Cell Medicine 3(1-3) Abstracts

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Cell Medicine, Vol. 3, pp. 3–11, 2012
2155-1790/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/215517912X639342
Copyright © 2012 Cognizant Comm. Corp.
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Review

In Vivo Bioimaging Rats for Translational Research in Cell and Tissue Transplantation

Takumi Teratani and Eiji Kobayashi

Division of Development of Advanced Therapy, Center for Development of Advanced Medical Technology, Jichi Medical University, Shimotsuke-shi, Tochigi, Japan

The rat is an excellent cell transplantation model. In accordance with the innovative development of in vivo bioimaging technology, over the last decade we have been developing an engineered rat system based on transgenic technology and have been demonstrating the usefulness of the system with genetically encoded imaging probes such as fluorescent and luminescent proteins. In cooperation with the Japan Society for Organ Preservation and Medical Biology (President: Professor T. Asano), we have also been using luciferase-Tg rats for research into organ preservation and cell transplantation. In this minireview, we introduce the results obtained recently by using these powerful experimental tools during international collaboration in cell transplantation research.

Key words: Cell transplantation; Living image; Translational research; Transgenic rat

Received January 31, 2011; final acceptance April 1, 2011. Online prepub date: May 15, 2012.
Address correspondence to Eiji Kobayashi, M.D., Ph.D., Division of Development Advanced Therapy, Center for Development of Advanced Medical Technology, Jichi Medical University, 3311-1, Yakushiji, Shimotsuke-shi, Tochigi 329-0498, Japan. Tel: +81-285-58-7583; Fax: +81-285-44-6219; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Medicine, Vol. 3, pp. 13–18, 2012
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DOI: http://dx.doi.org/10.3727/215517912X639469
Copyright © 2012 Cognizant Comm. Corp.
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Consideration of a Safe Protocol for Hepatocyte Transplantation Using Infantile Pigs

Shin Enosawa,* Wenji Yuan,* Masaharu Douzen,* Atsuko Nakazawa,† Takeshi Omasa,‡ Akinari Fukuda,§ Seisuke Sakamoto,§ Takanobu Shigeta,§ and Mureo Kasahara§

*Clinical Research Center, National Center for Child Health and Development, Tokyo, Japan
†Department of Pathology, National Center for Child Health and Development, Tokyo, Japan
‡Institute of Technology and Science, The University of Tokushima, Tokushima, Japan
§Division of Transplantation, National Center for Child Health and Development, Tokyo, Japan

Hepatocyte transplantation is hoped to be an alternative treatment to certain cases of liver transplantation. The most promising indication is for congenital metabolic diseases, especially in infants. To establish a safe protocol for hepatocyte transplantation into infants, we examined physiological changes during treatment using infantile pigs. Recipient pigs (domestic, crossbred with Large-Yorkshire, Landrace, and Duroc; 2.5 kg; 7–14 days old) were anesthetized with isoflurane; a midline incision of minimum length provided access to the superior mesenteric vein. A double lumen catheter was inserted through the vein to within 1 cm of the hepatic portal region. Physiological parameters such as heart rate, systemic blood pressure, and portal pressure were monitored. Cryopreserved porcine hepatocytes isolated from the same strain were suspended in physiological saline and transfused through the catheter. In experiments requiring tracing, cells were stained with fluorescent dye prior to transfusion. Recipient pigs were kept for 1 day and sacrificed for histological liver examination. After preliminary experiments, the optimized number and concentration for hepatocyte transplantation were determined to be 1 × 108 cells/kg and 1 × 107 cells/ml. The cell suspension was transfused at a rate of 0.67 ml/min. No marked anomaly of physiological parameters was observed, whereas light tachycardia occurred in preliminary trials when the transfusion rate was faster than our standard protocol. All five pigs transfused with the established method recovered from anesthesia and survived with good vital signs. Histology revealed that the transfused hepatocytes were integrated in the hepatic tissue.

Key words: Hepatocyte transplantation; Safe protocol; Infantile pigs

Received January 31, 2011; final acceptance April 1, 2011. Online prepub date: May 14, 2012.
Address correspondence to Shin Enosawa, Division Chief, Division for Advanced Medical Sciences, Clinical Research Center, National Center for Child Health and Development, 2-10-1 Ookura, Setagaya-ku, Tokyo 157-8534, Japan. Tel: 81-3-5494-8163; Fax: 81-3-3417-2864; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Medicine, Vol. 3, pp. 19–23, 2012
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DOI: http://dx.doi.org/10.3727/215517912X639478
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Construction of Artificial Hepatic Lobule-Like Spheroids on a Three-Dimensional Culture Device

Shin Enosawa,* Yoshitaka Miyamoto,* Hisayo Kubota,† Tomoko Jomura,‡ and Takeshi Ikeya‡

*Clinical Research Center, National Center for Child Health and Development, Tokyo, Japan
†National Institute of Occupational Safety and Health, Kanagawa, Japan
‡Transparent, Inc., Chiba, Japan

