Cell Transplantation 21(2-3) Abstracts

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Cell Transplantation, Vol. 21, pp. 387–399, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X605286
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Review

Regenerative Cells for Transplantation in Hepatic Failure

Tetsuya Ishikawa, Agnieszka Banas, Takumi Teratani, Hideki Iwaguro, and Takahiro Ochiya

Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, Tokyo, Japan

Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells have an enormous potential; however, their potential clinical application is being arrested due to various limitations such as teratoma formation followed by tumorigenesis, emergent usage, and the quality control of cells, as well as safety issues regarding long-term culture are also delaying their clinical application. In addition, human ES cells have two crucial issues: immunogenicity and ethical issues associated with their clinical application. The efficient generation of human iPS cells requires gene transfer, yet the mechanism underlying pluripotent stem cell induction has not yet been fully elucidated. Otherwise, although human adult regenerative cells including mesenchymal stem cells have a limited capacity for differentiation, they are nevertheless promising candidates for tissue regeneration in a clinical setting. This review highlights the use of regenerative cells for transplantation in hepatic failure.

Key words: Adipose tissue; Regenerative cells; Mesenchymal stem cells; Hepatic failure

Received March 31, 2010; final acceptance July 18, 2011.
Address correspondence to Takahiro Ochiya, Ph.D., Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, 1-1, Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan. Tel: (+81)3-3542-2511(ex 4800); Fax: (+81)3-5565-0727; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 401–410, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X605303
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
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Direct Oxygen Supply With Polydimethylsiloxane (PDMS) Membranes Induces a Spontaneous Organization of Thick Heterogeneous Liver Tissues From Rat Fetal Liver Cells In Vitro

Morgan Hamon,* Sanshiro Hanada,† Teruo Fujii,† and Yasuyuki Sakai†

*Laboratory for Integrated Micro-Mechatronic Systems (LIMMS/CNRS-IIS), Institute of Industrial Science, University of Tokyo, Tokyo, Japan
†Institute of Industrial Science (IIS), University of Tokyo, Tokyo, Japan

Oxygen is a vital nutrient for growth and maturation of in vitro cells (e.g., adult hepatocytes). We previously demonstrated that direct oxygenation through a polydimethylsiloxane (PDMS) membrane increases the oxygen supply to cell cultures and improves hepatocyte functions. In this study, we removed limits on oxygen supply to fetal rat liver cells through the use of direct oxygenation through a PDMS membrane to investigate in vitro growth and maturation. We chose fetal liver cells because they are considered a feasible source of liver progenitor cells for regenerative medicine therapy due to their highly efficient maturation and proliferation. Cells from 17-day-old pregnant rats were cultured under 5% and 21% oxygen atmospheres. Some cells were first cultured under 5% oxygen, and then switched to a 21% oxygen atmosphere. When oxygen supply was enhanced by a PDMS membrane, the rat fetal liver cells organized into a complex tissue composed of an epithelium of hepatocytes above a mesenchyme-like tissue. The thickness of this supportive tissue was directly correlated to oxygen concentration and was thicker under 5% oxygen. When cultures were switched from 5% to 21% oxygen, lumen-containing structures were formed in the thick mesenchymal-like tissue and the albumin secretion rate increased. In addition, cells adapted their glycolytic activity to the oxygen concentrations. This system promoted the formation of a functional and organized thick tissue suitable for use in regenerative medicine.

Key words: Polydimethylsiloxane (PDMS); Direct oxygenation; Oxygen concentration; Fetal liver cells; Hepatocyte progenitors

Received March 31, 2010; final acceptance July 18, 2011.
Address correspondence to Morgan Hamon, Ph.D. Laboratory for Integrated Micro-Mechatronic Systems (LIMMS/CNRS-IIS), Institute of Industrial Science, University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan. Tel: +81-(0)3-5452-6349; Fax: +81-(0)3-5452-6348; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it or This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 411–420, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X605312
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Cell Shape Regulation Based on Hepatocyte Sheet Engineering Technologies

Soichi Takagi,1 Maki Ohno,1,2 Kazuo Ohashi, Rie Utoh, Kohei Tatsumi, and Teruo Okano

Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, Tokyo, Japan

The de novo engineering of a uniform hepatocyte sheet in vitro is considered as a novel approach for liver-directed therapeutics. Hepatocytes can be cultured on a temperature-responsive culture dishes coated with poly(N-isopropylacrylamide) (PIPAAm). Following multiple days of culturing, the hepatocytes can be easily harvested as a uniform sheet by decreasing temperature from 37°C to 20°C. By modifying the sheet harvesting protocol, we have noticed that two different forms of the hepatocyte sheets, “extended” and “shrinking,” were obtained. This study describes the methods for harvesting the two different forms of sheets, and their cellular structure and hepatocyte-specific functions. To obtain an “extended sheet” form, a cluster of hepatocytes covered with a support membrane was harvested by the temperature reduction. For the “shrinking sheet” form, the hepatocyte sheet was floated after reducing the culture temperature, and the floating process allowed the sheet to shrink spontaneously. Histological analysis revealed that the hepatocytes in the extended sheet form were predominantly flat, whereas the shrinking sheet contained cuboidal shaped hepatocytes. The preservation of hepatocyte-specific ultrastructures was confirmed in both types of sheets. To investigate hepatocyte-specific functionality, the harvested hepatocyte sheets were recultured on Matrigel-coated dishes. Assessment of protein production levels and chemical metabolizing activities showed the similar functionalities for each form. In contrast, the recalculation of these values per sheet versus per square centimeter of sheet surface demonstrated that the function of the shrinking sheet was significantly higher than that of the extended sheets. This study demonstrated that the hepatocyte sheets created on the PIPAAm dish could spontaneously shrink in size, but retain their hepatocyte functionality. This type of hepatocyte sheet could be utilized for the engineering of liver tissue in limited areas that are unable to give adequate transplant space.

Key words: Tissue engineering; Cell sheet; Hepatocytes; Culture system; Bioartificial liver

Received March 31, 2010; final acceptance July 18, 2011.
1These authors provided equal contribution to this work.
2Present affiliation: Faculty of Pharmaceutical Sciences, Teikyo Heisei University, Chiba, Japan.
Address correspondence to Kazuo Ohashi, M.D., Ph.D., Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo, 162-8666, Japan. Tel: +81-3-3353-8111, ext. 66220; Fax: +81-3-3359-6046; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 421–428, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X605321
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
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Evaluation of a Hybrid Artificial Liver Module Based on a Spheroid Culture System of Embryonic Stem Cell-Derived Hepatic Cells

Hiroshi Mizumoto,* Shunsuke Hayashi,* Kinya Matsumoto,* Kaoru Ikeda,* Tomoaki Kusumi,* Masakazu Inamori,* Kohji Nakazawa,† Hiroyuki Ijima,* Kazumori Funatsu,* and Toshihisa Kajiwara*

*Department of Chemical Engineering, Faculty of Engineering, Kyushu University, Fukuoka, Japan
†Department of Life and Environment Engineering, Faculty of Environmental Engineering, The University of Kitakyushu, Kitakyushu, Japan

Hybrid artificial liver (HAL) is an extracorporeal circulation system comprised of a bioreactor containing immobilized functional liver cells. It is expected to not only serve as a temporary liver function support system, but also to accelerate liver regeneration in recovery from hepatic failure. One of the most difficult problems in developing a hybrid artificial liver is obtaining an adequate cell source. In this study, we attempt to differentiate embryonic stem (ES) cells by hepatic lineage using a polyurethane foam (PUF)/spheroid culture in which the cultured cells spontaneously form spherical multicellular aggregates (spheroids) in the pores of the PUF. We also demonstrate the feasibility of the PUF-HAL system by comparing ES cells to primary hepatocytes in in vitro and ex vivo experiments. Mouse ES cells formed multicellular spheroids in the pores of PUF. ES cells expressed liver-specific functions (ammonia removal and albumin secretion) after treatment with the differentiation-promoting agent, sodium butyrate (SB). We designed a PUF-HAL module comprised of a cylindrical PUF block with many medium-flow capillaries for hepatic differentiation of ES cells. The PUF-HAL module cells expressed ammonia removal and albumin secretion functions after 2 weeks of SB culture. Because of high proliferative activity of ES cells and high cell density, the maximum expression level of albumin secretion function per unit volume of module was comparable to that seen in primary mouse hepatocyte culture. In the animal experiments with rats, the PUF-HAL differentiating ES cells appeared to partially contribute to recovery from liver failure. This outcome indicates that the PUF module containing differentiating ES cells may be a useful biocomponent of a hybrid artificial liver support system.

