Cell Medicine 9(1-2) Abstracts

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Cell Medicine, Vol. 9, pp. 3-7, 2016
2155-1790/16 $90.00 + .00
DOI: http://dx.doi.org/10.3727/215517916X
693104
Copyright © 2016 Cognizant, LLC.
Printed in the USA. All rights reserved

Review

Cell Therapy for Liver Disease Using Bioimaging Rats

Junko Haga, Shin Enosawa, and Eiji Kobayashi

Department of Organ Fabrication, Keio University School of Medicine, Tokyo, Japan

Advances in stem cell research suggest that cell therapy is a potential alternative to liver transplantation. The use of individualized and minimally invasive cell therapy is desirable to avoid rejection and reduce patient burden. While allo-hepatocyte transplantation has been performed for metabolic hepatic disease, auto-bone marrow transplantation (BMT) has shifted toward mesenchymal stem cells (MSCs) transplantation for liver cirrhosis. In this article, an overview of cell transplantation research for liver disease is provided through our recent rat studies. We have developed various kinds of rat imaging models and have evaluated the effect of cell therapy for liver disease. Bone marrow cells (BMCs) of the Alb-DsRed2 rat were transplanted via the portal vein (PV) in acute and chronic liver damage models. The number of Alb-DsRed2+ albumin-producing cells increased, and the size of the cells increased in the chronic liver damage model as well as in the acute liver damage model. Luciferase transgenic (luc-Tg) rat hepatocytes were transplanted into the hepatectomized LEW rat via the PV. Luminescence intensity lasted for 2 months in the hepatectomized rat. BMCs obtained from green fluorescent protein (GFP) Tg rats were transplanted repeatedly via the PV using an implanted catheter with a port. Repeated BMT via the PV reduced the liver fibrosis. Adipocyte-derived MSCs from the luc-Tg rat were transplanted into the hepatectomized rat model via the PV after ischemic reperfusion. MSCs inhibited hepatocyte apoptosis and promoted liver regeneration. Transplanting the optimal number of cells by an effective and safe way is important for clinical application. Bioimaging rats are a powerful tool for cell transplantation research because it makes observation of the in vivo kinetics of transplanted cells possible. Cell transplantation research using bioimaging rats contributes greatly to evaluating effective methods of cell therapy.

Key words: Bioimaging rat; Bone marrow cells (BMCs); Cell transplantation; Liver; Mesenchymal stem cells (MSCs)

Received May 26, 2016; final acceptance October 18, 2016. Online prepub date: October 21, 2016.
Address correspondence to Eiji Kobayashi, M.D., Ph.D., Department of Organ Fabrication, Keio University School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo 1608582, Japan. Tel: +81-3-3353-1211, ext. 63968; Fax: +81-3-5315-4089; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Medicine, Vol. 9, pp. 9-14, 2016
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DOI: http://dx.doi.org/10.3727/215517916X693113
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Review

Challenges for Production of Human Transplantable Organ Grafts

Eiji Kobayashi

Department of Organ Fabrication, Keio University School of Medicine, Tokyo, Japan

The described research methods explain how you could generate a three-dimensional kidney, based on recent research results. The first method is to fabricate human organs in a pig body. The second is to transplant the so-called “organ bud” into a patient’s body for further development. The third method is to regenerate organs by filling cells into the cytoskeleton as a scaffold. Research for the in vitro fabrication of organ buds has been elaborately accelerated. The organ bud transplantation has been confronted with issues of continuity with the original organs, so the development of technology for achieving continuity between a transplanted organ bud and the existing organs is progressing well. The “organ fabrication” methodology, whereby cells are placed into completely decellularized organs, is supported by recent research results using pig organs taking the size of humans into consideration.