One major purpose of cell culture is the reconstruction of physiological structures. Using bovine aortic epithelium cell line HH (JCRB0099) as feeder cells and rat primary hepatocytes, we constructed hepatic lobule-like spheroids on a cell array plate designed for three-dimensional (3D) culture. Microfabricated patterning of the cell array with poly(ethyleneglycol) brushes promotes the formation of spheroids at 100-μm diameter at 100-μm intervals Our standard protocol is to seed with feeder HH cells and then seed with primary hepatic parenchymal cells. The composite cell spheroids thus obtained are called heterospheroids. Feeder cells that were attached to the plate migrated and encompassed the spheroidal hepatocyte mass. Electron microscopy revealed Disse spacelike structures characterized by hepatocyte-rooted microvilli rooted between hepatocyte and feeder epithelial HH cells. Differentiated hepatic functions such as albumin synthesis and cytochrome P450 subfamily CYP3A activities were maintained for 28 days in the heterospheroid versus monospheroid and monolayer cultures. In addition, glucuronide conjugation activity was maintained at a high level in heterospheroids. These results indicate that structurally similar hepatic lobules were formed in a microfabricated cell array coculture system and that the culture conditions are beneficial for maintaining differentiated hepatic functions.

Key words: Artificial hepatic lobules; Three-dimensional culture; Hepatic function

Received January 31, 2011; final acceptance April 1, 2011. Online prepub date: May 14, 2012.
Address correspondence to Shin Enosawa, Division Chief, Division for Advanced Medical Sciences, Clinical Research Center, National Center for Child Health and Development, 2-10-1 Ookura, Setagaya-ku, Tokyo 157-8534, Japan. Tel: +81-3-5494-8163; Fax: +81-3-3417-2864; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Medicine, Vol. 3, pp. 25–31, 2012
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DOI: http://dx.doi.org/10.3727/215517912X639496
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Hepatocyte Is a Sole Cell Type Responsible for the Production of Coagulation Factor IX In Vivo

Kohei Tatsumi,* Kazuo Ohashi,* Shigeki Mukobata,* Atsushi Kubo,† Fumikazu Koyama,‡ Yoshiyuki Nakajima,‡ Midori Shima,§ and Teruo Okano*

*Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, Shinjuku, Tokyo, Japan
†First Department Medicine, Nara Medical University, Kashihara, Nara, Japan
‡Department of Surgery, Nara Medical University, Kashihara, Nara, Japan
§Department of Pediatrics, Nara Medical University, Kashihara, Nara, Japan

Coagulation factor IX (FIX) is synthesized by hepatocytes, and the lack of this protein causes hemophilia B. Liver nonparenchymal cells, including liver sinusoidal endothelial cells (LSECs) and extrahepatic cells in the body, are scarcely shown to have an ability to synthesize and secrete FIX. The present study investigated the existence of cells responsible for synthesizing FIX other than hepatocytes in mice using gene expression analyses and FIX-specific clotting assays. Among the several organs investigated, including liver, lung, spleen, kidney, brain, intestine, and tongue, FIX mRNA expressions were observed only in the liver. From the liver, hepatocytes and LSECs were isolated. FIX mRNA expression and FIX protein secretion were observed exclusively in the hepatocytes. Furthermore, the clotting activity of FIX secreted from the cultured hepatocytes was found to be dependent on the concentration of vitamin K2. These findings indicated that the hepatocyte is the only cell type that biochemically produces functional FIX in vivo. This highlights the importance of hepatocytes or cells that are fully differentiated toward the hepatic lineage for possible application for regenerative medicine and for targeting gene delivery to establish new cell-based treatments for hemophilia B.

Key words: Factor IX; Hemophilia B; Hepatocyte; Nonparenchymal cell

Received January 31, 2011; final acceptance April 1, 2011. Online prepub date: May 14, 2012.
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. 30234; Fax: +81-3-3359-6046; E-mail address: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Medicine, Vol. 3, pp. 33–41, 2012
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DOI: http://dx.doi.org/10.3727/215517912X639388
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A Combined Continuous Density/Osmolality Gradient for Supplemental Purification of Human Islets

Hirofumi Noguchi,*†‡ Bashoo Naziruddin,†§ Masayuki Shimoda,¶ Daisuke Chujo,* Morihito Takita,* Koji Sugimoto,* Takeshi Itoh,* 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, Transplant and Surgical Oncology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
§Baylor Regional Transplant Institute, Dallas and Fort Worth, TX, USA
¶Division of Cardiology, Department of Internal Medicine, Baylor University Medical Center, Baylor Heart and Vascular Institute, Dallas, TX, USA

For islet transplantation, islet purification minimizes the risks associated with islet infusion through the portal vein. However, islet purification may result in decreased numbers of islets recovered from digested tissue. In this study, we evaluated the effectiveness of performing supplemental purification (SP) after regular purification (RP). We designed the densities of low- and high-density solutions based on the outcome of RP. Moreover, a combined continuous osmolality/continuous density gradient for the SP was used in this study. Low-density/osmolality (1.075–1.110 g/cm3/400–410 mOsm/kg) and high-density/osmolality (1.090–1.125 g/cm3/495–505 mOsm/kg) solutions were produced by changing the volumetric ratio of iodixanol, 10´ HBSS, and RP solutions. The percentage of islet recovery (postpurification IE/prepurification IE ´ 100) after RP was 77.3 ± 5.6%, and the percentage of islet recovery after addition of SP was 85.3 ± 5.4%. In vitro and in vivo assessments showed that islet viability and function were not altered by the additional purification step. These data suggest that the addition of SP could contribute approximately 8% to islet recovery with viability and potency comparable to that obtained by RP and, therefore, that usage of the combined continuous density and continuous osmolality gradient for SP could efficiently improve islet equivalents in the final preparation.

