ognizant Communication Corporation

CELL TRANSPLANTATION
The Regenerative Medicine Journal

ABSTRACTS
VOLUME 18, NUMBERS 5/6, 2009

Cell Transplantation, Vol. 18, pp. 491-496, 2009
0963-6897/09 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2009 Cognizant Comm. Corp.
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Regenerative Medicine for Diabetes Mellitus

Naoya Kobayashi,1 Takeshi Yuasa,1 and Teru Okitsu2

1Department of Gastroenterological Surgery, Transplant and Surgical Oncology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8558, Japan
2Transplant Unit, Kyoto University Hospital, Kyoto 606-8507, Japan

In diabetes, a loss of pancreatic β-cells causes insulin dependency. When insulin dependency is caused by type 1 diabetes or pancreatic diabetes, for example, pancreatic β-cells need to be regenerated for definitive treatment. The methods for generating pancreatic β-cells include a method of creating pancreatic β-cells in vitro and implanting them into the body and a method of regenerating pancreatic β-cells in the body via gene introduction or the administration of differential proliferation factors to the body. Moreover, the number of pancreatic β-cells is also low in type 2 diabetes, caused by the compounding factors of insulin secretory failure and insulin resistance; therefore, if pancreatic β-cells can be regenerated in a living body, then a further amelioration of the pathology can be expected. The development of pancreatic β-cell-targeting regenerative medicine can lead to the next generation of diabetes treatment.

Key words: Pancreatic β-cells; Insulin; Diabetes; Regenerative medicine

Address correspondence to Naoya Kobayashi, M.D., Ph.D., Department of Gastroenterological Surgery, Transplant and Surgical Oncology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama, 700-8558, Japan. Tel/Fax: (+81) 86-235-7485; E-mail: immortal@md.okayama-u.ac.jp




Cell Transplantation, Vol. 18, pp. 497-503, 2009
0963-6897/09 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2009 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Pancreas Preservation by the Two-Layer Method: Does it Have a Beneficial Effect Compared With Simple Preservation in University of Wisconsin Solution?

Hirofumi Noguchi,1 Marlon F. Levy,1Naoya Kobayashi,2 and Shinichi Matsumoto1

1Baylor Institute for Immunology Research/Baylor All Saints Medical Center, Baylor Research Institute, Dallas, TX, USA
2Department of Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8558, Japan

A large number of reports have shown 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 in islet transplantation. However, two recent large-scale studies showed no beneficial effect of TLM compared with UW storage in human islet transplantation. We reevaluated the effect of TLM by following three groups: group 1: UW simple storage; group 2: TLM performed by multiorgan procurement teams (not specialists of islet isolation); and group 3: TLM performed by specialists of islet isolation (Noguchi and Matsumoto). There were no significant differences between groups 1 and 2, whereas islet yields were significantly higher in group 3 compared with either group 1 or 2. Our data suggest that exact, complete performance of TLM could improve the outcome of islet isolation and transplantation. In this review, we describe the mechanisms of the TLM, the procedure of preoxygenated TLM, and the several possibilities for the reasons of the discrepancy.

Key words: Islet transplantation; Two-layer method; Islet isolation; Perfluorochemical; University of Wisconsin solution; Adenosine triphosphate

Address correspondence to Shinichi Matsumoto, M.D., Ph.D., Baylor Institute for Immunology Research, Baylor Research Institute, 3434 Live Oak, Suite 125, Dallas, TX 75204, USA. Tel: (817) 922-2570; Fax: (817) 922-4645; E-mail: shinichm@baylorhealth.edu or Hirofumi Noguchi, M.D., Ph.D., Baylor Institute for Immunology Research, Baylor Research Institute, 3434 Live Oak St., Dallas, TX 75204, USA. Tel: (214) 820- 9016; Fax: (214) 820-4952; E-mail: hirofumn@baylorhealth.edu




Cell Transplantation, Vol. 18, pp. 505-512, 2009
0963-6897/09 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2009 Cognizant Comm. Corp.
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Beneficial Storage Effects of Epigallocatechin-3-O-Gallate on the Articular Cartilage of Rabbit Osteochondral Allografts

Jung Yoon Bae,1 Kazuaki Matsumura,1Shigeyuki Wakitani,2 Amu Kawaguchi,2Sadami Tsutsumi,1 and Suong-Hyu Hyon1

1Department of Medical Simulation Engineering, Research Center for Nano Medical Engineering, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan
2Department of Orthopaedic Surgery, Osaka City University Graduate School of Medicine, Osaka 545-8585, Japan

A fresh osteochondral allograft is one of the most effective treatments for cartilage defects of the knee. Despite the clinical success, fresh osteochondral allografts have great limitations in relation to the short storage time that cartilage tissues can be well-preserved. Fresh osteochondral grafts are generally stored in culture medium at 4°C. While the viability of articular cartilage stored in culture medium is significantly diminished within 1 week, appropriate serology testing to minimize the chances for the disease transmission requires a minimum of 2 weeks. (-)-Epigallocatechin-3-O-gallate (EGCG) has differential effects on the proliferation of cancer and normal cells, thus a cytotoxic effect on various cancer cells, but a cytopreservative effect on normal cells. Therefore, a storage solution containing EGCG might extend the storage duration of articular cartilages. Rabbit osteochondral allografts were performed with osteochondral grafts stored at 4°C in culture medium containing EGCG for 2 weeks and then the clinical effects were examined with macroscopic and histological assessment after 4 weeks. The cartilaginous structure of an osteochondral graft stored with EGCG was well-preserved with high cell viability and glycosaminoglycan (GAG) content of the extracellular matrix (ECM). After an osteochondral allograft, the implanted osteochondral grafts stored with EGCG also provided a significantly better retention of the articular cartilage with viability and metabolic activity. These data suggest that EGCG can be an effective storage agent that allows long-term preservation of articular cartilage under cold storage conditions.

