Published Papers - Noguchi Hirofumi

Islet transplantation using donors after brain and cardiac death: A multicenter clinical trial in Japan

Anazawa, T; Marubashi, S; Kodama, S; Goto, M; Eguchi, H; Maruyama, M; Shimoda, M; Noguchi, H; Yamaguchi, T; Ito, T; Kenmochi, T; Gotoh, M

TRANSPLANTATION   107 ( 10 ) 84 - 84   2023.10 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Cytoprotective Effect of Pteryxin on Insulinoma MIN6 Cells Due to Antioxidant Enzymes Expression via Nrf2/ARE Activation

Junsei Taira, Ryuji Tsuda, Chika Miyagi-Shiohira, Hirofumi Noguchi, Takayuki Ogi

Antioxidants ( MDPI AG )  12 ( 3 ) 693 - 693   2023.03 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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The low-level antioxidant activity of pancreatic islets causes type 1 diabetes due to oxidative stress, which is also the cause of failure in the pancreatic islets' isolation and cell transplantation. In our previous study, pteryxin was found to be a natural product as a nuclear factor-erythroid-2-related factor (Nrf2) activator. This study focused on elucidation that the potentiality of pteryxin can activate the antioxidant enzymes, even under oxidative stress, by hydrogen peroxide (H2O2). Pteryxin treated with mouse insulinoma MIN6 cells was enhanced the antioxidant gene expressions in the ARE (antioxidant response element) region for HO-1 (Heme Oxygenase-1), GCLC (Glutamate-cysteine ligase catalytic subunit), SOD1 (Super Oxide dismutase1), and Trxr1 (Thioredoxin reductase1), and those enzymes were also expressed during the nuclei transference of cytoplasmic Nrf2. In fact, the cells exposed to H2O2 concentrations of a half-cell lethal in the presence of pteryxin were then induced main antioxidant enzymes, HO-1, GCLC, and Trxr1 in the ARE region. The increased glutathione (GSH) levels associated with the GCLC expression also suggested to be cytoprotective against oxidative stress by activating the redox-metabolizing enzymes involving their increased antioxidant activity in the cells. In addition, Akt is a modulator for Nrf2, which may be responsible for the Nrf2 activation. These results allowed us to consider whether pteryxin or its synthesized congeners, an Nrf2 activator, is a potential preservative agent against islet isolation during cell transplantation.

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Protocol for the generation of human induced hepatic stem cells using Sendai virus vectors

Noguchi, H; Nakashima, Y; Watanabe, M; Matsushita, M; Tsukahara, M; Saitoh, I; Miyagi-Shiohira, C

STAR PROTOCOLS ( STAR Protocols )  3 ( 4 ) 101884 - 101884   2022.12 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Our recent study demonstrated the generation of induced tissue-specific stem/progenitor (iTS/iTP) cells by the transient overexpression of reprogramming factors combined with tissue-specific selection. Here, we present a protocol to reprogram human hepatocytes to generate human induced tissue-specific liver stem (iTS-L) cells. Human hepatocytes are transfected with Sendai virus vectors (SeV) expressing OCT3/4, SOX2, KLF4, and c-MYC. iTS-L cells continuously express mRNA of hepatocyte-specific markers (HNF1β and HNF4α) and do not form teratomas. For complete details on the use and execution of this protocol, please refer to Nakashima et al. (2022).1.

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Functional Evaluation of 3D Liver Models Labeled with Polysaccharide Functionalized Magnetic Nanoparticles

Miyamoto, Y; Koshidaka, Y; Murase, K; Kanno, S; Noguchi, H; Miyado, K; Ikeya, T; Suzuki, S; Yagi, T; Teramoto, N; Hayashi, S

MATERIALS ( Materials )  15 ( 21 ) 7823 - 7823   2022.11 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Establishing a rapid in vitro evaluation system for drug screening is essential for the development of new drugs. To reproduce tissues/organs with functions closer to living organisms, in vitro three-dimensional (3D) culture evaluation using microfabrication technology has been reported in recent years. Culture on patterned substrates with controlled hydrophilic and hydrophobic regions (Cell-ableTM) can create 3D liver models (miniature livers) with liver-specific Disse luminal structures and functions. MRI contrast agents are widely used as safe and minimally invasive diagnostic methods. We focused on anionic polysaccharide magnetic iron oxide nanoparticles (Resovist®) and synthesized the four types of nanoparticle derivatives with different properties. Cationic nanoparticles (TMADM) can be used to label target cells in a short time and have been successfully visualized in vivo. In this study, we examined the morphology of various nanoparticles. The morphology of various nanoparticles showed relatively smooth-edged spherical shapes. As 3D liver models, we prepared primary hepatocyte–endothelial cell heterospheroids. The toxicity, CYP3A, and albumin secretory capacity were evaluated in the heterospheroids labeled with various nanoparticles. As the culture period progressed, the heterospheroids labeled with anionic and cationic nanoparticles showed lower liver function than non-labeled heterospheroids. In the future, there is a need to improve the method of creation of artificial 3D liver or to design a low-invasive MRI contrast agent to label the artificial 3D liver.

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Induced hepatic stem cells are suitable for human hepatocyte production

Nakashima, Y; Miyagi-Shiohira, C; Saitoh, I; Watanabe, M; Matsushita, M; Tsukahara, M; Noguchi, H

ISCIENCE ( iScience )  25 ( 10 ) 105052 - 105052   2022.10 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Human hepatocytes were transfected with Sendai virus vectors (SeV) expressing OCT3/4, SOX2, KLF4, and C-MYC to produce hepatocyte-derived induced pluripotent stem cells (iPSCs). The messenger RNA (mRNA) expression of undifferentiated markers (passage 19-21) and hepatocyte-specific markers (HSMs) (passage 0-20) in 48 established hepatocyte-derived iPSC-like colonies was examined. Among the 48 clones, 10 clones continuously expressed HSM mRNA (HNF1β and HNF4α) in passage 0-20. The colonies which expressed HSMs (iTS-L cells: induced tissue-specific stem cells from liver) showed a different tendency in microarray and methylation analyses to fibroblast-derived iPSCs (strain: 201B7). iTS-L cells were less likely to form teratomas in mice than iPSCs (He). The iTS-L cells were differentiated into hepatocyte-like cells more efficiently than iPSCs (He) or iPSCs (201B7). These data suggest that SeV expressing OCT3/4, SOX2, KLF4, and C-MYC induce the generation of iPSCs and iTS-L cells.

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The Role of Genetically Modified Human Feeder Cells in Maintaining the Integrity of Primary Cultured Human Deciduous Dental Pulp Cells

Ibano, N; Inada, E; Otake, S; Kiyokawa, Y; Sakata, K; Sato, M; Kubota, N; Noguchi, H; Iwase, Y; Murakami, T; Sawami, T; Kakihara, Y; Maeda, T; Terunuma, M; Terao, Y; Saitoh, I

JOURNAL OF CLINICAL MEDICINE ( Journal of Clinical Medicine )  11 ( 20 )   2022.10 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Tissue-specific stem cells exist in tissues and organs, such as skin and bone marrow. However, their pluripotency is limited compared to embryonic stem cells. Culturing primary cells on plastic tissue culture dishes can result in the loss of multipotency, because of the inability of tissue-specific stem cells to survive in feeder-less dishes. Recent findings suggest that culturing primary cells in medium containing feeder cells, particularly genetically modified feeder cells expressing growth factors, may be beneficial for their survival and proliferation. Therefore, the aim of this study was to elucidate the role of genetically modified human feeder cells expressing growth factors in maintaining the integrity of primary cultured human deciduous dental pulp cells. Feeder cells expressing leukemia inhibitory factor, bone morphogenetic protein 4, and basic fibroblast growth factor were successfully engineered, as evidenced by PCR. Co-culturing with mitomycin-C-treated feeder cells enhanced the proliferation of newly isolated human deciduous dental pulp cells, promoted their differentiation into adipocytes and neurons, and maintained their stemness properties. Our findings suggest that genetically modified human feeder cells may be used to maintain the integrity of primary cultured human deciduous dental pulp cells.

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AP39, a Mitochondrial-Targeted H₂S Donor, Improves Porcine Islet Survival in Culture

Shinzato, M; Miyagi-Shiohira, C; Kuwae, K; Nishime, K; Tamaki, Y; Yonaha, T; Sakai-Yonaha, M; Yamasaki, I; Otsuka, R; Saitoh, I; Watanabe, M; Noguchi, H

JOURNAL OF CLINICAL MEDICINE ( Journal of Clinical Medicine )  11 ( 18 )   2022.09 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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The rapid deterioration of transplanted islets in culture is a well-established phenomenon. We recently reported that pancreas preservation with AP39 reduces reactive oxygen species (ROS) production and improves islet graft function. In this study, we investigated whether the addition of AP39 to the culture medium could reduce isolated islet deterioration and improve islet function. Isolated islets from porcine pancreata were cultured with 400 nM AP39 or without AP39 at 37 °C. After culturing for 6-72 h, the islet equivalents of porcine islets in the AP39(+) group were significantly higher than those in the AP39(-) group. The islets in the AP39(+) group exhibited significantly decreased levels of ROS production compared to the islets in the AP39(-) group. The islets in the AP39(+) group exhibited significantly increased mitochondrial membrane potential compared to the islets in the AP39(-) group. A marginal number (1500 IEs) of cultured islets from each group was then transplanted into streptozotocin-induced diabetic mice. Culturing isolated islets with AP39 improved islet transplantation outcomes in streptozotocin-induced diabetic mice. The addition of AP39 in culture medium reduces islet deterioration and furthers the advancements in β-cell replacement therapy.

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Pancreas Preservation in Modified Histidine-lactobionate Solution Is Superior to That in University of Wisconsin Solution for Porcine Islet Isolation

Sakai-Yonaha, M; Miyagi-Shiohira, C; Kuwae, K; Tamaki, Y; Nishime, K; Yonaha, T; Saitoh, I; Watanabe, M; Noguchi, H

TRANSPLANTATION ( Transplantation )  106 ( 9 ) 1770 - 1776   2022.09 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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BACKGROUND: We previously reported that modified extracellular-type trehalose-containing Kyoto (MK) solution, which contains a trypsin inhibitor (ulinastatin), significantly improved the islet yield compared with University of Wisconsin (UW) preservation, which is the gold standard for organ preservation for islet isolation. In this study, we evaluated the efficiency of a modified histidine-lactobionate (MHL) solution in addition to UW or MK solution. The MHL solution has a high sodium-low potassium composition with low viscosity compared with the UW solution. Moreover, similar to MK solution, MHL solution also contains ulinastatin. METHODS: Porcine pancreata were preserved in UW, MK, or MHL solution, followed by islet isolation. An optimized number (1500 IE) of isolated islets from each group were then transplanted into streptozotocin-induced diabetic mice. RESULTS: The islet yield before and after purification was significantly higher in the MHL group than in the UW group. On the contrary, the islet yield before and after purification was not significantly different between the MHL and MK groups. Preserving the porcine pancreata in MHL solution improved the outcome of islet transplantation in streptozotocin-induced diabetic mice compared with that in UW solution. CONCLUSIONS: Pancreas preservation with MHL solution preserves islet function better than UW solution. The effect of MHL solution is similar to that of MK solution, suggesting that MHL solution can be used as an alternative to MK solution for pancreatic islet transplantation.

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Initiation of dialysis for kidney graft failure: A retrospective single-center cohort study

Taira, H; Noguchi, H; Ueki, K; Kaku, K; Tsuchimoto, A; Okabe, Y; Ohya, Y; Nakamura, M

THERAPEUTIC APHERESIS AND DIALYSIS ( Therapeutic Apheresis and Dialysis )  26 ( 4 ) 806 - 814   2022.08 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Pancreas Preservation with a Neutrophil Elastase Inhibitor, Alvelestat, Contributes to Improvement of Porcine Islet Isolation and Transplantation

Otsuka, R; Miyagi-Shiohira, C; Kuwae, K; Nishime, K; Tamaki, Y; Yonaha, T; Sakai-Yonaha, M; Yamasaki, I; Shinzato, M; Saitoh, I; Watanabe, M; Noguchi, H

JOURNAL OF CLINICAL MEDICINE ( Journal of Clinical Medicine )  11 ( 15 )   2022.07 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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For pancreatic islet transplantation, pancreas procurement, preservation, and islet isolation destroy cellular and non-cellular components and activate components such as resident neutrophils, which play an important role in the impairment of islet survival. It has been reported that inhibitors of neutrophil elastase (NE), such as sivelestat and α1-antitrypsin, could contribute to improvement of islet isolation and transplantation. In this study, we investigated whether pancreatic preservation with alvelestat, a novel NE inhibitor, improves porcine islet yield and function. Porcine pancreata were preserved with or without 5 μM alvelestat for 18 h, and islet isolation was performed. The islet yields before and after purification were significantly higher in the alvelestat (+) group than in the alvelestat (-) group. After islet transplantation into streptozotocin-induced diabetic mice, blood glucose levels reached the normoglycemic range in 55% and 5% of diabetic mice in the alvelestat (+) and alvelestat (-) groups, respectively. These results suggest that pancreas preservation with alvelestat improves islet yield and graft function and could thus serve as a novel clinical strategy for improving the outcome of islet transplantation.

