ognizant Communication Corporation

CELL TRANSPLANTATION
The Regenerative Medicine Journal

ABSTRACTS
VOLUME 19, NUMBERS 6-7, 2010

Cell Transplantation, Vol. 19, pp. 649-654, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X508744
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Review
Recent Advances in Protein Transduction Technology

Hirofumi Noguchi,1 Masayuki Matsushita,2 Naoya Kobayashi,3 Marlon F. Levy,1 and Shinichi Matsumoto1

1Baylor All Saints Medical Center, Baylor Research Institute, Fort Worth, TX, USA
2Mitsubishi Kagaku Institute of Life Sciences, Tokyo, Japan
3Department of Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan

During the past 15 years, a variety of peptides, known as protein transduction domains (PTDs), or cell-penetrating peptides (CPPs), have been characterized for their ability to translocate into live cells. There are now numerous examples of biologically active full-length proteins and peptides that have been successfully delivered to cells and tissues, both in vitro and in vivo. One of the principal mechanisms of protein transduction is via electrostatic interactions with the plasma membrane, subsequent penetration into the cells by macropinocytosis, and release into the cytoplasm and nuclei by retrograde transport. Recent reports have also now shown that some of the limitations of protein transduction technology have been overcome. In particular, the use of ubiquitination-resistant proteins has been demonstrated to be a more effective strategy for transduction because the half-life of these molecules is significantly increased. Moreover, the use of the NH2-terminal domain of the influenza virus hemagglutinin-2 subunit (HA2) or photosensitive PTDs has been shown to specifically enhance macropinosome escape. Hence, these and other recent advances in protein transduction technologies have created a number of possibilities for the development of new peptide-based drugs.

Key words: Protein transduction technology; Protein transduction domain; Cell penetrating peptide; Endocytosis

Address correspondence to Hirofumi Noguchi, M.D., Ph.D., Baylor All Saints Medical Center, Baylor Research Institute, 1400 8th Avenue, Fort Worth, TX 76104, USA. Tel: (214) 820-9016; Fax: (214) 820-4952; E-mail: hirofumn@baylorhealth.edu




Cell Transplantation, Vol. 19, pp. 655-665, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X508753
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Review
Cell Delivery: From Cell Transplantation to Organ Engineering

Alejandro Soto-Gutierrez,1* Hiroshi Yagi,1* Basak E. Uygun,1 Nalu Navarro-Alvarez,2 Korkut Uygun,1 Naoya Kobayashi,3 Yong-Guang Yang,2 and Martin L. Yarmush1,4

1Center for Engineering in Medicine and Department of Surgery, Massachusetts General Hospital, Harvard Medical School, and the Shriners Hospitals for Children, Boston, MA, USA
2Transplantation Biology Research Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA
3Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan
4Department of Biomedical Engineering, Rutgers University, Piscataway, NJ, USA

Cell populations derived from adult tissue and stem cells possess a great expectation for the treatment of several diseases. Great efforts have been made to generate cells with therapeutic impact from stem cells. However, it is clear that the development of systems to deliver such cells to induce efficient engraftment, growth, and function is a real necessity. Biologic and artificial scaffolds have received significant attention for their potential therapeutic application when use to form tissues in vitro and facilitate engraftment in vivo. Ultimately more sophisticated methods for decellularization of organs have been successfully used in tissue engineering and regenerative medicine applications. These decellularized tissues and organs appear to provide bioactive molecules and bioinductive properties to induce homing, differentiation, and proliferation of cells. The combination of decellularized organs and stem cells may dramatically improve the survival, engraftment, and fate control of transplanted stem cells and their ultimate clinical utility, opening the doors to a new era of organ engineering.

Key words: Cell transplant; Organ engineering; Decellularized matrices; Natural scaffolds

Address correspondence to Dr. Alejandro Soto-Gutierrez at his current address: Department of Surgery, Transplantation Section, Children's Hospital of Pittsburgh, 3511 Rangos Research Building 530 45th Street, Pittsburgh, PA 15201, USA. E-mail: alejandro.sotogutierrez@chp.edu or Martin L. Yarmush, M.D., Ph.D., Shriners Hospital for Children, 51 Blossom Street, Boston, MA 02114, USA. E-mail: ireis@sbi.org

*These authors provided equal contribution.




Cell Transplantation, Vol. 19, pp. 667-679, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X508762
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Review
Mesenchymal Stem Cells: Mechanisms of Immunomodulation and Homing

Hiroshi Yagi,1,2* Alejandro Soto-Gutierrez,1* Biju Parekkadan,1 Yuko Kitagawa,2 Ronald G. Tompkins,1 Naoya Kobayashi,3 and Martin L. Yarmush1,4

1Center for Engineering in Medicine and Surgical Services, Massachusetts General Hospital, Shriners Hospitals for Children and Harvard Medical School, Boston, MA, USA
2Department of Surgery, Keio University School of Medicine, Tokyo, Japan
3Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan
4Department of Biomedical Engineering, Rutgers University, Piscataway, NJ, USA

Mesenchymal stem cell (MSC) transplantation has been explored as a new clinical approach to repair injured tissue. A growing corpus of studies have highlighted two important aspects of MSC therapy: 1) MSCs can modulate T-cell-mediated immunological responses, and (2) systemically administered MSCs home to sites of ischemia or injury. In this review, we describe the known mechanisms of immunomodulation and homing of MSCs. First, we examine the low immunogenicity of MSCs and their antigen presentation capabilities. Next, we discuss the paracrine interactions between MSCs and innate [dendritic cells (DC)] and adaptive immune cells (T lymphocytes) with a focus on prostaglandin E2 (PGE2), indoleamine 2,3-dioxygenase (IDO), and toll-like receptor (TLR) signaling pathways. We transition to outline the steps of activation, rolling/adhesion, and transmigration of MSCs into target tissues during inflammatory or ischemic conditions. These aspects of MSC grafts-immunomodulation and homing-are contextualized to understand a reported side effect of MSC therapy, cancer development.

Key words: Immunosuppression; T-cell proliferation; Stem cell migration; Interferon-g(IFN-g); Nuclear factor-kB (NF-kb)

Address correspondence to Martin L. Yarmush, M.D., Ph.D., Shriners Hospitals for Children, 51 Blossom Street, Boston, MA 02114. Tel: 617-371-4882; Fax: 617-371-4950; E-mail: ireis@sbi.org

*These authors provided equal contribution.




Cell Transplantation, Vol. 19, pp. 681-689, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X508771
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Biological and Biomechanical Evaluations of Osteochondral Allografts Preserved in Cold Storage Solution Containing Epigallocatechin Gallate

Jung Yoon Bae,1 Dong-Wook Han,2 Shigeyuki Wakitani,3 Masashi Nawata,4 and Suong Hyu Hyon1

1Department of Medical Simulation Engineering, Research Center for Nano Medical Engineering, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan
2Department of Nanomedical Engineering, College of Nanoscience & Nanotechnology,Pusan National University, Busan, Korea
3Department of Orthopedic Surgery, Osaka City University Graduate School of Medicine, Osaka, Japan
4Department of Orthopaedic Surgery, Marunouchi Hospital, Matsumoto, Japan

The beneficial effects of (-)-epigallocatechin-3-O-gallate (EGCG) on the nonfrozen preservation of mammalian cells and tissues are generally not well understood. A storage solution containing EGCG was employed to test the hypothesis that EGCG is capable of extending the storage duration for the cold preservation of articular cartilages. Human articular cartilages were preserved in a storage solution composed of serum-free RPMI-1640 medium with 1% antibiotic-antimycotic solution and 1 mM EGCG at 4ºC for 1, 2, and 4 weeks. The chondrocyte viability (CCK-8 assay), biochemical and immunohistochemical composition [glycosaminoglycans (GAG) and (type II) collagen], and biomechanical property (compressive elastic modulus) were assessed. The chondrocyte viability of the cartilages preserved with EGCG was significantly well maintained for at least 2 weeks with high content of GAG and total collagen. These beneficial effects of EGCG were confirmed by the immunohistochemical observations of well-preserved cartilaginous structures and delayed denaturation of the extracellular matrix in preserved cartilages. There was no significant difference in the compressive elastic modulus (MPa) between the cartilages preserved with and without EGCG. These results suggest that EGCG may play an effective role in preserving osteochondral allografts, which can be exploited in devising strategies for the long-term preservation of other tissues under cold storage conditions.