Key words: Hybrid artificial liver (HAL); Embryonic stem (ES) cell; Hepatic differentiation; Spheroid; Polyurethane foam (PUF); Three-dimensional culture

Received March 31, 2010; final acceptance July 18, 2011.
Address correspondence to Toshihisa Kajiwara, Ph.D., Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, 819-0395, Japan. Tel: +81-92-802-2746; Fax: +81-92-802-2796; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 429–436, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X605330
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Liver Tissue Engineering Utilizing Hepatocytes Propagated in Mouse Livers In Vivo

Kazuo Ohashi,* Kohei Tatsumi,* Chise Tateno,†‡ Miho Kataoka,‡ Rie Utoh,* Katsutoshi Yoshizato,† and Teruo Okano*

*Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, Tokyo, Japan
†PhoenixBio, Co., Ltd., Hiroshima, Japan
‡Hiroshima Prefectural Institute of Industrial Science and Technology, Hiroshima, Japan

Recent advances in tissue engineering technologies have highlighted the ability to create functional liver systems using isolated hepatocytes in vivo. Considering the serious shortage of donor livers that can be used for hepatocyte isolation, it has remained imperative to establish a hepatocyte propagation protocol to provide highly efficient cell recovery allowing for subsequent tissue engineering procedures. Donor primary hepatocytes were isolated from human α-1 antitrypsin (hA1AT) transgenic mice and were transplanted into the recipient liver of urokinase-type plasminogen activator-severe combined immunodeficiency (uPA/SCID) mice. Transplanted donor hepatocytes actively proliferated within the recipient liver of the uPA/SCID mice. At week 8 or later, full repopulation of the uPA/SCID livers with the transplanted hA1AT hepatocytes were confirmed by blood examination and histological assessment. Proliferated hA1AT hepatocytes were recovered from the recipient uPA/SCID mice, and we generated hepatocyte sheets using these recovered hepatocytes for subsequent transplantation into the subcutaneous space of mice. Stable persistency of the subcutaneously engineered liver tissues was confirmed for up to 90 days, which was the length of our present study. These new data demonstrate the feasibility in propagating murine hepatocytes prior to the development of hepatic cells and bioengineered liver systems. The ability to regenerate and expand hepatocytes has potential clinical value whereby procurement of small amounts of tissue could be expanded to sufficient quantities prior to their use in hepatocyte transplantation or other hepatocyte-based therapies.

Key words: Liver tissue engineering; Cell proliferation; Cell sheet; Tissue engineering; Hepatocyte transplantation; Regenerative medicine

Received March 31, 2010; final acceptance July 18, 2011.
Address correspondence to Kazuo Ohashi, M.D., Ph.D., Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo, 162-8666, Japan. Tel: +81-3-3353-8111, ext. 66220; Fax: +81-3-3359-6046; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 437–445, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X605349
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
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Human Hepatocyte Propagation System in the Mouse Livers: Functional Maintenance of the Production of Coagulation and Anticoagulation Factors

Kohei Tatsumi,*† Kazuo Ohashi,* Chise Tateno,‡ Katsutoshi Yoshizato,‡ Akira Yoshioka,† Midori Shima,† and Teruo Okano*

*Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, Tokyo, Japan
†Department of Pediatrics, Nara Medical University, Nara, Japan
‡PhoenixBio. Co. Ltd., Hiroshima, Japan

We previously reported that cell-based therapies using isolated hepatocytes including hepatocyte transplantation and liver tissue engineering approaches provide therapeutic benefits to hemophilia. For clinical application of these approaches, it is important to establish an active hepatocyte proliferation system that enables providing a sufficient number of hepatocytes. We also reported that human hepatocytes, which were transplanted into the liver of urokinase-type plasminogen activator transgenic severe combined immunodeficiency (uPA/SCID) mice, were able to proliferate while retaining their ability to produce coagulation factor IX. The objective of this study was to explore the functionalities of other coagulation and anticoagulation factors of the propagated human hepatocytes in uPA/SCID mice. Human hepatocytes were transplanted into the liver of uPA/SCID mice, and the propagation status of human hepatocytes in the mice was monitored by the increase in serum human albumin levels and immunohistochemical evaluation on the liver sections. Using uPA/SCID livers with various stages of human hepatocyte propagation, we analyzed the gene expression levels of coagulation factors (prothrombin, factor VII, factor X, and factor VIII) and anticoagulation factors (protein C and protein S) by real-time polymerase chain reaction (PCR) using human-specific primers. As a result, the total amount of raw messenger RNA expression levels increased in all genes analyzed according to the progress of hepatocyte propagation and proliferation. Except for factor VIII, the gene expression levels of the highly repopulated uPA/SCID mouse livers with human hepatocyte showed higher levels than those of normal human livers, indicating that propagated human hepatocytes in the uPA/SCID system possess full functions to produce most of the coagulation-related factors. The current work demonstrated that human hepatocytes can be propagated in experimental animals while maintaining normal gene expression levels of coagulation-related factors. It could be speculated that the propagated cells serve as a cell source for the treatment of various types of coagulation factor deficiencies.

Key words: Hepatocyte; Cell therapy; Hepatocyte transplantation; Coagulation factor; Urokinase-type plasminogen activator transgenic severe combined immunodeficiency (uPA/SCID) mouse; Anticoagulation factor

Received March 31, 2010; final acceptance July 18, 2011.
Address correspondence to Kazuo Ohashi, M.D., Ph.D., Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo, 162-8666, Japan. Tel: +81-3-3353-8111, ext. 66214; Fax: +81-3-3359-6046; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 447–452, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X605358
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Repopulation of Human Origin Hepatocyte Progenitor-Like Cell Line, THLE-5b, in the SCID Mouse Liver Under p21-Mediated Cell Growth-Arresting Conditions

Shin Enosawa,* Taisuke Yamazaki,† Hitoshi Kohsaka,‡ and Takayoshi Tokiwa†

*Division for Advanced Medical Services, National Center for Child Health and Development, Tokyo, Japan
†Department of Liver Cell Biology, Kohno Clinical Medicine Research Institute, Tokyo, Japan
‡Department of Medicine and Rheumatology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan

The in vivo repopulation of hepatocytes depends on donor cell growth potential and recipient conditioning. We herein demonstrate the successful cell transplantation of a human hepatocyte cell line, THLE-5b, into the SCID mouse liver by means of a rather mild conditioning using a 55% hepatectomy and p21 transfection. Adult human liver-derived cells, THLE-5b, are SV40 T antigen-immortalized epithelial cells. A phenotypic examination of THLE-5b showed they expressed hepatic stem cell markers such as EpCAM, OCT3/4, and Thy-1, thus indicating the immature nature of the cells. A three-dimensional aggregate culture of THLE-5b showed a higher expression level of liver-specific genes such as albumin, α1-antitrypsin, and CYP3A4, thus suggesting that THLE-5b possess the capability to differentiate into hepatocytes. In a cell transplantation experiment, the cell cycle regulator p21 was transfected with adenoviral vector into the SCID mouse liver. On the next day, 8 × 105 cells of GFP-transfected THLE-5b were injected intrasplenically, together with the intraperitoneal administration of anti-asialo GM1 antibodies. The following day, a partial hepatectomy was performed. The GFP-THLE-5b cells were observed to have migrated and become integrated into the liver parenchyma 14 days after transplantation. The present protocol is thus considered to be a novel experimental model to elucidate the mechanism of hepatocyte repopulation and to develop efficient stem cell therapy in the liver.