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Clinical Islet Transplantation Covered by Health Insurance in Japan

Noguchi, H

JOURNAL OF CLINICAL MEDICINE ( Journal of Clinical Medicine )  11 ( 14 )   2022.07 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Pancreatic islet transplantation is a treatment option for patients with type 1 diabetes mellitus and has been performed in various countries [...].

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Tissue-Nonspecific Alkaline Phosphatase, a Possible Mediator of Cell Maturation: Towards a New Paradigm

Sato, M; Saitoh, I; Kiyokawa, Y; Iwase, Y; Kubota, N; Ibano, N; Noguchi, H; Yamasaki, Y; Inada, E

CELLS ( Cells )  10 ( 12 ) 3338 - 3338   2021.11 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Alkaline phosphatase (ALP) is a ubiquitous membrane-bound glycoprotein capable of providing inorganic phosphate by catalyzing the hydrolysis of organic phosphate esters, or removing inorganic pyrophosphate that inhibits calcification. In humans, four forms of ALP cDNA have been cloned, among which tissue-nonspecific ALP (TNSALP) (TNSALP) is widely distributed in the liver, bone, and kidney, making it an important marker in clinical and basic research. Interestingly, TNSALP is highly expressed in juvenile cells, such as pluripotent stem cells (i.e., embryonic stem cells and induced pluripotent stem cells (iPSCs)) and somatic stem cells (i.e., neuronal stem cells and bone marrow mesenchymal stem cells). Hypophosphatasia is a genetic disorder causing defects in bone and tooth development as well as neurogenesis. Mutations in the gene coding for TNSALP are thought to be responsible for the abnormalities, suggesting the essential role of TNSALP in these events. Moreover, a reverse-genetics-based study using mice revealed that TNSALP is important in bone and tooth development as well as neurogenesis. However, little is known about the role of TNSALP in the maintenance and differentiation of juvenile cells. Recently, it was reported that cells enriched with TNSALP are more easily reprogrammed into iPSCs than those with less TNSALP. Furthermore, in bone marrow stem cells, ALP could function as a "signal regulator" deciding the fate of these cells. In this review, we summarize the properties of ALP and the background of ALP gene analysis and its manipulation, with a special focus on the potential role of TNSALP in the generation (and possibly maintenance) of juvenile cells.

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In vivo evaluation of GG2-GG1/A2 element activity in the insulin promoter region using the CRISPR-Cas9 system

Noguchi, H; Miyagi-Shiohira, C; Kinjo, T; Saitoh, I; Watanabe, M

SCIENTIFIC REPORTS ( Scientific Reports )  11 ( 1 ) 20290 - 20290   2021.10 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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The insulin promoter is regulated by ubiquitous as well as pancreatic β-cell-specific transcription factors. In the insulin promoter, GG2-GG1/A2-C1 (bases - 149 to - 116 in the human insulin promoter) play important roles in regulating β-cell-specific expression of the insulin gene. However, these events were identified through in vitro studies, and we are unaware of comparable in vivo studies. In this study, we evaluated the activity of GG2-GG1/A2 elements in the insulin promoter region in vivo. We generated homozygous mice with mutations in the GG2-GG1/A2 elements in each of the Ins1 and Ins2 promoters by CRISPR-Cas9 technology. The mice with homozygous mutations in the GG2-GG1/A2 elements in both Ins1 and Ins2 were diabetic. These data suggest that the GG2-GG1/A2 element in mice is important for Ins transcription in vivo.

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Preservation of pancreas in the University of Wisconsin solution supplemented with AP39 reduces reactive oxygen species production and improves islet graft function

Nishime, K; Miyagi-Shiohira, C; Kuwae, K; Tamaki, Y; Yonaha, T; Sakai-Yonaha, M; Saitoh, I; Watanabe, M; Noguchi, H

AMERICAN JOURNAL OF TRANSPLANTATION ( American Journal of Transplantation )  21 ( 8 ) 2698 - 2708   2021.08 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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RNA analysis based on a small number of manually isolated fixed cells (RNA-snMIFxC) to profile stem cells from human deciduous tooth-derived dental pulp cells

Inada, E; Saitoh, I; Kubota, N; Iwase, Y; Kiyokawa, Y; Noguchi, H; Yamasaki, Y; Sato, M

BIOLOGICAL PROCEDURES ONLINE ( Biological Procedures Online )  23 ( 1 ) 12 - 12   2021.06 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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BACKGROUND: Expression of stemness factors, such as octamer-binding transcription factor 3/4 (OCT3/4), sex determining region Y-box 2 (SOX2), and alkaline phosphatase (ALP) in human deciduous tooth-derived dental pulp cells (HDDPCs) can be assessed through fixation and subsequent immuno- or cytochemical staining. Fluorescence-activated cell sorting (FACS), a powerful system to collect cells of interest, is limited by the instrument cost and difficulty in handling. Magnetic-activated cell sorting is inexpensive compared to FACS, but is confined to cells with surface expression of the target molecule. In this study, a simple and inexpensive method was developed for the molecular analysis of immuno- or cytochemically stained cells with intracellular expression of a target molecule, through isolation of a few cells under a dissecting microscope using a mouthpiece-controlled micropipette. RESULTS: Two or more colored cells (~ 10), after staining with a chromogen such a 3,3'-diaminobenzidine, were successfully segregated from unstained cells. Expression of glyceraldehyde 3-phosphate dehydrogenase, a housekeeping gene, was discernible in all samples, while the expression of stemness genes (such as OCT3/4, SOX2, and ALP) was confined to positively stained cells. CONCLUSION: These findings indicate the fidelity of these approaches in profiling cells exhibiting cytoplasmic or nuclear localization of stemness-specific gene products at a small-scale.

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Induced Tissue-Specific Stem Cells (iTSCs): Their Generation and Possible Use in Regenerative Medicine

Saitoh, I; Sato, M; Kiyokawa, Y; Inada, E; Iwase, Y; Ibano, N; Noguchi, H

PHARMACEUTICS ( Pharmaceutics )  13 ( 6 ) 780 - 780   2021.05 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Induced tissue-specific stem cells (iTSCs) are partially reprogrammed cells which have an intermediate state, such as progenitors or stem cells. They originate from the de-differentiation of differentiated somatic cells into pluripotent stem cells, such as induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs), or from the differentiation of undifferentiated cells. They show a limited capacity to differentiate and a morphology similar to that of somatic cell stem cells present in tissues, but distinct from that of iPSCs and ESCs. iTSCs can be generally obtained 7 to 10 days after reprogramming of somatic cells with Yamanaka's factors, and their fibroblast-like morphology remains unaltered. iTSCs can also be obtained directly from iPSCs cultured under conditions allowing cellular differentiation. In this case, to effectively induce iTSCs, additional treatment is required, as exemplified by the conversion of iPSCs into naïve iPSCs. iTSCs can proliferate continuously in vitro, but when transplanted into immunocompromised mice, they fail to generate solid tumors (teratomas), implying loss of tumorigenic potential. The low tendency of iTSCs to elicit tumors is beneficial, especially considering applications for regenerative medicine in humans. Several iTSC types have been identified, including iTS-L, iTS-P, and iTS-D, obtained by reprogramming hepatocytes, pancreatic cells, and deciduous tooth-derived dental pulp cells, respectively. This review provides a brief overview of iPSCs and discusses recent advances in the establishment of iTSCs and their possible applications in regenerative medicine.

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Pancreas preservation with amphotericin B deteriorates islet yield for porcine islet isolation

Sawada, S; Miyagi-Shiohira, C; Kuwae, K; Tamaki, Y; Nishime, K; Sakai-Yonaha, M; Yonaha, T; Saitoh, I; Watanabe, M; Noguchi, H

XENOTRANSPLANTATION ( Xenotransplantation )  28 ( 4 ) e12690   2021.04 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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BACKGROUND: Amphotericin B is a crucial agent in the management of serious systemic fungal infections. It is also known to be cytotoxic. In this study, we evaluated the effect of amphotericin B added to the preservation solution on islet yield during islet isolation. METHODS: Porcine pancreata were preserved in the preservation solution with or without amphotericin B (0.25 μg/mL) for approximately 18 hours at 4°C, and then islet isolation was performed. An optimized number (1750 IE) of isolated islets from each group were transplanted into streptozotocin-induced diabetic mice. The culture of isolated islets and acinar tissue with amphotericin B was also evaluated. RESULTS: The islet yield before and after purification in the amphotericin B (-) group was significantly higher than that in the amphotericin B (+) group. After islet transplantation into diabetic mice, blood glucose levels reached the normoglycemic range, with 50% and 0% of that of the diabetic mice in the amphotericin B (-) and amphotericin B (+) groups, respectively. In the culture study, amphotericin B was found to be cytotoxic to porcine islets and acinar tissue. CONCLUSIONS: Amphotericin B added to the preservation solution deteriorates islet yield during porcine islet isolation. Thus, the use of amphotericin B should be considered carefully for the preservation of the pancreas for islet isolation and islet culture before islet transplantation.

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Pancreas preservation in extracellular-type p38 inhibitor-containing solution improves islet yield for porcine islet isolation

Yonaha, T; Miyagi-Shiohira, C; Kuwae, K; Tamaki, Y; Nishime, K; Sakai-Yonaha, M; Saitoh, I; Watanabe, M; Noguchi, H

XENOTRANSPLANTATION ( Xenotransplantation )  28 ( 2 ) e12661   2021.03 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Gene Expression in Pancreatic Cancer-Like Cells and Induced Pancreatic Stem Cells Generated by Transient Overexpression of Reprogramming Factors

Miyagi-Shiohira, C; Saitoh, I; Watanabe, M; Noguchi, H

JOURNAL OF CLINICAL MEDICINE ( Journal of Clinical Medicine )  10 ( 3 ) 1 - 12   2021.01 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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We previously reported that transient overexpression of reprogramming factors can be used to generate induced pluripotent stem (iPS) cells, induced tissue-specific stem (iTS) cells, and fibroblast-like (iF) cells from pancreatic tissue. iF cells have tumorigenic ability and behave similarly to pancreatic cancer cells. In this study, we analyzed gene expression in iF cells and iTS-P cells (iTS cells from pancreatic tissue) via microarray analysis and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The expression levels of the Mybl2 and Lyn genes, which are reported to be oncogenes, were significantly higher in iF cells than in iTS-P cells. The expression level of Nestin, which is expressed in not only pancreatic progenitor cells but also pancreatic ductal adenocarcinomas, was also higher in iF cells than in iTS-P cells. Itgb6 and Fgf13, which are involved in the pathogenesis of diseases such as cancer, exhibited higher expression levels in iF cells than in iTS-P cells. Unexpectedly, the expression levels of genes related to epithelial-mesenchymal transition (EMT), except Bmp4, were lower in iF cells than in iTS-P cells. These data suggest that the Mybl2, Lyn, Nestin, Itgb6, and Fgf13 genes could be important biomarkers to distinguish iTS-P cells from iF cells.

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Drug-Induced Naïve iPS Cells Exhibit Better Performance than Primed iPS Cells with Respect to the Ability to Differentiate into Pancreatic β-Cell Lineage.

Kiyokawa Y, Sato M, Noguchi H, Inada E, Iwase Y, Kubota N, Sawami T, Terunuma M, Maeda T, Hayasaki H, Saitoh I

Journal of clinical medicine   9 ( 9 )   2020.09 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Schumannione, a new butenolide derivative isolated from Schumannianthus dichotomus as a potential phytotoxic agent

Rob M.M.

Tetrahedron Letters ( Tetrahedron Letters )  61 ( 30 )   2020.07 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Mutations in the C1 element of the insulin promoter lead to diabetic phenotypes in homozygous mice.

Noguchi H, Miyagi-Shiohira C, Nakashima Y, Kinjo T, Saitoh I, Watanabe M

Communications biology ( Communications Biology )  3 ( 1 ) 309   2020.06 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Novel cell-permeable p38-MAPK inhibitor efficiently prevents porcine islet apoptosis and improves islet graft function.

Noguchi H, Miyagi-Shiohira C, Nakashima Y, Saitoh I, Watanabe M

American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons ( American Journal of Transplantation )  20 ( 5 ) 1296 - 1308   2020.05 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Tree fern cyathea lepifera may survive by its phytotoxic property

Ida N.

Plants ( Plants )  9 ( 1 )   2020.01 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Reduced glycemic variability and flexible graft function after islet transplantation: A case report

Toshihiro Nakamura, Junji Fujikura, Takayuki Anazawa, Ryo Ito, Masahito Ogura, Hideaki Okajima, Hirofumi Noguchi, Shinji Uemoto, Nobuya Inagaki

Journal of Diabetes Investigation     2020 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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© 2020 The Authors. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd To date, studies of patients with islet transplantation addressing intermittently scanned continuous glucose monitoring profile and the flexibility of the graft islet function under different doses of insulin administration, both of which reflect the real daily life of patients, are quite limited. Here, we report a case of a 46-year-old woman who received islet transplantation after kidney transplantation. The patient was followed up over a period of 2 years after initial islet transplantation. Our results show that intermittently scanned continuous glucose monitoring can be useful for monitoring the reduction of glycemic variability, and suggest the appropriate regulation of insulin secretion from graft islets during mixed-meal test by using different doses of exogenous insulin administration. Additionally, during the 2-year observational period, glucagon elevation was detected only at hypoglycemia, whereas the level was within the normal range at normoglycemia or hyperglycemia.