Key words: Osteochondral allografts; Articular cartilages; Epigallocatechin gallate; Cold preservation

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




Cell Transplantation, Vol. 19, pp. 691-699, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X508780
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Polyampholytes as Cryoprotective Agents for Mammalian Cell Cryopreservation

Kazuaki Matsumura, Jung Yoon Bae, and Suong Hyu Hyon

Department of Medical Simulation Engineering,Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan

Cryoprotective agents (CPAs) such as dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, and propylene glycol have been used for the cryopreservation of cells and tissues. DMSO is the most effective CPA but shows high cytotoxicity and can effect differentiation. <gk>?-Poly-L-lysine (PLL) derivatives show higher cryopreservation efficiency than the conventional CPAs. Culture medium solutions with 7.5 w/w% of PLL whose amino groups of more than 50 mol% were converted to carboxyl groups by succinic anhydride showed higher postthaw survival efficiency of L929 cells than those of current CPAs without the addition of any proteins. In addition, rat mesenchymal stem cells were cryopreserved more effectively than with DMSO and fully retained the potential for proliferation and differentiation. Furthermore, many kinds of cells could be cryopreserved with PLL having the appropriate ratio of COOH groups, regardless of the cell types, including adhesive and floating cells, human- and mouse-derived cells, primary cells, and established cell lines. The properties might be associated with the antifreeze protein properties. These results indicate that these polymeric extracellular CPAs may replace current CPAs and the high viability after thawing and nonnecessity of serum ensure that these CPAs may be used in various preservation fields.

Key words: Cryopreservation; Poly-lysine; Dimethyl sulfoxide; Mesenchymal stem cell; Antifreeze protein

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




Cell Transplantation, Vol. 19, pp. 701-706, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X508799
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

An Improvement in the Attaching Capability of Cryopreserved Human Hepatocytes by a Proteinaceous High Molecule, Sericin, in the Serum-Free Solution

Yoshitaka Miyamoto,1* Naozumi Teramoto,2 Shuji Hayashi,3 and Shin Enosawa1

1Clinical Research Center, National Center for Child Health and Development, Tokyo, Japan
2Department of Life and Environmental Sciences, Faculty of Engineering, Chiba Institute of Technology, Chiba, Japan
3Department of Advanced Medicine in Biotechnology and Robotics, Nagoya University Graduate School of Medicine, Nagoya, Japan

The methodology of cryopreservation of human hepatocytes remains unsatisfactory. Even when the viability of thawed cells is tolerable, the cells often lose the attaching capability to a culture dish, resulting in the cells' inability to survive. Previously, we described the effectiveness of maltose on the attachment of hepatocytes. This article demonstrates that a silk-derived high molecular protein, sericin, improves the cell-attaching capability in the serum-free freezing medium. When human hepatocytes [initial viability: 60.9 ± 3.1% (mean ± SD, n = 3)] were frozen with serum-free Dulbecco's modified Eagle medium (DMEM) containing 10% dimethyl sulfoxide (DMSO), the viability was 29.4 ± 3.2% and the cell-attaching capability 20.4 ± 4.1%. On the other hand, DMEM containing 10% DMSO and 1% sericin increased the values to 45.0 ± 0.8% and 26.2 ± 3.2%. Moreover, the addition of 0.1 mol/L maltose to the sericin-containing medium improved to 42.2 ± 3.2% and 51.1 ± 1.0%, as we demonstrated in a previous report. The present results indicated that sericin combined with maltose is a novel additive in the serum-free freezing medium for human hepatocytes.

Key words: Cryopreservation; Human hepatocytes; Sericin

Address correspondence to Shin Enosawa, Clinical Research Center, National Center for Child Health and Development, 2-10-1 Ookura, Setagayaku, Tokyo 157-8535, Japan. Tel: +81-3-3416-0181; Fax: +81-3-3417-2864; E-mail: senosawa@nch.go.jp

*Present address: Department of Advanced Medicine in Biotechnology and Robotics, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan.




Cell Transplantation, Vol. 19, pp. 707-712, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X508807
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Seventy-Two-Hour Preservation, Resuscitation, and Transplantation of an Isolated Rat Heart With High Partial Pressure Carbon Monoxide Gas (PCO = 400 hPa) and High Partial Pressure Carbon Dioxide (PCO2 = 100 hPa)

Naoyuki Hatayama, Yu Yoshida, and Kunihiro Seki

Resonance Club Co., Ltd., Tokyo, Japan

The cardiac cavity of an isolated rat heart was filled with a Krebs-Henseleit (KH) solution, and the heart was hung in a high-pressure chamber. After the high-pressure chamber had been filled with a mixed gas (PCO = 400 hPa, PCO2 = 100 hPa, PO2 = 900 hPa, PHe = 5600 hPa) and preserved for 72 h, we performed a cervical ectopic heart transplantation on a recipient rat and resuscitated the preserved heart. This is the first incidence in the world of a mammalian organ having been successfully preserved and resuscitated after 72 h via a desiccation method.

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

Address correspondence to Naoyuki Hatayama, Resonance Club Co., Ltd., 5-1, Nihonbashi Kabutocho, Chuo-ku, Tokyo, 103-0046, Japan. Tel: 03-3863-2557; Fax: 03-5847-7901; E-mail: nao_h416@yahoo.co.jp




Cell Transplantation, Vol. 19, pp. 713-721, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X508816
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Renal Protective Effects of Erythropoietin on Ischemic Reperfusion Injury

Manabu T. Moriyama, Tatsuro Tanaka, Nobuyo Morita, Takeo Ishii, Ippei Chikazawa, Kodai Suga, Katsuhito Miyazawa, and Koji Suzuki

Department of Urogenital Surgery, Knazawa Medical University, Ishikawa, Japan

While the problem of organ shortage has not yet been solved, the number of patients who need to be treated with dialysis due to end-stage renal disease (ESRD) is increasing each year. With the aim of eliminating dialytic therapy as much as possible, the opportunities for organ donation from expansive criteria donor (ECD) or marginal donors due to cardiac death have been increasing. With the purpose of extracting organs in a state in which the function is preserved as much as possible, we reexamined the conditions of tissue disorders resulting from temporary ischemia of the organs as well as changes in tissue function and the effects on the preservation of renal function over time by using rat models in order to clinically utilize erythropoietin, which has inhibitory effects on ischemia-reperfusion disorder, as has been conventionally reported. With 8- to 9-week-old Wister male rats, after the right kidney had been resected under general anesthesia, the left renal artery was clamped to inhibit the blood flow for 45 min. At 30 min before inhibiting the blood flow and after releasing the inhibited blood flow, 100 U/kg of recombinant human erythropoietin (rhEPO) was administered via the inferior vena cava and the abdominal cavity, and then the tissues and blood samples were extracted at 6 and 24 h after the release. The renal tissue specimens were evaluated using H&E staining and TUNEL staining in order to observe differences in the expression of apoptosis as well as the renal function and changes in the emergence of active oxygen were investigated by using samples that had been obtained from drawn blood. Moreover, we examined the degree of renal dysfunction by means of neutrophil gelatinase-associated lipocalin (NGAL) in the spot urine samples. The changes in renal function, which were observed according to the serum creatinine level, showed that the renal function was preserved with a significant difference in the rhEPO administration group. The liver deviation enzymes, which had also shown increases in the serum as well as the occurrence of renal dysfunction, showed clear decreases in the serum, even though changes with a significant difference were not observed in the rhEPO administration group. The active oxygen did not show changes before and after ischemia-reperfusion nor changes due to the rhEPO administration. When examining the status of apoptosis in the tissues, apoptosis was shown to be inhibited due to the rhEPO administration. It is believed that the main preservation effects of rhEPO are the elimination of cytopathy/cell death, as derived from the resulting ischemic condition that extends to the target organ before ischemia occurs. In this examination, no direct effects of rhEPO administration on the emergence of active oxygen were observed. It is therefore suggested that there is a possibility of preserving the renal function in marginal donors with a longer agonal stage by effectively using rhEPO.