Key words: Human hepatocytes; THLE-5b; SCID mouse liver; Repopulation; p21

Received March 31, 2010; final acceptance July 18, 2011.
Address correspondence to Shin Enosawa, Division for Advanced Medical Services, National Center 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: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 453–464, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X605367
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
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Matrix Metalloproteinase-9 Contributes to the Mobilization of Bone Marrow Cells in the Injured Liver

Kengo Kawai,* Feng Xue,*† Terumi Takahara,* Hiroshi Kudo,* Yutaka Yata,*‡ Wei Zhang,* and Toshiro Sugiyama*

*Third Department of Internal Medicine, Faculty of Medicine, University of Toyama, Toyama, Japan
†Transplantation Center, Renji Hospital Affiliated to Jiao Tong University, Shanghai, P.R. China
‡Saiseikai-Maebashi Hospital, Gunma, Japan

Effective mobilization of hematopoietic stem cells (HSCs) in injured organs has not been established. Matrix metalloproteinase-9 (MMP-9) is known to release HSCs from bone marrow (BM) into the peripheral blood, but its role in the recruitment of HSCs to injured organs is unclear. In this study we tried to clarify the role of the host MMP-9 in trafficking of HSCs toward the injured liver, especially the relation of MMP-9 with the chemokine receptor 4 (CXCR4)–chemokine ligand 12 (CXCL12) axis, and to examine whether MMP-9 deficiency affects BM cell trafficking to the injured liver in mice. In vitro, we investigated the effect of MMP-9 on migration activity and CXCR4 expression on lineage-negative (Lin) BM cells. In vivo, we induced acute and chronic liver injury in MMP-9 knockout (KO) and control mice by inoculation of carbon tetrachloride, followed by transplantation of Lin BM cells obtained from enhanced green fluorescent protein (EGFP)-transgenic mice, and counted the BM cells mobilized in the injured liver. In a migration assay, active MMP-9, but not proMMP-9, increased the number of migrated Lin BM cells, which was inhibited by tissue inhibitor of metalloproteinase-1 or a MMP inhibitor. This chemoattractant function by MMP-9 was synergistic when cotreated with CXCL12. CXCR4 expression on Lin BM cells was dose- and time-dependently increased by active MMP-9. At the same time, treatment with MMP-9 enhanced CXCL12 expression, and CXCL12 reciprocally increased MMP-9 expression in BM cells. In in vivo studies, many EGFP+ cells were seen in control recipient mice. In contrast, few EGFP+  cells were observed in MMP-9 KO mice. BM cells tended to differentiate into desmin+ cells. In conclusion, MMP-9 contributes to the mobilization of BM cells in the injured liver by upregulating the expression of CXCR4 on Lin BM cells and attracting M cells along its gradient of CXCL12. Therefore, host MMP-9 plays an important role in BM cell migration in the injured liver.

Key words: Stromal-derived factor 1α (SDF-1α); Chemokine ligand 12 (CXCL12); Chemokine ligand receptor 4 (CXCR4); Lineage-negative cell; Bone marrow (BM) transplantation

Received March 31, 2010; final acceptance July 18. 2011.
Address correspondence to Terumi Takahara, M.D., Ph.D., Third Department of Internal Medicine, Faculty of Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan. Tel: (81)-76-434-7301; Fax: (81)-76-434-5027; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 465–471, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X605376
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
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Improvement of Porcine Islet Isolation by Inhibition of Trypsin Activity During Pancreas Preservation and Digestion Using α1-Antitrypsin

Masayuki Shimoda,* Hirofumi Noguchi,† Yasutaka Fujita,‡ Morihito Takita,† Tetsuya Ikemoto,† Daisuke Chujo,§ Bashoo Naziruddin,¶ Marlon F. Levy,†¶ Naoya Kobayashi,# Paul A. Grayburn,* and Shinichi Matsumoto†

*Baylor University Medical Center, Dallas, TX, USA
†Baylor Research Institute, Fort-Worth, TX, USA
‡Otsuka Pharmaceutical Factory Inc., Naruto, Japan
§Baylor Institute for Immunology Research, Dallas, TX, USA
¶Baylor Simmons Transplant Institute, Dallas, TX, USA
#Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan

Porcine islets are considered to be a promising resource for xenotransplantation. However, it is difficult to isolate porcine islets because of the marked fragility and rapid dissociation. Endogenous trypsin is one of the main factors to damage islets during the isolation procedure. Recent studies have suggested that trypsin inhibitors during the preservation of pancreas or the collagenase digestion can improve the result of islet isolation. In this study, we examined whether α1-antitrypsin (Aralast™), which inhibits several endogenous proteases and has immunomodulatory properties, can protect islets from the proteases and improve the results of porcine islet isolation. Twelve porcine pancreata were divided into three groups: without Aralast group (standard, n = 5), preserved with Aralast using the ductal injection (DI) method (DI, n = 3), and with Aralast using the DI method and in the collagenase solution (DI+C, n = 4). Efficacy of islet isolation was assessed by islet yields, purity, and viability. The trypsin activity of the preservation and the digestion solution during the isolation procedure was measured. During islet isolation, the trypsin activity in DI+C group was significantly inhibited compared to the standard group, whereas DI group showed less effect than DI+C group. The average of postpurification islet equivalents (IEQ) per pancreas weight in the DI+C group was significantly higher than the standard group (standard: 3516 ± 497 IEQ/g, DI: 4607 ± 1090 IEQ/g, DI+C: 7097 ± 995 IEQ/g; p = 0.017 between standard and DI+C). In the DI+C group, stimulation index was higher than in other groups, although there was no significant difference. The presence of Aralast in both DI solution and collagenase solution markedly inhibited trypsin activity during pancreas digestion procedure and improved the porcine islet isolation. Inhibition of trypsin activity by Aralast could improve porcine islet isolation.

Key words: Islet transplantation; Xenotransplantation; α1-Antitrypsin; Porcine islets; Trypsin inhibitor

Received March 31, 2010; final acceptance July 18, 2011.
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: This e-mail address is being protected from spambots. You need JavaScript enabled to view it or Bashoo Naziruddin, Ph.D., Baylor Simmons Transplant Institute, 3410 Worth Street Suite 950, Dallas, TX 75246, USA. Tel: 214-820-2662; Fax: 214-820-7142; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 473–482, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X605385
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
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A Model to Evaluate Toxic Factors Influencing Islets During Collagenase Digestion: The Role of Serine Protease Inhibitor in the Protection of Islets

Manabu Tsukada,* Takuro Saito,* Kazuya Ise,* Akira Kenjo,* Takashi Kimura,* Yoshihiro Satoh,* Takaharu Saito,* Takayuki Anazawa,* Ikuro Oshibe,* Shigeya Suzuki,† Yasuhiro Hashimoto,‡ and Mitsukazu Gotoh*

*Department of Surgery, Fukushima Medical University, Fukushima City, Fukushima, Japan
†Research and Development Division, Kikkoman Co. Ltd, Noda City, Chiba, Japan
‡Department of Biochemistry, Fukushima Medical University, Fukushima City, Fukushima, Japan

The recovery of all of the islets contained in a pancreas is the goal of islet isolation for transplantation. This study reveals an environment that injures the isolated islets during digestion and proposes a new model for optimal islet isolation. Islets were isolated from Wistar rat pancreases by stationary collagenase digestion while the digestion time was varied at 15, 30, 60, and 120 min. The digested pancreas and islets were analyzed histologically and adenosine nucleotides were measured. Overnight cultured islets (40 islets) were cocultured for 30 min with the supernatants obtained from pancreatic collagenase digestion at different digestion periods in order to assess the toxic environment. The peak yields of islets were obtained at 30 min of digestion. The histological study of digested pancreas showed that the exocrine cells lost their cellular integrity at 120 min of digestion, but the islet cells were left intact. Accordingly, the ATP levels of the pancreatic tissue decreased during the digestion period. The coculture experiment demonstrated that the islets cultured with the supernatants from the collagenase digestion showed digestion time-dependent disruption of the cellular integrity of islets in accordance with a rapid decrease of ATP levels in the islets. The addition of serine protease inhibitors into this coculture clearly showed protection of islets, which maintained high ATP levels in association with intact membrane integrity as assessed by AO/PI staining. Morphological deterioration of islets as well as a marked ATP decrease was evident in the entire digested pancreas as well as in islets cocultured in the supernatants from the collagenase digestion. Various factors toxic to the islets can therefore be analyzed in future experiments using this coculture model for obtaining a good yield of viable islets.