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Potential use of Schumannianthus dichotomus waste: the phytotoxic activity of the waste and its identified compounds

Rob M.M.

Journal of Environmental Science and Health - Part B Pesticides, Food Contaminants, and Agricultural Wastes ( Journal of Environmental Science and Health - Part B Pesticides, Food Contaminants, and Agricultural Wastes )    2020 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Regulation of c-Jun NH₂-Terminal Kinase for Islet Transplantation.

Noguchi H

Journal of clinical medicine   8 ( 11 )   2019.10 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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piggyBac Transposon-Based Immortalization of Human Deciduous Tooth Dental Pulp Cells with Multipotency and Non-Tumorigenic Potential.

Inada E, Saitoh I, Kubota N, Iwase Y, Kiyokawa Y, Shibasaki S, Noguchi H, Yamasaki Y, Sato M

International journal of molecular sciences ( International Journal of Molecular Sciences )  20 ( 19 )   2019.10 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Excellent Islet Yields after 18-h Porcine Pancreas Preservation by Ductal Injection, Pancreas Preservation with MK Solution, Bottle Purification, and Islet Purification Using Iodixanol with UW Solution and Iodixanol with MK Solution.

Kuwae K, Miyagi-Shiohira C, Hamada E, Tamaki Y, Nishime K, Sakai M, Yonaha T, Makishi E, Saitoh I, Watanabe M, Noguchi H

Journal of clinical medicine   8 ( 10 )   2019.09 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Adipose tissue-derived mesenchymal stem cells ameliorate bone marrow aplasia related with graft-versus-host disease in experimental murine models.

Nishi Y, Murakami A, Murayama Y, Tsukahara N, Okamoto S, Nakachi S, Morichika K, Tamaki K, Noguchi H, Matsushita M, Karube KN, Fukushima T, Morishima S, Kishimoto H, Masuzaki H

Transplant immunology ( Transplant Immunology )  55   101205   2019.08 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Suppressor of Fused regulates the proliferation of postnatal neural stem and precursor cells via a Gli3-dependent mechanism.

Gomez HG, Noguchi H, Castillo JG, Aguilar D, Pleasure SJ, Yabut OR

Biology open   8 ( 6 )   2019.06 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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A culture substratum with net-like polyamide fibers promotes the differentiation of mouse and human pluripotent stem cells to insulin-producing cells.

Kaitsuka T, Kojima R, Kawabe M, Noguchi H, Shiraki N, Kume S, Tomizawa K

Biomedical materials (Bristol, England) ( Biomedical Materials (Bristol) )  14 ( 4 ) 045019   2019.06 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Induction of Expandable Tissue-Specific Progenitor Cells from Human Pancreatic Tissue through Transient Expression of Defined Factors.

Noguchi H, Miyagi-Shiohira C, Nakashima Y, Kinjo T, Kobayashi N, Saitoh I, Watanabe M, Shapiro AMJ, Kin T

Molecular therapy. Methods & clinical development ( Molecular Therapy - Methods and Clinical Development )  13   243 - 252   2019.06 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Identification of Proteins Differentially Expressed by Adipose-derived Mesenchymal Stem Cells Isolated from Immunodeficient Mice.

Nakashima Y, Nahar S, Miyagi-Shiohira C, Kinjo T, Kobayashi N, Kitamura S, Saitoh I, Watanabe M, Fujita J, Noguchi H

International journal of molecular sciences ( International Journal of Molecular Sciences )  20 ( 11 )   2019.05 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Suppressor of fused controls perinatal expansion and quiescence of future dentate adult neural stem cells.

Noguchi H, Castillo JG, Nakashima K, Pleasure SJ

eLife   8   2019.04 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Repeated human deciduous tooth-derived dental pulp cell reprogramming factor transfection yields multipotent intermediate cells with enhanced iPS cell formation capability.

Miki Soda, Issei Saitoh, Tomoya Murakami, Emi Inada, Yoko Iwase, Hirofumi Noguchi, Shinji Shibasaki, Mie Kurosawa, Tadashi Sawami, Miho Terunuma, Naoko Kubota, Yutaka Terao, Hayato Ohshima, Haruaki Hayasaki, Masahiro Sato

Scientific reports ( Scientific Reports )  9 ( 1 ) 1490 - 1490   2019.02 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Human tissue-specific stem cells (hTSCs), found throughout the body, can differentiate into several lineages under appropriate conditions in vitro and in vivo. By transfecting terminally differentiated cells with reprogramming factors, we previously produced induced TSCs from the pancreas and hepatocytes that exhibit additional properties than iPSCs, as exemplified by very low tumour formation after xenogenic transplantation. We hypothesised that hTSCs, being partially reprogrammed in a state just prior to iPSC transition, could be isolated from any terminally differentiated cell type through transient reprogramming factor overexpression. Cytochemical staining of human deciduous tooth-derived dental pulp cells (HDDPCs) and human skin-derived fibroblasts following transfection with Yamanaka's factors demonstrated increased ALP activity, a stem cell marker, three weeks after transfection albeit in a small percentage of clones. Repeated transfections (≤3) led to more efficient iPSC generation, with HDDPCs exhibiting greater multipotentiality at two weeks post-transfection than the parental intact HDDPCs. These results indicated the utility of iPSC technology to isolate TSCs from HDDPCs and fibroblasts. Generally, a step-wise loss of pluripotential phenotypes in ESCs/iPSCs occurs during their differentiation process. Our present findings suggest that the reverse phenomenon can also occur upon repeated introduction of reprogramming factors into differentiated cells such as HDDPCs and fibroblasts.

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A Novel Preservation Solution Containing a JNK Inhibitory Peptide Efficiently Improves Islet Yield for Porcine Islet Isolation.

Noguchi H, Miyagi-Shiohira C, Nakashima Y, Ebi N, Hamada E, Tamaki Y, Kuwae K, Kitamura S, Kobayashi N, Saitoh I, Watanabe M

Transplantation ( Transplantation )  103 ( 2 ) 344 - 352   2019.02 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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著明なQOLの改善が得られた，遺伝性膵炎に対する自家膵島移植の本邦第1例

富丸 慶人, 後藤 邦仁, 小林 省吾, 永野 浩昭, 森 正樹, 土岐 祐一郎, 江口 英利, 伊藤 壽記, 野口 洋文, 宮下 和幸, 川本 弘一, 岩上 佳史, 秋田 裕史, 野田 剛広

移植 ( 一般社団法人 日本移植学会 )  54 ( 4 ) 217 - 222   2019 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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<p>Total pancreatectomy with islet autotransplantation is an ideal surgical procedure used to relieve chronic pain derived from the pancreatitis, while insulin-dependent diabetes is inevitably induced after the surgery. Islet autotransplantation combined with a total pancreatectomy can reduce the risk of severe diabetes by preserving available islet cells. We experienced a case with hereditary pancreatitis, which is a rare inherited condition characterized by recurrent acute pancreatitis and/or chronic pancreatitis. The case was that of a female in her thirties who was introduced to our hospital due to treatment-refractory abdominal pain derived from repeated pancreatitis even after surgical treatments. At the introduction, the pain was uncontrollable even by opioid use, and oral intake was impossible due to increase of the pain after the intake. She was genetically diagnosed with hereditary pancreatitis. For relieving the pancreatitis-derived pain, total remnant pancreatectomy with islet autotransplantation was planned. After the pancreatectomy, islet cells were extracted from the excised remnant pancreas, and injected into the portal vein for liver autotransplantation. The pain was completely relieved for her after the surgery, suggesting improvement of her quality-of-life. A glucose tolerance test and glucagon loading test performed one month after the surgery showed C-peptide secretion as the blood glucose rose. However, the serum c-peptide level and SUIT index had been gradually decreased after the surgery, and she is now treated with insulin. The case suggested that total pancreatectomy with islet autotransplantation is useful for improvement of the patient's quality of life by controlling the pain in cases with hereditary pancreatitis.</p>

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膵島移植・再生医療の現状と展望

野口 洋文

生体医工学 ( 公益社団法人 日本生体医工学会 )  57 ( 0 ) S195_2 - S195_2   2019 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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<p>現在、糖尿病に対する移植療法として、膵臓移植と膵島移植がある。膵島移植は、局所麻酔下にて膵臓から分離した膵島を経門脈的に注入するため、膵臓移植に比べ低侵襲である。現在までに欧米で1000例以上の移植報告がなされており、日本でも2004年より臨床膵島移植が開始されている。発表者の野口はハーバード大学で膵島分離技術を習得したのち、日本初となる心停止ドナー膵島移植（2004年）、世界初成功例となる生体膵島移植（2005年）、日本初となる脳死ドナー膵島移植（2013年）を実施した。過去5年間に実施された膵島移植の約85%を発表者が実施している。しかしながら、本邦ではドナー数が圧倒的に少なく、脳死ドナー数をどのようにして増やすかが膵島移植の成功へのカギとなる。脳死ドナー不足の解消には時間がかかる現状を考慮し、別の治療法を模索する動きも活発化している。特に、再生医療研究が世界中で活発に行われているが、インスリン分泌細胞への分化誘導法が確立されておらず、いまだ研究段階であるのが現状である。本シンポジウムでは膵島移植の現状と問題点を示すとともに、最先端の糖尿病治療研究について紹介する。</p>

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A Liquid Chromatography with Tandem Mass Spectrometry-Based Proteomic Analysis of Primary Cultured Cells and Subcultured Cells Using Mouse Adipose-Derived Mesenchymal Stem Cells.

Nakashima Y, Nahar S, Miyagi-Shiohira C, Kinjo T, Kobayashi N, Saitoh I, Watanabe M, Fujita J, Noguchi H

Stem cells international ( Stem Cells International )  2019   7274057   2019 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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A Comparison of Proteins Expressed between Human and Mouse Adipose-Derived Mesenchymal Stem Cells by a Proteome Analysis through Liquid Chromatography with Tandem Mass Spectrometry.

Nahar S, Nakashima Y, Miyagi-Shiohira C, Kinjo T, Kobayashi N, Saitoh I, Watanabe M, Noguchi H, Fujita J

International journal of molecular sciences ( International Journal of Molecular Sciences )  19 ( 11 )   2018.11 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Induction of Expandable Adipose-Derived Mesenchymal Stem Cells from Aged Mesenchymal Stem Cells by a Synthetic Self-Replicating RNA.

Miyagi-Shiohira C, Nakashima Y, Kobayashi N, Kitamura S, Saitoh I, Watanabe M, Noguchi H

International journal of molecular sciences ( International Journal of Molecular Sciences )  19 ( 11 )   2018.11 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Cytokines in adipose-derived mesenchymal stem cells promote the healing of liver disease.

Nahar S, Nakashima Y, Miyagi-Shiohira C, Kinjo T, Toyoda Z, Kobayashi N, Saitoh I, Watanabe M, Noguchi H, Fujita J

World journal of stem cells ( World Journal of Stem Cells )  10 ( 11 ) 146 - 159   2018.11 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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A Liquid Chromatography with Tandem Mass Spectrometry-Based Proteomic Analysis of the Proteins Secreted by Human Adipose-Derived Mesenchymal Stem Cells.

Nakashima Y, Nahar S, Miyagi-Shiohira C, Kinjo T, Toyoda Z, Kobayashi N, Saitoh I, Watanabe M, Fujita J, Noguchi H

Cell transplantation ( Cell Transplantation )  27 ( 10 ) 1469 - 1494   2018.10 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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The Healing Effect of Human Milk Fat Globule-EGF Factor 8 Protein (MFG-E8) in A Rat Model of Parkinson's Disease.

Nakashima Y, Miyagi-Shiohira C, Noguchi H, Omasa T

Brain sciences ( Brain Sciences )  8 ( 9 )   2018.08 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Comparison Between Modified Extracellular-Type Trehalose-Containing Kyoto Solution and University of Wisconsin Solution in 18-Hour Pancreas Preservation for Islet Transplantation.

Hamada E, Ebi N, Miyagi-Shiohira C, Tamaki Y, Nakashima Y, Kobayashi N, Saitoh I, Watanabe M, Kinjo T, Noguchi H

Pancreas ( Pancreas )  47 ( 7 ) e46 - e47   2018.08 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Characterization of induced tissue-specific stem cells from pancreas by a synthetic self-replicative RNA.

Miyagi-Shiohira C, Nakashima Y, Kobayashi N, Saitoh I, Watanabe M, Noguchi H

Scientific reports ( Scientific Reports )  8 ( 1 ) 12341   2018.08 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Atorvastatin Inhibits the HIF1α-PPAR Axis, Which Is Essential for Maintaining the Function of Human Induced Pluripotent Stem Cells.

Nakashima Y, Miyagi-Shiohira C, Noguchi H, Omasa T

Molecular therapy : the journal of the American Society of Gene Therapy ( Molecular Therapy )  26 ( 7 ) 1715 - 1734   2018.07 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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A Liquid Chromatography with Tandem Mass Spectrometry-Based Proteomic Analysis of Cells Cultured in DMEM 10% FBS and Chemically Defined Medium Using Human Adipose-Derived Mesenchymal Stem Cells.

Nakashima Y, Nahar S, Miyagi-Shiohira C, Kinjo T, Kobayashi N, Saitoh I, Watanabe M, Fujita J, Noguchi H

International journal of molecular sciences ( International Journal of Molecular Sciences )  19 ( 7 )   2018.07 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Modified cell-permeable JNK inhibitors efficiently prevents islet apoptosis and improves the outcome of islet transplantation.