Key words: Islet transplantation; Islet isolation; Supplemental purification; Regular purification; Osmolality; Density

Received January 31, 2011; final acceptance April 1, 2011. Online prepub date: May 8, 2012.
Address correspondence to Hirofumi Noguchi, 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: +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 or This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Medicine, Vol. 3, pp. 43–49, 2012
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DOI: http://dx.doi.org/10.3727/215517912X639397
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Novel Positive-Charged Nanoparticles for Efficient Magnetic Resonance Imaging of Islet Transplantation

Koichi Oishi,* Hirofumi Noguchi,*† Hiroaki Saito,‡ Hiroshi Yukawa,* Yoshitaka Miyamoto,* Kenji Ono,§ Katsutoshi Murase,‡ Makoto Sawada,§ and Shuji Hayashi*

*Department of Advanced Medicine in Biotechnology and Robotics, Nagoya University Graduate School of Medicine, Nagoya, Japan
†Department of Gastroenterological Surgery, Transplant and Surgical Oncology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
‡Nagoya Research Laboratory, MEITO Sangyo Co., Ltd., Nagoya, Japan
§Department of Brain Functions, Division of Stress Adaptation and Protection, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan

Significant graft loss immediately after islet transplantation occurs due to immunological and nonimmunological events. Magnetic resonance imaging (MRI) is an attractive potential tool for monitoring islet mass in vivo. Although an efficient uptake of MRI contrast agent is required for islet cell labeling, commercially available magnetic nanoparticles are not efficiently transduced into cells. In this study, we developed six kinds of novel magnetic iron oxide nanoparticles, which are electrically charged by cationic end-group substitution of dextran. Each of the nanoparticles consisted of a small monocrystalline, superparamagnetic iron oxide core that is stabilized by a cross-linked aminated dextran coating to improve stability. We also used three different commercially available nanoparticles for controls. The labeling efficiency of the novel nanoparticles was evaluated, and the feasibility of the imaging by MRI was assessed. The positive-charged nanoparticles were transduced into a b-cell line, MIN6 cells, but not three commercially available nanoparticles. MRI showed a marked decrease in signal intensity on T1- and T2-weighted images at the site of the labeled cells in vitro. These data suggest that novel positive-charged nanoparticles could be useful MRI contrast agents to monitor islet mass after transplantation.

Key words: Cationic nanoparticles; Islet transplantation; Magnetic resonance imaging (MRI); In vivo imaging; Dextran

Received January 31, 2011; final acceptance April 1, 2011. Online prepub date: May 8, 2012.
Address correspondence to Hirofumi Noguchi, 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: +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 or This e-mail address is being protected from spambots. You need JavaScript enabled to view it

Cell Medicine, Vol. 3, pp. 51–61, 2012
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DOI: http://dx.doi.org/10.3727/215517912X639487
Copyright © 2012 Cognizant Comm. Corp.
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Differentiation of Mouse Pancreatic Stem Cells Into Insulin-Producing Cells by Recombinant Sendai Virus-Mediated Gene Transfer Technology

Hiroshi Yukawa,*† Hirofumi Noguchi,‡ Koichi Oishi,*§ Yoshitaka Miyamoto,*¶ Makoto Inoue,# Mamoru Hasegawa,# Shuji Hayashi,*,** and Yoshinobu Baba†,††‡‡

*Department of Advanced Medicine in Biotechnology and Robotics, Nagoya University Graduate School of Medicine, Higashi-ku, Nagoya, Japan
†FIRST Research Center for Innovative Nanobiodevices, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Japan
‡Baylor All Saints Medical Center and Baylor Research Institute, Dallas, TX, USA
§Research Institute of Environmental Medicine, Stress Adaption and Protection, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Japan
¶Department of Oral Disease Research, National Center for Geriatrics and Gerontology, Obu-shi, Aichi, Japan
#DNAVEC Corporation, Tsukuba-shi, Ibaragi, Japan
**The Foundation for Promotion of State of the Art in Medicine and Health Care, Chikusa-ku, Nagoya, Japan
††Department of Applied Chemistry, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Japan
‡‡Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Hayashi-cho, Takamatsu, Japan