Key words: Articular cartilage; Cold preservation; Storage solution; Epigallocatechin-3-O-gallate; Osteochondral allograft

Address correspondence to Prof. Suong-Hyu Hyon, Ph.D., Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. E-mail: biogen@frontier.kyoto-u.ac.jp




Cell Transplantation, Vol. 18, pp. 513-519, 2009
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Copyright © 2009 Cognizant Comm. Corp.
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Long-Term Preservation of Rat Skin Tissue by Epigallocatechin-3-O-Gallate

HakHee Kim,1 Takeshi Kawazoe,2 Kazuaki Matsumura,1 Shigehiko Suzuki,3 and Suong-Hyu Hyon1

1Department of Medical Simulation Engineering, Research Center for Nano Medical Engineering, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan
2Department of Plastic and Reconstructive Surgery, Kijunkai, Yoshikawa Hospital, Kyoto 606-8392, Japan
3Department of Plastic and Reconstructive Surgery, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan

Skin grafts can be preserved by cryopreservation and refrigerated storage at 4°C. Epigallocatechin-3-O-gallate (EGCG) enhances the viability of stored skin grafts and also extends the storage time up to 7 weeks at 4°C. EGCG, the major polyphenolic constituent present in green tea, has potent antioxidant, antimicrobial, antiproliferative, and free radical scavenging effects. This study examined the effects of EGCG on skin cryopreservation. Skin sample biopsy specimens from GFP rats were previously treated with/without EGCG then moved to -196°C. Skin samples were transplanted to nude mice after 2, 8, and 24 weeks of preservation. Glucose consumption was measured after thawing to assess the metabolic activity. Two weeks later the transplanted skin grafts were excised and histologically analyzed. Histological examinations revealed the degeneration of the epidermal and dermal layers in all groups. In the EGCG groups, the grafts showed higher integrity in the epidermal layer and dermal matrix. The present findings suggest the future clinical usefulness of EGCG for skin preservation; however, the mechanism by which EGCG promotes skin preservation still remains unclear.

Key words: Polyphenol; Epigallocatechin-3-O-gallate (EGCG); Skin; Skin grafts; Cryopreservation

Address correspondence to Suong-Hyu Hyon, Ph.D., Associate Professor, Department of Medical Simulation Engineering, Research Center for Nano Medical Engineering, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. Tel: +81 75 751 4125; Fax: +81 75 751 4141; E-mail: biogen@frontier.kyoto-u.ac.jp




Cell Transplantation, Vol. 18, pp. 521-528, 2009
0963-6897/09 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2009 Cognizant Comm. Corp.
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Preservation of Platelets by Adding Epigallocatechin-3-O-Gallate to Platelet Concentrates

Kazuaki Matsumura,1 Hiroshi Takayama,2Jung Yoon Bae,1 Mitsuru Kurihara,2Sadami Tsutsumi,1 and Suong-Hyu Hyon1

1Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan
2Department of Health/Nutrition, School of Human Cultures, The University of Shiga Prefecture, Shiga 522-8533, Japan

The effect of epigallocatechin-3-O-gallate (EGCG), a major component of green tea, on platelet preservation was evaluated. Single donor platelets (N = 10) were collected and preserved by the standard method. EGCG was added to the platelet concentrates before preservation and then the functional and biochemical parameters were monitored throughout the storage period. After 6 days of preservation, the aggregability of the platelets was significantly maintained by addition of 50 and 100 µg/ml of EGCG. Platelet prothrombinase activity was also significantly retained by the addition of EGCG. The accumulation of P-selectin and RANTES in the plasma preserved with EGCG was less than those preserved without EGCG, which indicated that EGCG might inhibit platelet activation. Furthermore, EGCG reduced the increase of LDH in plasma during preservation and inhibited the activation of caspase-3 and cleavage of gelsolin, thereby showing that EGCG could inhibit the apoptosis of platelets. These results suggest that EGCG may play an effective role in preserving platelets by inhibiting the activation and apoptosis of platelets.

Key words: Platelet; Preservation; Green tea polyphenol; Transfusion

Address correspondence to Suong-Hyu Hyon, Ph.D., Associate Professor, Department of Medical Simulation Engineering, Research Center for Nano Medical Engineering, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. Tel: +81 75 751 4125; Fax: +81 75 751 4141; E-mail: biogen@frontier.kyoto-u.ac.jp




Cell Transplantation, Vol. 18, pp. 529-534, 2009
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E-ISSN 1555-3892
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A Study on the Perfusion Preservation, Resuscitation, and Transplantation of a Rat Heart Isolated for 96 Hours

Naoyuki Hatayama,1 Yu Yoshida,1 and Kunihiro Seki1

1Kanagawa University Faculty of Science, Hiratsukashi, 259-1293, Japan

Krebs-Henseleit (KH) solution was used to fill the heart chamber of an isolated rat heart before it was immersed in perfluorocarbon (PFC), which is an inert fluid. A gas mixture (PCO2 = 150 hPa and PO2 = 850 hPa) was then aerated at a constant rate into the PFC solution, and the isolated heart was thereafter preserved for 96 h with KH solution perfused continuously at a rate of 0.1 ml/h from the aorta of the isolated heart through a cannula. After preservation, the preserved heart was heterotopically transplanted into the neck of a recipient rat and then it was resuscitated. Using this method for preserving mammalian organs, we attained reproducibility after perfusion preservation for 96 h.

Key words: Isolated rat heart; Perfluorocarbon (PFC); Preservation; Resuscitation; Heterotrophic transplantation; Perfusion preservation

Address correspondence to Kaoyuki Hatayama, Kanagawa University Faculty of Science, 2946, Tsuchiya, Hiratsukashi, 259-1293 Japan. Tel: 0463-59-4111, Post 2513; Fax: 0463-58-9684; E-mail: nao_h416@yahoo.co.jp




Cell Transplantation, Vol. 18, pp. 535-540, 2009
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E-ISSN 1555-3892
Copyright © 2009 Cognizant Comm. Corp.
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Study on the Preservation With CO (PCO = 200-2,000 hPa), Resuscitation, and Heterotopic Transplantation of an Isolated Rat Heart

Yu Yoshida,1 Naoyuki Hatayama,1 and Kunihiro Seki1

1Kanagawa University Faculty of Science,Hiratsukashi, 259-1293 Japan

In this experiment, CO was used as a gas mixture in a reversible relationship with O2. CO was added in a gas form mixed with O2. An isolated donor rat heart was obtained, exposed to a gas mixture such as PO2 = 1,800 hPa and PCO = 200 hPa, and PO2 = 1,000 hPa and PCO = 1,000 hPa in a 2 ATA high-pressure chamber and preserved in a refrigerator at 4°C. This report demonstrates that significant reproducibility has been verified. The heart was removed from the refrigerator 24 h later and heterotopic heart transplantation was performed in the right neck of a recipient rat and the pulsating of the transplanted heart was detected by an electrocardiogram.