Noguchi H, Miyagi-Shiohira C, Nakashima Y, Ebi N, Hamada E, Tamaki Y, Kuwae K, Kobayashi N, Saitoh I, Watanabe M

Scientific reports ( Scientific Reports )  8 ( 1 ) 11082   2018.07 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Comparison of Tissue Loading Before and After the Creation of a Continuous Density Gradient in Porcine Islet Purification

Miyagi-Shiohira Chika, Nakashima Yoshiki, Ebi Nana, Hamada Eri, Tamaki Yoshihito, Kuwae Kazuho, Kobayashi Naoya, Saitoh Issei, Watanabe Masami, Kinjo Takao, Noguchi Hirofumi

CELL MEDICINE   10   2155179018781343   2018.06 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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A Comparison of Pancreatic Islet Purification using Iodixanol with University of Wisconsin Solution and with Na-Lactobionate and Histidine Solution

Nakashima Yoshiki, Miyagi-Shiohira Chika, Ebi Nana, Hamada Eri, Tamaki Yoshihito, Kuwae Kazuho, Kobayashi Naoya, Saitoh Issei, Watanabe Masami, Kinjo Takao, Noguchi Hirofumi

CELL MEDICINE   10   2155179018775071   2018.06 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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The Development of Cancer through the Transient Overexpression of Reprogramming Factors

Miyagi-Shiohira Chika, Nakashima Yoshiki, Kobayashi Naoya, Saitoh Issei, Watanabe Masami, Noguchi Yasufumi, Kinjo Takao, Noguchi Hirofumi

CELL MEDICINE   10   2155179017733172   2018.05 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Evaluation of Islet Purification Methods for Making a Continuous Density Gradient and Loading Tissue

Ebi Nana, Miyagi-Shiohira Chika, Hamada Eri, Tamaki Yoshihito, Masamoto Mariko, Makishi Erika, Nakashima Yoshiki, Kobayashi Naoya, Saitoh Issei, Watanabe Masami, Noguchi Yasufumi, Kinjo Takao, Noguchi Hirofumi

CELL MEDICINE   10   2155179017733090   2018.05 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Adhesion characteristics of porcine pancreatic islets and exocrine tissue to coating materials

Yoshiki Nakashima, Chika Miyagi-Shiohira, Naoya Kobayashi, Issei Saitoh, Masami Watanabe, Hirofumi Noguchi

Islets ( Taylor and Francis Inc. )  10 ( 3 ) e1460294   2018.05 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Since the report of the Edmonton protocol in 2000, islet transplantation has been implemented worldwide, and xenotransplantation using porcine islets has also been reported. In addition, many basic experiments using pancreatic islets and exocrine tissue after isolation have been reported. Recently, exocrine cells have been found to be essential for inducing the differentiation of pancreatic islets. Therefore, the importance of the culture conditions for pancreatic tissue when conducting experiments using pancreatic tissue is also increasing. In this study, we focused on the coat material and examined the adhesive properties of porcine pancreatic islets and exocrine tissue after isolation. Porcine islet isolation was performed, and isolated islets (purity ≥95%) and exocrine tissue (purity ≥99%) were used to achieve adhesion to several extracellular matrixes, fibronectin, collagen type I, collagen type IV, laminin I, fibrinogen, and bovine serum albumin (BSA). DMEM with 0.5% FBS was used as the assay medium. For exocrine tissue, the adhesion was promoted in fibronectin, collagen type I, laminin I, and fibrinogen. The adhesive ability to fibronectin was more than twice that to BSA, while the adhesive ability to collagen type I, laminin I, and fibrinogen was less than twice that to BSA. For islets, the adhesive ability to fibronectin was weaker than that of exocrine tissue. Furthermore, the adhesion effect in fibronectin was obtained within 30 minutes and in medium containing little serum for both islets and exocrine tissues. These data suggest that fibronectin may be useful for the adhesion of pancreatic tissue.

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Np95/Uhrf1 regulates tumor suppressor gene expression of neural stem/precursor cells, contributing to neurogenesis in the adult mouse brain.

Murao N, Matsubara S, Matsuda T, Noguchi H, Mutoh T, Mutoh M, Koseki H, Namihira M, Nakashima K

Neuroscience research   143   31 - 43   2018.05 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Induced tissue-specific stem cells and epigenetic memory in induced pluripotent stem cells

Hirofumi Noguchi, Chika Miyagi-Shiohira, Yoshiki Nakashima

International Journal of Molecular Sciences ( MDPI AG )  19 ( 4 )   2018.04 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Induced pluripotent stem (iPS) cells have significant implications for overcoming most of the ethical issues associated with embryonic stem (ES) cells. The pattern of expressed genes, DNA methylation, and covalent histone modifications in iPS cells are very similar to those in ES cells. However, it has recently been shown that, following the reprogramming of mouse/human iPS cells, epigenetic memory is inherited from the parental cells. These findings suggest that the phenotype of iPS cells may be influenced by their cells of origin and that their skewed differentiation potential may prove useful in the generation of differentiated cell types that are currently difficult to produce from ES/iPS cells for the treatment of human diseases. Our recent study demonstrated the generation of induced tissue-specific stem (iTS) cells by transient overexpression of the reprogramming factors combined with tissue-specific selection. iTS cells are cells that inherit numerous components of epigenetic memory from donor tissue and acquire self-renewal potential. This review describes the “epigenetic memory” phenomenon in iPS and iTS cells and the possible clinical applications of these stem cells.

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Role of Egr1 on Pancreatic Endoderm Differentiation.

Tsugata T, Nikoh N, Kin T, Miyagi-Shiohira C, Nakashima Y, Saitoh I, Noguchi Y, Ueki H, Watanabe M, Kobayashi N, Shapiro AMJ, Noguchi H

Cell medicine   10   2155179017733177   2018 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Long-term Cryopreservation of Human and other Mammalian Cells at -80 °C for 8 Years.

Miyamoto Y, Ikeuchi M, Noguchi H, Hayashi S

Cell medicine   10   2155179017733148   2018 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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A Comparison of the Preservation of Mouse Adipose Tissue-Derived Mesenchymal Stem Cells Using the University of Wisconsin Solution and Hank's Balanced Salt Solution.

Nahar S, Nakashima Y, Miyagi-Shiohira C, Kinjo T, Kobayashi N, Saitoh I, Watanabe M, Noguchi H, Fujita J

Stem cells international ( Stem Cells International )  2018   1625464   2018 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Synergistic induction of astrocytic differentiation by factors secreted from meninges in the mouse developing brain

Yoichiro Kawamura, Sayako Katada, Hirofumi Noguchi, Hiroyuki Yamamoto, Tsukasa Sanosaka, Koji Iihara, Kinichi Nakashima

FEBS Letters ( Wiley Blackwell )  591 ( 22 ) 3709 - 3720   2017.11 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Astrocytes, which support diverse neuronal functions, are generated from multipotent neural stem/precursor cells (NS/PCs) during brain development. Although many astrocyte-inducing factors have been identified and studied in vitro, the regions and/or cells that produce these factors in the developing brain remain elusive. Here, we show that meninges-produced factors induce astrocytic differentiation of NS/PCs. Consistent with the timing when astrocytic differentiation of NS/PCs increases, expression of astrocyte-inducing factors is upregulated. Meningeal secretion-mimicking combinatorial treatment of NS/PCs with bone morphogenetic protein 4, retinoic acid and leukemia inhibitory factor synergistically activate the promoter of a typical astrocytic marker, glial fibrillary acidic protein. Taken together, our data suggest that meninges play an important role in astrocytic differentiation of NS/PCs in the developing brain.

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Exogenous DKK-3/REIC inhibits Wnt/β-catenin signaling and cell proliferation in human kidney cancer KPK1.

Xu J, Sadahira T, Kinoshita R, Li SA, Huang P, Wada K, Araki M, Ochiai K, Noguchi H, Sakaguchi M, Nasu Y, Watanabe M

Oncology letters ( Oncology Letters )  14 ( 5 ) 5638 - 5642   2017.11 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Isolation and characterization of lymphoid enhancer factor-1-positive deciduous dental pulp stem-like cells after transfection with a piggyBac vector containing LEF1 promoter-driven selection markers

Tomoya Murakami, Issei Saitoh, Masahiro Sato, Emi Inada, Miki Soda, Masataka Oda, Hisanori Domon, Yoko Iwase, Tadashi Sawami, Kazunari Matsueda, Yutaka Terao, Hayato Ohshima, Hirofumi Noguchi, Haruaki Hayasaki

ARCHIVES OF ORAL BIOLOGY ( PERGAMON-ELSEVIER SCIENCE LTD )  81   110 - 120   2017.09 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Objective: Lymphoid enhancer-binding factor-1 (LEF1) is a 48-kD nuclear protein that is expressed in pre-B and T cells. LEF1 is also an important member of the Wnt/beta-catenin signaling pathway that plays important roles in the self-renewal and differentiation of embryonic stem cells. We speculated that LEF1 might function in the stem cells from human exfoliated deciduous teeth (SHED). In this study, we attempted to isolate such LEF1-positive cells from human deciduous dental pulp cells (HDDPCs) by genetic engineering technology, using the human LEF1 promoter. Design: A piggyBac transposon plasmid (pTA-LEN) was introduced into HDDPCs, using the Neon (R) transfection system. After G418 selection, the emerging colonies were assessed for EGFP-derived fluorescence by fluorescence microscopy. Reverse transcription polymerase chain reaction (RT-PCR) analysis was performed using RNA isolated from these colonies to examine stem cell-specific transcript expression. Osteoblastic or neuronal differentiation was induced by cultivating the LEF1-positive cells with differentiation-inducing medium. Results: RT-PCR analysis confirmed the expression of several stem cell markers, including OCT3/4, SOX2, REX1, and NANOG, in LEF1-positive HDDPCs, which could be differentiated into osteoblasts and neuronal cells. Conclusions: The isolated LEF1-positive HDDPCs exhibited the properties of stem cells, suggesting that LEF1 might serve as a marker for SHED.

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Hypoxia Epigenetically Confers Astrocytic Differentiation Potential on Human Pluripotent Cell-Derived Neural Precursor Cells

Tetsuro Yasui, Naohiro Uezono, Hideyuki Nakashima, Hirofumi Noguchi, Taito Matsuda, Tomoko Noda-Andoh, Hideyuki Okano, Kinichi Nakashima

STEM CELL REPORTS ( CELL PRESS )  8 ( 6 ) 1743 - 1756   2017.06 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Human neural precursor cells (hNPCs) derived from pluripotent stem cells display a high propensity for neuronal differentiation, but they require long-term culturing to differentiate efficiently into astrocytes. The mechanisms underlying this biased fate specification of hNPCs remain elusive. Here, we show that hypoxia confers astrocytic differentiation potential on hNPCs through epigenetic gene regulation, and that this was achieved by cooperation between hypoxia-inducible factor 1 alpha and Notch signaling, accompanied by a reduction of DNA methylation level in the promoter region of a typical astrocyte-specific gene, Glial fibrillary acidic protein. Furthermore, we found that this hypoxic culture condition could be applied to rapid generation of astrocytes from Rett syndrome patient-derived hNPCs, and that these astrocytes impaired neuronal development. Thus, our findings shed further light on the molecular mechanisms regulating hNPC differentiation and provide attractive tools for the development of therapeutic strategies for treating astrocyte-mediated neurological disorders.

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RCAN-11R peptide provides immunosuppression for fully mismatched islet allografts in mice

Hirofumi Noguchi, Koji Sugimoto, Chika Miyagi-Shiohira, Yoshiki Nakashima, Naoya Kobayashi, Issei Saitoh, Masami Watanabe, Yasufumi Noguchi

SCIENTIFIC REPORTS ( NATURE PUBLISHING GROUP )  7 ( 1 ) 3043   2017.06 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Calcineurin inhibitors have been used for transplant therapy. However, the inhibition of calcineurin outside the immune system has a number of side effects. We previously developed a cell-permeable inhibitor of NFAT (nuclear factor of activated T cells) using the polyarginine peptide delivery system. This peptide (11R-VIVIT) selectively interferes with calcineurin-NFAT interaction without affecting the activity of calcineurin phosphatase and provides immunosuppression for fully mismatched islet allografts in mice. However, our recent study showed that 11R-VIVIT affected cell viability in vitro when it was used at higher concentration because of the VIVIT sequence. The aim of this study is to develop a safer NFAT inhibitor (RCAN-11R) that does not affect cell viability, and which is less toxic than calcineurin inhibitors. The minimal sequence of the protein family of regulators of calcineurin (RCAN) that is responsible for the inhibition of calcineurin-NFAT signaling was recently characterized. The peptide could selectively interfere with the calcineurin-NFAT interaction without affecting the activity of calcineurin phosphatase, similar to 11R-VIVIT. RCAN-11R did not affect cell viability when it was used at a higher concentration than the toxic concentration of 11R-VIVIT. RCAN-11R could therefore be useful as a therapeutic agent that is less toxic than current drugs or 11R-VIVIT.

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Facilitating Transplantation.

Noguchi H

Cell medicine   9 ( 1-2 ) 1 - 2   2017.01 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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The Evaluation of Islet Purification Methods That Use Large Bottles to Create a Continuous Density Gradient.