Islet transplantation, including b-cells, has proven to be effective for diabetes in many recent studies; however, this treatment strategy requires sufficient organ donors. One attractive approach for the generation of b-cells is to utilize the expansion and differentiation of cells from pancreatic stem cells (PSCs), which are closely associated to the b-cells lineage. In this study, we investigated whether important transcription factors (Pdx-1, Ngn3, NeuroD, and MafA) in islet cells could be efficiently transduced into mouse PSCs (mPSCs) using Sendai virus (SeV) vectors and found that the transduced cells were differentiated into insulin-producing pancreatic b-cells. The mPSCs transduced with single transcription factors using SeV vectors could not express the insulin-2 mRNA. When combinations of two transcription factors were transduced using the SeV vectors, including combinations of Pdx-1 + NeuroD, Pdx-1 + MafA, and NeuroD + MafA, the expression of insulin-2 mRNA was low but could be detected. When combinations of three or more transcription factors were transduced using SeV vectors, the expression of insulin-2 mRNA could be detected. In particular, the transduction of the combination of PDX-1, NeuroD, and MafA produced the most effective for the expression of insulin-2 mRNA out of all of the different combinations examined. These data suggest that the transduction of transcription factors using SeV vectors facilitates mPSC differentiation into insulin-producing cells and showed the possibility of regenerating b-cells by using transduced PSCs.

Key words: Pancreatic stem cells (PSCs); Sendai virus (SeV); Insulin-producing cells; Islet transplantation

Received January 31, 2011; final acceptance April 1, 2011. Online prepub date: May 14, 2012.
Address correspondence to Hiroshi Yukawa, FIRST Research Center for Innovative Nanobiodevices, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan. Tel: +81-52-789-5654; Fax: +81-52-789-5117; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it



Cell Medicine, Vol. 3, pp. 63–74, 2012
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DOI: http://dx.doi.org/10.3727/215517912X639379
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Eicosapentenoic Acid Attenuates Allograft Rejection in an HLA-B27/EGFP Transgenic Rat Cardiac Transplantation Model

Zhong Liu,*§1 Naoyuki Hatayama,*1 Lin Xie,* Ken Kato,*† Ping Zhu,* Takahiro Ochiya,‡ Yukitoshi Nagahara,† Xiang Hu,§ and Xiao-Kang Li*

*Division of Radiation Safety and Immune Tolerance, National Research Institute for Child Health and Development, Tokyo, Japan
†Department of Biomedical Sciences, Tokyo Denki University, Saitama, Japan
‡Section for Studies on Metastasis, National Cancer Center Research Institute, Tokyo, Japan
§Department of General Surgery, First Affiliated Hospital of Dalian Medical University, Dalian, China

The development of an animal model bearing definite antigens is important to facilitate the evaluation and modulation of specific allo-antigen responses after transplantation. In the present study, heterotopic cardiac transplantation was performed from F344/EGFPTg and F344/HLA-B27Tg rats to F344 rats. The F344 recipients accepted the F344/EGFPTg transplants, whereas they rejected the cardiac tissue from the F344/HLA-B27Tg rats by 39.4 ± 6.5 days, due to high production of anti-HLA-B27 IgM- and IgG-specific antibodies. In addition, immunization of F344 rats with skin grafts from F344/HLA-B27Tg rats resulted in robust production of anti-HLA-B27 IgM and IgG antibodies and accelerated the rejection of a secondary cardiac allograft (7.4 ± 1.9 days). Of interest, the F344 recipients rejected cardiac grafts from double transgenic F344/HLA-B27&EGFPTg rats within 9.0 ± 3.2 days, and this was associated with a significant increase in the infiltration of lymphocytes by day 7, suggesting a role for cellular immune rejection. Eicosapentenoic acid (EPA), one of the w-3 polyunsaturated fatty acids in fish oil, could attenuate the production of anti-HLA IgG antibodies and B-cell proliferation, significantly prolonging double transgenic F344HLA-B27&EGFPTg to F344 rat cardiac allograft survival (36.1 ± 13.6 days). Moreover, the mRNA expression in the grafts was assessed by quantitative reverse transcription polymerase chain reaction (qRT-PCR), revealing an increase in the expression of the HO-1, IL-10, TGF-b, IDO, and Foxp3 genes in the EPA-treated group. Hence, our data indicate that HLA-B27 and/or GFP transgenic proteins are useful for establishing a unique animal transplantation model to clarify the mechanism underlying the allogeneic cellular and humoral immune response, in which the transplant antigens are specifically presented. Furthermore, we also demonstrated that EPA was effective in the treatment of rat cardiac allograft rejection and may allow the development of novel immunomodulatory strategies for organ transplantation.

Key words: Allograft; GFP; HLA-B27; Regulatory T cell; Tolerance; Transgenic rat

Received January 31, 2011; final acceptance April 1, 2011. Online prepub date: May 8, 2012.
1These authors provide equal contribution to this work.
Address correspondence to Xiao-Kang Li, M.D., Ph.D., Division of Radiation Safety and Immune Tolerance, National Research Institute for Child Health and Development, 2-10-1 Okura, Setagaya-ku, Tokyo 157-8535 Japan. Tel: +81-3-3416-0181; Fax: +81-3-3417-2864; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Medicine, Vol. 3, pp. 75–80, 2012
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DOI: http://dx.doi.org/10.3727/215517912X639423
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Higher Sensitivity of Peripheral Blood Lymphocytes to Endogenous Glucocorticoid in Renal Transplant Recipients Treated With Tacrolimus, as Compared to Those Treated With Cyclosporine