Key words: Organ fabrication; Organ bud; In vivo bioreactor; Experimental pig

Received May 26, 2016; final acceptance October 18, 2016. Online prepub date: October 21, 2016.
Address correspondence to Eiji Kobayashi M.D., Ph.D., Department of Organ Fabrication, Keio University School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo 1608582, Japan. Tel: +81-3-3353-1211, ext. 63968; Fax: +81-3-5315-4089; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Medicine, Vol. 9, pp. 15-20, 2016
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DOI: http://dx.doi.org/10.3727/215517916X693122
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Evaluation of Serum-Free, Xeno-Free Cryopreservation Solutions for Human Adipose-Derived Mesenchymal Stem Cells

Chika Miyagi-Shiohira,* Naoya Kobayashi,† Issei Saitoh,‡ Masami Watanabe,§ Yasufumi Noguchi,¶ Masayuki Matsushita,# and Hirofumi Noguchi*

*Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan
†Okayama Saidaiji Hospital, Okayama, Japan
‡Division of Pediatric Dentistry, Graduate School of Medical and Dental Science, Niigata University, Niigata, Japan
§Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
¶Department of Socio-environmental Design, Hiroshima International University, Hiroshima, Japan
#Department of Molecular and Cellular Physiology, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan

Adipose-derived mesenchymal stem cells (ASCs) have the potential to differentiate into cells of mesodermal origin, such as osteoblasts, adipocytes, myocytes, and chondrocytes, and cryopreservation is currently performed as a routine method for preserving ASCs to safely acquire large numbers of cells. For clinical application of ASCs, serum-free, xeno-free cryopreservation solutions should be used. This study determined the viability and adipo-osteogenic potential of cryopreserved ASCs using four cryopreservation solutions: 10% DMSO, Cell Banker 2 (serum free), Stem Cell Banker (=Cell Banker 3: serum free, xeno free), and TC protector (serum free, xeno free). The viability of the cryopreserved ASCs was over 80% with all cryopreservation solutions. No difference in the adipo-osteogenic potential was found between the cells that did or did not undergo cryopreservation in these cryopreservation solutions. These data suggest that Cell Banker 3 and TC protector are comparable with 10% DMSO and Cell Banker 2 for ASCs, and cryopreserved as well as noncryopreserved ASCs could be applied for regenerative medicine.

Key words: Adipose-derived mesenchymal stem cells (ASCs); Cryopreservation; Dimethyl sulfoxide (DMSO); Cell Banker 3; TC protector

Received May 26, 2016; final acceptance August 12, 2016. Online prepub date: September 1, 2016.
Address correspondence to Hirofumi Noguchi, M.D., Ph.D., Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903-0215, Japan. Tel: +81-98-895-3331; Fax: +81-98-895-3331; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Medicine, Vol. 9, pp. 21-33, 2016
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DOI: http://dx.doi.org/10.3727/215517916X693159
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Immunomodulatory Effects of Adipose Tissue-Derived Stem Cells on Concanavalin A-Induced Acute Liver Injury in Mice

Yasuma Yoshizumi,* Hiroshi Yukawa,†‡ Ryoji Iwaki,§ Sanae Fujinaka,¶ Ayano Kanou,* Yuki Kanou,* Tatsuya Yamada,* Shingo Nakagawa,* Tomomi Ohara,* Kenta Nakagiri,* Yusuke Ogihara,‡ Yoko Tsutsui,† Yumi Hayashi,* Masatoshi Ishigami,# Yoshinobu Baba,†‡ and Tetsuya Ishikawa*

*Department of Radiological and Medical Laboratory Sciences, Nagoya University Graduate School of Medicine, Higashi-ku, Nagoya, Japan
ImPACT Research Center for Innovative Nanobiodevices, Nagoya University, Chikusa-ku, Nagoya, Japan
‡Department of Applied Chemistry, Nagoya University Graduate School of Engineering, Chikusa-ku, Nagoya, Japan
§Kinuura-Tobu Health Care Center, Kariya, Japan
¶Department of Clinical Laboratories, Aichi Cancer Center Hospital, Chikusa-ku, Nagoya, Japan
#Department of Gastroenterology and Hepatology, Nagoya University Graduate School of Medicine, Showa-ku, Nagoya, Japan