Key words: Islet isolation; Collagenase digestion; Adenosine triphosphate (ATP); Trypsin; Pefabloc; Ulinastatin

Received March 31, 2010; final acceptance July 18, 2011.
Address correspondence to Mitsukazu Gotoh, M.D., Ph.D., Department of Surgery 1, Fukushima Medical University, 1 Hikarigaoka, Fukushima 960-1295, Japan. Tel: 81-24-547-1254; Fax: 81-24-548-2735; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 483–491, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X605394
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
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Adenine Nucleotide Levels in a Closed Enzymatic Digestion System for Porcine Islet Isolation

Ikuro Oshibe,* Takuro Saito,* Yoshihiro Sato,* Takaharu Saito,* Manabu Tsukada,* Kazuya Ise,* Akira Kenjo,* Takashi Kimura,* Takayuki Anazawa,* Shigeya Suzuki,† Yasuhiro Hashimoto,‡ and Mitusukazu Gotoh*

*Department of Surgery I, Fukushima Medical University, Fukushima City, Japan
†Biochemical Department, Kikkoman Food Products Company, Noda-shi, Chiba, Japan
‡Department of Biochemistry, Fukushima Medical University, Fukushima City, Japan

Obtaining viable islets is a crucial step for successful islet transplantation. Adenosine triphosphate (ATP) is a marker of cell viability. However, little is known about any changes in the energy status of the tissues that are being digested during the digestion phase. We herein examined whether the ATP content in serially digested pancreatic tissue samples could be specific objective parameters that signal the optimal point to stop the digestion process. We obtained partial pancreata (body to tail) from 4- to 5-year-old pigs from a slaughterhouse. The tissue samples were preserved in M-Kyoto solution for less than 3 h. They were digested using an automated enzymatic and mechanical dissociation system at 37°C for 90 min following intraductal injection of Liberase HI. Samples were collected from the digestive circuit every 5 or 10 min to determine the ATP level, total adenine nucleotide (TAN) level, islet count (count/g), and yield of islet equivalent (IEQ) in the serial digestive fluids. The ATP and TAN levels, IEQ and islet count were increased and then decreased during digestion process. The profile of these parameters differed from case to case. However, when ATP changing ratio (respective value/precedent value) was compared with IEQ changing ratio, a greater than threefold increase in the ATP changing ratio followed by an increase in the islet count changing ratio within 5 min was consistently observed, indicating the optimal time to stop the digestion. The ATP levels of the handpicked islets in the digested samples were lower in the overdigested phase in comparison to those in the earlier digested phase. These results indicate that the ATP level in digested fluid could be an effective indicator to estimate the viability of cells as well as determine the optimal time to terminate the digestion process in order to obtain viable islets.

Key words: Islet isolation; Enzymatic digestion; Adenine nucleotide

Received March 31, 2010; final acceptance July 18, 2011.
Address correspondence to Mitsukazu Gotoh, M.D., Department of Surgery I, Fukushima Medical University, 1 Hikarigaoka Fukushima, 960-1295, Japan. Tel: +81-24-547-1252; Fax: +81-24-548-2735; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 493–500, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X605402
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Evaluation of Osmolality of Density Gradient for Human Islet Purification

Hirofumi Noguchi,*†‡ Bashoo Naziruddin,†§ Masayuki Shimoda,¶ Yasutaka Fujita,* Daisuke Chujo,* Morihito Takita,* Han Peng,† Koji Sugimoto,* Takeshi Itoh,* Naoya Kobayashi,‡ Nicholas Onaca,§ Marlon F. Levy,*§ and Shinichi Matsumoto*

*Baylor All Saints Medical Center, Baylor Research Institute, Fort Worth, TX, USA
†Institute of Biomedical Studies, Baylor University, Waco, TX, USA
‡Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
§Baylor Regional Transplant Institute, Dallas & Fort Worth, TX, USA
¶Division of Cardiology, Department of Internal Medicine, Baylor University Medical Center, Baylor Heart and Vascular Institute, Dallas, TX, USA

For pancreatic islet transplantation, the most common method of islet purification is density gradient centrifugation because of the differences in density between islets and acinar tissue. The density of islets/acinar tissue depends on several conditions, such as osmolality of purification solution. In this study, we evaluated the osmolality of iodixanol-controlled density gradients (400, 450, and 500 mOsm/kg) on the islet purification step. The density of the purification solutions was controlled by changing the volumetric ratio of iodixanol and the purification solutions (iodixanol-Kyoto solutions; IK solutions). The osmolality of density gradients was controlled by addition of 10× Hanks balanced salt solution (HBSS) solution. Density of both islets and acinar tissue increased relative to increase of the osmolality of purification solutions. There were no significant differences among the three groups on islet yield after density-adjusted purification and the rate of postpurification recovery. In vitro and in vivo assays suggest that the quality of islets was similar among the three groups. Our data suggest that efficacy of purification and quality of isolated islets is similar when the osmolality of purification solutions is between 400 and 500 mOsm/kg and density adjustment is applied. Since the density of islet and acinar tissue is changed according to osmolality, the density adjustment is important when using several osmolality solutions.

Key words: Islet transplantation; Islet isolation; Iodixanol-Kyoto (IK) solution; Purification; Osmolality

Received March 31, 2010; final acceptance July 18, 2011.
Address correspondence to Hirofumi Noguchi, M.D., Ph.D., Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. Tel: +81-86-235-7257; Fax: +81-86-221-8775; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 501–508, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X605411
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Islet Purification Method Using Large Bottles Effectively Achieves High Islet Yield From Pig Pancreas

Masayuki Shimoda,* Hirofumi Noguchi,† Yasutaka Fujita,‡ Morihito Takita,† Tetsuya Ikemoto,† Daisuke Chujo,§ Bashoo Naziruddin,¶ Marlon F. Levy,*¶ Naoya Kobayashi,# Paul A. Grayburn,* and Shinichi Matsumoto†

*Baylor University Medical Center, Dallas, TX, USA
†Baylor Research Institute, Fort-Worth, TX, USA
‡Otsuka Pharmaceutical Factory Inc, Naruto, Japan
§Baylor Institute for Immunology Research, Dallas, TX, USA
¶Baylor Simmons Transplant Institute, Dallas, TX, USA
#Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan

Porcine islets are a promising resource for xenotransplantation. However, low efficacy of islet isolation because of their marked fragility remains a problem. Recently we found that the standard purification method using COBE 2991 cell processor (COBE) with Ficoll density gradient solution damaged islets mechanically by high shearing force. In this study, we evaluated our new purification method using large plastic bottles for the efficacy of islet purification. Ten porcine pancreata were used. The average warm ischemic time was over 40 min; therefore, these pancreata were considered to be in a marginal condition. After digestion, the digested tissue was divided into three groups. Each group was purified using either top loading method with bottle (top group) or bottom loading method with bottle (bottom group) or standard COBE method (COBE group). Islet yield per pancreas weight (IEQ/g) and the rate of postpurification recovery in the top group were significantly higher than the COBE group (top: 8060 ± 1652 IEQ/g, bottom: 4572 ± 614 IE/g, COBE: 3900 ± 734 IE/g. p < 0.02 in top vs. COBE; top percentage of recovery: 99.3 ± 12.3%, bottom: 62.6 ± 8.8%, COBE: 49.5 ± 6.7%, p < 0.02 in top vs. bottom and COBE). The average sizes of purified islets in the top and bottom groups were significantly larger than COBE group (Average diameter top: 156 ± 8 μm, bottom: 147 ± 6 μm, COBE: 119 ± 6 μm, p < 0.01 in top vs. COBE and in bottom vs. COBE), which indicated that bottle method can reduce shear force during purification. Our new purification using top loading bottle method enabled us to obtain a high yield of porcine islets from marginal pancreata.