Gulimire Muhetaer,*† Hironori Takeuchi,‡ Sogo Akizuki,‡ Hitoshi Iwamoto,* Motohide Shimazu,* Sakae Unezaki,* and Toshihiko Hirano§

*Department of 5th Surgery, Hachioji Medical Center, Tokyo Medical University, Tokyo, Japan
†Department of Surgery, Uygur Autonomous Region People’s Hospital, Xinjiang Uyghur Autonomous Region, China
‡Department of Practical Pharmacy, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan
§Department of Clinical Pharmacology, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan

Lymphocyte sensitivity to endogenous glucocorticoid cortisol could be a biological marker for safe reduction and withdrawal of steroids in renal transplant recipients. We compared peripheral lymphocyte sensitivity with cortisol between transplant recipients treated with tacrolimus (Tac) and those treated with cyclosporine. The suppressive efficacies of cortisol against T-cell mitogen-stimulated proliferation of peripheral lymphocytes were investigated in 44 renal transplant patients, who either had reduced or been withdrawn from steroid treatment. Twenty of the 44 patients were treated with Tac, and the other 24 patients were treated with cyclosporine A (CyA). The lymphocyte sensitivity to cortisol was compared between these two patient groups. The cortisol IC50 values in the Tac and CyA groups were 0.09 ± 0.12 and 14.2 ± 12.7 ng/ml, respectively. Lymphocyte sensitivity to cortisol in the Tac-treated group was significantly higher than that in the CyA-treated group (p = 0.0283). On the other hand, incidences of steroid withdrawal syndrome and increases in serum creatinine concentration were not significantly different between the Tac and CyA groups. Lymphocyte sensitivity to cortisol was higher in the Tac-treated patients than that in the CyA-treated ones. Since the cortisol sensitivity of peripheral lymphocytes is suggested to be a predictive marker for safe steroid withdrawal, Tac administration shows promise in aiding successful withdrawal of steroid treatment in long-term renal transplant recipients.

Key words: Tacrolimus; Cyclosporine; Renal transplantation; Peripheral blood mononuclear cells

Received January 31, 2011; final acceptance April 1, 2011. Online prepub date: May 14, 2012.
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: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Medicine, Vol. 3, pp. 81–88, 2012
2155-1790/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/215517912X639360
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Clinical Significance of the Pharmacological Efficacy of Tacrolimus Estimated by the Lymphocyte Immunosuppressant Sensitivity Test (LIST) Before and After Renal Transplantation

Kentaro Sugiyama,* Kazuya Isogai,† 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 Sciences, 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

The lymphocyte immunosuppressant sensitivity test (LIST) with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay procedure can predict the pharmacological efficacy of immunosuppressive agents. A previous study reported the pharmacological efficacy of tacrolimus evaluated by LIST just before renal transplantation significantly correlated with the incidence of acute rejection episodes. However, the pharmacological efficacy of tacrolimus has not been estimated after renal transplantation. Therefore, the present study evaluated the pharmacological efficacy of tacrolimus by LIST using the MTT assay procedure before and 1, 3, and 12 months after transplantation in 17 renal transplant recipients that received tacrolimus-based immunosuppressive therapy. The tacrolimus pharmacological efficacies before and after the procedure were also compared with incidence of acute rejection and cytomegalovirus (CMV) infection episodes. The individual values of tacrolimus 50% inhibition of lymphocyte proliferation (IC50) varied widely before transplantation, and the mean value of the IC50 was 126.4 ± 337.7 ng/ml. The patients were divided into two groups according to the tacrolimus IC50 values evaluated before transplantation. The rate of acute rejection episodes in the tacrolimus high-sensitivity group was significantly lower than that in the tacrolimus low-sensitivity group (p = 0.005). The tacrolimus IC50 deviation between patients expanded further at one and three months after surgery. However, the sensitivity deviation almost converged at 1 year after surgery. Moreover, the pharmacological efficacy of tacrolimus evaluated at 1, 3, and 12 months after transplantation did not significantly correlate with the incidence of acute rejection episodes. The pharmacological efficacies of tacrolimus evaluated at both before and after surgery were not significantly correlated with the episodes of CMV infection. These findings suggest that the pharmacological efficacy of tacrolimus evaluated with LIST before surgery is a useful biomarker for predicting the occurrence of acute allograft rejection in renal transplantation.

Key words: Tacrolimus; Renal transplantation; Lymphocyte immunosuppressant-sensitivity test (LIST); 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT); Peripheral-blood mononuclear cells (PBMCs)

Received January 31, 2011; final acceptance April 1, 2011. Online prepub date: June 15, 2012.
Address correspondence to Kentaro Sugiyama, Ph.D., Associate Professor, Department of Clinical Pharmacology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji City, Tokyo 192-0392, Japan. Tel: +81-426-76-5111; Fax: +81-426-76-5796; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Medicine, Vol. 3, pp. 89–95, 2012
2155-1790/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/215517912X639405
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Cryopreservation of Induced Pluripotent Stem Cells

Yoshitaka Miyamoto,*†‡ Hirofumi Noguchi,§ Hiroshi Yukawa,* Koichi Oishi,* Kenji Matsushita,† Hisashi Iwata,¶ and Shuji Hayashi*