Cell therapy with adipose tissue-derived stem cells (ASCs) is expected to be a candidate for the treatment of fulminant hepatic failure (FHF), which is caused by excessive immune responses. In order to evaluate the therapeutic effects of ASCs on FHF, the in vitro and in vivo immunomodulatory effects of ASCs were examined in detail in the mouse model. The in vitro effects of ASCs were examined by assessing their influence on the proliferation of lymphomononuclear cells (LMCs) stimulated with three kinds of mitogens: phorbol 12-myristate 13-acetate (PMA) plus ionomycin, concanavalin A (ConA), and lipopolysaccharide (LPS). The proliferation of LMCs was efficiently suppressed in a dose-dependent manner by ASCs in the cases of PMA plus ionomycin stimulation and ConA stimulation, but not in the case of LPS stimulation. The in vivo effects of transplanted ASCs were examined in the murine FHF model induced by ConA administration. The ALT levels and histological inflammatory changes in the ConA-administered mice were apparently relieved by the transplantation of ASCs. The analysis of mRNA expression patterns in the livers indicated that the expressions of the cytokines such as Il-6Il-10Ifn, and Tnf, and the cell surface markers such as Cd3γCd4Cd8αCd11b, and Cd11c were downregulated in the ASC-transplanted mice. The immunomodulatory and therapeutic effects of ASCs were confirmed in the mouse model both in vitro and in vivo. These suggest that the cell therapy with ASCs is beneficial for the treatment of FHF.

Key words: Adipose tissue-derived stem cells (ASCs); Immunomodulatory effect; Fulminant hepatic failure (FHF); Concanavalin A (Con A)

Received May 26, 2016; final acceptance September 30, 2016. Online prepub date: October 6, 2016. Address correspondence to Hiroshi Yukawa, ImPACT 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  or Tetsuya Ishikawa, Department of Radiological and Medical Laboratory Sciences, Nagoya University Graduate School of Medicine, Higashi-ku, Nagoya 461-8673, Japan. Tel/Fax: +81-52-719-1561; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Medicine, Vol. 9, pp. 35-44, 2016
2155-1790/16 $90.00 + .00
DOI: http://dx.doi.org/10.3727/215517916X693096
Copyright © 2016 Cognizant, LLC.
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Enhanced Adipogenic Differentiation of Human Adipose-Derived Stem Cells in an In Vitro Microenvironment: The Preparation of Adipose-Like Microtissues Using a Three-Dimensional Culture

Yoshitaka Miyamoto,*†1 Masashi Ikeuchi,†‡1 Hirofumi Noguchi,§ Tohru Yagi,¶ and Shuji Hayashi*

*Department of Advanced Medicine in Biotechnology and Robotics, Nagoya University Graduate School of Medicine, Showa-ku, Nagoya, Japan
†Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan
‡PRESTO, Japan Science and Technology (JST), Saitama, Japan
§Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan
¶School of Information Science and Engineering, Tokyo Institute of Technology, Tokyo, Japan

The application of stem cells for cell therapy has been extensively studied in recent years. Among the various types of stem cells, human adipose tissue-derived stem cells (ASCs) can be obtained in large quantities with relatively few passages, and they possess a stable quality. ASCs can differentiate into a number of cell types, such as adipose cells and ectodermal cells. We therefore focused on the in vitro microenvironment required for such differentiation and attempted to induce the differentiation of human stem cells into microtissues using a microelectromechanical system. We first evaluated the adipogenic differentiation of human ASC spheroids in a three-dimensional (3D) culture. We then created the in vitro microenvironment using a 3D combinatorial TASCL device and attempted to induce the adipogenic differentiation of human ASCs. The differentiation of human ASC spheroids cultured in maintenance medium and those cultured in adipocyte differentiation medium was evaluated via Oil red O staining using lipid droplets based on the quantity of accumulated triglycerides. The differentiation was confirmed in both media, but the human ASCs in the 3D cultures contained higher amounts of triglycerides than those in the 2D cultures. In the short culture period, greater adipogenic differentiation was observed in the 3D cultures than in the 2D cultures. The 3D culture using the TASCL device with adipogenic differentiation medium promoted greater differentiation of human ASCs into adipogenic lineages than either a 2D culture or a culture using a maintenance medium. In summary, the TASCL device created a hospitable in vitro microenvironment and may therefore be a useful tool for the induction of differentiation in 3D culture. The resultant human ASC spheroids were “adipose-like microtissues” that formed spherical aggregation perfectly and are expected to be applicable in regenerative medicine as well as cell transplantation.