Key words: Isolated rat heart; Perfluorocarbon (PFC); Desiccation; Preservation; Resuscitation; Heterotrophic transplantation

Address correspondence to Yu Yoshida, Kanagawa University Faculty of Science, 2946, Tsuchiya, Hiratsukashi, 259-1293, Japan. Tel: 0463-59-4111, Post 2513; Fax: 0463-58-9684; E-mail: yoshiy412@hotmail.co.jp




Cell Transplantation, Vol. 18, pp. 541-547, 2009
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E-ISSN 1555-3892
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Comparison of Trypsin Inhibitors in Preservation Solution for Islet Isolation

Hirofumi Noguchi,1,2,3,4 Michiko Ueda,4Shuji Hayashi,3 Naoya Kobayashi,5 Teru Okitsu,1 Yasuhiro Iwanaga,1 Hideo Nagata,4Xiaoling Liu,4 Hiroki Kamiya,4 Marlon F. Levy,2 and Shinichi Matsumoto2,4

1Transplantation Unit, Kyoto University Hospital, Kyoto 606-8507, Japan
2Baylor Institute for Immunology Research/Baylor All Saints Medical Center, Baylor Research Institute, Dallas, TX 75204, USA
3Department of Advanced Medicine in Biotechnology and Robotics, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
4Second Department of Surgery, Fujita Health University, Aichi 470-1192, Japan
5Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama 700-8558, Japan

Islet transplantation has recently emerged as an effective therapy and potential cure for type 1 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 ETKyoto solution with ulinastatin for clinical islet transplantation.

Key words: Islet transplantation; Islet isolation; MK solution; Trypsin inhibitor; Preservation solution

Address correspondence to Hirofumi Noguchi, M.D., Ph.D., Baylor Institute for Immunology Research, Baylor Research Institute, 3434 Live Oak St., Dallas, TX 75204, USA. Tel: (214) 820-9016; Fax: (214) 820-4952; E-mail: hirofumn@baylorhealth.edu




Cell Transplantation, Vol. 18, pp. 549-556, 2009
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E-ISSN 1555-3892
Copyright © 2009 Cognizant Comm. Corp.
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Estimation of Donor Usability for Islet Transplantation in the United States With the Kyoto Islet Isolation Method

Shinichi Matsumoto,1 Hirofumi Noguchi,1Nobuyo Hatanaka,2 Masayuki Shimoda,3Naoya Kobayashi,4 Andrew Jackson,1Nicholas Onaca,1 Bashoo Naziruddin,1 and Marlon F. Levy1

1Baylor All Saints Medical Center, Baylor Research Institute, Fort Worth, TX 76104, USA
2The Institute of Medical Science, The University of Tokyo, Tokyo, 108-8639, Japan
3Baylor University Medical Center, Dallas, TX 75246, USA
4Department of Surgery, Okayama University, Okayama, 700-8530, Japan

The quality of donor pancreata is important for successful islet isolation. However, in some countries like Japan, the number of donor pancreata is very low; therefore, marginal donors have been used with less restrictive donor criteria. In order to use marginal donor pancreata, we established the Kyoto islet isolation method (KIIM). According to United Network for Organ Sharing (UNOS) in 2005, more than 6,000 pancreata were not clinically used in the US. In this study, we applied the KIIM for brain-dead donors and reevaluated donor usability based on the Japanese islet donor criteria. Islets were isolated with the Ricordi method using pancreata stored in University of Wisconsin (UW) solution (UW group) or by the two-layer method (TLM group) or the TLM combined with ductal injection (DI group). We implemented the KIIM (KIIM group) to confirm the effect of the KIIM on brain-dead donors. Donor charts in Texas from 2005 to 2006 were reviewed. If pancreata were not used clinically, the reason was reviewed and donors were reevaluated based on Japanese criteria. There were no significant differences of islet yield, viability, and purity between the UW and TLM groups. The DI group significantly improved islet yields and isolations were further improved in the KIIM group [UW: 251,663 ± 60,217 islet equivalent (IE); TLM: 243,738 ± 54,170 IE; DI: 498,639 ± 28,853 IE; KIIM: 678,286 ± 55,853]. The KIIM provided high-quality islets in high numbers from islet isolations from brain-dead donors. A total of 236 donor charts were reviewed and 194 pancreata (82%) were not used. Of these, 185 cases identified the reasons that the pancreata were not used. When we applied the Japanese criteria, an additional 82 cases out of 185 (44%) seem to be suitable for islet isolations. With the KIIM, more than 2,500 additional donor pancreata can be used for islet isolation in the US every year when the Japanese criteria are applied.

Key words: Donor; Ductal injection; Islet transplantation; Kyoto islet isolation method; Kyoto solution; Two-layer method

Address correspondence to Shinichi Matsumoto, M.D., Ph.D., Baylor All Saints Medical Center, Baylor Research Institute, 1400 8th Avenue, Fort Worth, TX 76104, USA. Tel: 817-922-2570; Fax: 817-922-4645; E-mail: shinichi41@mac.com




Cell Transplantation, Vol. 18, pp. 557-562, 2009
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SUITO Index for Evaluation of Efficacy of Single Donor Islet Transplantation

Shinichi Matsumoto,1 Hirofumi Noguchi,1Nobuyo Hatanaka,2 Masayuki Shimoda,3Naoya Kobayashi,4 Andrew Jackson,1Nicholas Onaca,1 Bashoo Naziruddin,1 and Marlon F. Levy1

1Baylor All Saints Medical Center, Baylor Research Institute, Fort Worth, TX 76104, USA
2The Institute of Medical Science, The University of Tokyo, Tokyo, 108-8639, Japan
3Baylor University Medical Center, Dallas, TX 75246, USA
4Department of Surgery, Okayama University, Okayama, 700-8530, Japan

Evaluation of engrafted islets mass is important for clinical care of patients after islet transplantation. Recently, we developed the secretory unit of islet transplant objects (SUITO) index, which reflected engrafted islet mass. In this study, we evaluated the SUITO index for the prediction of clinical outcome after single islet transplantation. Single islet transplantations were performed into six type 1 diabetic patients. Isolated islets were quantitatively assessed at the time of transplantation. The SUITO index was calculated as follows: fasting C-peptide (ng/dl)/[fasting blood glucose (mg/dl) - 63] × 1500. Islet yield/recipient's body weight and SUITO index were evaluated, along with HbA1C, relative insulin dose (insulin dose posttransplant/pretransplant), and M-values. HbA1C improved in all cases, irrespective of the SUITO index score or islet yield/body weight. The average SUITO index from postoperative days 3 to 30 (R2 = 0.728, p < 0.04), but not islet yield/body weight (R2 = 0.259, p = 0.303), correlated with relative insulin dose. The daily SUITO index strongly correlated with the daily relative insulin dose (R2 = 0.558, p < 0.0001) and weakly correlated with the daily M-values (R2 = 0.207, p < 0.02). A SUITO index score of less than 10 was associated with increasing insulin dose even after islet transplantation. The SUITO index seems to be a better predictor of success of islet transplantations than islet yield/body weight. SUITO index is recommended to assess clinical outcome of islet transplantation.