Miyagi-Shiohira C, Kobayashi N, Saitoh I, Watanabe M, Noguchi Y, Matsushita M, Noguchi H

Cell medicine   9 ( 1-2 ) 45 - 51   2017.01 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Comparison of Purification Solutions With Different Osmolality for Porcine Islet Purification.

Miyagi-Shiohira C, Kobayashi N, Saitoh I, Watanabe M, Noguchi Y, Matsushita M, Noguchi H

Cell medicine   9 ( 1-2 ) 53 - 59   2017.01 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Evaluation of Serum-Free, Xeno-Free Cryopreservation Solutions for Human Adipose-Derived Mesenchymal Stem Cells.

Miyagi-Shiohira C, Kobayashi N, Saitoh I, Watanabe M, Noguchi Y, Matsushita M, Noguchi H

Cell medicine   9 ( 1-2 ) 15 - 20   2017.01 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Enhanced Adipogenic Differentiation of Human Adipose-Derived Stem Cells in an In Vitro Microenvironment: The Preparation of Adipose-Like Microtissues Using a Three-Dimensional Culture.

Miyamoto Y, Ikeuchi M, Noguchi H, Yagi T, Hayashi S

Cell medicine   9 ( 1-2 ) 35 - 44   2017.01 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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心停止下膵島移植に向けた持続冷却灌流保存法による膵臓保存

圷 尚武, 野口 洋文, 丸山 通広, 大月 和宣, 石田 健倫, 齋藤 友永, 西郷 健一, 長谷川 正行, 青山 博道, 剣持 敬

Organ Biology ( 一般社団法人　日本臓器保存生物医学会 )  24 ( 2 ) 175 - 179   2017 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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1型糖尿病に対する膵島移植が国内で始まったものの，依然として大きな問題はドナー不足である．その解決手段のひとつとして，心停止下ドナーのような，条件の悪いいわゆるマージナルドナーより採取された膵臓の有効な利用である．LifePort^TM（LP）はOrgan Recovery Systems社により開発された臓器搬送用の持続冷却灌流装置であり，現在欧米において障害を伴った腎臓の搬送に臨床応用され，その有効性が確認されている．本装置はマージナルドナーから採取された臓器移植，特に虚血再潅流障害を受けやすい膵臓の保存に有用で，膵島移植の安全性や成績の向上に有効であると考えられる．本研究では，ビーグル犬を使用し，障害膵モデルより膵臓を摘出し，LPにより持続冷却灌流保存することにより，膵臓の障害が最小限に抑えられ，従来法であるUW液や2層法による浸漬保存に比べて分離膵島の収量や機能が改善でき，臨床膵島移植に応用し，成績向上に寄与することを目的とした．［方法］ビーグル犬から30分の温阻血時間をおいて全膵を摘出し，LPにて24時間持続冷却灌流保存（LP群），UW液にて24時間冷却浸漬保存（UW群），2層法にて24時間冷却浸漬保存（2層法群）し，膵島分離を行い，その形態・機能を比較検討した．［結果］形態的には，LP群でより大きく，形の良い膵島が分離できた．また，膵島収量については，純化前後でLP群においてUW群や2層法群に比べて良好であった．また，分離膵島のインスリン分泌能を評価するStatic Incubationの結果でも，LP群でUW群や2層法群に比べて良好な結果であった．［結論］心停止下ドナーよりの膵島移植を想定したモデルにおいて，LPによる障害膵の持続冷却灌流保存は，UW液や2層法による冷却浸漬保存に比べて，分離膵島の収量や機能保持に優れていることが示唆された．しかし，臨床応用のためには，さらなる保存条件や膵島分離法の改善が必要であると考えられた．

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A proteome analysis of pig pancreatic islets and exocrine tissue by liquid chromatography with tandem mass spectrometry

Yoshiki Nakashima, Chika Miyagi-Shiohira, Naoya Kobayashi, Issei Saitoh, Masami Watanabe, Hirofumi Noguchi

ISLETS ( TAYLOR & FRANCIS INC )  9 ( 6 ) 159 - 176   2017 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Liquid chromatography with tandem mass spectrometry (LC-MS/MS) is a proteome analysis method, and the shotgun analysis by LC-MS/MS comprehensively identifies proteins from tissues and cells with high resolving power. In this study, we analyzed the protein expression in pancreatic tissue by LC-MS/MS. Islets isolated from porcine pancreata (purity 95%) and exocrine tissue (purity 99%) were used in this study. LC-MS/MS showed that 13 proteins were expressed in pancreatic islets only (Group I), 43 proteins were expressed in both islets and exocrine tissue (Group I&E), and 102 proteins were expressed in exocrine tissue only (Group E). Proteins involved in islet differentiation and cell proliferation were identified in Group I (e.g. CLUS, CMGA, MIF). In addition, various functional proteins (e.g. SCG2, TBA1A) were identified in islet by using the new method of principal component analysis (PCA)'. However, the function of such proteins on islets remains unclear. EPCAM was identified in Group E. Group E was found to include proteins involved in clinical inflammatory diseases such as pancreatitis (e.g. CBPA1, CGL, CYTB, ISK1 and PA21B). Many of these identified proteins were reported less frequently in previous studies, and HS71B, NEC2, PRAF3 and SCG1 were newly detected in Group I while CPNS1, DPEP1, GANAB, GDIB, GGT1, HSPB1, ICTL, VILI, MUTA, NDKB, PTGR1, UCHL3, VAPB and VINC were newly detected in Group E. These results show that comprehensive expression analysis of proteins by LC-MS/MS is useful as a method to investigate new factors constructing cellular component, biological process, and molecular function.

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Targeted DNA demethylation in vivo using dCas9-peptide repeat and scFv-TET1 catalytic domain fusions.

Morita S, Noguchi H, Horii T, Nakabayashi K, Kimura M, Okamura K, Sakai A, Nakashima H, Hata K, Nakashima K, Hatada I

Nature biotechnology   34 ( 10 ) 1060 - 1065   2016.10 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Tissue-Specific Stem Cells Obtained by Reprogramming of Non-Obese Diabetic (NOD) Mouse-Derived Pancreatic Cells Confer Insulin Production in Response to Glucose

Issei Saitoh, Masahiro Sato, Miki Soda, Emi Inada, Yoko Iwase, Tomoya Murakami, Hayato Ohshima, Haruaki Hayasaki, Hirofumi Noguchi

PLOS ONE ( PUBLIC LIBRARY SCIENCE )  11 ( 9 ) e0163580   2016.09 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Type 1 diabetes occurs due to the autoimmune destruction of pancreatic beta-cells in islets. Transplantation of islets is a promising option for the treatment of patients with type 1 diabetes that experience hypoglycemic unawareness despite maximal care, but the present shortage of donor islets hampers such transplantation. Transplantation of insulin-producing cells derived from the patients themselves would be one of the most promising approaches to cure type 1 diabetes. Previously, we demonstrated that insulin-producing cells could be produced by transfecting murine pancreatic cells with Yamanaka's reprogramming factors. Non-obese diabetic (NOD) mice are naturally occurring mutant mice defective in insulin production due to autoimmune ablation of pancreatic beta-cells. In this study, we showed that glucose-sensitive insulin-producing cells are successfully generated by transfecting primary pancreatic cells from NOD mice (aged 6 months old) with a plasmid harboring the cDNAs for Oct-3/4, Sox2, Klf4, and c-Myc. Transfection was repeated 4 times in a 2 day-interval. Sixty-five days after final transfection, cobblestone-like colonies appeared. They proliferated in vitro and expressed pluripotency-related genes as well as Pdx1, a transcription factor specific to tissue-specific stem cells for the beta-cell lineage. Transplantation of these cells into nude mice failed to produce teratoma unlike induced pluripotent stem cells (iPSCs). Induction of these cells to the pancreatic beta-cell lineage demonstrated their capability to produce insulin in response to glucose. These findings suggest that functional pancreatic beta-cells can be produced from patients with type 1 diabetes. We call these resultant cells as "induced tissue-specific stem cells from the pancreas" (iTS-P) that could be valuable sources of safe and effective materials for cell-based therapy in type 1 diabetes.

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DNA Methyltransferase 1 Is Indispensable for Development of the Hippocampal Dentate Gyrus.

Noguchi H, Murao N, Kimura A, Matsuda T, Namihira M, Nakashima K

The Journal of neuroscience : the official journal of the Society for Neuroscience   36 ( 22 ) 6050 - 6068   2016.06 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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書評:Molecular Biology of the Cell（細胞の分子生物学）

野口 洋文

Organ Biology ( 一般社団法人　日本臓器保存生物医学会 )  23 ( 2 ) 196 - 197   2016 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Prenatal deletion of DNA methyltransferase 1 in neural stem cells impairs neurogenesis and causes anxiety-like behavior in adulthood.

Noguchi H, Kimura A, Murao N, Namihira M, Nakashima K

Neurogenesis (Austin, Tex.)   3 ( 1 ) e1232679   2016 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Creating a Future of Transplantation.

Noguchi H

Cell medicine   8 ( 1-2 ) 1   2015.12 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Spheroid Formation and Evaluation of Hepatic Cells in a Three-Dimensional Culture Device.

Miyamoto Y, Ikeuchi M, Noguchi H, Yagi T, Hayashi S

Cell medicine   8 ( 1-2 ) 47 - 56   2015.12 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Choice of Feeders Is Important When First Establishing iPSCs Derived From Primarily Cultured Human Deciduous Tooth Dental Pulp Cells.

Saitoh I, Inada E, Iwase Y, Noguchi H, Murakami T, Soda M, Kubota N, Hasegawa H, Akasaka E, Matsumoto Y, Oka K, Yamasaki Y, Hayasaki H, Sato M

Cell medicine   8 ( 1-2 ) 9 - 23   2015.12 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Islet Culture/Preservation Before Islet Transplantation.

Noguchi H, Miyagi-Shiohira C, Kurima K, Kobayashi N, Saitoh I, Watanabe M, Noguchi Y, Matsushita M

Cell medicine   8 ( 1-2 ) 25 - 29   2015.12 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Cryopreservation of Adipose-Derived Mesenchymal Stem Cells.

Miyagi-Shiohira C, Kurima K, Kobayashi N, Saitoh I, Watanabe M, Noguchi Y, Matsushita M, Noguchi H

Cell medicine   8 ( 1-2 ) 3 - 7   2015.12 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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A case report of a type 1 diabetes patient maintaining C-peptide secretion for ten years after islet transplantation using donation after cardiocirculatory death (DCD) donors

Yasuhiro Iwanaga, Shinichi Matsumoto, Teru Okitsu, Hideo Nagata, Hirofumi Noguchi, Yukihide Yonekawa, Michiya Kawaguchi, Takayuki Anazawa, Yoshiya Kawaguchi, Hideaki Okajima, Shinji Uemoto

XENOTRANSPLANTATION ( WILEY-BLACKWELL )  22   S97 - S97   2015.11 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

A CASE REPORT OF A TYPE 1 DIABETES PATIENT MAINTAINING C-PEPTIDE SECRETION FOR TEN YEARS AFTER ISLET TRANSPLANTATION USING DONATION AFTER CARDIOCIRCULATORY DEATH (DCD) DONORS.

Yasuhiro Iwanaga, Shinichi Matsumoto, Teru Okitsu, Hideo Nagata, Hirofumi Noguchi, Yukihide Yonekawa, Michiya Kawaguchi, Takayuki Anazawa, Yoshiya Kawaguchi, Hideaki Okajima, Shinji Uemoto

TRANSPLANTATION ( LIPPINCOTT WILLIAMS & WILKINS )  99 ( 11 ) S158 - S158   2015.11 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

Expression of DNMT1 in neural stem/precursor cells is critical for survival of newly generated neurons in the adult hippocampus.

Noguchi H, Kimura A, Murao N, Matsuda T, Namihira M, Nakashima K

Neuroscience research   95   1 - 11   2015.06 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Basic and Clinical Science for Organ Biology.

Noguchi H

Cell medicine   7 ( 2 ) 49   2015.02 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Three-Dimensional In Vitro Hepatic Constructs Formed Using Combinatorial Tapered Stencil for Cluster Culture (TASCL) Device.

Miyamoto Y, Ikeuchi M, Noguchi H, Yagi T, Hayashi S

Cell medicine   7 ( 2 ) 67 - 74   2015.02 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Potential Factors for the Differentiation of ESCs/iPSCs Into Insulin-Producing Cells.

Tsugata T, Nikoh N, Kin T, Saitoh I, Noguchi Y, Ueki H, Watanabe M, James Shapiro AM, Noguchi H

Cell medicine   7 ( 2 ) 83 - 93   2015.02 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Induction of tissue-specific stem cells by reprogramming factors, and tissue-specific selection.