*Department of Advanced Medicine in Biotechnology and Robotics, Nagoya University Graduate School of Medicine, Higashi-ku, Nagoya, Japan
†Department of Oral Disease Research, National Center for Geriatrics and Gerontology, Aichi, Japan
‡Clinical Research Center, National Center for Child Health and Development, Tokyo, Japan
§Department of Gastroenterological Surgery, Transplant and Surgical Oncology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
¶Department of Biomedical Sciences, Chubu University College of Life and Health Sciences, Aichi, Japan

Induced pluripotent stem (iPS) cells have attracted attention as a promising cell source for medical treatment that could replace marrow stromal cells (MSCs) and adipose tissue-derived stem cells (ASCs). These pluripotent cells can be induced in vitro and in vivo to differentiate into various tissues and organs. The cells will be useful for regenerative medicine, cell therapy, and drug screening. Vitrification is used, as well as a rapid-freeze method, for colony-forming iPS cells. However, the method requires a high degree of technical skill. We herein report a more convenient method for freezing iPS cells in suspension. We examined the proliferation potency of cryopreserved mouse iPS cells using culture medium, 10% DMSO, 10% glycerol, 5% DMSO, 5% glycerol, 5% DMSO + 5% glycerol, cell-freezing medium-DMSO, cell-freezing medium-glycerol, Cell Banker 1, Cell Banker 1+, Cell Banker 2, and Cell Banker 3 as cryopreservation solutions. Among them, Cell Banker 3 showed the highest efficacy in terms of the proliferation of mouse iPS cells. The mouse iPS cells cryopreserved in Cell Banker 3 at –80°C for 12 months maintained a high proliferation rate and an undifferentiated status. The formation of teratomas was also examined. In conclusion, Cell Banker 3 allows for freezing of iPS cells in suspension.

Key words: Induced pluripotent stem (iPS) cells; Pluripotency; Cryopreservation; Slow freezing

Received January 31, 2011; final acceptance April 1, 2011. Online prepub date: May 8, 2012.
Address correspondence to Hirofumi Noguchi, 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: +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 or This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Medicine, Vol. 3, pp. 97–102, 2012
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DOI: http://dx.doi.org/10.3727/215517912X639414
Copyright © 2012 Cognizant Comm. Corp.
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Generation of Mouse STO Feeder Cell Lines That Confer Resistance to Several Types of Selective Drugs

Issei Saitoh,* Masahiro Sato,† Yoko Iwase,‡ Emi Inada,* Toshiki Nomura,* Eri Akasaka,* Youichi Yamasaki,* and Hirofumi Noguchi§

*Department of Pediatric Dentistry, Kagoshima University Graduate School of Medical and Dental Sciences, Sakuragaoka, Kagoshimashi, Kagoshima, Japan
†Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, Korimoto, Kagoshima, Kagoshima, Japan
‡Department of Dental Anesthesia, Kagoshima University Medical and Dental Hospital, Sakuragaoka, Kagoshimashi, Kagoshima, Japan
§Department of Gastroenterological Surgery, Transplant and Surgical Oncology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Shikata-cho, Okayama, Japan

Feeder cells are generally required for establishment and maintenance of embryonic stem (ES)/induced pluripotent stem (iPS) cells. Increased demands for generation of those cells carrying various types of vectors (i.e., KO vectors and transgenes) also require feeder cells that confer resistance to any types of preexisting selective drugs. Unfortunately, the use of the feeders that are resistant to various drugs appears to be limited to a few laboratories. Here we generated a set of gene-engineered STO feeder cells that confer resistance to several commercially available drugs. The STO cells, which have long been used as a feeder for mouse ES and embryonal carcinoma (EC) cells, were transfected with pcBIH [carrying bleomycin resistance gene (ble) and hygromycin B phosphotransferase gene (Hyg)], pcBIP [carrying ble and puromycin resistance gene (puro)], or pcBSN [carrying ble and neomycin resistance gene (neo)]. The resulting stably transfectants (termed SHB for pcBIH, SPB for pcBIP, and SNB for pcBSN) exhibited bleomycin/hygromycin, bleomycin/puromycin, or bleomycin/neomycin, as expected. The morphology of these cells passaged over 18 generations was indistinguishable from that of parental STO cells. Of isolated clones, the SHB3, SPB3, and SNB2 clones successfully supported the growth of mouse ES cells in an undifferentiated state, when coculture was performed. PCR analysis revealed the presence of the selective markers in these clones, as expected. These SHB3, SPB3, and SNB2 cells will thus be useful for the acquisition and maintenance of genetically manipulated ES/iPS cells.