Key words: Adipose-derived stem cells (ASCs); Spheroid; Three-dimensional (3D) culture; Tapered stencil for cluster culture (TASCL); Adipogenic differentiation; Microenvironment

Received May 26, 2016; final acceptance September 5, 2016. Online prepub date: September 14, 2016.
1These authors provided equal contribution to this work.
Address correspondence to Yoshitaka Miyamoto, Ph.D., Department of Advanced Medicine in Biotechnology and Robotics, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, 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


Cell Medicine, Vol. 9, pp. 45-51, 2016
2155-1790/16 $90.00 + .00
DOI: http://dx.doi.org/10.3727/215517916X693131
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The Evaluation of Islet Purification Methods That Use Large Bottles to Create a Continuous Density Gradient

Chika Miyagi-Shiohira,* Naoya Kobayashi,† Issei Saitoh,‡ Masami Watanabe,§ Yasufumi Noguchi,¶ Masayuki Matsushita,# and Hirofumi Noguchi*

*Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan
†Okayama Saidaiji Hospital, Okayama, Japan
‡Division of Pediatric Dentistry, Graduate School of Medical and Dental Science, Niigata University, Niigata, Japan
§Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
¶Department of Socio-environmental Design, Hiroshima International University, Hiroshima, Japan
#Department of Molecular and Cellular Physiology, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan

Islet purification is one of the most important steps of islet isolation for pancreatic islet transplantation. The most common method of islet purification is density gradient centrifugation using a COBE 2991 cell processor. However, this method can damage islets mechanically through its high shearing force. We recently reported that a new purification method using large plastic bottles effectively achieves a high yield of islets from the porcine pancreas. In the present study, we evaluated the methods of making a continuous density gradient. The gradient was produced with a gradient maker and two types of candy cane-shaped stainless steel pipes. One method was to use a “bent-tipped” stainless steel pipe and to load from a high-density solution to a low-density solution, uploading the stainless steel pipe. The other method was to use a regular stainless steel pipe and to load from a low-density solution to a high-density solution, leaving the stainless steel pipe in place. There were no significant differences between the two solutions in terms of the islet yield, rate of viability or purity, score, or the stimulation index after purification. Furthermore, there were no differences in the attainability or suitability of posttransplantation normoglycemia. Our study shows the equivalency of these two methods of islet purification.

Key words: Islet transplantation; Islet isolation; Islet purification; Bottle purification; Continuous density gradient

Received May 26, 2016; final acceptance August 26, 2016. Online prepub date: September 1, 2016.
Address correspondence to Hirofumi Noguchi, M.D., Ph.D., Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903-0215, Japan. Tel: +81-98-895-1696; Fax: +81-98-895-1684; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Medicine, Vol. 9, pp. 53-59, 2016
2155-1790/16 $90.00 + .00
DOI: http://dx.doi.org/10.3727/215517916X693140
Copyright © 2016 Cognizant, LLC.
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Comparison of Purification Solutions With Different Osmolality for Porcine Islet Purification

Chika Miyagi-Shiohira,* Naoya Kobayashi,† Issei Saitoh,‡ Masami Watanabe,§ Yasufumi Noguchi,¶ Masayuki Matsushita,# and Hirofumi Noguchi*

*Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan
†Okayama Saidaiji Hospital, Okayama, Japan
‡Division of Pediatric Dentistry, Graduate School of Medical and Dental Science, Niigata University, Niigata, Japan
§Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
¶Department of Socio-environmental Design, Hiroshima International University, Hiroshima, Japan
#Department of Molecular and Cellular Physiology, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan

The osmolality of the purification solution is one of the most critical variables in human islet purification during islet isolation. We previously reported the effectiveness of a combined continuous density/osmolality gradient for the supplemental purification of human islets. We herein applied a combined continuous density/osmolality gradient for regular purification. The islets were purified with a continuous density gradient without osmolality preparation [continuous density/normal osmolality (CD/NO)] or continuous density/osmolality solution with osmolality preparation by 10× Hank’s balanced salt solution (HBSS) [continuous density/continuous osmolality (CD/CO)]. The osmolality of the low-density solution was 400 mOsm/kg in both groups and that of the high-density solution was 410 mOsm/kg in the CD/NO group and 500 mOsm/kg in the CD/CO group. Unexpectedly, we noted no significant differences between the two solutions in terms of the islet yield, rate of viability and purity, score, stimulation index, or the attainability and suitability of posttransplantation normoglycemia. Despite reports that the endocrine and exocrine tissues of pancreata have distinct osmotic sensitivities and that high-osmolality solutions result in greater purification efficiency, the isolation and transplant outcomes did not markedly differ between the two purification solutions with different osmolalities in this study.

Key words: Islet transplantation; Islet isolation; Islet purification; Osmolality

Received May 26, 2016; final acceptance August 26, 2016. Online prepub date: September 1, 2016.
Address correspondence to Hirofumi Noguchi, M.D., Ph.D., Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903-0215, Japan. Tel: +81-98-895-1696; Fax: +81-98-895-1684; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Medicine, Vol. 9, pp. 61-66, 2016
2155-1790/16 $90.00 + .00
DOI: http://dx.doi.org/10.3727/215517916X693087
Copyright © 2016 Cognizant, LLC.
Printed in the USA. All rights reserved

Measurement of DNA Length Changes Upon CpG Hypermethylation by Microfluidic Molecular Stretching

Daisuke Onoshima,*† Naoko Kawakita,†‡ Daiki Takeshita,†‡ Hirohiko Niioka,§ Hiroshi Yukawa,†‡ Jun Miyake,§ and Yoshinobu Baba*†‡¶

*Institute of Innovation for Future Society, Nagoya University, Chikusa-ku, Nagoya, Japan
ImPACT Research Center for Advanced Nanobiodevices, Nagoya University, Chikusa-ku, Nagoya, Japan
‡Department of Applied Chemistry, Graduate School of Engineering, Nagoya University, Chikusa-ku, Nagoya, Japan
§Graduate School of Engineering Science, Osaka University, Toyonaka, Osaka, Japan
¶Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Takamatsu, Japan

Abnormal DNA methylation in CpG-rich promoters is recognized as a distinct molecular feature of precursor lesions to cancer. Such unintended methylation can occur during in vitro differentiation of stem cells. It takes place in a subset of genes during the differentiation or expansion of stem cell derivatives under general culture conditions, which may need to be monitored in future cell transplantation studies. Here we demonstrate a microfluidic device for investigating morphological length changes in DNA methylation. Arrayed polymer chains of single DNA molecules were fluorescently observed by parallel trapping and stretching in the microfluidic channel. This observational study revealed that the shortened DNA length is due to the increased rigidity of the methylated DNA molecule. The trapping rate of the device for DNA molecules was substantially unaffected by changes in the CpG methylation.

Key words: DNA methylation; Cytosine-guanine dinucleotides (CpG); Microfluidic device; Single-molecule detection

Received May 26, 2016; final acceptance September 12, 2016. Online prepub date: September 14, 2016.
Address correspondence to Daisuke Onoshima, Institute of Innovation for Future Society, 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