the transferred SV40T gene was achieved in virtually 100% of HNNT-1 and HNNT-2 cells after infection with a recombinant adenovirus expressing the Cre recombinase fused to a nuclear localization signal and subsequent selection with G418. Reverted cells maintained their differentiated EC phenotype. This study extends the utility of the reversible immortalization procedure and provides a means to expand primary human ECs of various sources for basic studies and possible cell and gene therapies.

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Cre/loxP-based reversible immortalization of human hepatocytes

N Kobayashi, H Noguchi, KA Westerman, T Watanabe, T Matsumura, T Totsugawa, T Fujiwara, P Leboulch, N Tanaka

CELL TRANSPLANTATION ( COGNIZANT COMMUNICATION CORP )  10 ( 4-5 ) 383 - 386   2001 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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An ideal alternative to the primary human hepatocytes for hepatocyte transplantation would be to use a clonal. cell line that grows economically in culture and exhibits the characteristics of differentiated, nontransformed hepatocytes following transplantation. The purpose of the present studies was to establish a reversibly immortalized human hepatocyte cell line. Human hepatocytes were immortalized with a retroviral vector SSR#69 expressing simian virus 40 large T antigen (SV40Tag) gene flanked by a pair of loxP recombination targets. One of the resulting clones, NKNT-3, showed morphological characteristics of liver parenchymal cells and expressed the genes of differentiated liver functions. NKNT-3 cells offered unlimited availability. After an adenoviral delivery of Cre recombinase and subsequent differential selection, efficient removal of SV40Tag from NKNT-3 cells was performed. Here we represent that elimination of the retrovirally transferred SV40Tag gene can be excised by adenovirus-mediated site-specific recombination.

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A new approach to develop a biohybrid artificial liver using a tightly regulated human hepatocyte cell line.

Kobayashi N, Noguchi H, Watanabe T, Matsumura T, Totsugawa T, Fujiwara T, Taguchi T, Urata H, Kishimoto N, Hayashi N, Nakaji S, Westerman KA, Leboulch P, Murakami T, Tanaka N

Human cell   13 ( 4 ) 229 - 235   2000.12 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Establishment of a highly differentiated immortalized adult human hepatocyte cell line by retroviral gene transfer

N Kobayashi, H Noguchi, T Fujiwara, KA Westerman, P Leboulch, N Tanaka

TRANSPLANTATION PROCEEDINGS ( ELSEVIER SCIENCE INC )  32 ( 7 ) 2368 - 2369   2000.11 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

Efficient Cre/loxP site-specific recombination in a HepG2 human liver cell line

N Kobayashi, H Noguchi, KA Westerman, T Matsumura, T Watanabe, T Totsugawa, T Fujiwara, P Leboulch, N Tanaka

CELL TRANSPLANTATION ( COGNIZANT COMMUNICATION CORP )  9 ( 5 ) 737 - 742   2000.09 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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A worldwide shortage of donor livers is a limiting factor of the clinical application of hepatocyte transplantation (HTX). To resolve this issue, we focused on a reversible immortalization system that allows temporary expansion of primary hepatocyte populations by transfer of an oncogene that can be subsequently excised. As a preliminary test toward this goal, we examined the efficacy of Cre/loxP site-specific recombination in a transformed human liver cell line, HepG2. The present study utilized retroviral transfer of a prototypical immortalizing gene, simian virus 40 large T antigen (SV40Tag), flanked by a pair of loxP recombination targets and adenovirus-mediated Cre/loxP recombination. Here we report that complete elimination of the retroviral transferred oncogene was achieved by site-specific recombination using a replication-deficient recombinant adenovirus vector producing Cre recombinase (Ad-Cre).

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Establishment of a reversibly immortalized human hepatocyte cell line by using Cre/LoxP site-specific recombination

N Kobayashi, H Noguchi, T Fujiwara, N Tanaka

TRANSPLANTATION PROCEEDINGS ( ELSEVIER SCIENCE INC )  32 ( 5 ) 1121 - 1122   2000.08 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

A case of thyroid cancer involving the trachea: Treatment by partial tracheal resection and repair with a latissimus dorsi musculocutaneous flap

K Shigemitsu, Y Naomoto, M Haisa, T Yamatsuji, H Noguchi, M Kataoka, Y Kamikawa, N Tanaka

JAPANESE JOURNAL OF CLINICAL ONCOLOGY ( OXFORD UNIV PRESS )  30 ( 5 ) 235 - 238   2000.05 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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A 65 year-old man had undergone left thyroidectomy for thyroid cancer. The cancer had directly invaded the cervical esophagus and trachea and the patient was referred to our hospital for radical resection and reconstruction. Cervical computed tomography showed a mass at the left-posterior wall of the trachea. Cervical esophagectomy, resection of the left half of the trachea (6 x 3 cm) including seven rings and cervical lymph node dissection were performed. The tracheal defect was covered by a latissimus dorsi musculocutaneous flap. The patient did not lose vocal function and remains alive and well 3 years after surgery without any evidence of recurrence. Latissimus dorsi muscle flap coverage of tracheal defects seems to be a useful technique in the combined resection of the trachea.

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Establishment of a highly differentiated immortalized human hepatocyte cell line as a source of hepatic function in the bioartificial liver

N Kobayashi, M Miyazaki, K Fukaya, Y Inoue, M Sakaguchi, H Noguchi, N Tanaka, M Namba

TRANSPLANTATION PROCEEDINGS ( ELSEVIER SCIENCE INC )  32 ( 2 ) 237 - 241   2000.03 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

Establishment of a highly differentiated immortalized human hepatocyte cell line with tight regulation

N. Kobayashi, H. Noguchi, T. Fujiwara, N. Tanaka

ASAIO Journal ( Lippincott-Raven Publ )  46 ( 2 ) 229   2000 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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To determine an ideal cell line for bioartificial liver (BAL) therapy, primary human hepatocytes were immortalized with a plasmid SV3neo containing the genes of simian virus 40 large T antigen and neomycin phosphototransferase by a method of lipofection. As a safeguard in clinical application, HSV-tk gene was retrovirally introduced into the immortal cells. The gene expression of liver-specific functions and in vitro sensitivity to ganciclovir (GCV) were examined. To investigate the in-vivo potential of cells, intrasplenic transplantation (Isp-Tx) was performed in rats that underwent 90% hepatectomy. The results indicate that the cells can offer the advantages of uniformity, sterility, unlimited availability, and freedom from infectious pathogens and may be useful for BAL.

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