Key words: Erythropoietin; Marginal donor; Ischemia; Reperfusion injury; Kidney transplantation

Address correspondence to Manabu T. Moriyama, Department of Urogenital Surgery, Knazawa Medical University, 1-1, Uchinada, Kahoku, Ishikawa, 9200293, Japan. Tel: +81-76-218-8145; Fax: 81-76-286-5516; E-mail: moriyama@kanazawa-med.ac.jp




Cell Transplantation, Vol. 19, pp. 723-729, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X508825
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Serum Tissue Inhibitor of Metalloproteinases 1 (TIMP-1) Predicts Organ Recovery From Delayed Graft Function After Kidney Transplantation From Donors After Cardiac Death

Mamoru Kusaka, Yoko Kuroyanagi, Manabu Ichino, Hitomi Sasaki, Takahiro Maruyama, Kunihiro Hayakawa, Ryoichi Shiroki, Atsushi Sugitani, Hiroki Kurahashi, and Kiyotaka Hoshinaga

Department of Urology, Division of Molecular Genetics, Department of Organ Transplantation and Regenerative Medicine, Fujita Health University School of Medicine, Toyoake, Aichi, Japan

Donors after cardiac death (DCD) have recently become an important source of renal transplants to alleviate the shortage of renal grafts in kidney transplantation (KTx), although DCD kidneys often have complications associated with a delayed graft function (DGF). A microarray-based approach using renal biopsy samples obtained at 1 h after KTx from DCD identified the tissue inhibitor of metalloproteinases 1 (TIMP-1) gene as a potential predictive marker for DGF. The current study measured serum TIMP-1 in patients undergoing KTx and analyzed the time course after KTx. The average serum TIMP-1 level before KTx was 240 ± 10 ng/ml (n = 34). In patients undergoing KTx from a living donor (n = 23), the serum TIMP-1 levels showed no increase after KTx (POD1: 226 ± 12, POD2: 211 ± 12, and POD3: 195 ± 10 ng/ml), but in one case, the only patient who required post-KTx HD due to DGF, the level on POD1 was the highest among subjects (361 ng/ml). In contrast, patients undergoing KTx from DCDs (n = 11), the serum TIMP-1 levels increased rapidly after a KTx (POD1: 418 ± 32, POD2: 385 ± 42, and POD3: 278 ± 25 ng/ml). However, two patients who avoided post-KTx HD due to the immediate function of the graft did not show increased levels (<370 ng/ml) on either POD1 or POD2. The peak serum TIMP-1 values appeared to correlate to the post-KTx dialysis period. Furthermore, the increment of serum TIMP-1 on the early POD was found to be predictive of immediate or delayed function of the grafts. These data suggest that monitoring of serum TIMP-1 levels allow the prediction of graft recovery and the need for HD after a KTx from a DCD.

Key words: Donation after cardiac death (DCD); Delayed graft function (DGF); Kidney transplantation; Tissue inhibitor of metalloproteinases 1 (TIMP-1)

Address correspondence to Mamoru Kusaka, M.D., Department of UrologyFujita Health University School of Medicine, 1-98 Dengakugakubo, Kutsukake-cho, Toyoake, Aichi, 470-1192 Japan. Tel: +81-562-93-2181; Fax: +81-562-93-7863; E-mail: mkusaka@fujita-hu.ac.jp




Cell Transplantation, Vol. 19, pp. 731-741, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X508834
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Assessment of Islet Quality Following International Shipping of More Than 10,000 km

Tetsuya Ikemoto,1,2,3 Shinichi Matsumoto,1,2 Takeshi Itoh,2,4 Hirofumi Noguchi,1,2,5 Yoshiko Tamura,6 Andrew M. Jackson,5,6 Masayuki Shimoda,7 Bashoo Naziruddin,6 Nicholas Onaca,6 Yohichi Yasunami,4 and Marlon F. Levy6

1Baylor Institute for Immunology Research, Baylor Research Institute, Dallas, TX, USA
2Baylor All Saints Islet Cell Laboratory, Fort Worth, TX, USA
3Department of Digestive and Pediatric Surgery, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan
4Department of Regenerative Medicine & Transplantation, Faculty of Medicine, Fukuoka University, Fukuoka, Japan
5Institute of Biomedical Studies, Baylor University, Waco, TX, USA
6Baylor Regional Transplantation Institute, Dallas, TX, USA
7Division of Cardiology, Department of Internal Medicine, Baylor Heart and Vascular Institute, Baylor University Medical Center, Dallas, TX, USA

Islet transplantation is an attractive therapy for type 1 diabetes, although some issues remain. One of them is the severe donor shortage in some countries. In this study, we investigated the possibility of international islet shipping beyond 10,000 km to supply islets to countries with donor shortages. Human islets were isolated from six cadaver donors and cultured until shipment. Islets were packed in either gas-permeable bags or in non-gas-permeable bags and shipped from Baylor Research Institute (Dallas, TX, USA) to Fukuoka University (Fukuoka, Japan). Pre- and postshipment islet number, purity, viability, and stimulation index (by glucose stimulation test) were assessed. Shipped 1,500 IE islets were transplanted into streptozotocininduced diabetic nude mice for in vivo assay. The distance of our shipment was 11,148.4 km, and the mean duration of the shipments was 48.2 ± 8.2 h. The islet number recovery rate (postshipment/preshipment) was significantly higher in gas-permeable bags (56.4 ± 10.1% vs. 20.5 ± 20.6%, p < 0.01). Islet purity was significantly reduced during shipment in non-gas-permeable bags (from 47.7 ± 18.6% to 40.2 ± 28.2 in gaspermeable bags vs. from 50.4 ± 6.4% to 25.9 ± 15.6% in non-gas-permeable bags, p < 0.05). Islet viability and stimulation index did not change significantly between pre- and postshipping, in either gas-permeable bags or in non-gas-permeable bags. One of three diabetic nude mice (33.3%) converted to normoglycemia. It is feasible to ship human islet cells internationally in gas-permeable bags. This strategy would promote basic and preclinical research for countries with donor shortages, even though the research centers are remote (over 10,000 km from the islet isolation center).

Key words: Islet transplantation; International shipping; Gas-permeable bag; Basic and preclinical research

Address correspondence to Shinichi Matsumoto, M.D., Ph.D., Baylor All Saints Islet Cell Laboratory, 1400 Eight Ave, Fort Worth, TX 76104, USA. Tel: 817-922-2570; E-mail: shinichm@baylorhealth.edu or Tetsuya Ikemoto, M.D., Ph.D., Department of Regenerative Medicine & Transplantation, Faculty of Medicine, Fukuoka University, Fukuoka, 814-0180, Japan. Tel: +81-88-633-7139; E-mail: tikemoto@clin.med.tokushimau.ac.jp




Cell Transplantation, Vol. 19, pp. 743-750, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X508843
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Improved Yield and Functional Parameters of Rat Pancreas Islets Isolated Under Intramuscular Anesthesia

Joon Ye Kim,1* Jeong-Ik Lee,1,5* Jin Ho Jeong,1,2 Yuhui Fang,1,2 Man Ki Ju,1,4 Soo Jin Kim,1,4 Kyu Ha Huh,1,4 Myoung Soo Kim,1,4 and Yu Seun Kim1,2,3,4

1The Research Institute for Transplantation, Yonsei University, Seoul, South Korea
2Graduate Program of Nanoscience and Technology, Yonsei University, Seoul, South Korea
3BK21 for Medical Science, Yonsei University, College of Medicine, Seoul, South Korea
4Department of Transplantation Surgery, Yonsei University Health System, Seoul, South Korea
5Department of Biomedical Science & Technology, IBST, Konkuk University, Seoul, South Korea

Intraperitoneal (IP) anesthesia is commonly used for laboratory animal experiments including rat islet isolation. However, the direct effects of anesthetics on pancreatic islets have been neglected. This study compared the islet function and recovery yield from rats that were anesthetized using IP and intramuscular (IM) injection. In addition, the lag time required to lose deep pain was measured in the following anesthetics combinations. Lewis rats were anesthetized using ketamine and xylazine (K/X) or zoletil and xylazine (Z/X). A glucose challenge test was performed on each group of prepared islets. The effect of the anesthetic agents (e.g., ketamine, zoletil, xylazine alone, and the combination of K/X and Z/X) on cell lines (rat insulinoma; RIN-5F) was investigated by determining their effect on the cell viability, the amount of insulin, and insulin mRNA expression levels of RIN-5F. The time needed for deep anesthesia in IM anesthesia was significantly shortened in comparison to IP [K/X (IM: 313 ± 66 s, IP: 371 ± 84 s) and Z/X (IM: 206 ± 76 s, IP: 245 ± 92 s)]. In addition, number of isolated islet yield by IM anesthesia was significantly improved [K/X (IM: 1530 ± 242, IP: 1245 ± 149) and Z/X (IM: 1136 ± 226, IP: 511 ± 154)]. The functions of fresh islets, indicated by the stimulation index, acquired under IM anesthesia was better preserved than that of IP. The viability and the insulin secretion of RIN-5F were decreased at 24 and 48 h. Insulin gene expression levels were decreased at 24 h as well. Anesthetics may be absorbed through the pancreas surface to the islets and have a direct effect, resulting in islet exposure and deterioration during isolation. In conclusion, for rodent islet isolation, IM anesthesia is simpler and safer in comparison to IP anesthesia.