Key words: Feeder; STO cell; Selective drug; Plasmid; ES cell; iPS cell

Received January 31, 2011; final acceptance April 1, 2011. Online prepub date: May 8, 2012.
Address correspondence to Hirofumi Noguchi, 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: +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 or This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Medicine, Vol. 3, pp. 103–112, 2012
2155-1790/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/215517912X639351
Copyright © 2012 Cognizant Comm. Corp.
Printed in the USA. All rights reserved

A Simplified In Vitro Teratoma Assay for Pluripotent Stem Cells Injected Into Rodent Fetal Organs

Shigeo Masuda,*1 Takashi Yokoo,†‡1 Naomi Sugimoto,†§ Masako Doi,†§ Shuh-hei Fujishiro,* Kengo Takeuchi,¶ Eiji Kobayashi,†§ and Yutaka Hanazono*

*Division of Regenerative Medicine, Center for Molecular Medicine, Jichi Medical University, Tochigi, Japan
Division of Development of Advanced Treatment (DDAT), Center for Development of Advanced Medical Technology (CDAMTec), Jichi Medical University, Tochigi, Japan
Project Laboratory for Kidney Regeneration, Institute of DNA Medicine, Jikei University School of Medicine, Tokyo, Japan
§Research and Development Center, Otsuka Pharmaceutical Factory, Inc., Tokushima, Japan
Pathology Project for Molecular Targets, Cancer Institute, Japanese Foundation for Cancer Research, Tokyo, Japan

Teratoma formation assays are established methods for evaluating the pluripotency of embryonic stem (ES) cells and induced pluripotent stem (iPS) cells. Teratoma formation in immunodeficient mice takes approximately 2 months. Here, we have developed a novel assay system for developing teratomas in vitro from ES cells and iPS cells in a short period. In vitro culture of ES, iPS, and mesenchymal stem cells (MSCs) in fetal rat metanephroi for 1 week resulted in distinct cell-dependent distribution patterns: Pluripotent cells (ES and iPS cells) formed aggregated masses, whereas MSCs showed disseminated distribution. The aggregated masses that had developed from ES cells and iPS cells after 2 weeks of culture comprised teratomas, though they were largely composed of immature components. Furthermore, in vitro organ culture for 1 week followed by relay transplantation into immunodeficient mice resulted in considerably rapid growing teratomas (teratomas developed in 4 weeks) having similar pathological features as of the teratomas developed using conventional 7-week in vivo teratoma formation assays. In addition, the initial cell number required in the in vitro assay was 1 × 103 cells, which was about 1% of the number of cells required in the conventional in vivo teratoma formation assays. These results suggest that the in vitro teratoma assay is a rapid and convenient screening system and might be an alternative method for developing teratomas for investigating the pluripotency of ES cells and iPS cells.

Key words: iPS cells; ES cells; Teratoma formation; Organ culture; Fetal rat metanephros

Received January 31, 2011; final acceptance April 1, 2011. Online prepub date: May 8, 2012.
1These authors provide equal contribution to this work.
Address correspondence to Eiji Kobayashi, M.D., Ph.D., Division of Development of Advanced Treatment (DDAT), Center for Development of Advanced Medical Technology (CDAMTec), Jichi Medical University, 3311-1 Yakushiji, Shimotsuke-shi, Tochigi 329-0498, Japan. Tel: +81-285-58-7496; Fax: +81-285-44-6219; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it or Yutaka Hanazono, M.D., Ph.D., Division of Regenerative Medicine, Center for Molecular Medicine, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke-shi, Tochigi 329-0498, Japan. Tel: +81-285-58-7450; Fax: +81-285-44-5205; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Medicine, Vol. 3, pp. 113–119, 2012
2155-1790/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/215517912X639432
Copyright © 2012 Cognizant Comm. Corp.
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Cell Therapy Using Adipose-Derived Stem Cells for Chronic Liver Injury in Mice

Kazuo Ohashi,* Yoshinori Matsubara,† Kohei Tatsumi,* Ayako Kohori,† Rie Utoh,* Hiroshi Kakidachi,† Akihiro Horii,† Masahiro Tsutsumi,‡ and Teruo Okano*

*Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, Shinjuku, Tokyo, Japan
†Corporate R&D Center, Olympus Corporation, Hachioji, Tokyo, Japan
‡Department of Pathology, Saiseikai Chuwa Hospital, Sakurai, Nara, Japan

The present study investigated whether transplantation of autologous adipose-derived stem cells (ASCs) administered into the systemic circulation of a mouse with chronic liver injury provides therapeutic efficacy in the absence of any undesirable side effects. The ASCs used were isolated from mice with the same genetic background as the recipient mice and expanded in vitro. For the induction of chronic liver injury, mice were repetitively administered twice a week with CCl4, a well-known hepatotoxin, for a period of 4 weeks. One day after the eighth dose of CCl4, ASC transplantation was performed by tail vein injection and subsequently followed by two additional doses of CCl4 administration. The recipient mice were divided into four groups (vehicle control, 1.5 ´ 103, 1.5 ´ 104, and 1.5 ´ 105 ASCs per mouse). One day after the final CCl4 administration, all mice were sacrificed to assess serum markers and liver histology. The level of serum markers for liver injury and hepatic function did not differ among the four groups. Similarly, no difference was observed in the liver histology between groups. Cell transplantation with ASCs in our model of chronic liver failure did not result in any observable side effects, but from our results, a single application of ASCs seems to be ineffective in improving liver injury.