Key words: SUITO index; Islet transplantation; Single donor; M-value

Address correspondence to Shinichi Matsumoto, M.D., Ph.D., Baylor All Saints Medical Center, Baylor Research Institute, 1400 8th Avenue, Fort Worth, TX 76104, USA. Tel: 817-922-2570; Fax: 817-922-4645; E-mail: shinichm@baylorhealth.edu




Cell Transplantation, Vol. 18, pp. 563-571, 2009
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E-ISSN 1555-3892
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Establishment of Mouse Pancreatic Stem Cell Line

Hirofumi Noguchi,1 Koichi Oishi,2Michiko Ueda,3 Hiroshi Yukawa,2 Shuji Hayashi,2 Naoya Kobayashi,4 Marlon F. Levy,1 and Shinichi Matusmoto1

1Baylor Institute for Immunology Research/Baylor All Saints Medical Center, Baylor Research Institute, Dallas, TX 75204, USA
2Department of Advanced Medicine in Biotechnology and Robotics, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
3Transplantation Unit, Kyoto University Hospital, Kyoto 606-8507, Japan
4Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama 700-8558, Japan

&beta;-Cell replacement therapy via islet transplantation is a promising possibility for the optimal treatment of type 1 diabetes. However, such an approach is severely limited by the shortage of donor organs. Pancreatic stem/progenitor cells could become a useful target for &beta;-cell replacement therapy in diabetic patients because the cells are abundantly available in the pancreas of these patients and in donor organs. In this study, we established a mouse pancreatic stem cell line without genetic manipulation. The duct-rich population after islet isolation was inoculated into 96-well plates in limiting dilution. From over 200 clones, 15 clones were able to be cultured for over 3 months. The HN#13 cells, which had the highest expression of insulin mRNA after induction, expressed PDX-1 transcription factor, glucagon-like peptide-1 (GLP-1) receptor, and cytokeratin- 19 (duct-like cells). These cells continue to divide actively beyond the population doubling level (PDL) of 300. Exendin-4 treatment and transduction of PDX-1 and NeuroD proteins by protein transduction technology in HN#13 cells induced insulin and pancreas-related gene expression. This cell line could be useful for analyzing pancreatic stem cell differentiation. Moreover, the isolation technique might be useful for identification and isolation of human pancreatic stem/progenitor cells.

Key words: Pancreatic stem cell; PDX-1; BETA2/NeuroD; Exendin-4; Pancreatic duct; Protein transduction domain

Address correspondence to Hirofumi Noguchi, M.D., Ph.D., Baylor Institute for Immunology Research, Baylor Research Institute, 3434 Live Oak St., Dallas, TX 75204, USA. Tel: (214) 820-9016; Fax: (214) 820-4952; E-mail: hirofumn@baylorhealth.edu




Cell Transplantation, Vol. 18, pp. 573-580, 2009
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E-ISSN 1555-3892
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Recombinant Sendai Virus-Mediated Gene Transfer to Mouse Pancreatic Stem Cells

Koichi Oishi,1 Hirofumi Noguchi,2Hiroshi Yukawa,1 Makoto Inoue,3 Soichi Takagi,1 Hisashi Iwata,4 Mamoru Hasegawa,3 and Shuji Hayashi1

1Department of Advanced Medicine in Biotechnology and Robotics, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
2Baylor Institute for Immunology Research/Baylor All Saints Medical Center, Baylor Research Institute, Dallas, TX 75204, USA
3DNAVEC Corporation, Ibaraki, 305-0856, Japan
4Department of Biomedical Sciences, Chubu University College of Life and Health Sciences, Aichi 487-8501, Japan

Efficient gene transfer into stem cells is essential for the basic research and for therapeutic applications in gene-modified regenerative medicine. Adenovirus (AdV) vectors, one of the most commonly used types of vectors, can mediate high, albeit transient, levels of expression of the transgene in pancreatic stem/progenitor cells. However, high multiplicity of infection (MOI) with AdV vectors can result in cellular toxicity. Therefore, AdV vectors have been of limited usefulness in clinical applications. In this study, we investigated the in vitro gene transfer efficiency of Sendai virus (SeV) vectors, a paramyxovirus vector that can efficiently introduce foreign genes without toxicity into several cell types, including pancreatic stem cells. The dosedependent GFP expression of pancreatic stem cells transfected with SeV vectors after 48 h of culture at 37°C was observed. The transfection of pancreatic stem cells with SeV vectors and AdV vectors results in equal expression of the transgene (GFP expression) in the cells after 48 h of culture at 37°C. Although the transfection of pancreatic stem cells with AdV vectors at high MOIs was cytotoxic, transfection with SeV vectors at high MOIs was rarely cytotoxic. In addition, pancreatic stem cells transfected with SeV maintained their differentiation ability. These data suggest that SeV could provide advantages with respect to safety issues in gene-modified regenerative medicine.

Key words: Pancreatic stem cells; Differentiation; Regenerative medicine; Sendai virus; Adenovirus

Address correspondence to Hirofumi Noguchi, M.D., Ph.D., Baylor Institute for Immunology Research, Baylor Research Institute, 3434 Live Oak St., Dallas, TX 75204, USA. Tel: (214) 820-9016; Fax: (214) 820-4952; E-mail: hirofumn@baylorhealth.edu




Cell Transplantation, Vol. 18, pp. 581-589, 2009
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Differential Ability of Somatic Stem Cells

Koichi Oishi,1 Hirofumi Noguchi,2Hiroshi Yukawa,1 and Shuji Hayashi1

1Department of Advanced Medicine in Biotechnology and Robotics, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
2Baylor Institute for Immunology Research/Baylor All Saints Medical Center, Baylor Research Institute, Dallas, TX 75204, USA

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;-glycerophosphate 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.

Key words: Somatic stem cells; Adipose tissue-derived stem/progenitor cells (ASCs); Pancreatic stem cells; Differential ability

Address correspondence to Hirofumi Noguchi, M.D., Ph.D., Baylor Institute for Immunology Research, Baylor Research Institute, 3434 Live Oak St., Dallas, TX 75204, USA. Tel: (214) 820-9016; Fax: (214) 820-4952; E-mail: hirofumn@baylorhealth.edu




Cell Transplantation, Vol. 18, pp. 591-599, 2009
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E-ISSN 1555-3892
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Quantum Dots for Labeling Adipose Tissue-Derived Stem Cells

Hiroshi Yukawa,1 Shogo Mizufune,2Chiharu Mamori,2 Yukimasa Kagami,2Koichi Oishi,1 Noritada Kaji,2 Yukihiro Okamoto,2 Manabu Takeshi,2 Hirofumi Noguchi,3 Yoshinobu Baba,2,4,5,6,7 Michinari Hamaguchi,8 Nobuyuki Hamajima,9 and Shuji Hayashi1

