Published Papers - Noguchi Hirofumi

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 < 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 < 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 < 23 kg/m(2). n = 5) and high BMI group (BMI >= 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 < 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<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<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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