Key words: Cell transplantation; Adipose-derived stem cells; Chronic liver injury; Liver; Animal model

Received January 31, 2011; final acceptance April 1, 2011. Online prepub date: May 14, 2012.
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 Medicine, Vol. 3, pp. 121–126, 2012
2155-1790/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/215517912X639441
Copyright © 2012 Cognizant Comm. Corp.
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Isolation of Hepatic Progenitor Cells From Human Liver With Cirrhosis Secondary to Biliary Atresia Using EpCAM or Thy-1 Markers

Taisuke Yamazaki,* Shin Enosawa,† Mureo Kasahara,‡ Akinari Fukuda,‡ Seisuke Sakamoto,‡ Takanobu Shigeta,‡ Atsuko Nakazawa,§ and Takayoshi Tokiwa*

*Department of Liver Cell Biology, Kohno Clinical Medicine Research Institute, Shinagawa-ku, Tokyo, Japan
†Division for Advanced Medical Science, National Center for Child Health and Development, Tokyo, Japan
‡Division of Surgery, National Center for Child Health and Development, Tokyo, Japan
§Department of Clinical Pathology, National Center for Child Health and Development, Tokyo, Japan

We sought to determine whether hepatic progenitor cells can be isolated from cirrhotic liver using epithelial cell adhesion molecule (EpCAM) or Thy-1 markers. Liver tissue with cirrhosis secondary to biliary atresia (BA) was collagenase digested, and nonparenchymal cells (NPCs) were cultivated for 24 h. Noncirrhotic NPCs derived from patients with carbamyl phosphate synthetase and ornithine transcarbamylase deficiencies were used as controls. Flow cytometric analysis demonstrated that the percentages of EpCAM- and Thy-1-positive cells were significantly higher in NPC populations derived from BA liver than in those derived from control liver. Reverse transcription polymerase chain reaction analysis revealed that EpCAM-positive sorted cells expressed EpCAM, Thy-1, albumin, and CK-19, whereas Thy-1-positive sorted cells expressed Thy-1, albumin, and CK-19. These findings indicate that EpCAM- or Thy-1-positive hepatic progenitor cells can be more efficiently isolated from BA liver than from control liver and suggest that the properties of EpCAM-positive cells are somewhat different from those of Thy-1-positive cells.

Key words: Biliary atresia; Cirrhosis; Hepatic progenitor cells; Flow cytometry; EpCAM; Thy-1

Received January 31, 2011; final acceptance April 1, 2011. Online prepub date: June 15, 2012.
Address correspondence to Dr. Takayoshi Tokiwa, Department of Liver Cell Biology, Kohno Clinical Medicine Research Institute, 1-28-15, Kitashinagawa, Shinagawa-ku, Tokyo 140-0001, Japan. Tel/Fax: +81-3-3474-1833; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Medicine, Vol. 3, pp. 127–135, 2012
2155-1790/12 $90.00 + .00
DOI: http://dx.doi.org/10.3727/215517912X639450
Copyright © 2012 Cognizant Comm. Corp.
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Side Population Cells From an Immortalized Human Liver Epithelial Cell Line Exhibit Hepatic Stem-Like Cell Properties

Takayoshi Tokiwa,* Taisuke Yamazaki,* and Shin Enosawa†

*Department of Liver Cell Biology, Kohno Clinical Medicine Research Institute, Tokyo Japan
†Department of Regeneration Surgery, Research Institute, National Center for Child Health and Development, Tokyo Japan

The existence of hepatic stem cells in human livers is controversial. We investigated whether the side population (SP) cells derived from an immortalized human liver epithelial cell line THLE-5b possess the properties of hepatic stem-like cells. SP cells derived from THLE-5b were isolated using flow cytometry and were assayed for the expression of phenotypic markers by reverse transcription polymerase chain reaction and immunostaining. THLE-5b SP cells retained the capacity to generate both SP and non-SP cells, showed a capacity for self-renewal, and were more efficient in colony formation than non-SP cells. Neither the SP nor the non-SP cells formed tumors when transplanted into athymic nude mice or severe combined immunodeficient mice. The expression level of stem cell-associated markers such as an ATP-binding cassette membrane transporter, epithelial cell adhesion molecule, c-kit, Thy-1, and octomer binding transcription factor 4 was higher in SP cells than in non-SP cells. When cultivated as rotation-mediated aggregates, the expression of liver-specific genes including tryptophan oxygenase and CYP3A4 was up-regulated in SP cells, suggesting that THLE-5b SP cells have the ability to differentiate into a hepatocyte phenotype. One of the clonal cell lines derived from the SP cells expressed stem cell-associated markers. These results indicate that SP cells derived from THLE-5b possess hepatic stem-like cell properties and suggest that THLE-5b can be used as a model of normal human liver progenitor or stem cell line.

Key words: Side population, Immortalized cell line, Hepatic stem/progenitor cell

Received January 31, 2011; final acceptance April 1, 2011. Online prepub date: June 15, 2012.
Address correspondence to Dr. Takayoshi Tokiwa, Department of Liver Cell Biology, Kohno Clinical Medicine Research Institute, 1-28-15,Kitashinagawa, Shinagawa, Tokyo 140-0001, Japan. Tel/Fax: +81-3-3474-1833; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it