Key words: Islet transplantation; Xenotransplantation; Porcine islets; Islet purification

Received March 31, 2010; final acceptance July 18, 2011.

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: This e-mail address is being protected from spambots. You need JavaScript enabled to view it or Bashoo Naziruddin, Ph.D., Baylor Simmons Transplant Institute, 3410 Worth Street Suite 950, Dallas, TX 75246, USA. Tel: 214-820-2662; Fax: 214-820-7142; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 509–516, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X605420
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

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,*‡ and Shinichi Matsumoto*

*Baylor All Saints Medical Center, Baylor Research Institute, Fort Worth, TX, USA
†Institute of Biomedical Studies, Baylor University, Waco, TX, USA
‡Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
§Baylor Regional Transplant Institute, Dallas & Fort Worth, TX, USA
¶Division of Cardiology, Department of Internal Medicine, Baylor University Medical Center, Baylor Heart and Vascular Institute, Dallas, TX, USA
#Second Department of Surgery, Fujita Health University, Toyoake, Aichi, Japan
**Transplantation Unit, Kyoto University Hospital, Kyoto, Japan

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 ETKyoto 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.

Key words: Islet transplantation; Islet isolation; Modified ET-Kyoto (MK) solution; Trypsin inhibitor; Preservation solution

Received March 31, 2010; final acceptance July 18, 2011.
Address correspondence to Hirofumi Noguchi, M.D., Ph.D., Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. Tel: +81-86-235-7257; Fax: +81-86-221-8775; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 517–523, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X605439
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Fresh Islets Are More Effective for Islet Transplantation Than Cultured Islets

Hirofumi Noguchi,*†‡ Bashoo Naziruddin,†§ Andrew Jackson,†§ Masayuki Shimoda,¶ Tetsuya Ikemoto,* Yasutaka Fujita,* Daisuke Chujo,* Morihito Takita,* Han Peng,† Koji Sugimoto,* Takeshi Itoh,* Naoya Kobayashi,‡ Nicholas Onaca,§ Marlon F. Levy,*§ and Shinichi Matsumoto*

*Baylor All Saints Medical Center, Baylor Research Institute, Fort Worth, TX, USA
†Institute of Biomedical Studies, Baylor University, Waco, TX, USA
‡Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
§Baylor Regional Transplant Institute, Dallas & Fort Worth, TX, USA
¶Division of Cardiology, Department of Internal Medicine, Baylor University Medical Center, Baylor Heart and Vascular Institute, Dallas, TX, USA

For clinical islet transplantation, isolated islets deteriorate rapidly in culture, although culturing islets prior to transplantation provides flexibility for evaluation of isolated islets and pretreatment of patients. In the present study, we compared human fresh islets to cultured islets with in vitro and in vivo assays. After culture for 24, 48, and 72 h, islet yield significantly decreased from 2,000 to 1,738 ± 26 (13% loss), 1,525 ± 30 (24% loss), or 1,298 ± 18 IEQ (35% loss), respectively. The ATP contents were significantly higher in the 6-h cultured group (near fresh group) than in 48-h culture groups. The stimulation index was relatively higher in the 6-h cultured group than in 48-h cultured group. Human islets with or without culture were transplanted into diabetic nude mice. The attainability of posttransplantation normoglycemia was significantly higher in fresh group than in the culture groups. Intraperitoneal glucose tolerance testing (IPGTT) showed that the blood glucose levels of mice transplanted with fresh islets were significantly lower than with cultured islets at 30, 60, 90, and 120 min after injection. These data suggest that human islet transplantation without culture could avoid the deterioration of islets during culture and improve the outcome of islet transplantation. Based on these data, we have transplanted fresh islets without culture for our current clinical islet transplantation protocol.

Key words: Islet transplantation; Culture; Fresh islets; Cultured islets

Received March 31, 2010; final acceptance July 18, 2011.
Address correspondence to Hirofumi Noguchi, M.D., Ph.D., Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. Tel: +81-86-235-7257; Fax: +81-86-221-8775; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 525–533, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X605448
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Immunoisolation Effect of Polyvinyl Alcohol (PVA) Macroencapsulated Islets in Type 1 Diabetes Therapy

Zhi Qi,*† Chizuru Yamamoto,† Naomi Imori,† Ayano Kinukawa,† Kai-chiang Yang,† Goichi Yanai,† Etsuko Ikenoue,† Yanna Shen,‡ Yasumasa Shirouzu,† Akihito Hiura,† Kazutomo Inoue,§ and Shoichiro Sumi†

*Department of Histology and Embryology, Nankai University School of Medicine, Tianjin, China
†Department of Organ Reconstruction, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan
‡Department of Microbiology, Kyoto University Graduate School of Medicine, Kyoto, Japan
§Inoue Clinic Diabetes Center, Kyoto, Japan

Islet transplantation has shown great success in the treatment of type 1 diabetes since the Edmonton protocol was established. However, it still has two major problems to overcome: the lack of organ donors and the side effects of immunosuppression. Encapsulated islets have emerged as a potential option for islet transplantation because it can, at least partly, overcome these two problems. Wistar rat islets suspended in 3% polyvinyl alcohol (PVA) hydrogel were frozen-thawed to make macroencapsulated islets (MEIs). The recovery rate, insulin content, and morphological change in culture medium with/without fresh human plasma (FHP) were measured in MEIs and free islets in vitro. In vivo, MEIs of either Wistar or Lewis rats were transplanted into the peritoneal cavity of streptozotocin (STZ)-induced diabetic Lewis rats and nonfasting blood glucose (NFBG), body weight, and histological evaluations were processed. FHP destroyed rat free islets but did not affect the islet morphology, islet recovery rate, or insulin content of rat MEIs. The transplantation of MEIs decreased the NFBG level and prevented body weight loss without a significant difference between the donor strains. Insulin-positive islets were observed in PVA MEIs 24 weeks after allotransplantation. These results suggest that PVA MEIs may be used as a cure for type 1 diabetes.