Hirofumi Noguchi, Issei Saitoh, Takako Tsugata, Hitomi Kataoka, Masami Watanabe, Yasufumi Noguchi

Cell Death and Differentiation ( Cell Death and Differentiation )  22 ( 1 ) 145 - 155   2015.01 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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再生医療

小林 英司, 野口 洋文

Organ Biology ( 一般社団法人　日本臓器保存生物医学会 )  22 ( 2 ) 134 - 135   2015 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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A New Method for Generating Insulin-Secreting Cells from Human Pancreatic Epithelial Cells After Islet Isolation Transformed by NeuroD1

Masayuki Shimoda, Shuyuan Chen, Hirofumi Noguchi, Morihito Takita, Koji Sugimoto, Takeshi Itoh, Daisuke Chujo, Shuichi Iwahashi, Bashoo Naziruddin, Marlon F. Levy, Shinichi Matsumoto, Paul A. Grayburn

HUMAN GENE THERAPY METHODS ( MARY ANN LIEBERT, INC )  25 ( 3 ) 206 - 219   2014.06 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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The generation of insulin-secreting cells from nonendocrine pancreatic epithelial cells (NEPEC) has been demonstrated for potential clinical use in the treatment of diabetes. However, previous methods either had limited efficacy or required viral vectors, which hinder clinical application. In this study, we aimed to establish an efficient method of insulin-secreting cell generation from NEPEC without viral vectors. We used nonislet fractions from both research-grade human pancreata from brain-dead donors and clinical pancreata after total pancreatectomy with autologous islet transplantation to treat chronic pancreatitis. It is of note that a few islets could be mingled in the nonislet fractions, but their influence could be limited. The NeuroD1 gene was induced into NEPEC using an effective triple lipofection method without viral vectors to generate insulin-secreting cells. The differentiation was promoted by adding a growth factor cocktail into the culture medium. Using the research-grade human pancreata, the effective method showed high efficacy in the differentiation of NEPEC into insulin-positive cells that secreted insulin in response to a glucose challenge and improved diabetes after being transplanted into diabetic athymic mice. Using the clinical pancreata, similar efficacy was obtained, even though those pancreata suffered chronic pancreatitis. In conclusion, our effective differentiation protocol with triple lipofection method enabled us to achieve very efficient insulin-secreting cell generation from human NEPEC without viral vectors. This method offers the potential for supplemental insulin-secreting cell transplantation for both allogeneic and autologous islet transplantation.

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Establishment of a pancreatic stem cell line from fibroblast-derived induced pluripotent stem cells

Takashi Kuise, Hirofumi Noguchi, Hiroshi Tazawa, Takashi Kawai, Masaya Iwamuro, Issei Saitoh, Hitomi Usui Kataoka, Masami Watanabe, Yasufumi Noguchi, Toshiyoshi Fujiwara

BIOMEDICAL ENGINEERING ONLINE ( BIOMED CENTRAL LTD )  13 ( 1 ) 64   2014.05 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Background: For cell therapies to treat diabetes, it is important to produce a sufficient number of pancreatic endocrine cells that function similarly to primary islets. Induced pluripotent stem (iPS) cells represent a potentially unlimited source of functional pancreatic endocrine cells. However, the use of iPS cells for laboratory studies and cell-based therapies is hampered by their high tumorigenic potential and limited ability to generate pure populations of differentiated cell types in vitro. The purpose of this study was to establish a pancreatic stem cell line from iPS cells derived from mouse fibroblasts. Methods: Mouse iPS cells were induced to differentiate into insulin-producing cells by a multi-step differentiation protocol, which was conducted as described previously with minor modifications. Selection of the pancreatic stem cell was based on morphology and Pdx1 expression. The pancreatic potential of the pancreatic stem cells was evaluated using a reverse transcription PCR, real-time PCR, immunofluorescence, and a glucose challenge test. To assess potential tumorigenicity of the pancreatic stem cells, the cells were injected into the quadriceps femoris muscle of the left hindlimb of nude mice. Results: The iPS-derived pancreatic stem cells expressed the transcription factor -Pdx1- a marker of pancreatic development, and continued to divide actively beyond passage 80. Endocrine cells derived from these pancreatic stem cells expressed insulin and pancreatic genes, and they released insulin in response to glucose stimulation. Mice injected with the pancreatic stem cells did not develop tumors, in contrast to mice injected with an equal number of iPS cells. Conclusion: This strategy provides a new approach for generation of insulin-producing cells that is more efficient and safer than using iPS cells. We believe that this approach will help to develop a patient-specific cell transplantation therapy for diabetes in the near future.

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Comparison of Incubation Solutions Prior to the Purification of Porcine Islet Cells.

Kawai T, Noguchi H, Kuise T, Nakatsuka A, Katayama A, Imagawa N, Kataoka HU, Saitoh I, Noguchi Y, Watanabe M, Fujiwara T

Cell medicine   6 ( 1-2 ) 9 - 14   2013.12 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Development of Canine Models of Type 1 Diabetes With Partial Pancreatectomy and the Administration of Streptozotocin.

Seita M, Noguchi H, Kubota Y, Kawamoto H, Nakaji S, Kobayashi N, Fujiwara T

Cell medicine   6 ( 1-2 ) 25 - 31   2013.12 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Comparison of New Preservation Solutions, HN-1 and University of Wisconsin Solution, in Pancreas Preservation for Porcine Islet Isolation.

Katayama A, Noguchi H, Kuise T, Nakatsuka A, Hirota D, Kataoka HU, Kawai T, Inoue K, Imagawa N, Saitoh I, Noguchi Y, Watanabe M, Wada J, Fujiwara T

Cell medicine   6 ( 1-2 ) 3 - 8   2013.12 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Corrigendum to Embryonic body formation using the tapered soft stencil for cluster culture device [Biomaterials 32 (2011) 3729-3738]

Hiroshi Yukawa, Hirofumi Noguchi, Shuji Hayashi

Biomaterials   34 ( 33 ) 8531   2013.11 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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ER Stress and β-Cell Pathogenesis of Type 1 and Type 2 Diabetes and Islet Transplantation.

Kataoka HU, Noguchi H

Cell medicine   5 ( 2-3 ) 53 - 57   2013.11 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Culture Conditions for Mouse Pancreatic Stem Cells.

Noguchi H, Saitoh I, Kataoka HU, Watanabe M, Noguchi Y, Fujiwara T

Cell medicine   5 ( 2-3 ) 63 - 68   2013.11 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Cancer stem cell-like characteristics of a CD133+ subpopulation in the J82 human bladder cancer cell line.

Peng Huang, Masami Watanabe, Haruki Kaku, Hideo Ueki, Hirofumi Noguchi, Morito Sugimoto, Takeshi Hirata, Hiroshi Yamada, Kohji Takei, Shaobo Zheng, Kai Xu, Yasutomo Nasu, Yasuyuki Fujii, Chunxiao Liu, Hiromi Kumon

Molecular and clinical oncology   1 ( 1 ) 180 - 184   2013.01 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Cancer stem cells (CSCs) are thought to be crucial for understanding the biological roots of cancer, and are of increasing importance as a target for new anticancer agents. According to an expression analysis of the cell surface antigens of various types of cancer, CD133 is considered to be a potential marker of cancer stemness. In this study, a human urinary bladder cancer cell line (J82) was used to analyze the cancer stem cell-like characteristics of CD133+ bladder cancer cells in vitro and in vivo. The CD133 expression in the J82 cells was examined and the cells were immunomagnetically categorized into positive and negative subsets. The CD133- and CD133+ subsets were phenotypically divergent with regard to the cell growth pattern, while CD133+ cells tended to colonize during their growth. In CD133+ cells, the pluripotent stem cell factors Oct-4 and Sox-2 were upregulated, and a statistically significant proliferation increase was observed when compared to CD133- cells. The CD133+ subpopulation was more tolerant to the chemotherapeutic agent cisplatin, and Bacillus Calmette-Guérin (BCG), an agent instilled intravesically to treat bladder cancer. In addition, CD133+ J82 cells were more resistant to radiation treatment when compared to CD133- cells. The in vivo tumorigenesis of the CD133- and CD133+ subsets of J82 cancer cells was also examined by subcutaneously injecting them into nude mice. The tumor growth was more aggressive in the CD133+ subpopulation, showing a significant difference in the tumorigenic potential in these subsets. In conclusion, J82 human bladder cancer cells include CD133- and CD133+ subpopulations, while the CD133 molecule is a potential marker of the potential malignancy of human bladder cancer. In the present study, the CD133+ subpopulation was herein demonstrated to have certain characteristics consistent with those of cancer stem cells.

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A novel gene expression system for detecting viable bladder cancer cells

Hideo Ueki, Masami Watanabe, Haruki Kaku, Peng Huang, Shun-Ai Li, Kazuhiko Ochiai, Takeshi Hirata, Hirofumi Noguchi, Hiroshi Yamada, Kohji Takei, Yasutomo Nasu, Yuji Kashiwakura, Hiromi Kumon

INTERNATIONAL JOURNAL OF ONCOLOGY ( SPANDIDOS PUBL LTD )  41 ( 1 ) 135 - 140   2012.07 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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A novel transcriptional system was developed that can robustly enhance cancer-specific gene expression. In the system, hTERT promoter-driven gene expression was enhanced by an advanced two-step transcriptional amplification (TSTA). This construct was used to develop a novel system for detection of bladder cancer cells. The current study evaluated the advanced TSTA system by examining the cancer-specific gene transcription in various bladder cancer cell lines. The system significantly enhanced cancer-specific luciferase gene expression in the bladder cancer cell lines in comparison to the previous expression system of one-step or conventional TSTA. The fold gain of the enhancement was significantly correlated to the telomerase activity of the cell lines. A green fluorescent protein (GFP) gene encoding plasmid vector was constructed where hTERT promoter-driving transcription is enhanced by the advanced TSTA to utilize the system for the imaging and detection of viable bladder cancer cells. The advanced TSTA-hTERT-GFP plasmid successfully induced cancer-specific gene expression, showing robust GFP expression in human bladder cancer cell lines, but no visible GFP expression in normal bladder urothelial cells. The control GFP plasm id with a CMV promoter yielded GFP expression in both normal bladder cells and cancer cells. The advanced TSTA-hTERT-GFP plasmid allowed selective visualization of viable human bladder cancer cells in mixed cell culture containing 10- and 100-fold more normal bladder urothelial cells. These findings indicate that the advanced TSTA-hTERT expressional system is a valuable tool for detecting viable bladder cancer cells. The current system can be applied for in vitro detection of bladder cancer cells in urine and other types of cancer cells disseminated in vivo.

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A Combined Continuous Density/Osmolality Gradient for Supplemental Purification of Human Islets.

Noguchi H, Naziruddin B, Shimoda M, Chujo D, Takita M, Sugimoto K, Itoh T, Onaca N, Levy MF, Matsumoto S

Cell medicine   3 ( 1-3 ) 33 - 41   2012.01 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Adverse Events in Clinical Islet Transplantation: One Institutional Experience

Morihito Takita, Shinichi Matsumoto, Hirofumi Noguchi, Masayuki Shimoda, Tetsuya Ikemoto, Daisuke Chujo, Yoshiko Tamura, Greg S. Olsen, Bashoo Naziruddin, Kerri Purcell, Nicholas Onaca, Marlon F. Levy

CELL TRANSPLANTATION ( COGNIZANT COMMUNICATION CORP )  21 ( 2-3 ) 547 - 551   2012 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Islet transplantation is one of the most promising treatments for an unstable form of type 1 diabetes. However, islet transplantation still has some obstacles, such as low success rate of islet isolation, difficulty to obtain long-term insulin freedom, and adverse events related to transplant protocol. We describe the adverse events of current clinical islet transplantation at our institute in this report. Nine type 1 diabetic patients received 17 islet infusions from March 2005 to October 2008. The islet infusion procedure and immunosuppression regimen were based on a modified Edmonton protocol. Severe adverse events (SAEs) were defined as events that were more than grade 3 according to the Terminology Criteria for Adverse Events in Trials of Adult Pancreatic Islet Transplantation, version 4.1 (Collaborative Islet Transplant Registry, CITR). Sixteen events were reported as SAEs and among them 12 events were probably or definitely related to transplant protocols; all occurred within 1 year after infusion except for one. Five adverse events (31%) occurred within 10 days after transplantation and were related to infusion procedures. Seven events (44%) occurred after 50 days and were related to immunosuppressive therapy. SAEs related to the protocol included three events of elevated liver enzymes, two of hemorrhage into gall bladder or peritoneal cavity, two of neutropenia, two of infection, one of vomiting, one of diarrhea, and one of renal dysfunction. All events were grade 3, except for one case that was grade 4 of neutropenia. All SAEs resolved with no sequelae. Neoplasms and deaths were not observed in our study. The present study suggests need to improve both infusion procedure and immunosuppressive strategy from the view of preventing SAEs.

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Attenuation of Murine Graft-Versus-Host Disease by a Tea Polyphenol

Jun Kanamune, Yasuhiro Iwanaga, Tatsuo Kina, Hirofumi Noguchi, Kazuaki Matsumura, Shinji Uemoto, Suong-Hyu Hyon

CELL TRANSPLANTATION ( COGNIZANT COMMUNICATION CORP )  21 ( 5 ) 909 - 918   2012 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Since donor T-cells' allorecognition of host antigens is a prerequisite for the onset of graft-versus-host disease (GVHD), blocking their cellular signaling pathways can decrease the severity of GVHD. We hypothesized that epigallocatechin-3-gallate (EGCG), due to its strong affinity to macromolecules, would adhere to surface molecules of donor T cells, inhibit their allorecognition, and attenuate GVHD in the recipient. We tested the hypothesis by treating donor splenocytes with EGCG in both in vitro and in vivo murine GVHD models. EGCG treatment decreased the proliferation of donor cells in MLR cultures and secretion of IL-2 and INF-gamma. It also reduced the epitope detection of CD3 epsilon, CD4, and CD28 but did not downregulate the protein expression of these molecules, suggesting blockage of cell surface stimulatory signals. Similarly, EGCG treatment did not decrease mRNA expression for some of these molecules but decreased mitogen-induced cell proliferation, indicating that EGCG did not interfere the transcription of these genes but affected cell proliferation pathways. Furthermore, EGCG-treated donor splenocytes, when transplanted into immuno-compromized recipient mice, decreased of proliferation, and the treatment extended the recipients' survival at least during the early stage of GVHD. These results strongly suggest that EGCG attenuates GVHD by both blocking specific cell surface molecules and affecting the donor T-cell proliferation pathways.