Key words: Rat islet; RIN-5F; Intraperitoneal anesthesia; Intramuscular anesthesia

Address correspondence to Yu Seun Kim, M.D., Department of Surgery, Yonsei University College of Medicine, 134 Shinchon-Dong, Seodaemun-Ku, Seoul 120-752, South Korea. Tel: +82-2-2228-2115; Fax: +82-2-313-8289; E-mail: yukim@yuhs.ac or yukim@yumc.yonsei.ac.kr

*These two authors provided equal contribution to this work and should be considered co-first authors.




Cell Transplantation, Vol. 19, pp. 751-758, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368909X508852
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Comparison of Modified Celsior Solution and M-Kyoto Solution for Pancreas Preservation in Human Islet Isolation

Hirofumi Noguchi,1,2 Bashoo Naziruddin,3 Nicholas Onaca,3 Andrew Jackson,2,3 Masayuki Shimoda,4 Tetsuya Ikemoto,1 Yasutaka Fujita,1 Naoya Kobayashi,5 Marlon F. Levy,1,2 and Shinichi Matsumoto1

1Baylor All Saints Medical Center, Baylor Research Institute, Fort Worth, TX, USA
2Institute of Biomedical Studies, Baylor University, Waco, TX, USA
3Baylor Regional Transplant Institute, Dallas-Fort Worth, TX, USA
4Division of Cardiology, Department of Internal Medicine, Baylor Heart and Vascular Institute, Baylor University Medical Center, Dallas, TX, USA
5Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan

Since the successful demonstration of the Edmonton protocol, islet transplantation has advanced significantly on several fronts, including improved pancreas preservation systems. In this study, we evaluated two different types of organ preservation solutions for human islet isolation. Modified Celsior (Celsior solution with hydroxyethyl starch and nafamostat mesilate; HNC) solution and modified Kyoto (MK) solution were compared for pancreas preservation prior to islet isolation. Islet yield after purification was significantly higher in the MK group than in the HNC group (MK = 6186 ± 985 IE/g; HNC = 3091 ± 344 IE/g). The HNC group had a longer phase I period (digestion time), a higher volume of undigested tissue, and a higher percentage of embedded islets, suggesting that the solution may inhibit collagenase. However, there was no significant difference in ATP content in the pancreata or in the attainability of posttransplant normoglycemia in diabetic nude mice between the two groups, suggesting that the quality of islets was similar among the two groups. In conclusion, MK solution is better for pancreas preservation before islet isolation than HNC solution due to the higher percentage of islets that can be isolated from the donor pancreas. MK solution should be the solution of choice among the commercially available solutions for pancreatic islet isolation leading to transplantation.

Key words: Islet transplantation; Preservation solution; Modified Kyoto solution; Modified Celsior solution

Address correspondence to Shinichi Matsumoto, M.D., Ph.D., Baylor All Saints Medical Center, Baylor Research Institute, 1400 8th Avenue, Fort Worth, TX 76104, USA. Tel: (817) 922-2570; Fax: (817) 922-4645; E-mail: shinichm@baylorhealth.edu or Hirofumi Noguchi, M.D., Ph.D., Baylor All Saints Medical Center / Baylor Institute for Immunology Research, Baylor Research Institute, 3434 Live Oak St., Dallas, TX 75204, USA. Tel: (214) 820-9014; Fax: (214) 820-4952; E-mail: hirofumn@baylorhealth.ed




Cell Transplantation, Vol. 19, pp. 759-764, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X508861
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Expression of Phosphatidylserine-Specific Phospholipase A1 mRNA in Human THP-1-Derived Macrophages

Hiroyuki Hosono,1 Masato Homma,2 Yoko Ogasawara,2 Kumiko Makide,3 Junken Aoki,3 Hideaki Niwata,1 Machiko Watanabe,1 Keizo Inoue,1 Nobuhiro Ohkohchi,4 and Yukinao Kohda2

1Faculty of Pharmaceutical Sciences, Teikyo University, Kanagawa, Japan
2Department of Pharmaceutical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Ibaraki, Japan
3Department of Molecular & Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Miyagi, Japan
4Department of Surgery, Advanced Biomedical Applications, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Ibaraki, Japan

The expression of phosphatidylserine-specific phospholipase A1 (PS-PLA1) is most upregulated in the genes of peripheral blood cells from chronic rejection model rats bearing long-term surviving cardiac allografts. The expression profile of PS-PLA1 in peripheral blood cells responsible for the immune response may indicate a possible biological marker for rejection episodes. In this study, PS-PLA1 mRNA expression was examined in human THP-1-derived macrophages. The effects of several immunosuppressive agents on this expression were also examined in in vitro experiments. A real-time RT-PCR analysis revealed that PS-PLA1 mRNA expression was found in human THP-1-derived macrophages. This expression was enhanced in the cells stimulated with lipopolysaccharide (LPS), a toll-like receptor (TLR) 4 ligand. Other TLR ligands (TLR2, 3, 5, 7, and 9) did not show a significant induction of PS-PLA1 mRNA. The time course of the mRNA expression profiles was different between PS-PLA1 and tumor necrosis factor-a(TNF-a), which showed a maximal expression at 12 and 1 h after LPS stimulation, respectively. Among the observed immunosuppressive agents, corticosteroids, prednisolone, 6a-methylprednisolone, dexamethasone, and beclomethasone inhibited PS-PLA1 expression with half-maximal inhibitory concentrations less than 3.0 nM, while methotrexate, cyclosporine A, tacrolimus, 6-mercaptopurine, and mycophenoic acid showed either a weak or moderate inhibition. These results suggest that the expression of PS-PLA1 mRNA in THP-1-derived macrophages is activated via TLR4 and it is inhibited by corticosteroids, which are used at high dosages to suppress chronic allograft rejection.

Key words: Phosphatidylserine-specific phospholipase A1 (PS-PLA1); THP-1-derived macrophages; mRNA expression; Corticosteroids; Chronic rejection

Address correspondence to Hiroyuki Hosono, Faculty of Pharmaceutical Sciences, Teikyo University, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan. Tel: +81-3-3964-1211 (ext. 7486); Fax: +81-3-3964-9425; E-mail: hhosono@pharm.teikyo-u.ac.jp




Cell Transplantation, Vol. 19, pp. 765-774, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X508870
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Prevention of Graft-Versus-Host Diseases by In Vivo supCD28mAb-Expanded Antigen-Specific nTreg Cells

Yusuke Kitazawa,1,2 Xiao-Kang Li,1 Zhong Liu,1 Hiromitsu Kimura,1 Yoshitaka Isaka,2 Thomas Hünig,3 and Shiro Takahara2

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

Naturally occurring CD4+CD25+ Treg cells (nTregs) can be exploited to establish an immunologic tolerance to non-self-antigens. The in vivo administration of a single superagonistic CD28-specific monoclonal antibody (supCD28mAb) to naive rat preferentially expanded the nTregs, which induced a potent inhibition of lethality of the graft-versus-host (GvH) diseases. The appearance of increased Foxp3 molecules was accompanied with a polarization towards a Th2 cytokine profile with a decreased production of IFN-g and increased production of IL-4 and IL-10 in the serum of the antibody-treated rat. The peripheral Foxp3 nTregs are decreased in acute GvHD, while supCD28mAb administration showed that nTregs were preferentially proliferating in vivo, thus resulting in the significant prevention of the GvH disease. Furthermore, antigen-specific nTregs could suppress conventional T-cell proliferation stimulated with alloantigen in vitro. Taken together, our findings demonstrate that the potent regulatory functions of the Tregs for the treatment of GvHD are antigen specific. These data also provide evidence that GvHD is associated with decrease of Tregs in the periphery of the host. The determination of the Foxp3 Tregs can be a helpful tool to discriminate GvHD severity and lethality after allogeneic stem cell transplantation.