1Department of Advanced Medicine in Biotechnology and Robotics, Nagoya University Graduate School of Medicine, Nagoya 461-0047, Japan
2Department of Applied Chemistry, Nagoya University Graduate School of Engineering, Nagoya 464-8603, Japan
3Baylor Institute for Immunology Research, Baylor Research Institute, Dallas, TX 75204, USA
4MEXT Innovative Research Center for Preventive Medical Engineering, Nagoya University, Nagoya 464-8603, Japan
5Plasma Nanotechnology Research Center, Nagoya University, Nagoya 464-8603, Japan
6Health Technology Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Takamatsu 761-0395, Japan
7Institute for Molecular Science, National Institutes of Natural Sciences, Okazaki 444-8585, Japan
8Division of Cancer Biology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
9Department of Preventive Medicine, Biostatistics and Medical Decision Making, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan

Adipose tissue-derived stem cells (ASCs) have a self-renewing ability and can be induced to differentiate into various types of mesenchymal tissue. Because of their potential for clinical application, it has become desirable to label the cells for tracing transplanted cells and for in vivo imaging. Quantum dots (QDs) are novel inorganic probes that consist of CdSe/ZnS-core/shell semiconductor nanocrystals and have recently been explored as fluorescent probes for stem cell labeling. In this study, negatively charged QDs655 were applied for ASCs labeling, with the cationic liposome, Lipofectamine. The cytotoxicity of QDs655-Lipofectamine was assessed for ASCs. Although some cytotoxicity was observed in ASCs transfected with more than 2.0 nM of QDs655, none was observed with less than 0.8 nM. To evaluate the time dependency, the fluorescent intensity with QDs655 was observed until 24 h after transfection. The fluorescent intensity gradually increased until 2 h at the concentrations of 0.2 and 0.4 nM, while the intensity increased until 4 h at 0.8 nM. The ASCs were differentiated into both adipogenic and osteogenic cells with red fluorescence after transfection with QDs655, thus suggesting that the cells retain their potential for differentiation even after transfected with QDs655. These data suggest that QDs could be utilized for the labeling of ASCs.

Key words: Adipose tissue-derived stem cells (ASCs); Semiconductor; Quantum dots (QDs); Cell labeling

Address correspondence to Hiroshi Yukawa, Department of Advanced Medicine in Biotechnology and Robotics, Nagoya University Graduate School of Medicine, Higashi-ku, Nagoya 461-0047, Japan. Tel: 81-52-719-1975; Fax: 81-52-719-1977; E-mail: hiroshiy@med.nagoya-u.ac.jp




Cell Transplantation, Vol. 18, pp. 601-609, 2009
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E-ISSN 1555-3892
Copyright © 2009 Cognizant Comm. Corp.
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Comparison of Sendai Virus-Mediated Gene Transfer Efficiency to Adhesive and Floating Adipose Tissue-Derived Stem Cells

Hiroshi Yukawa,1 Hirofumi Noguchi,2Koichi Oishi,1 Makoto Inoue,3 Mamoru Hasegawa,3 Michinari Hamaguchi,4 Nobuyuki Hamajima,5 and Shuji Hayashi1

1Department of Advanced Medicine in Biotechnology and Robotics, Nagoya University Graduate School of Medicine, Nagoya 461-0047, Japan
2Baylor All Saints Medical Center, Baylor Research Institute, Dallas, TX 75204, USA
3DNAVEC Corporation, Tsukuba-city, Ibaraki, 305-0856, Japan
4Division of Cancer Biology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
5Department of Preventive Medicine, Biostatistics and Medical Decision Making, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan

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.

Key words: Adipose tissue-derived stem cells (ASCs); Sendai virus (SeV); Gene therapy; Differentiation; Floating state

Address correspondence to Hiroshi Yukawa, Department of Advanced Medicine in Biotechnology and Robotics, Nagoya University Graduate School of Medicine, Higashi-ku, Nagoya 461-0047, Japan. Tel: 81-52-719-1975; Fax: 81-52-719-1977; E-mail: hiroshiy@med.nagoya-u.ac.jp




Cell Transplantation, Vol. 18, pp. 611-618, 2009
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E-ISSN 1555-3892
Copyright © 2009 Cognizant Comm. Corp.
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Cell Transplantation of Adipose Tissue-Derived Stem Cells in Combination With Heparin Attenuated Acute Liver Failure in Mice

Hiroshi Yukawa,1 Hirofumi Noguchi,1Koichi Oishi,1 Soichi Takagi,1 Michinari Hamaguchi,1 Nobuyuki Hamajima,1 and Shuji Hayashi1

1Department of Advanced Medicine in Biotechnology and Robotics, Nagoya University Graduate School of Medicine, Nagoya 461-0047, Japan
2Baylor All Saints Medical Center, Baylor Research Institute, Dallas, TX 75204, USA
3Division of Cancer Biology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
4Department of Preventive Medicine, Biostatistics and Medical Decision Making, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan

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 × 106 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.

Key words: Cell transplantation; Adipose tissue-derived stem cells (ASCs); Acute liver failure; Heparin

Address correspondence to Hiroshi Yukawa, Department of Advanced Medicine in Biotechnology and Robotics, Nagoya University Graduate School of Medicine, Higashi-ku, Nagoya 461-0047, Japan. Tel: 81-52-719-1975; Fax: 81-52-719-1977; E-mail: hiroshiy@med.nagoya-u.ac.jp




Cell Transplantation, Vol. 18, pp. 619-626, 2009
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E-ISSN 1555-3892
Copyright © 2009 Cognizant Comm. Corp.
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Cryopreservation In Situ of Cell Monolayers on Collagen Vitrigel Membrane Culture Substrata: Ready-to-Use Preparation of Primary Hepatocytes and ES Cells

Yoshitaka Miyamoto,1,2 Shin Enosawa,1Tomoyo Takeuchi,2 and Toshiaki Takezawa2

1Department of Innovative Surgery, National Research Institute for Child Health and Development, Tokyo, Japan
2Transgenic Animal Research Center, National Institute of Agrobiological Sciences, Ibaraki, Japan

Cryopreservation is generally performed on cells in suspension. In the case of adherent cells such as hepatocytes, a loss of their ability to attach is a more serious problem than a decreased viability after cryopreservation. We herein report a novel technology of direct in situ cryopreservation of cells cultured on collagen vitrigel membranes, which have excellent mechanical strength and can be easily handled by tweezers even when coated with cultured cells. Rat primary hepatocytes, mitomycin C-treated mouse fibroblasts (feeder cells for ES cells), and mouse ES cells on the feeder cells were cultured on collagen vitrigel membranes for 1 day. The membranes with cells attached were then plucked up from the dish, soaked in cryopreservation medium containing 10% dimethyl sulfoxide, frozen using a controlled-rate freezer, and transferred to liquid nitrogen. The cells cultured on plastic cell culture dishes were also frozen as controls. After storage in liquid nitrogen for periods from 1 week to 3 months, the cryopreserved membranes with the cells still attached were thawed by adding warmed culture medium. Cell viability estimated by morphology and functional staining with calcein showed significant improvement in comparison to cells cryopreserved without the collagen vitrigel membrane. The recoveries of living cells after cryopreservation were 26.7%, 76.2%, and 58.6% for rat hepatocytes, mitomycin C-treated mouse fibroblasts, and mouse ES cells on collagen vitrigel membranes, respectively. In contrast, essentially no cells at all remained on the plastic cell culture dishes after thawing. Because adherent cell storage under these conditions is very convenient, the use of this technique employing collagen vitrigel membranes should be generally applicable to the cryopreservation of adherent cells that are otherwise problematic to store as frozen stocks.