Key words: Polyvinyl alcohol (PVA); Macroencapsulated islets; Type 1 diabetes; Islet transplantation

Received March 31, 2010; final acceptance July 18, 2011.
Address correspondence to Shoichiro Sumi, M.D., Ph.D., Department of Organ Reconstruction, Institute for Frontier Medical Sciences, Kyoto University, 53 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. Tel: 81-75-751-4848; Fax: 81-75-751-4145; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 535–545, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X605457
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Mizoribine as Sole Immunosuppressive Agent in Islet Xenotransplantation Models: A Candidate Immunosuppressant Causing no Adverse Effects on Islets

Michitoshi Yamashita, Takuro Saito, Kazuya Ise, Show Ishii, Yoshihiro Satoh, Takaharu Saito, Ikuro Oshibe, Hirofumi Shimizu, Akira Kenjo, Takashi Kimura, and Mitsukazu Gotoh

Department of Surgery I, School of Medicine, Fukushima Medical University, Fukushima, Japan

Mizoribine (MZ) inhibits the differentiation and proliferation of helper T and B cells after antigen recognition by suppressing the purine biosynthesis pathway and nucleic acid synthesis. MZ has been used in kidney transplantation, but distinct data are unavailable for islet transplantation. The present study investigated the efficacy of MZ for islet xenotransplantation. Immunosuppressive effects of MZ were determined by mixed lymphocyte reaction (MLR) assay in vitro. Toxicities for Wistar rat islets were determined by adenosine triphosphate (ATP) contents of islets during 3-day culture and stimulation index in response to glucose after culture. Immunosuppressive effects in vivo were tested in a Wistar-to-B6 islet xenotransplantation model. MZ was administered continuously for 28 days subcutaneously or intramuscularly. MZ inhibited MLR response by approximately 50% at 0.1 μg/ml. ATP contents decreased with MZ >100 μg/ml, while stimulation index was maintained. Continuous infusion of MZ at 10 mg/kg maintained blood concentrations at 0.13–0.19 μg/ml, while intramuscular injection of MZ at 100 mg/kg/day (peak 520 μg/ml at 1 h postinjection) resulted in below measurable levels (<0.03 μg/ml) within 24 h. Graft survival was significantly prolonged following continuous infusion of 10 mg/kg/day compared to controls (31.0 ± 9.5 vs. 13.2 ± 5.2 days; p = 0.002). Furthermore, animals with intramuscular injection at doses of 3.2, 10, or 100 mg/kg/day showed significantly longer graft survival (20.0 ± 7.5, 22.0 ± 7.31, and 24.5 ± 8.1 days, respectively; p < 0.05 each). Histological examination showed significant suppression of lymphocyte infiltration by MZ administration. MZ showed immunosuppressive effects in an experimental islet xenotransplantation model without adverse effects on endocrine function of islet grafts.

Key words: Islet xenotransplantation; Mizoribine (MZ); Immunosuppression

Received March 31, 2010; final acceptance July 18, 2011.
Address correspondence to Mitsukazu Gotoh, M.D., First Department of Surgery, Fukushima Medical University, 1 Hikarigaoka, Fukushima 960-1295, Japan. Tel: +81-24-547-1111, ext. 2330; Fax: +81-24-548-2735; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 547–551, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X605466
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

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,§ and Marlon F. Levy§

*Baylor Research Institute Fort Worth Campus, Fort Worth, TX, USA
†Baylor University Medical Center, Dallas, TX, USA
‡Baylor Institute for Immunology Research, Dallas, TX, USA
§Baylor Simmons Transplant Institute, Dallas, TX, USA

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.

Key words: Islet transplantation; Side effects; Infusion procedure; Immunosuppression

Received March 31, 2010; final acceptance July 18, 2011.
Address correspondence to Shinichi Matsumoto, M.D., Ph.D., Baylor Research Institute Fort Worth Campus, Islet Cell Laboratory, 1400 8th Avenue, Fort Worth, TX 76104, USA. Tel: 817-922-2570; Fax: 817-922-4645; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it or Morihito Takita, M.D., Baylor Research Institute Fort Worth Campus, Islet Cell Laboratory, 1400 8th Avenue, Fort Worth, TX 76104, USA. Tel: 817-922-2573; Fax: 817-922-4655; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 453–458, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X605475
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Improved Pancreatic Islet Isolation Outcome in Autologous Transplantation for Chronic Pancreatitis

Bashoo Naziruddin,* Shinichi Matsumoto,† Hirofumi Noguchi,† Morihito Takita,† Masayuki Shimoda,‡ Yasutaka Fujita,† Daisuke Chujo,§ Chad Tate,‡ Nicholas Onaca,* Jeffrey Lamont,‡ Naoya Kobayashi,¶ and Marlon F. Levy*

*Baylor Simmons Transplant Institute, Dallas, TX, USA
†Baylor Research Institute, Fort Worth Campus, Fort Worth, TX, USA
‡Baylor University Medical Center, Dallas, TX, USA
§Baylor Institute for Immunology Research, Dallas, TX, USA
¶Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan

Total or partial pancreatectomy followed by autologous islet transplantation is a therapeutic option for the treatment of refractory chronic pancreatitis (CP). Maximization of islet yields from fibrotic and inflamed organs is crucial for prevention of posttransplant diabetes. We adapted technical advancements developed for islet allotransplantation toward islet autotransplantation. Eight patients (two men, six women; ages 24–58 years) underwent total (n = 7) or partial (n = 1) pancreatectomy for the treatment of CP refractory to maximal medical management. Pancreata were preserved in UW solution (UW group) in initial three cases and the last five pancreata were preserved with pancreatic ductal injection followed by ET-Kyoto/oxygenated PFC solutions (DI+TLM group). Islets were isolated by modified Ricordi method and were purified only in one case. All islet infusions were performed under general anesthesia via direct vein injection into the portal venous system with pressure monitoring. Total islet yields (129,314 ± 51,627 vs. 572,841 ± 116,934 IEQ, p < 0.04), islet yield/pancreas weight (1,233 ± 359 vs. 6,848 ± 847 IEQ/g, p < 0.003), and islet yield/patient body weight (1,951 ± 762 vs. 7,305 ± 1,531 IEQ/kg, p < 0.05) were significantly higher in the DI+TLM group when compared to the UW group. Pellet size was also higher (5.3 ± 0.3 vs. 13.5 ± 3.4 ml) in the DI+TLM group, suggesting that this method of preservation effectively protected pancreatic tissue against autolysis. First month posttransplant basal C-peptide and the secretory unit of islet transplant objects (SUITO) index were also higher in the DI+TLM group when compared to the UW group (2.0 ± 0.3 vs. 1.4 ± 0.4 ng/ml and 42.6 ± 12.7 vs. 14.6 ± 5.6, respectively). There were no technical complications related to the infusion. Our results suggest that higher islet yields can be achieved even from chronically inflamed and fibrotic organs using DI+TLM. The techniques applied for islet isolations from normal pancreata are showing promise for fibrotic pancreata from CP patients.

Key words: Islet autotransplantation; Chronic pancreatitis; Two-layer method; Ductal injection; ET-Kyoto solution

Received March 31, 2010; final acceptance July 18, 2011.
Address correspondence to Shinichi Matsumoto, M.D., Ph.D., Baylor Research Institute, Fort Worth Campus, 1400 8th Avenue, Fort Worth, TX 76104, USA. Tel: +1-817-922-2570; Fax: +1-817-922-4645; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it or Bashoo Naziruddin, Ph.D., Baylor Simmons Transplant Institute, 3410 Worth Street Suite 950, Dallas, TX 75246, USA. Tel: 214-820-2662; Fax: 214-820-7142; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 559–563, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X605484
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Results of Islet Isolation and Their Relationship to the Clinical Outcome of Kidney Transplantation in Cases Where Both Grafts Are Harvested From the Same Non-Heart-Beating Donor

Michihiro Maruyama, Takashi Kenmochi, Kenichi Saigo, Akutsu Naotake, Chikara Iwashita, Kazunori Otsuki, and Taihei Ito

Department of Surgery, Chiba-East National Hospital, Chiba City, Japan

Grafts from non-heart-beating donors (NHBDs) are used because of the limited availability of heart-beating brain-dead donors. These grafts sustain ischemic damage, and the severity of this damage varies among different areas of an organ. This study determined whether the results of islet isolation were correlated with the clinical outcomes of kidney transplantations in cases where both grafts were harvested from the same NHBD. Islets we isolated from the pancreata of 23 NHBDs between February 2004 and March 2007. Forty-six kidneys were also harvested from these NHBDs. The recipients of kidney transplants were divided into the successful isolation (n = 14) and failed isolation (n = 32) groups depending on the results of islet isolation. The clinical outcomes of kidney transplantation were compared between the recipients in these two groups. The immediate graft function rate and the 1-year graft survival rate after kidney transplantation in both groups were similar. Hemodialysis after transplantation was required for 6.0 days (SD, 5.2 days) in the successful isolation group and for 12.7 days (13.1 days) in the failed isolation group (p < 0.05). The serum creatinine concentrations at 1, 3, 6, and 12 months after transplantation were elevated in the failed isolation group (p < 0.05). The islet yield was inversely correlated with the requirement of hemodialysis (days) and the serum creatinine level at 1 month after transplantation. However, hemodialysis was required for only 7 days in the recipients of six kidneys that were obtained from NHBDs from whom <40,000 IEQ were obtained (extreme failure of islet isolation). The results of islet isolation were found to correlate with the kidney function after transplantation when both grafts are harvested from the same NHBD. However, the marginal conditions of NHBDs affect the results of islet isolation more than they do the posttransplantation kidney function.