Key words: Graft-versus-host disease; nTregs; Antigen-specific nTregs

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




Cell Transplantation, Vol. 19, pp. 775-782, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X508889
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Brain Death in Combination With Warm Ischemic Stress During Isolation Procedures Induces the Expression of Crucial Inflammatory Mediators in the Isolated Islets

Yukihiko Saito,1 Masafumi Goto,1,2 Kozue Maya,2 Norihiko Ogawa,1 Keisei Fujimori,3 Yoshimochi Kurokawa,4 and Susumu Satomi1

1Division of Advanced Surgical Science and Technology, Tohoku University, Sendai, Japan
2Tohoku University International Advanced Research and Education Organization, Tohoku University, Sendai, Japan
3Medical Safety Management Office, Tohoku University, Sendai, Japan
4Tohoku University Innovation of New Biomedical Engineering Center, Tohoku University, Sendai, Japan

Tissue factor (TF) and monocyte chemoattractant protein-1 (MCP-1) expressed on the islets have been identified as the main trigger of the instant blood-mediated inflammatory reaction (IBMIR) in islet transplantation. Because the key steps that directly induce TF and MCP-1 remain to be determined, we focused on the influence of brain death (BD) on TF and MCP-1 expression in the pancreatic tissues and isolated islets using a rodent model. TF and MCP-1 mRNA levels in the pancreatic tissues were similar between the BD and the control group. However, TF and MCP-1 mRNA in the fresh islets of the BD group were significantly higher than that of the control group (p < 0.01). BD may thus be suggested to be of great importance as an initiator of TF and MCP-1 induction in the isolated islets. Furthermore, the upregulation of crucial inflammatory mediators induced by BD could be exacerbated by warm ischemic damage during digestion procedures. In the present study, the islet yield and purity were affected by BD. However, almost no influences were observed with respect to islet viability, indicating that the expression of inflammatory mediators rather than islet viability is more susceptible to BD. According to the change in time course of TF and MCP-1 expression in the isolated islets, the selected time point for islet infusion in current clinical islet transplantation was thus shown to be at its worst level, at least with respect to the damage caused by BD and ischemic stress. In conclusion, BD in combination with warm ischemic stress during isolation procedures induces a high expression of TF and MCP-1 in the isolated islets. In order to reduce the expression of crucial inflammatory mediators in the islet grafts, the management of the pancreas from brain-dead donors with early anti-inflammatory treatments is thus warranted.

Key words: Islets; Transplantation; Brain death; Tissue factor (TF); Monocyte chemoattractant protein-1 (MCP-1)

Address correspondence to Masafumi Goto, M.D., Ph.D., Tohoku University International Advanced Research and Education Organization, Tohoku University, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, Japan. Tel: +81 22 717 7895; Fax: +81 22 717 7899; E-mail: gotokichi@aol.com




Cell Transplantation, Vol. 19, pp. 783-789, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X508898
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Human Immune Reactivity Against Liver Sinusoidal Endothelial Cells From GalTa(1,3)GalT-Deficient Pigs

Daan van Poll,1 Yakoov Nahmias,1 Alejandro Soto-Gutierrez,1 Mitra Ghasemi,2 Hiroshi Yagi,1 Naoya Kobayashi,3 Martin L. Yarmush,1,4 and Martin Hert1,2

1Center for Engineering in Medicine and Department of Surgery, Massachusetts General Hospital, Harvard Medical School, and the Shriners Hospitals for Children, Boston, MA, USA
2Department of Surgery, Transplantation Unit, Massachusetts General Hospital, Boston, MA, USA
3Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan
44Department of Biomedial Engineering, Rutgers University, Piscataway, NJ, USA

Elimination of galactose-a(1,3)galactose (Gal) expression in pig organs has been previously shown to prevent hyperacute xenograft rejection. However, naturally present antibodies to non-Gal epitopes activate endothelial cells, leading to acute humoral xenograft rejection. Still, it is unknown whether xenogeneic pig liver sinusoidal endothelial cells (LSECs) from a(1,3)galactosyltransferase (GalT)-deficient pigs are damaged by antibody and complement-mediated mechanisms. The present study examined the xeno-antibody response of LSECs from GalT-deficient and wild pigs. Isolated LSEC from wild-type and GalT pigs were expose to human and baboon sera; IgM and IgG binding was analyzed by flow cytometry. Complement activation (C3a and CH50) was quantified in vitro from serum-exposed LSEC cultures using Enzyme-Linked Immuno-Sorbent assay (ELISA). Levels of complement-activated cytotoxicity (CAC) were also determined by a fluorescent Live-Dead Assay and by the quantification of LDH release. IgM binding to GalT knockout (KO) LSECs was significantly lower (80% human and 87% baboon) compare to wild-type pig LSEC. IgG binding was low in all groups. Moreover, complement activation (C3a and CH50) levels released following exposure to human or baboon sera were importantly reduced (42% human and 52% baboon), CAC in GalT KO LSECs was reduced by 60% in human serum and by 72% in baboon serum when compared to wild-type LSECs, and LDH release levels were reduced by 37% and 57%, respectively. LSECs from GalT KO pigs exhibit a significant protection to humoral-induced cell damage compared to LSECs from wild pigs when exposed to human serum. Although insufficient to inhibit xenogeneic reactivity completely, transgenic GalT KO expression on pig livers might contribute to a successful application of clinical xenotransplantation in combination with other protective strategies.

Key words: Xenotransplantation; Liver endothelial cells; GalTa(1,3)GalT-knockout pigs

Address correspondence to Martin Hertl, M.D., Transplant Unit, Massachusetts General Hospital, 55 Fruit Street, White 546, Boston, MA 02114, USA. Tel: 617-726-3664; Fax: 617-724-5993; E-mail: mhertl@partners.org




Cell Transplantation, Vol. 19, pp. 791-797, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X508906
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Bone Repair Using a Hybrid Scaffold of Self-Assembling Peptide PuraMatrix and Polyetheretherketone Cage in Rats

Hiroyuki Nakahara,1 Haruo Misawa,1 Aki Yoshida,1 Takayuki Hayashi,1 Masato Tanaka,1 Takayuki Furumatsu,1 Noriaki Tanaka,2 Naoya Kobayashi,2 and Toshifumi Ozaki1

1Department of Orthopaedic Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
2Department of Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan

Self-assembling peptide scaffold (SAPS) is well known to have very good bone conduction properties. However, the intensity of SAPS is too weak to actually use it for a clinical bone regeneration. Therefore, we have produced a hybrid scaffold system that involves fabricating a cage from polyetheretherketone (PEEK) that has high intensity, filling the interior of this cage with SAPS, and then transplanted this hybrid scaffold to bone defects in rat femurs. After 28 days, soft X-ray radiographs and histological assessment revealed that good new bone formation was clearly observed in the defects transplanted the PEEK cage with SAPS, but not in the PEEK cage only. The PEEK cage maintained a form and osteoconduction ability of internal SAPS, and SAPS promoted bone formation inside the PEEK; therefore, each was in charge of intensity and bone regeneration separately. The present study suggests that hybrid scaffolds made from PEEK cages and SAPS can be useful tools for the regeneration of load-bearing bones, based on the idea that it should be possible to develop ideal bone filler materials by combining the strength of artificial bone with the bone regeneration and bone conduction properties of SAPS.

Key words: Self-assembling peptide scaffold (SAPS); Bone repair; Polyetheretherketone (PEEK); Bone conduction

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: tozaki@md.okayamau.ac.jp




Cell Transplantation, Vol. 19, pp. 799-806, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X508915
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Treatment of Acute Liver Failure in Mice by Hepatocyte Xenotransplantation

Tsuyoshi Yamamoto,1 Nalú Navarro-Alvarez,1 Alejandro Soto-Gutierrez,1 Takeshi Yuasa,1 Masaya Iwamuro,2 Yasuhiro Kubota,1 Masayuki Seita,1 Hironobu Kawamoto,1 Shahid M. Javed,1 Eisaku Kondo,3 Hirofumi Noguchi,4 Satoru Kobayashi,5 Shuhei Nakaji,6 and Naoya Kobayashi1

1Department of Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
2Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
3Department of Pathology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
4Baylor Research Institute, Islet Cell Transplantation Laboratory, Baylor Institution for Immunology Research, Dallas, TX, USA
53-DMatrix, Ltd., Tokyo, Japan
6Department of Biomedical Engineering, School of Engineering, Okayama University of Science, Okayama, Japan

Liver diseases still have a high mortality even though liver transplantation has become a standard treatment. Currently, hepatocyte transplantation has been proposed as another promising strategy. One limitation is the availability of human livers as a source of hepatocytes. Because of an unlimited supply, the use of porcine hepatocytes might address this problem. Regardless of the source, once isolated hepatocytes lose specific functionality due to the loss of the natural microenvironment. For this reason, we tested the ability of a self-assembling peptide nanofiber (SAPNF) to provide a provisional three-dimensional (3D) support to interact with cells to control their function in vivo. Isolated porcine hepatocytes were embedded in SAPNF, or collagen type I and transplanted by direct injection into the splenic pulp of SCID mice suffering from acute liver failure (ALF) by 90% hepatectomy. SAPNF porcine hepatocyte transplantation produced engraftment that was far superior to that obtained using collagen and prolonged the survival of mice with ALF, in contrast with controls. An ultrastructural evaluation using transmission electron microscopy indicated extensive cell-cell communication and preservation of hepatocyte architecture. The transplanted SAPNF hepatocytes showed higher expression of albumin and PAS and lower apoptotic events assessed by TUNEL staining. Hepatocytes culture in a truly 3D network allows in vivo maintaining of differentiated functions, and once transplanted between widely divergent species can function to correct acute liver failure in mice and prolong their survival.