Key words: Cryopreservation; Hepatocytes; ES cell; Collagen vitrigel

Address correspondence to Shin Enosawa, Department of Innovative Surgery, National Research Institute for Child Health and Development, 2-10-1 Ookura, Setagaya-ku, Tokyo 157-8535, Japan. Tel: 81-3-3416-0181; Fax: 81-3-3417-2864; E-mail: senosawa@nch.go.jp




Cell Transplantation, Vol. 18, pp. 627-637, 2009
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E-ISSN 1555-3892
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Superagonist CD28 Antibody Preferentially Expanded Foxp3-Expressing nTreg Cells and Prevented Graft-Versus-Host Diseases

Yusuke Kitazawa,1,4 Masayuki Fujino,1,2Xiao-Kang Li,1 Lin Xie,1,4 Naotsugu Ichimaru,3 Masayoshi Okumi,3 Norio Nonomura,3 Akira Tsujimura,3 Yoshitaka Isaka,4 Hiromitsu Kimura,1 Thomas Hünig,5 and Shiro Takahara4

1Laboratory of Transplantation Immunology, National Research Institute for Child Health and Development, Tokyo, Japan
2AIDS Research Center, National Institute of Infectious Diseases, Tokyo, Japan
3Department of Urology, Osaka University Graduate School of Medicine, Osaka, Japan
4Department of Advanced Technology for Transplantation, Osaka University Graduate School of Medicine, Osaka, Japan
5Institute for Virology and Immunobiology, University of Wüurzburg, Wüurzburg, Germany

Regulatory lymphocytes play a pivotal role in preventing organ-specific autoimmune disease and in induction and maintenance of tolerance in various experimental transplantation models. The enhancement of the number and activity of peripheral CD4+CD25+ Treg cells is an obvious goal for the treatment of autoimmunity and for the suppression of alloreactions. The present study demonstrates that naturally occurring CD4+CD25+ Treg (nTreg) cells preferentially proliferate to a fourfold increase within 3 days in response to the administration of a single superagonistic CD28-specific monoclonal antibody (supCD28 mAb). The appearance of increased Foxp3 molecules was accompanied with polarization toward a Th2 cytokine profile with decreased production of IFN-&gamma; and increased production of IL-4 and IL-10 in the expanded Treg subset. Adoptive transfer of supCD28 mAb-expanded cells in a graft-versus-host disease (GvHD) model induced a potent inhibition of lethality. These results suggest that this therapeutic effect is mediated by the in vivo expansion of nTreg cells. Taken together, these data demonstrate that supCD28-mAb may target nTreg cells in vivo and maintain and enhance their potent regulatory functions for the treatment GvHD.

Key words: Superagonist CD28 antibody; Graft-versus-host disease; Treg; nTreg

Address correspondence to Xiao-Kang Li, M.D., Ph.D., Laboratory of Transplantation Immunology, National Research Institute for Child Health and Development, 2-10-1 Okura, Setagaya-ku, Tokyo 157-8535, Japan. Tel: 81-3-3416-0181; Fax: 81-3417-2864; E-mail: sri@nch.go.jp




Cell Transplantation, Vol. 18, pp. 639-646, 2009
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E-ISSN 1555-3892
Copyright © 2009 Cognizant Comm. Corp.
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Comparative Study of the Cellular Pharmacodynamics of Calcineurin Inhibitors Between Patients With Chronic Renal Failure Awaiting Renal Transplantation and Cirrhosis Patients Awaiting Liver Transplantation

Yu Kihara,1 Naoto Matsuno,1 Abuduxukuer Mijiti,1 Takeshi Nagao,1 Hironori Takeuchi,2Sakae Unezaki,2 and Toshihiko Hirano3

1Department of 5th Surgery, Hachioji Medical Center, Tokyo Medical University, Tokyo, Japan
2Department of Practical Pharmacy, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan
3Department of Clinical Pharmacology, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan

The in vitro response of peripheral blood mononuclear cells (PBMCs) to the suppressive effects of calcineurin inhibitors is known to correlate with the clinical efficacy of drugs used in renal transplantations. The present study was conducted to examine the differences of PBMC responses to calcineurin inhibitors between chronic renal failure (CRF) patients awaiting renal transplantation and cirrhosis patients awaiting liver transplantation. The study included 99 CRF patients awaiting renal transplantation and 27 cirrhosis patients awaiting liver transplantation. Twenty milliliters of venous blood was taken 1-7 days before transplantation. The in vitro drug concentrations giving 50% inhibition of PBMC blastogenesis stimulated with concanavalin A (IC50s) were calculated. The suppressive effects of tacrolimus against PBMC blastogenesis were more than 10-100 times stronger than those of cyclosporine. The median IC50 value for cyclosporine against the CRF PBMCs was not significantly different from the median IC50 value against the cirrhosis PBMCs. In contrast, tacrolimus sensitivity in cirrhosis PBMCs is approximately seven times higher than that in CRF PBMCs. The median IC50 value for tacrolimus against cirrhosis PBMCs was significantly lower and therefore the effect was stronger in comparison to the CRF PBMCs (p < 0.001). These data suggest that the PBMCs of cirrhosis patients, in comparison to those of CRF patients, are highly sensitive to the suppressive effect of tacrolimus. However, PBMC sensitivity to cyclosporine was not significantly different between the CRF and cirrhosis patients. These observations raise the possibility that treatment with tacrolimus, rather than cyclosporine, may therefore be a better choice to reduce the risks of allograft rejection in liver transplantation.