Key words: Islet isolation; Kidney transplantation; Non-heart-beating donor; Correlation

Received March 31, 2010; final acceptance July 18, 2011.
Address correspondence to Michihiro Maruyama, Department of Surgery, Chiba-East National Hospital, 673 Nitona, Chuo ku, Chiba City 2608712, Japan. Tel: +81 43 261 5171; Fax: +81 43 264 3269; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 565–570, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X605493
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Comparative Study of the Cellular Pharmacodynamics of Tacrolimus in Renal Transplant Recipients Treated With and Without Basiliximab

Kentaro Sugiyama,* Kazuya Isogai,† Satoshi Horisawa,† Akira Toyama,† Hiroshi Satoh,† Kazuhide Saito,‡ Yuki Nakagawa,‡ Masayuki Tasaki,‡ Kota Takahashi,‡ and Toshihiko Hirano*

*Department of Clinical Pharmacology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Tokyo, Japan
†Division of Pharmacy, Niigata University Medical and Dental Hospital, Niigata, Japan
‡Division of Urology, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan

Basiliximab is a recently developed immunosuppressive agent for the prevention of acute allograft rejection in renal transplant recipients. The combination use of basiliximab and a calcineurin inhibitor was suggested to be more effective in comparison to immunosuppressive therapy using calcineurin inhibitor without basiliximab. Cyclosporine has been generally administered with basiliximab for renal transplant recipients. However, in cases of tacrolimus-based immunosuppressive regimen, the clinical efficacy and safety of combined use of tacrolimus and basiliximab remains to be elucidated. This study evaluated the tacrolimus pharmacological efficacy using a lymphocyte immunosuppressant sensitivity test (LIST) with MTT assay procedures in 16 cases of renal transplant recipients treated by tacrolimus without basiliximab and in 13 cases treated by tacrolimus in combination with basiliximab. The rate of acute rejection episodes in the recipients treated with tacrolimus plus basiliximab was 1/13 (7.7%), whereas the rate in the recipients treated with tacrolimus without basiliximab was 6/16 (37.5%). The recipients were divided into two groups according to their peripheral blood mononuclear cell (PBMC) sensitivity to tacrolimus [i.e., including a tacrolimus high sensitivity group (IC50 <1.0 ng/ml) and a low sensitivity group (IC50 >1.0 ng/ml). In the recipients treated with tacrolimus without basiliximab, the rate of acute rejection episodes in the tacrolimus high sensitivity group was 1/10 (10.0%), which was significantly lower than the rate in the low sensitivity group of 5/6 (83.3%; p = 0.008). The incidence of cytomegalovirus infection was not significantly different between the tacrolimus high and the low sensitivity groups of the recipients treated with tacrolimus with and without basiliximab. Therefore, in the case of selected tacrolimus-based immunosuppressive therapy for renal transplant recipients, the tacrolimus pharmacological efficacy should be evaluated using LIST at a time just before the transplant procedure in order to accurately predict allograft rejection. The data also suggested that low tacrolimus sensitivity recipients should be treated with tacrolimus-based immunosuppressive therapy in combination with basiliximab.

Key words: Tacrolimus; Basiliximab; Renal transplantation; Lymphocyte immunosuppressant sensitivity test (LIST); Peripheral blood mononuclear cells (PBMCs)

Received March 31, 2010; final acceptance July 18, 2011.
Address correspondence to Kentaro Sugiyama, Ph.D., Department of Clinical Pharmacology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji City, Tokyo 192-0392, Japan. Tel: +81-42-676-5111; Fax: +81-42-676-5798; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 571–580, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X605501
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Carbamylated Erythropoietin Ameliorates Cyclosporine Nephropathy Without Stimulating Erythropoiesis

Toyofumi Abe,* Yoshitaka Isaka,† Ryoichi Imamura,* Yoichi Kakuta,* Masayoshi Okumi,* Koji Yazawa,* Naotsugu Ichimaru,* Hidetoshi Tsuda,† Norio Nonomura,* Shiro Takahara,† and Akihiko Okuyama*

*Department of Specific Organ Regulation (Urology), Osaka University Graduate School of Medicine, Osaka, Japan
†Department of Advanced Technology for Transplantation, Osaka University Graduate School of Medicine, Osaka, Japan

The introduction of cyclosporine (CsA) has improved graft survival, but it causes nephropathy, which limits its clinical utility. Recently, we reported that carbamylated erythropoietin (CEPO) protected kidneys from ischemia reperfusion injury as well as EPO. To investigate the clinical applications of CEPO, we next evaluated the long-term therapeutic effect of CEPO using a CsA-induced nephropathy model. CsA caused renal dysfunction, while EPO/CEPO administration significantly improved renal function. EPO treatment significantly increased Hb concentration, while CEPO treatment neither enhanced nor reduced Hb concentration. CsA treatment induced tubular apoptosis, while EPO/CEPO administration inhibited it and increased PI3 kinase activation and Akt phosphorylation. In parallel, morphological assessment revealed that EPO/CEPO significantly reduced CsA-induced interstitial fibrosis and inhibited interstitial macrophage infiltration. In addition, real-time RT-PCR demonstrated that cortical mRNA levels of TGF-β1 and type I collagen were suppressed in the EPO/CEPO group. These results suggest a new therapeutic approach using CEPO to protect kidneys from CsA-induced nephropathy.

Key words: Cyclosporine nephropathy; Carbamylated erythropoietin (CEPO)

Received March 31, 2010; final acceptance July 18, 2011.
Address correspondence to Yoshitaka Isaka, M.D., Ph.D., Associate Professor, Department of Advanced Technology for Transplantation, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. Tel: +81-6-6879-3746; Fax: +81-6-6879-3749; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 581–590, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X605510
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Bone Marrow-Derived Conventional, But Not Cloned, Mesenchymal Stem Cells Suppress Lymphocyte Proliferation and Prevent Graft-Versus-Host Disease in Rats

Yusuke Kitazawa,*† Xiao-Kang Li,* Lin Xie,*† Ping Zhu,* Hiromitsu Kimura,* and Shiro Takahara†

*Laboratory of Transplantation Immunology, National Research Institute for Child Health and Development, Tokyo, Japan
†Department of Advanced Technology for Transplantation, Osaka University Graduate School of Medicine, Osaka, Japan

Bone marrow-derived mesenchymal stem cells (MSCs) could exert a potent immunosuppressive effect, and therefore may have a therapeutic potential in T-cell-dependent pathologies. In the present study, we aimed to determine whether MSCs could be used to control graft-versus-host disease (GvHD), a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). MSCs were isolated from Lewis rat bone morrow and then cultured in 10% FBS DMEM at 37°C for 4 weeks. The enriched conventional MSCs and macrophages were purified by auto-MACS. Cloned MSCs were obtained by cloning using the limiting dilution method and expanded up to more than 6 months. The identity of MSCs was confirmed by their typical spindle-shaped morphology and immunophenotypic criteria, based on the absence of expression of CD45 and CD11b/c molecules. Both types of MSCs were also tested for their ability to differentiate into adipocytes. We showed that MSCs, like macrophages, exhibit immunomodulatory properties capable of inhibiting T-cell proliferation stimulated by alloantigens, anti-CD3e/CD28 mAbs, and ConA in a dose-dependent manner in vitro. After performing adoptive transfer, MSCs suppressed systemic Lewis to (Lewis × DA)F1 rat GvHD. In contrast to the immunosuppressive activities of conventional MSCs, the cloned MSCs enhanced T-cell proliferation in vitro and yielded no clinical benefit in regard to the incidence or severity of GvHD. Therefore, these rat models have shown intriguing differences in the suppression effects of lymphocyte proliferation and GvHD prevention between short-term cultured conventional MSCs and cloned MSCs.