Key words: Hepatocytes; Acute liver failure; Hepatocyte transplantation; Xenotransplantation; Self-assembling peptide nanofiber

Address correspondence to Naoya Kobayashi, M.D., Ph.D., Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama, 700-8558, Japan. Tel: (+81) 86-235-7255; Fax: (+81) 86-235-7485; E-mail: immortal@md.okayama-u.ac.jp




Cell Transplantation, Vol. 19, pp. 807-813, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X508924
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Engineering Liver Tissues Under the Kidney Capsule Site Provides Therapeutic Effects to Hemophilia B Mice

Kazuo Ohashi,1 Kohei Tatsumi,1,2 Rie Utoh,1 Soichi Takagi,1 Midori Shima,2 and Teruo Okano1

1Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, Tokyo, Japan
2Department of Pediatrics, Nara Medical University, Nara, Japan

Recent advances in liver tissue engineering have encouraged further investigation into the evaluation of therapeutic benefits based on animal disease models. In the present study, liver tissues were engineered in coagulation factor IX knockout (FIX-KO) mice, a mouse model of hemophilia B, to determine if the tissue engineering approach would provide therapeutic benefits. Primary hepatocytes were isolated from the liver of wild-type mice and suspended in a mixture of culture medium and extracellular matrix components. The hepatocyte suspension was injected into the space under the bilateral kidney capsules of the FIX-KO mice to engineer liver tissues. The plasma FIX activities (FIX:C) of the untreated FIX-KO mice were undetectable at any time point. In contrast, the liver tissue engineered FIX-KO mice achieved 1.5-2.5% of plasma FIX activities (FIX:C) and this elevated FIX:C level persisted throughout the 90 day experimental period. Significant FIX mRNA expression levels were found in the engineered liver tissues at levels similar to the wild-type livers. The present study demonstrates that liver tissue engineering could provide therapeutic benefits in the treatment of hemophilia B.

Key words: Hemophilia B; Liver tissue engineering; Cell therapy; Blood clotting factors; Drug delivery system; Regenerative medicine

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: +81-3-3353-8112, ext. 66220; Fax: +81-3-3359-6046; E-mail: ohashi@abmes.twmu.ac.jp




Cell Transplantation, Vol. 19, pp. 815-822, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X508933
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Engineering of an Hepatic Organoid to Develop Liver Assist Devices

Alejandro Soto-Gutierrez,1,2* Nalú Navarro-Alvarez,1*# Hiroshi Yagi,2 Yakoov Nahmias,2 Martin L. Yarmush,2,3 and Naoya Kobayashi1

1Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan
2Center for Engineering in Medicine and Department of Surgery, Massachusetts General Hospital, Harvard Medical School, and the Shriners Hospitals for Children, Boston, MA, USA
3Department of Biomedical Engineering, Rutgers University, Piscataway, NJ, USA

Cell-based technologies to support/restore liver function represent one of the most promising opportunities in the treatment of acute liver failure. However, the understanding of the constituent cell types that interact to achieve liver-specific structure and function has not been achieved in the development of liver assist devices (LADs). Here we show that hepatocytes migrated toward and adhered and formed sinusoids-like structures in conjunction with liver nonparenchymal cells, and that this liver organoid formed sophisticated tissue after 7 days in an implanted LAD in rodents. Hepatocytes only or in combination with human nonparenchymal liver cell lines (endothelial, cholangiocytes, and stellate cells) were cultured in Matrigel. Ultrastructural analysis showed that the hepatocyte-decorated endothelial vascular structures resemble in vivo sinusoids containing plate-like structures, bile canaliculi, and lumen. The sinusoid-like structures retained albumin secretion and drug metabolism capabilities. In addition, LADs containing cocultures of human liver nonparenchymal cells were transplanted in animals for a week; the liver tissue formed sophisticated structures resembling the liver. These results demonstrate the importance of nonparenchymal cells in the cellular composition of LADs. The novelty of the culture's sinusoid-like organization and function strongly support the integration of liver nonparenchymal units into hepatocyte coculture-based LADs as a potential destination therapy for liver failure.

Key words: Organoid; Liver support; Liver cell therapy; Liver failure

Address correspondence to Alejandro Soto-Gutierrez, M.D., Ph.D., Department of Surgery, Transplantation Section, Children's Hospital of Pittsburgh, 3511 Rangos Research Building, 530 45th Street, Pittsburgh, PA 15201, USA. Tel: (412) 692-5562; Fax: (412) 692-6599; E-mail: alejandro.sotogutierrez@chp.edu

*These authors provided equal contribution.
#Present address: Bone Marrow Transplantation Section, Transplantation Biology Research Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.




Cell Transplantation, Vol. 19, pp. 823-830, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X508942
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Bone Marrow Mesenchymal Stromal Cells Attenuate Organ Injury Induced by LPS and Burn

Hiroshi Yagi,1* Alejandro Soto-Gutierrez,1* Yuko Kitagawa,2 Arno W. Tilles,1 Ronald G. Tompkins,1 and Martin L. Yarmush1,3

1Department of Surgery, Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School and Shriners Hospitals for Children, Boston, MA, USA
2Department of Surgery, Keio University School of Medicine, Tokyo, Japan
3Department of Biomedical Engineering, Rutgers University, Piscataway, NJ, USA

Bone marrow mesenchymal stromal cells (MSCs) suppress immune cell responses and have beneficial effects in various inflammatory-related immune disorders. A therapeutic modality for systemic inflammation and its consequences is not available yet. Thus, this work investigates the therapeutic effects of MSCs in injury models induced by lipopolysaccharide (LPS) or burn. Gene expression was analyzed in MSCs when exposed to inflammatory serum from injured animals and it showed remarkable alterations compared to normal culture. In addition, injured animals were transplanted intramuscularly with MSCs. Forty-eight hours after cell transplantation, kidney, lung, and liver were analyzed for infiltration of inflammatory cells and TUNEL-expressing cells. Results showed that MSCs attenuate injury by reducing the infiltration of inflammatory cells in various target organs and by reducing cell death. These data suggest that MSCs emerge as key regulators of immune/inflammatory responses in vivo and as attractive candidates for cell-based treatments for systemic inflammatory-based disorders.

Key words: Anti-inflammation; Antiapoptosis; Sepsis; Burn; Lipopolysaccharide (LPS); Systemic inflammatory response syndrome (SIRS); Multiple organ injury

Address correspondence to Martin L. Yarmush, M.D., Ph.D., Shriners Hospitals for Children, 51 Blossom Street, Boston, MA 02114, USA. Tel: 617-371-4882; Fax: 617-371-4950; E-mail: ireis@sbi.org

*These authors provided equal contribution.