Key words: Calcineurin inhibitors; Peripheral blood mononuclear cells; Chronic renal failure; Cirrhosis; Renal transplantation; Liver transplantation

Address correspondence to Toshihiko Hirano, Ph.D., Department of Clinical Pharmacology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan. Tel: 81-426-76-5796; Fax: 81-426-76-5798; E-mail: hiranot@ps.toyaku.ac.jp




Cell Transplantation, Vol. 18, pp. 647-656, 2009
0963-6897/09 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2009 Cognizant Comm. Corp.
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Global Expression Profiles in 1-Hour Biopsy Specimens of Human Kidney Transplantation From Donors After Cardiac Death

Mamoru Kusaka,1 Yoko Kuroyanagi,1Terumi Mori,1 Kayuri Nagaoka,1 Hitomi Sasaki,1 Takahiro Maruyama,1 Kunihiro Hayakawa,1 Ryoichi Shiroki,1 Hiroki Kurahashi,1 and Kiyotaka Hoshinaga1

1Department of Urology, Division of Molecular Genetics, Institute for Comprehensive Medical Science and 21st Century COE Program, Development Center for Targeted and Minimally Invasive Diagnosis and Treatment, Fujita Health University School of Medicine, Aichi 470-1192, Japan

Because of the worldwide shortage of renal grafts, kidney transplantation (KTx) from donors after cardiac death (DCD) is an alternative way to obtain KTx from brain-dead donors. Although the prognosis of DCD KTx is gradually improving, the graft often undergoes delayed graft function (DGF), rendering the control of DGF essential for post-KTx patient care. In an attempt to characterize etiology of DGF, genome-wide gene expression profiling was performed using renal biopsy samples performed at 1 h after KTx from DCD and the data were compared with those of KTx from living donors (LD). A total of 526 genes were differentially expressed between them. Genes involved in acute inflammation were activated, while metabolic pathways were consistently downregulated in DCD. These findings imply the inferior performance of the DCD grafts relative to LD grafts. Several genes were identified where the expression levels were correlated well with parameters indicating short- and long-term prognosis of the DCD patients. In addition, several genes encoding secretory proteins were identified that might reflect the performance of the graft and be potential noninvasive biomarkers. These data provide a good source for candidates of biomarkers that are potentially useful for the control of DGF.

Key words: Donation after cardiac death; Delayed graft function; Kidney transplantation; Gene expression

Address correspondence to Mamoru Kusaka, M.D., Department of Urology, Fujita Health University School of Medicine, 1-98 Dengakugakubo, Kutsukake-cho, Toyoake, Aichi, 470-1192 Japan. Tel: 81-562-93-9257; Fax: 81-562-93-7863; E-mail: mkusaka@fujita-hu.ac.jp




Cell Transplantation, Vol. 18, pp. 657-664, 2009
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E-ISSN 1555-3892
Copyright © 2009 Cognizant Comm. Corp.
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Clinical Significance of the Cellular Pharmacodynamics of Tacrolimus in Living-Donor Liver Transplantation

Abuduxukuer Mijiti,1,2 Naoto Matsuno,1Hironori Takeuchi,3 Sakae Unezaki,3Takeshi Nagao,1 and Toshihiko Hirano4

1Department of 5th Surgery, Hachioji Medical Center, Tokyo Medical University, Tokyo 193-0944, Japan
2Department of Surgery, Kashgar First People's Hospital, Xinjiang Uyghur Autonomous Region, China
3Department of Practical Pharmacy, Tokyo University of Pharmacy and Life Sciences, Tokyo 192-0392, Japan
4Department of Clinical Pharmacology, Tokyo University of Pharmacy and Life Sciences, Tokyo 192-0392, Japan

Successful immunosuppressive therapy is critical for liver transplantation; however, a considerable number of patients experience fatal rejection or alternatively exhibit serious infection resulting from excessive immunosuppression. The in vitro tacrolimus response of peripheral blood mononuclear cells (PBMCs) before transplantation was compared to the clinical outcome up to 4 weeks after operation in 28 living-donor liver transplant recipients treated with tacrolimus. The tacrolimus IC50 values against concanavalin A-induced PBMC blastogenesis in vitro were calculated. These recipients were classified into two groups with the mean tacrolimus IC50 (0.18 ng/ml) as the cutoff point, after which the clinical outcome between the patient groups was compared. The allograft rejection incidence in the low-sensitivity group (IC50 < 0.18 ng/ml; n = 16) was 6/12 (50.0%), which was significantly higher than the incidence of 2/16 (12.5%) in the highsensitivity group (IC50 > 0.18 ng/ml; n = 12) (p = 0.0297). In contrast, the infection incidence in the highsensitivity group was 6/16 (37.5%), which was significantly higher than that of the low-sensitivity group (1/12; 8.3%) (p = 0.0401). These data suggest that patients exhibiting a low PBMC sensitivity to tacrolimus have a risk of rejection, whereas highly sensitive patients have a risk of infection in living-donor liver transplantations under tacrolimus therapy.

Key words: Tacrolimus; Peripheral blood mononuclear cells; Allograft rejection; Infection; Living-donor liver transplantation

Address correspondence to Toshihiko Hirano, Ph.D., Department of Clinical Pharmacology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan. Tel: 81-426-76-5796; Fax: 81-426-76-5798; E-mail: hiranot@ps.toyaku.ac.jp




Cell Transplantation, Vol. 18, pp. 665-675, 2009
0963-6897/09 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2009 Cognizant Comm. Corp.
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Hepatocytes From Fibrotic Liver Possess High Growth Potential in Vivo

Manabu Nishie,1,2 Chise Tateno,1Rie Utoh,1 Toshihiko Kohashi,3 Norio Masumoto,1,3 Naoya Kobayashi,2 Toshiyuki Itamoto,3 Noriaki Tanaka,2 Toshimasa Asahara,3 and Katsutoshi Yoshizato1,4

1Yoshizato Project, CLUSTER, Hiroshima Prefectural Institute of Industrial Science and Technology, Hiroshima 739-0046, Japan
2Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama 700-8558, Japan
3Department of Surgery, Division of Frontier Medical Science, Program for Biomedical Research, and Hiroshima University 21st Century COE Program for Advanced Radiation Casualty Medicine, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8551, Japan
4Developmental Biology Laboratory and Hiroshima University 21st Century COE Program for Advanced Radiation Casualty Medicine, Department of Biological Science, Graduate School of Science, Hiroshima University, Hiroshima 739-8526, Japan

Hepatocyte transplantation is effective for treating liver failure, but healthy donors as a source of hepatocytes are quite limited. The livers of patients with hepatic fibrosis could be an alternative source; however, few reports have examined the nature of hepatocytes from fibrotic livers (f-hepatocytes). In this study, we compared the growth of f-hepatocytes and hepatocytes from normal livers (n-hepatocytes). Hepatocytes were isolated from normal and CCl4-treated wild-type Fischer rats that express dipeptidyl dipeptidase IV (DPPIV) gene (DPPIV+). The n- and f-hepatocytes proliferated in culture at similar rates. Both types of hepatocytes were transplanted into DPPIV- mutant Fischer rats that had been treated with retrorsine to injure the liver and were partially hepatectomized (PHx) before transplantation. Both n- and f-DPPIV+-hepatocytes proliferated and formed colonies. The colony sizes of f-hepatocytes 21 days posttransplantation were approximately three times those of n-hepatocytes. The hepatocytes were analyzed using a fluorescence activated cell sorter (FACS). The FACS profile differed between f- and n-hepatocytes: f-hepatocytes were less granular, less autofluorescent, and smaller than n-hepatocytes. These characteristics of f-hepatocytes resembled those reported for small-sized n-hepatocytes (SHs), which are highly proliferative and preferentially express a unique set of 10 SH genes. However, f-hepatocytes preferentially expressed only five of the SH genes. The expression profile of f-hepatocytes was rather similar to that of proliferating n-hepatocytes in the regenerating liver after PHx. The f-hepatocytes were morphologically normal and did not show any preneoplastic phenotype. These normal and proliferative natures of f-hepatocytes in vivo suggest the fibrotic liver as a source of hepatocytes for transplantation.