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Comparison of Ulinastatin, Gabexate Mesilate, and Nafamostat Mesilate in Preservation Solution for Islet Isolation

Hirofumi Noguchi, Bashoo Naziruddin, Andrew Jackson, Masayuki Shimoda, Yasutaka Fujita, Daisuke Chujo, Morihito Takita, Han Peng, Koji Sugimoto, Takeshi Itoh, Naoya Kobayashi, Michiko Ueda, Teru Okitsu, Yasuhiro Iwanaga, Hideo Nagata, Xiaoling Liu, Hiroki Kamiya, Nicholas Onaca, Marlon F. Levy, Shinichi Matsumoto

CELL TRANSPLANTATION ( COGNIZANT COMMUNICATION CORP )  21 ( 2-3 ) 509 - 516   2012 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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For islet transplantation, maintaining organ viability after pancreas procurement is critically important for optimal graft function and survival. We recently reported that islet yield was significantly higher in the modified ET-Kyoto (MK) solution, which includes a trypsin inhibitor (ulinastatin), compared with the UW solution, and that the advantages of MK solution are trypsin inhibition and less collagenase inhibition. In this study, we compared ulinastatin with other trypsin inhibitors, gabexate mesilate, and nafamostat mesilate, in preservation solution for islet isolation. Ulinastatin was easily dissolved in ET-Kyoto solution, while ET-Kyoto with gabexate mesilate and nafamostat mesilate became cloudy immediately after addition. Although there were no significant differences in islet yield among the three groups, viability was significantly higher for the MK group than for the GK group or the NK group. The stimulation index was significantly higher for the MK group than for the GK group. In summary, there are no other trypsin inhibitors that are more effective than ulinastatin. Based on these data, we now use ET-Kyoto solution with ulinastatin for clinical islet transplantation.

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Cryopreservation of induced pluripotent stem cells.

Miyamoto Y, Noguchi H, Yukawa H, Oishi K, Matsushita K, Iwata H, Hayashi S

Cell Medicine   3   89-95   2012 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

Cryopreservation of Human Adipose Tissue-Derived Stem/Progenitor Cells Using the Silk Protein Sericin

Yoshitaka Miyamoto, Koichi Oishi, Hiroshi Yukawa, Hirofumi Noguchi, Masahiro Sasaki, Hisashi Iwata, Shuji Hayashi

CELL TRANSPLANTATION ( COGNIZANT COMMUNICATION CORP )  21 ( 2-3 ) 617 - 622   2012 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Adipose tissue-derived stem/progenitor cells (ASCs) have attracted attention as a cell source that replaces marrow stromal cells (MSCs); ASCs may thus have applications in both regenerative medicine and cell transplantation. These medical treatments, however, require a high-quality supply of human ASCs. Therefore, the cryopreservation methods have been improved by changing a component of a cryopreservation medium. Sericin, a protein hydrolysate (with an average molecular weight of 30 kDa) is very rich in serine. The viability and the adipogenic/osteogenic potential of human ASCs were tested after freezing in a cryopreservation medium containing sericin. After thawing, the viability of the human ASCs frozen in the cryopreservation medium was found to be more than 95%. The proliferation rate of human ASCs frozen in CELLBANKER 2, and DMEM/Ham's F-12 medium (serum free) + 10% DMSO, 0.1 mol/L maltose, and 1% sericin was higher than that of the cells frozen in the maintenance medium + 10% DMSO. The adipogenic/osteogenic differentiation capabilities of frozen human ASCs were examined by Oil Red 0 staining/Von Kossa's method. The human ASCs were frozen using CELLBANKER 2, and DMEM/Ham's F-12 medium (serum free) + 10% DMSO, 0.1 mol/L maltose, and 1% sericin were positive. In conclusion, the cryopreservation medium containing sericin is therefore considered to have a beneficial effect on freezing human ASCs. This serum-free cryopreservation medium should be widely used in regenerative medicine, cell transplantation, and biological research.

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[Islet transplantation].

Noguchi H, Yamada T, Tanaka K

Nihon rinsho. Japanese journal of clinical medicine   69 ( 12 ) 2209 - 2213   2011.12 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Cluster Analysis of Self-Monitoring Blood Glucose Assessments in Clinical Islet Cell Transplantation for Type 1 Diabetes

Morihito Takita, Shinichi Matsumoto, Hirofumi Noguchi, Masayuki Shimoda, Daisuke Chujo, Takeshi Itoh, Koji Sugimoto, Jeffery A. SoRelle, Nicholas Onaca, Bashoo Naziruddin, Marlon F. Levy

DIABETES CARE ( AMER DIABETES ASSOC )  34 ( 8 ) 1799 - 1803   2011.08 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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OBJECTIVE-Cluster analysis was performed on the results of self-monitoring of blood glucose (SMBG) to discriminate islet graft function after islet cell transplantation (ICT) in patients with type 1 diabetes. RESEARCH DESIGN AND METHODS-Eleven islet recipients were included in this study. The patients visited our clinic monthly after ICT and provided blood samples for fasting C-peptide (n = 270), which were used to evaluate islet graft function. They also provided their SMBG data through an automatic data collection system. The SMBG data for 3 days immediately before each clinic visit were evaluated using the following assessments: M value, mean amplitude of glycemic excursions, J index, index of glycemic control, average daily risk range, and glycemic risk assessment diabetes equation. The cluster analysis was performed for both SMBG assessments and samples. Multivariate logistic regression analysis was used to evaluate the clusters of SMBG for assessing islet graft function. RESULTS-Analysis for SMBG assessments revealed five types of clusters, which showed similar patterns according to functional or dysfunctional islet graft phase. Two clusters, the euglycemia cluster (P &lt; 0.001) and the hypoglycemia cluster (P = 0.001), were significant factors in the logistic model for islet graft function. The SMOG clusters had significant correlations with clinical graft indexes (P &lt; 0.001). CONCLUSIONS-Cluster analysis of SMBG data as part of an automated data quality system could allow discrimination of islet graft dysfunction after ICT. This approach should be considered for islet recipients.

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Embryonic body formation using the tapered soft stencil for cluster culture device

Hiroshi Yukawa, Hirofumi Noguchi, Shuji Hayashi

BIOMATERIALS ( ELSEVIER SCI LTD )  32 ( 15 ) 3729 - 3738   2011.05 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Induced pluripotent stem (iPS) cells are expected to provide a source of tissue, a renewable cell source for tissue engineering, and a method for in vitro drug screening for patient-specific or disease-specific treatment. A simple technology by which iPS cells can be differentiated effectively and in large quantities is strongly desired. In this paper, a new device (Tapered Soft Stencil for Cluster Culture: TASCL) is proposed for the easy and efficient formation of EBs which can be used in regenerative medicine. This device was compared with the two major methods currently being evaluated, namely the HD method and the Terasaki (R) plate (MWC substitution), in terms of the efficiency, morphology and acquired number of EB formation. Using the TASCL device, the shape of the EBs formed was almost a perfect sphere, and the formation was also faster than for the two other methods. There was little variability in the number of cells. Moreover, EBs formed using the TASCL device had the ability to differentiate into all three germ layers, and differentiation of EBs from the TASCL culture into hepatic cells was confirmed. In conclusion, it appears that the TASCL device can be utilized for EB formation to generate cells for regenerative medicine applications. (C) 2011 Elsevier Ltd. All rights reserved.

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Body Mass Index Reflects Islet Isolation Outcome in Islet Autotransplantation for Patients With Chronic Pancreatitis

Morihito Takita, Bashoo Naziruddin, Shinichi Matsumoto, Hirofumi Noguchi, Masayuki Shimoda, Daisuke Chujo, Takeshi Itoh, Koji Sugimoto, Yoshiko Tamura, Greg S. Olsen, Nicholas Onaca, Jeffrey Lamont, Luis F. Lara, Marlon F. Levy

CELL TRANSPLANTATION ( COGNIZANT COMMUNICATION CORP )  20 ( 2 ) 313 - 322   2011 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Total pancreatectomy with autologous islet cell transplantation (TP with AIT) is an effective treatment for chronic pancreatitis patients with severe abdominal pain. Body mass index (BMI) of the pancreatic donor is proven to be a useful predictor for islet isolation and transplantation outcomes in allogenic islet transplantation. However, the association between BMI and islet isolation outcome and/or metabolism after AIT was previously unclear. Twelve patients who received TP with AIT at our hospital were included in this study. All pancreata were preserved with both pancreatic ductal injection and oxygen-charged static two-layer method using ET-Kyoto solution. The cohort was divided into two groups: low BMI group (BMI &lt; 23 kg/m(2). n = 5) and high BMI group (BMI &gt;= 23, n = 7). The high BMI group had a significantly higher islet yield per gram than the low BMI group both in pancreas postdigestion and in final product (postdigestion: 7330 +/- 539 vs. 3509 +/- 563 IE/g; p &lt; 0.001; final product: 6555 +/- 585 vs. 3476 +/- 546 IE/g; p = 0.004). For islet yield in final product per patient body weight, the high BMI group also had significantly higher islet yield than the low BMI group (7997 +/- 779 vs. 4175 +/- 750 IE/kg, p = 0.007). Insulin independence rate in the high BMI group (71%) was also higher than that low BMI group (40%), but it did not reach statistical significance. Pancreata from patients with higher BMI could obtain higher islet yield in the setting of autologous islet cell transplantation for chronic pancreatitis.

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Assessment of Human Islet Isolation With Four Different Collagenases

M. Shimoda, H. Noguchi, B. Naziruddin, Y. Fujita, D. Chujo, M. Takita, H. Peng, Y. Tamura, G. S. Olsen, K. Sugimoto, T. Itoh, N. Onaca, M. F. Levy, P. A. Grayburn, S. Matsumoto

TRANSPLANTATION PROCEEDINGS ( ELSEVIER SCIENCE INC )  42 ( 6 ) 2049 - 2051   2010.07 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Background. The isolation of islets from the human pancreas critically depends on the efficiency of the digestive enzymes. Liberase HI had been used as a standard preparation until the issues concerning bovine spongiform encephalopathy. Thus, we must now use other collagenases for clinical islet transplantation, four of which we have evaluated herein. Methods. The digestion of each of 17 pancreata from brain-dead donors was performed using the following collagenases: Liberase HI (HI; Roche, n = 9); Liberase MTF C/T (MTF; Roche, n = 4); Collagenase NB1 Premium Grade (NB1; Serva, n = 7); or Clzyme Collagenase HA (CI, VitaCyte, n = 4). Islet isolations were based on the Edmonton protocol for HI, whereas our modified islet isolation method was used for the three new enzymes (MTF, NB1, and CI). Results. There were no significant differences in donor age, body mass index, pancreas size, and cold ischemic time among the four groups. The phase I time in the NB1 group was significantly shorter than in the CI group (P = .0014). The prepurification IEQ/g in the HI group was significantly lower than the others (P = .0003 vs MTF,.0007 vs NB1, and .0009 vs CI, respectively). The postpurification IEQ/g in the MTF group was significantly higher than in the HI group (P = .006). The viability in the NB1 group was significantly greater than the HI group (P = .003). Conclusion. Three new enzymes (MTF, NB1, and CI) may enable us to obtain higher islet yields than with HI.

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Comparison of Fresh and Cultured Islets From Human and Porcine Pancreata

H. Noguchi, B. Naziruddin, M. Shimoda, Y. Fujita, D. Chujo, M. Takita, H. Peng, K. Sugimoto, T. Itoh, Y. Tamura, G. S. Olsen, N. Kobayashi, N. Onaca, M. F. Levy, S. Matsumoto

TRANSPLANTATION PROCEEDINGS ( ELSEVIER SCIENCE INC )  42 ( 6 ) 2084 - 2086   2010.07 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Introduction. For clinical islet transplantation, many centers have recently introduced of human islet cultures prior to transplantation. They provide flexibility to evaluate isolated islets and pretreat patients. However, isolated islets deteriorate rapidly in culture. In the present study, we compared fresh human and porcine islets with cultured islets for c-Jun NH(2)-terminal kinase (JNK) activity. Materials and methods. Islet isolations from human and porcine pancreata were performed using the standard Ricordi technique with a modified Edmonton protocol. Isolated islets cultured for 24 hours at 37 degrees C with 5% CO(2) in culture medium were evaluated for counts and JNK activity. Results. After 24 hours of culture, the percentages of surviving islets were 86.9% for human and 47.3% for porcine sources. JNK activity in isolated islets declined to a low baseline level after 24-hour culture. Conclusion. Both human and porcine islets deteriorated rapidly in 24-hour cultures, although the in vitro conditions did not induce JNK activation.