Cell Transplantation, Vol. 19, pp. 831-839, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X508951
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Comparative Analysis of Endoderm Formation Efficiency Between Mouse ES Cells and iPS Cells

Masaya Iwamuro,1 Toshiyuki Komaki,2 Yasuhiro Kubota,3 Masayuki Seita,3 Hironobu Kawamoto,3 Takeshi Yuasa,3 Javed M. Shahid,3 Reham A. R. A. Hassan,3 Wael A. R. A. Hassan,4 Shuhei Nakaji,5 Yuriko Nishikawa,6 Eisaku Kondo,6 Kazuhide Yamamoto,1 and Naoya Kobayashi3

1Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
2Okayama University Medical School, Okayama, Japan
3Department of Gastroenterological Surgery, Transplant and Surgical Oncology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
4Department of Pathophysiology-Periodontal Science, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
5Department of Biomedical Engineering, School of Engineering, Okayama University of Science, Okayama, Japan
6Department of Pathology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan

Definitive endoderm (DE) derived from stem cells holds potential to differentiate into hepatocytes. Stem cell therapy using those cells has potential for a treatment of liver disease. To date, various ways of inducing hepatocytes from embryonic stem (ES) cells have been reported by researchers. However, it has not been proved enough that induced pluripotent stem (iPS) cells behave in the same manner as ES cells in endoderm differentiation. The purpose of this study was to establish an efficient method to induce DE from iPS cells, through comparatively analyzing the efficacy of endoderm formation from mouse ES cells. Furthermore, the efficiency of a serum-free medium in the differentiation into DE was investigated. Mouse ES cells and iPS cells were floated in culture medium for 2 or 5 days and embryoid bodies (EB) were formed. Subsequently, DE was induced with 100 ng/ml activin A and 100 ng/ml basic fibroblast growth factor (bFGF). RT-PCR and real-time PCR analyses were carried out at each step to determine the gene expression of EB markers. The difference in cellular proliferation between serum-containing and serum-free media was examined by an MTS assay in EB and DE induction. iPS cells showed the paralleled mRNA expression to ES cells in each step of differentiation into EB, but the levels of expression of Sox17 and Foxa2 were relatively higher in ES cell-derived DE, whereas Cxcr4 expression was higher in iPS cell-derived DE. The utilization of serum-free medium for iPS cells showed significantly favorable cellular proliferation during EB formation and subsequent DE induction. Forming EB for 5 days and subsequently DE induction with activin A and bFGF with serum-free medium was an appropriate protocol in iPS cells. This may represent an important step for generating hepatocytes from iPS cells for the development of cell therapy.

Key words: Induced pluripotent stem cells (iPS); Embryonic stem cells; Definitive endoderm (DE)

Address correspondence to Masaya Iwamuro, M.D., Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-Ku, Okayama 700-8558, Japan. Tel: +81-86-235-7219; Fax: +81-86-225-5991; E-mail: iwamuromasaya@yahoo.co.jp




Cell Transplantation, Vol. 19, pp. 841-847, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X508960
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Hepatic Differentiation of Mouse iPS Cells In Vitro

Masaya Iwamuro,1 Toshiyuki Komaki,2 Yasuhiro Kubota,3 Masayuki Seita,3 Hironobu Kawamoto,3 Takeshi Yuasa,3 Javed M. Shahid,3 Reham A. R. A. Hassan,3 Wael A. R. A. Hassan,4 Shuhei Nakaji,5 Yuriko Nishikawa,6 Eisaku Kondo,6 Kazuhide Yamamoto,1 Ira J. Fox,7 and Naoya Kobayashi3

1Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
2Okayama University Medical School, Okayama, Japan
3Department of Gastroenterological Surgery, Transplant and Surgical Oncology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
4Department of Pathophysiology-Periodontal Science, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
5Department of Biomedical Engineering, School of Engineering, Okayama University of Science, Okayama, Japan
6Department of Pathology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
7Department of Surgery and Pediatric Transplantation, University of Pittsburgh School of Medicine, Pittsburg, PA, USA

Induced pluripotent stem (iPS) cells are pluripotent and are able to unlimitedly proliferate in vitro. This technical breakthrough in creating iPS cells from somatic cells has noteworthy implications for overcoming the immunological rejection and the ethical issues associated with the derivation of embryonic stem cells from embryos. In the current work, we present an efficient hepatic differentiation of mouse iPS cells in vitro. iPS cells were cultured free floating to induce the formation of embryoid bodies (EB) for 5 days. EB were transferred to a gelatin-coated plate and treated with 100 ng/ml activin A and 100 ng/ml basic fibroblast growth factor (bFGF) for 3 days to induce definitive endoderm. Cells were further cultured for 8 days with 100 ng/ml hepatocyte growth factor (HGF) to generate hepatocytes. Characterization was performed by RTPCR assay. Functional analysis for albumin secretion and ammonia removal was also carried out. iPS cell-derived hepatocyte-like cells (iPS-Heps) were obtained at the end of the differentiation program. Expression levels of a gestational hepatocyte gene and lineage-specific hepatic genes intensified in iPS-Heps. The production of albumin increased in a time-dependent manner. iPS-Heps were capable of metabolizing ammonia. We present here instant hepatic differentiation of mouse iPS cells using combined 3-day treatments of activin A and bFGF with subsequent 8-day HGF. Our study will be an important step to generate hepatocytes from human iPS cells as a new source for liver-targeted cell therapies.

Key words: Induced pluripotent stem (iPS) cells; Hepatic differentiation; Hepatocytes

Address correspondence to Masaya Iwamuro, M.D., Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-Ku, Okayama 700-8558, Japan. Tel: +81-86-235-7219; Fax: +81-86-225-5991l; E-mail: iwamuromasaya@yahoo.co.jp




Cell Transplantation, Vol. 19, pp. 849-856, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X508979
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Establishment of an Immortalized Porcine Liver Cell Line JSNK-1 With Retroviral Transduction of SV40T

Javed M. Shahid,1 Masaya Iwamuro,2 Hiromi Sasamoto,1 Yasuhiro Kubota,1 Masayuki Seita,1 Hironobu Kawamoto,1 Shuhei Nakaji,3 Hirofumi Noguchi,4 Kazuhide Yamamoto,2 and Naoya Kobayashi1

1Department of Gastroenterological Surgery, Transplant and Surgical Oncology, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan
2Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan
3Department of Biomedical Engineering, School of Engineering, Okayama University of Science, Okayama, Japan
4Baylor Research Institute, Islet Cell Transplantation Laboratory, Baylor Institution for Immunology Research, Dallas, TX, USA

Maintenance of freshly isolated porcine liver cells in vitro is limited for a short period of time. Therefore, establishment of easy handling cell lines is extremely important for in vitro study for liver cells and their possible utilization for cell differentiation and growth of stem cells. Porcine liver cells were transduced with a retroviral vector SSR#69 expressing SV40T, one of SSR#69-immortalized porcine liver cell lines, JSNK-1, was established and characterized. Morphology of JSNK-1 cells was spindle shaped. When the cells became confluent, JSNK-1 cells revealed hills-and-valleys pattern. In the presence of vitamin A, JSNK-1 cells showed big droplets inside the cytoplasm, which were positive with PAS staining. JSNK-1 cells showed the gene expression of collagen type 1a1, collagen type 1a2, FLT-1, b-actin, and SV40T. Immunostaining study revealed that JSNK-1 cells produced collagen, vimentin, and a-smooth muscle actin. JSNK-1 cells possessed the characteristics of the liver stellate cells. JSNK-1 cells produced hepatocyte growth factor (HGF) in a time-dependent manner. When cocultured with iPS cells towards the hepatic differentiation, JSNK-1 cells facilitated their hepatic differentiation in terms of albumin production. In conclusion, JSNK-1 cells would be valuable in the study of liver stellate cell pathophysiology and contribute to the optimization of hepatic differentiation of iPS cells.