Key words: Hepatocyte transplantation; Liver regeneration; Hepatocyte proliferation; Hepatectomy; Retrorsine; Carbon tetrachloride

Address correspondence to Katsutoshi Yoshizato, Ph.D., PhoenixBio. Co. Ltd., 3-4-1 Kagamiyama, Higashishiroshima, Hiroshima, 739-0046, Japan. Tel: 81-82-431-0016; Fax: 81-82-431-0017; E-mail: katsutoshi.yoshizato@phoenixbio.co.jp




Cell Transplantation, Vol. 18, pp. 677-681, 2009
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E-ISSN 1555-3892
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Preconditioned Cell Array Optimized for a Three-Dimensional Culture of Hepatocytes

Yoshitaka Miyamoto,1 Takeshi Ikeya,2 and Shin Enosawa1

1Department of Innovative Surgery, National Research Institute for Child Health and Development, Tokyo, Japan
2Toyo Gosei Co., Ltd., Chiba, Japan

Three-dimensional culture procedures have attracted attention in various fields of cell biology. A newly developed cell array assisted in the formation of hepatocyte spheroids by two innovations: 1) micropatterning by a hydrophilic polymer, and 2) the use of bovine carotid artery-derived HH cells as feeder cells. The former contributes to the standardization of the spheroid size and the latter to the maintenance of the spheroids. We created a way to provide a ready-to-use cell array by cryopreservation of an HH feeder cell cultured array. After inoculation of HH cells on the cell array, the culture medium was replaced by freezing medium containing dimethyl sulfoxide. Thereafter, the array was frozen and stored in a -80°C deep freezer. At the start of the hepatocyte culture, the cryopreserved HH cell array was thawed by adding warmed (37°C) culture medium. The morphology and biological activities of the cryopreserved HH cells were intact, as confirmed by phase contrast microscopy and functional staining with calcein and formazan. The rat hepatocytes formed perfect spheroids on the cryopreserved HH cell array without any differences from those on the freshly prepared HH cell array. The CYP3A drug metabolism activities of the hepatocytes were well maintained on the cryopreserved and fresh cell arrays. The present protocol greatly shortened the time and labor required to prepare a cell array for culturing hepatocytes.

Key words: Cryopreservation; Cell array; Three-dimensional culture; Heptocytes

Address correspondence to Shin Enosawa, Department of Innovative Surgery, National Research Institute for Child Health and Development, 2-10-1 Ookura, Setagaya-ku, Tokyo 157-8535, Japan. Tel: 81-3-3416-0181; Fax: 81-3-3417-2864; E-mail: senosawa@nch.go.jp




Cell Transplantation, Vol. 18, pp. 683-688, 2009
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E-ISSN 1555-3892
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Neovascularization Induced Around an Artificial Device Implanted in the Abdomen by the Use of Gelatinized Fibroblast Growth Factor 2

Takeshi Yuasa,1 Jorge D. Rivas-Carrillo,1Nalú Navarro-Alvarez,1 Alejandro Soto-Gutierrez,1Yasuhiro Kubota,1 Yasuhiko Tabata,2Teru Okitsu,3 Hirofumi Noguchi,5,6Shinichi Matsumoto,5,6 Shuhei Nakaji,6Noriaki Tanaka,1 and Naoya Kobayashi1

1Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama 700-8558, Japan
2Department of Organ Reconstruction and Biomaterials, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan
3Department of Transplant Surgery, Kyoto University Hospital, Kyoto 606-8507, Japan
4Baylor All Saints Islet Cell Laboratory, Fort Worth, TX, USA
5Baylor Institution for Immunology Research, Islet Cell Transplantation Laboratory, Baylor Research Institute, Dallas, TX, USA
6Department of Biomedical Engineering, Okayama University of Science, Okayama 700-0005, Japan

The development of a bioartificial pancreas (BAP) with immunoisolating fashion has been gaining attention as a new method for treating diabetes. We have been proceeding with the development of a bag-type BAP that can be easily implanted and that allows for the optional injection or rejection of cells at any time. If fibrosis develops around a BAP device, then the permeability of substances transmitted through a semipermeable membrane will decrease, thereby reducing the reactivity with glucose, so it is necessary for the material of the device to have an excellent histocompatibility. Furthermore, in order to improve the efficacy of BAP treatment, it is important to maintain an environment of ample blood flow around the device. We have created a bag-type device for BAP that is 20 × 20 mm in size and comprises two layers of membranes. We have used an EVAL membrane for the outer membrane of the two layers. The EVAL membrane is a semipermeable membrane with good insulin permeability, which functions as an immunoisolation membrane. The inner membrane consists of PAU-coated HD-PE (nonwoven material processed with polyaminourethan) and it is designed to function as a scaffold for cells. We used Lewis rats to determine whether the effectiveness of fibroblast growth factor 2 (bFGF) can be improved by concomitantly using bFGF with a capacity for blood vessel regeneration as well as bFGF immersed in a sheet of gelatin. We placed the BAP in the abdominal cavity and covered it with the greater omentum. We were able to significantly increase the blood flow and the number of new blood vessels in the tissue surrounding the BAP device by using gelatinized bFGF. There were only a few instances of fibrosis as a biological reaction to the EVAL membrane, and the infiltration of inflammatory cells was mild. There were no adverse effects related to implantation of the device. We confirmed in this study that the use of an implantable BAP device and bFGF allowed for a better blood flow around the BAP device. There were only minor instances of fibrosis and inflammation reaction around the BAP, thus indicating the BAP that we are currently developing to have an excellent histocompatibility.

Key words: Bioartificial pancreas; Neovascularization; Fibroblast growth factor 2

Address correspondence to Naoya Kobayashi, M.D., Ph.D., Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama, 700-8558, Japan. Tel: (81) 86-235-7485; Fax: (81) 86-235-7485; E-mail: immortal@md.okayama-u.ac.jp