Key words: Allorejection; Cell-based therapy; Graft-versus-host disease; Hemopoietic stem cell transplantation; Mesenchymal stem cells (MSCs)

Received March 31, 2010; final acceptance July 18, 2011.
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: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 591–600, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X605529
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Transplantation of Osteogenically Differentiated Mouse iPS Cells for Bone Repair

Takahiro Hayashi,* Haruo Misawa,† Hiroyuki Nakahara,* Hirofumi Noguchi,‡ Aki Yoshida,* Naoya Kobayashi,§ Masato Tanaka,* and Toshifumi Ozaki*

*Department of Orthopeadic Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
†Department of Intelligent Orthopaedic System, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
‡Department of Gastroenterological Surgery, Transplant and Surgical Oncology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
§Department of Surgery, Okayama Saidaiji Hospital, Saidaiji Nakano Honmachi, Japan

Induced pluripotent stem (iPS) cells are a type of undifferentiated cell that can be obtained from differentiated cells and have the pluripotent potential to differentiate into the musculoskeletal system, the myocardium, vascular endothelial cells, neurons, and hepatocytes. We therefore cultured mouse iPS cells in a DMEM containing 15% FBS, 10−7 M dexamethasone, 10 mM β-glycerophosphate, and 50 μg/ml ascorbic acid for 3 weeks, in order to induce bone differentiation, and studied the expression of the bone differentiation markers Runx2 and osteocalcin using RT-PCR in a time-dependent manner. Osteocalcin, a bone differentiation marker in bone formation, exhibited the highest expression in the third week. In addition, the deposition of calcium nodules was observed using Alizarin red S staining. iPS cells cultured for bone differentiation were transplanted into severe combined immunodeficiency (SCID) mice, and the osteogenic potential exhibited after 4 weeks was studied. When bone differentiation-induced iPS cells were transplanted into SCID mice, bone formation was confirmed in soft X-ray images and tissue specimens. However, teratoma formation was confirmed in 20% of the transplanted models. When mouse iPS cells were treated with irradiation of 2 Gray (Gy) prior to transplantation, teratoma formation was inhibited. When mouse iPS cells treated in a likewise manner were xenotransplanted into rats, bone formation was confirmed but teratoma formation was not observed. It is believed that irradiation before transplantation is an effective way to inhibit teratoma formation.

Key words: Induced pulripotent stem (iPS) cells; Bone differentiation; Osteogenic potential

Received March 31, 2010; final acceptance July 18, 2011.
Address correspondence to Toshifumi Ozaki, M.D., Ph.D., Department of Orthopaedic Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. Tel/Fax: +81-86-235-7272; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it or Naoya Kobayashi, M.D., Ph.D., Department of Surgery, Okayama Saidaiji Hospital, 8-41 Saidaiji Nakano Honmachi 704-8192, Japan. Tel: +81-86-943-2211; Fax: +81-86-943-2212; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 601–607, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X605538
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Tissue Augmentation by White Blood Cell-Containing Platelet-Rich Plasma

Takeshi Kawazoe* and Hak Hee Kim†

*Department of Plastic Reconstructive and Aesthetic Surgery, Kijunkai, Yoshikawa Hospital, Kyoto, Japan
†Institute for Frontier Medical Science, Kyoto University, Kyoto, Japan

Platelet-rich plasma (PRP) is a matrix of fibrin and platelets that releases cytokines that are important in wound healing. PRP is produced from the patient’s blood and therefore has less risk of allergic reaction and infection. We have obtained PRP with an enhanced white blood cell component (W-PRP) by optimizing the centrifugal separation of PRP from plasma. Here we show that injection of W-PRP into the auricle of nude mice gave greater tissue augmentation compared to PRP. Further augmentation occurred when bFGF was added to W-PRP, and there was a significant increase in the number of α-smooth muscle actin-positive cells in mice treated with W-PRP+bFGF. Our results suggest that W-PRP may have value in cosmetic surgery aimed at rejuvenation of wrinkled and sagging skin. W-PRP injection constitutes a new concept in cell transplantation, in which cells required for tissue regeneration are induced by cytokines released from the transplanted cells.

Key words: Tissue augmentation; White blood cell; Platelet-rich plasma (PRP)

Received March 31, 2010; final acceptance July 18, 2011.
Address correspondence to Takeshi Kawazoe, M.D., Ph.D., Director, Department of Plastic Reconstructive and Aesthetic Surgery, Kijunkai, Yoshikawa Hospital, 1 Sanno-cho, Shogoin, Sakyo-ku, Kyoto 606-8392, Japan. Tel: (+81) 75-761-0316; Fax: (+81) 75-771-0528; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 609–615, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X605547
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

Preservation by Desiccation of Isolated Rat Hearts for 48 Hours Using Carbon Monoxide (PCO = 4,000 hPa) and Oxygen (PO2 = 3,000 hPa)

Naoyuki Hatayama,* Munekazu Naito,* Shuichi Hirai,* Yu Yoshida,† Tomohiro Kojima,† Kunihiro Seki,‡ Xiao-Kang Li,§ and Masahiro Itoh*

*Department of Anatomy, Tokyo Medical University, Tokyo, Japan
†Resonance Club Co., Tokyo, Japan
‡Faculty of Science, Kanagawa University, Kanagawa, Japan
§National Research Institute for Child Health and Development, Tokyo, Japan

It is currently said that CO has anti-inflammatory and antiapoptosis effects and it has attracted attention as a medical gas. We used CO for rat hearts and conducted a preservation experiment. We isolated rat hearts, placed them into a specially made chamber, filled the chamber with a gas mixture of PCO (4,000 hPa) and PO2 (3,000 hPa), and preserved the hearts in a refrigerator at 4°C for 48 h. We then performed a heterotrophic transplantation on the neck of each recipient rat and resuscitated the preserved hearts. We herein report our findings.

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

Received March 31, 2010; final acceptance July 18, 2011.
Address correspondence to Naoyuki Hatayama, Department of Anatomy, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan. E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it or This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Transplantation, Vol. 21, pp. 617–622, 2012
0963-6897/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/096368911X605556
E-ISSN 1555-3892
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

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,§ and Shuji Hayashi*

*Department of Advanced Medicine in Biotechnology and Robotics, Nagoya University Graduate School of Medicine, Nagoya, Japan
†Baylor Institute for Immunology Research, Baylor Research Institute, Dallas, TX, USA
‡Technical Center, Seiren Co. Ltd., Fukui, Japan
§Department of Biomedical Sciences, Chubu University College of Life and Health Sciences, Aichi, Japan

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 O staining/ Von Kossa’s method. The huma 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.

Key words: Adipose tissue-derived stem cells (ASCs); Refreezing; Cryopreservation; Sericin; Adipogenic/osteogenic potential

Received March 31, 2010; final acceptance July 18, 2011.
Address correspondence to Yoshitaka Miyamoto, Department of Advanced Medicine in Biotechnology and Robotics, Nagoya University Graduate School of Medicine, Higashi-ku, Nagoya 461-0047, Japan. Tel: 81-52-719-1873; Fax: 81-52-719-1977; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it or This e-mail address is being protected from spambots. You need JavaScript enabled to view it