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Assessment of Islet Quality Following International Shipping of More Than 10,000 km

Tetsuya Ikemoto, Shinichi Matsumoto, Takeshi Itoh, Hirofumi Noguchi, Yoshiko Tamura, Andrew M. Jacksong, Masayuki Shimoda, Bashoo Naziruddin, Nicholas Onaca, Yohichi Yasunami, Marlon F. Levy

CELL TRANSPLANTATION ( SAGE PUBLICATIONS INC )  19 ( 6-7 ) 731 - 741   2010 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Islet transplantation is an attractive therapy for type I diabetes, although some issues remain. One of them is the severe donor shortage in some countries. In this study, we investigated the possibility of international islet shipping beyond 10,000 km to supply islets to countries with donor shortages. Human islets were isolated from six cadaver donors and cultured until shipment. Islets were packed in either gas-permeable bags or in non-gas-permeable bags and shipped from Baylor Research Institute (Dallas, TX, USA) to Fukuoka University (Fukuoka, Japan). Pre- and postshipment islet number, purity, viability, and stimulation index (by glucose stimulation test) were assessed. Shipped 1,500 IE islets were transplanted into streptozotocin-induced diabetic nude mice for in vivo assay. The distance of our shipment was 11,148.4 km, and the mean duration of the shipments was 48.2 +/- 8.2 h. The islet number recovery rate (postshipment/preshipment) was significantly higher in gas-permeable bags (56.4 +/- 10.1% vs. 20.5 +/- 20.6%, p&lt;0.01). Islet purity was significantly reduced during shipment in non-gas-permeable bags (from 47.7 +/- 18.6% to 40.2 +/- 28.2 in gas-permeable bags vs. from 50.4 +/- 6.4% to 25.9 +/- 15.6% in non-gas-permeable bags, p&lt;0.05). Islet viability and stimulation index did not change significantly between pre- and postshipping, in either gas-permeable bags or in non-gas-permeable bags. One of three diabetic nude mice (33.3%) converted to normoglycemia. It is feasible to ship human islet cells internationally in gas-permeable bags. This strategy would promote basic and preclinical research for countries with donor shortages, even though the research centers are remote (over 10,000 km from the islet isolation center).

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Cell Labeling With a Novel Contrast Agent of Magnetic Resonance Imaging

Koichi Oishi, Hirofumi Noguchi, Hiroaki Saito, Hiroshi Yukawa, Yoshitaka Miyamoto, Katsutoshi Murase, Shuji Hayashi

CELL TRANSPLANTATION ( COGNIZANT COMMUNICATION CORP )  19 ( 6-7 ) 887 - 892   2010 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Cell therapy is a proven and efficient method for treating multiple diseases. For both basic research and clinical practice, the development of noninvasive in vivo imaging methods is essential for monitoring the trafficking or homing of transplanted cells. One attractive approach for the effective imaging of transplanted cells is the efficient labeling of cells with a contrast agent. In this study, we developed a novel contrast agent of magnetic resonance imaging (MRI), TMADM-02. TMADM-02 was efficiently transduced into cells without toxicity. However, the aggregation of TMADM-02 was observed because of its low stability in culture medium. Therefore, TMADM-02 may have led to a false-positive test result. In future studies, we should verify not only the efficiency of labeling cells but also the stability of the contrast agent of MRI for clinical applications.

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Efficient Transfection of Sendai Virus Vector to Mouse Pancreatic Stem Cells in the Floating State

Koichi Oishi, Hirofumi Noguchi, Hiroshi Yukawa, Makoto Inoue, Yoshitaka Miyamoto, Hisashi Iwata, Mamoru Hasegawa, Shuji Hayashi

CELL TRANSPLANTATION ( COGNIZANT COMMUNICATION CORP )  19 ( 6-7 ) 893 - 900   2010 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Sendai virus (SeV) vectors can efficiently introduce foreign genes without toxicity into various organs and are expected to be clinically applicable. We previously compared the transfectional efficiency of SeV and adenovirus (AdV) vectors by assessing the transfer of the green fluorescent protein (GFP) gene to pancreatic stem cells. Although the gene transfer efficiency was similar between these vectors, SeV vector had a lower toxicity in comparison to the AdV vector. In this study, we assessed the gene transfer efficiency of SeV vector in the floating state to pancreatic stem cells. The efficiency of gene transfer was much higher at all time points and at all concentrations in the floating state versus in the adhesion state. In addition, the pancreatic stem cells transfected with SeV in the floating state maintained their differentiation ability. These data suggest that SeV transfection to pancreatic stem cells in the floating state may be useful in gene transfer technology.

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Comparison of Modified Celsior Solution and M-Kyoto Solution for Pancreas Preservation in Human Islet Isolation

Hirofumi Noguchi, Bashoo Naziruddin, Nicholas Onaca, Andrew Jackson, Masayuki Shimoda, Tetsuya Ikemoto, Yasutaka Fujita, Naoya Kobayashi, Marlon F. Levy, Shinichi Matsumoto

CELL TRANSPLANTATION ( COGNIZANT COMMUNICATION CORP )  19 ( 6-7 ) 751 - 758   2010 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Since the successful demonstration of the Edmonton protocol, islet transplantation has advanced significantly on several fronts, including improved pancreas preservation systems. In this study, we evaluated two different types of organ preservation solutions for human islet isolation. Modified Celsior (Celsior solution with hydroxyethyl starch and nafamostat mesilate; HNC) solution and modified Kyoto (MK) solution were compared for pancreas preservation prior to islet isolation. Islet yield after purification was significantly higher in the MK group than in the HNC group (MK = 6186 +/- 985 IE/g; HNC = 3091 +/- 344 IE/g). The HNC group had a longer phase I period (digestion time), a higher volume of undigested tissue, and a higher percentage of embedded islets, suggesting that the solution may inhibit collagenase. However, there was no significant difference in ATP content in the pancreata or in the attainability of posttransplant normoglycemia in diabetic nude mice between the two groups, suggesting that the quality of islets was similar among the two groups. In conclusion, MK solution is better for pancreas preservation before islet isolation than HNC solution due to the higher percentage of islets that can be isolated from the donor pancreas. MK solution should be the solution of choice among the commercially available solutions for pancreatic islet isolation leading to transplantation.

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Characterization of Human Pancreatic Progenitor Cells

Hirofumi Noguchi, Bashoo Naziruddin, Andrew Jackson, Masayuki Shimoda, Tetsuya Ikemoto, Yasutaka Fujita, Daisuke Chujo, Morihito Takita, Naoya Kobayashi, Nicholas Onaca, Shuji Hayashi, Marlon F. Levy, Shinichi Matsumoto

CELL TRANSPLANTATION ( COGNIZANT COMMUNICATION CORP )  19 ( 6-7 ) 879 - 886   2010 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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beta-Cell replacement therapy via islet transplantation is an effective treatment for diabetes mellitus, but its widespread use is severely limited by the shortage of donor organs. Because pancreatic stem/progenitor cells are abundantly available in the pancreas of these patients and in donor organs, the cells could become a useful target for beta-cell replacement therapy. We previously established a mouse pancreatic stem cell line without genetic manipulation. In this study, we used the techniques to identify and isolate human pancreatic stem/progenitor cells. The cells from a duct-rich population were cultured in 23 kinds of culture media, based on media for mouse pancreatic stem cells or for human embryonic stem cells. The cells in serum-free media formed "cobblestone" morphologies, similar to a mouse pancreatic stem cell line. On the other hand, the cells in serum-containing medium and the medium for human embryonic stem cells formed "fibroblast-like" morphologies. The cells divided actively until day 30, and the population doubling level (PDL) was 6-10. However, the cells stopped dividing after 30 days in any culture conditions. During the cultures, the nucleus/cytoplasm (N/C) ratio decreased, suggesting that the cells entered senescence. Exendin-4 treatment and transduction of PDX-1 and NeuroD proteins by protein transduction technology into the cells induced insulin and pancreas-related gene expression. Although the duplications of these cells were limited, this approach could provide a potential new source of insulin-producing cells for transplantation.

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Bone repair by transplantation of hTERT-immortalized human mesenchymal stem cells in mice.

Hiroyuki Nakahara, Haruo Misawa, Takahiro Hayashi, Eisaku Kondo, Takeshi Yuasa, Yasuhiro Kubota, Masayuki Seita, Hironobu Kawamoto, Wael A R A Hassan, Reham A R A Hassan, Shahid M Javed, Masato Tanaka, Hirosuke Endo, Hirofumi Noguchi, Shinichi Matsumoto, Katsuyoshi Takata, Yuichi Tashiro, Shuhei Nakaji, Toshifumi Ozaki, Naoya Kobayashi

Transplantation   88 ( 3 ) 346 - 53   2009.08 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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BACKGROUND: Human mesenchymal stem cells (hMSCs) are multipotent stem cells found in the adult bone marrow that have the capacity to differentiate into various mesenchymal cell types. The hMSCs may provide a potential therapy to restore damaged tissues or organs of mesenchymal origin; however, a drawback is their limited life span in vitro. METHODS: We immortalized normal hMSCs with retrovirally transmitted human telomerase reverse transcriptase cDNA. One of the immortalized clones (YKNK-12) was established, and the biological characteristics were investigated in vitro and in vivo. RESULTS: YKNK-12 cells were capable of differentiating adipocytes, osetoblasts, and chondrocytes. Osteogenically differentiated YKNK-12 cells produced significant levels of growth factors BMP4, BMP6, FGF6, FGF7, transforming growth factor-beta1, and transforming growth factor-beta3.. Microcomputer tomography T and soft X-ray assays showed an excellent calvarial bone healing in mice after transplantation of osteogenically differentiated YKNK-12 cells. These cells expressed human-specific osteocalcin and increased the gene expression of runt-related transcription factor 2, alkaline phosphatase, osteocalcin, and osterix in the bone regenerating area. YKNK-12 cell transplant corrected the bone defect without inducing any adverse effects. CONCLUSIONS: We conclude that hMSCs immortalized by transduction with human telomerase reverse transcriptase may provide an unlimited source of cells for therapeutic use in bone regeneration.

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Ductal Injection of JNK Inhibitors Before Pancreas Preservation Prevents Islet Apoptosis and Improves Islet Graft Function

Hirofumi Noguchi, Shinichi Matsumoto, Nicholas Onaca, Bashoo Naziruddin, Andrew Jackson, Tetsuya Ikemoto, Masayuki Shimoda, Yasutaka Fujita, Daisuke Chujo, Yasuhiro Iwanaga, Hideo Nagata, Teru Okitsu, Naoya Kobayashi, Hideki Ueno, Damien Chaussabel, Paul Grayburn, Jacques Banchereau, Marlon F. Levy

HUMAN GENE THERAPY ( MARY ANN LIEBERT INC )  20 ( 1 ) 73 - 85   2009.01 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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Human islet isolation to obtain high-quality islets is still challenging. This study investigates how c-Jun NH(2)-terminal kinase (JNK) is activated during human and porcine islet isolation. We also investigated whether ductal injection of preservation solution with JNK inhibitors improves islet isolation results by preventing apoptosis of islet cells. A low molecular weight inhibitor (SP600125) and a cell-permeable peptide inhibitor, the latter introduced by protein transduction technology, were used in porcine and human studies, respectively. JNK activity progressively increased during the isolation procedure. The addition of 10 mu M JNK inhibitors into the ductal preservation solution prevented JNK activation during the isolation procedure and prevented islet apoptosis immediately after isolation. We incubated islets (2000 islet equivalents) for 24-48 hr and then transplanted them below the kidney capsule of streptozotocin-induced diabetic mice. The blood glucose levels reached normoglycemia in more than 80% of the JNK inhibitor-positive group, whereas less than 20% of the JNK inhibitor-negative group achieved normoglycemia. These findings suggest that the JNK pathway is the major mediator of islet deterioration during/immediately after isolation and that JNK inhibition before islet isolation could improve outcomes after pancreatic islet transplantation.

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Cell Transplantation of Adipose Tissue-Derived Stem Cells in Combination With Heparin Attenuated Acute Liver Failure in Mice

Hiroshi Yukawa, Hirofumi Noguchi, Koichi Oishi, Soichi Takagi, Michinari Hamaguchi, Nobuyuki Hamajima, Shuji Hayashi

CELL TRANSPLANTATION ( SAGE PUBLICATIONS INC )  18 ( 5-6 ) 611 - 618   2009 [ Peer Review Accepted ]

Type of publication: Research paper (scientific journal)

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The effect of adipose tissue-derived stem cells (ASCs) in combination with heparin transplantation on acute liver failure mice with carbon tetrachloride (CCl4) injection was investigated. CCl4 is a well-known hepatotoxin and induces hepatic necrosis. Heparin did not affect the viability of ASCs for at least 24 h. The injection of heparin into the caudal tail vein decreased slightly the activities of the alanine aminotransferase (ALT), asparate aminotransferase (AST), and lactate dehydrogenase (LDH) in plasma. In the transplantation of ASCs (1 x 10(6) cells) group, there was a trend toward decreased activities of all markers. However, four out of six mice died of the lung infarction. In the transplantation of ASCs in combination with heparin group, there was also a trend toward decreased activities of all markers. In addition, all mice survived for at least the duration of the study period. In conclusion, the transplantation of ASCs in combination with heparin was thus found to effectively treat acute liver failure.

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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 &lt; .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&apos; 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&apos; 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 &lt; 0.05) and from 8.5 mM (4.8-11.7) to 3.3 mM (2.0-4.5, P &lt; 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 &lt; 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 &lt; 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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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 &lt; 0.01) and the Ricordi method (317.073 +/- 86.093 IE, p &lt; 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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