Key words: Porcine liver cells; Stellate cells; Immortalization; SV40T; Induced pluripotent stem (iPS) cells

Address correspondence to Javed M. Shahid, M.B.B.S., Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama, 700-8558, Japan. Tel: +81-86-235-7255; Fax: +81-86-221-8775; E-mail: drshjaved@gmail.com or drshjaved@yahoo.com




Cell Transplantation, Vol. 19, pp. 857-864, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X508988
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Characteristics of CD133+ Human Colon Cancer SW620 Cells

Hironobu Kawamoto,1 Takeshi Yuasa,1 Yasuhiro Kubota,1 Masayuki Seita,1 Hiromi Sasamoto,1 Javed M. Shahid,1 Takahiro Hayashi,2 Hiroyuki Nakahara,2 Reham Hassan,1 Masaya Iwamuro,3 Eisaku Kondo,4 Shuhei Nakaji,5 Noriaki Tanaka,1 and Naoya Kobayashi1

1Department of Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
2Department of Orthopeadic Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
3Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
4Department of Pathology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
5Department of Biomedical Engineering, Okayama University of Science School, Okayama, Japan

Worldwide, colorectal cancer is the third most common type of cancer affecting both sexes. It has been proposed that a small subset of cancer cells (cancer stem cells) within each tumor is able to initiate tumor growth. In 2007, two research groups simultaneously identified a colon cancer stem cell population in human tumors by the use of CD133 expression. In the present study, we used a human colon cancer cell line, SW620, to analyze the cancer stem cell-like characteristics of CD133+ cells in vitro and in vivo. In vitro, CD133+ SW620 cells had a higher proliferative capacity, were more irradiation- and chemotherapy-resistant, and had a higher expression of b-catenin compared with CD133- cells. Injections of either CD133+ or CD133- cells into the skin or rectal mucosa of NOD/SCID mice led to tumors; however, injection of CD133+ cells resulted in the formation of larger tumors. Tumors derived from injections of CD133- cells did not contain any CD133+ cells, whereas tumors derived from injections of CD133+ cells did contain CD133+ cells, suggesting self-renewing capability. However, the proportion of CD133+ cells in the newly formed tumors in vivo was lower than the proportion of CD133+ cells in vitro. In conclusion, the human colon cancer cell line, SW620, contains both CD133+ and CD133- phenotypes, and the CD133+ phenotype has characteristics consistent with those of cancer stem cells.

Key words: CD133; Colon cancer; Tumorigenesis

Address correspondence to Hironobu Kawamoto, M.D., Ph.D., Department of Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. E-mail: hi_ro_no_bu-kw@rk9.so-net.ne.jp




Cell Transplantation, Vol. 19, pp. 865-877, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X508997
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Isolation and Propagation of a Human CD133- Colon Tumor-Derived Cell Line With Tumorigenic and Angiogenic Properties

Nalú Navarro-Alvarez,1*# Eisaku Kondo,2# Hironobu Kawamoto,1 Wael Hassan,3 Takeshi Yuasa,1 Yasuhiro Kubota,1 Masayuki Seita,1 Hiroyuki Nakahara,4 Takahiro Hayashi,4 Yuriko Nishikawa,2 Reham A. R. A. Hassan,1 Shahid M. Javed,1 Hirofumi Noguchi,5 Shinichi Matsumoto,5 Shuhei Nakaji,6 Noriaki Tanaka,1 Naoya Kobayashi,1 and Alejandro Soto-Gutierrez1,7#

1Department of Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
2Department of Pathology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
3Department of Pathophysiology-Periodontal Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
4Department of Orthopedic Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
5Baylor Research Institute, Islet Cell Transplantation Laboratory, Baylor Institution for Immunology Research, Dallas, TX, USA
6Department of Biomedical Engineering, School of Engineering, Okayama University of Science, Okayama, Japan
7Center for Innovative Regenerative Therapies, Department of Surgery, Transplantation Section, Children's Hospital of Pittsburgh, McGowan Institute for Regenerative Medicine and University of Pittsburgh Medical School, Pittsburgh, PA, USA

It has been proposed in human colorectal cancers (CRC) a minority subset of cancer cells within tumors able to initiate tumor growth, defined as cancer stem cells (CSC). Solid human primary colonic and its ovarian metastatic cancer tissues were collected from fresh surgical samples and subsequent xenografts were established in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. The resulting tumors were disaggregated into single-cell suspensions and a CD133- cell line (NANK) was newly established and analyzed by flow cytometry. Surface markers of progenitor cells were immunophenotypically analyzed, and expression of stem cell and cancer-related genes was characterized. Secreted angiogenesis-associated molecules were investigated by proteomic array technology. Finally, different numbers of NANK were implanted and their tumor-initiating properties were investigated in NOD/SCID mice. Intraperitoneal injection of NANK in NOD/SCID mice induced tumors with developing progressive peritoneal dissemination and ascites. NANK cells maintained a differentiated phenotype and reproduced the full morphologic and phenotypic heterogeneity of their parental lesions. Noticeably, NANK lacked the expression of conventional CSC markers CD133 and CD44, self-renewal genes Oct-4 and Nanog, but showed the expression of an important gastrointestinal development marker CDX-2 and BMI-1 that is essential in regulating the proliferative activity of normal and leukemic stem cells. In addition, NANK secreted high amounts of important angiogeneic cytokines. These results provide a novel and extensive model in human CSC for studying the generation and maintenance of phenotypic heterogeneity in CRC.

Key words: Cell line; Cancer; Metastasis; Intraperitoneal dissemination; Gene expression; Cytokines; Angiogenesis

Address correspondence to Alejandro Soto-Gutierrez, M.D., Ph.D., Center for Innovative Regenerative Therapies, Department of Surgery, Transplantation Section, Children's Hospital of Pittsburgh, McGowan Institute for Regenerative Medicine and University of Pittsburgh, 3511 Rangos Research Building, 530 45th Street, Pittsburgh, PA 15201, USA. Tel: (412) 692-5562; Fax: (412) 692-6599; E-mail: alejandro.sotogutierrez@chp.edu

*Present address: Bone Marrow Transplantation Section, Transplantation Biology Research Center, Massachusetts General Hospital, Harvard Medical, Boston, MA 02129, USA.
#These authors provided equal contribution to this work.




Cell Transplantation, Vol. 19, pp. 879-886, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X509004
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Characterization of Human Pancreatic Progenitor Cells

Hirofumi Noguchi,1,2 Bashoo Naziruddin,2,3 Andrew Jackson,2,3 Masayuki Shimoda,4 Tetsuya Ikemoto,1 Yasutaka Fujita,1 Daisuke Chujo,1 Morihito Takita,1 Naoya Kobayashi,5 Nicholas Onaca,3 Shuji Hayashi,6 Marlon F. Levy,1,3 and Shinichi Matsumoto1

1Baylor All Saints Medical Center, Baylor Research Institute, Fort Worth, TX, USA
2Institute of Biomedical Studies, Baylor University, Waco, TX, USA
3Baylor Regional Transplant Institute, Dallas, TX and Fort Worth, TX, USA
4Division of Cardiology, Department of Internal Medicine, Baylor University Medical Center, Baylor Heart and Vascular Institute, Dallas, TX, USA
5Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan
6Department of Advanced Medicine in Biotechnology and Robotics, Nagoya University Graduate School of Medicine, Nagoya, Japan

b-Cell replacement therapy via islet transplantation is an effective treatment for diabetes mellitus, but its widespread use is severely limited by the shortage of donor organs. Because pancreatic stem/progenitor cells are abundantly available in the pancreas of these patients and in donor organs, the cells could become a useful target for b-cell replacement therapy. We previously established a mouse pancreatic stem cell line without genetic manipulation. In this study, we used the techniques to identify and isolate human pancreatic stem/progenitor cells. The cells from a duct-rich population were cultured in 23 kinds of culture media, based on media for mouse pancreatic stem cells or for human embryonic stem cells. The cells in serum-free media formed "cobblestone" morphologies, similar to a mouse pancreatic stem cell line. On the other hand, the cells in serum-containing medium and the medium for human embryonic stem cells formed "fibroblast-like" morphologies. The cells divided actively until day 30, and the population doubling level (PDL) was 6-10. However, the cells stopped dividing after 30 days in any culture conditions. During the cultures, the nucleus/cytoplasm (N/C) ratio decreased, suggesting that the cells entered senescence. Exendin-4 treatment and transduction of PDX-1 and NeuroD proteins by protein transduction technology into the cells induced insulin and pancreas-related gene expression. Although the duplications of these cells were limited, this approach could provide a potential new source of insulin-producing cells for transplantation.

Key words: Human pancreatic progenitor cell; PDX-1; BETA2/NeuroD; Exendin-4; Duct-rich population; Protein transduction domain

Address correspondence to Hirofumi Noguchi, M.D., Ph.D., Baylor All Saints Medical Center, Baylor Research Institute, 1400 8th Avenue, Fort Worth, TX 76104, USA. Tel: (214) 820-9016; Fax: (214) 820-4952; E-mail: hirofumn@baylorhealth.edu




Cell Transplantation, Vol. 19, pp. 887-892, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X509013
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Cell Labeling With a Novel Contrast Agent of Magnetic Resonance Imaging

Koichi Oishi,1 Hirofumi Noguchi,1,2 Hiroaki Saito,3 Hiroshi Yukawa,1 Yoshitaka Miyamoto,1 Katsutoshi Murase,3 and Shuji Hayashi1

1Department of Advanced Medicine in Biotechnology and Robotics, Nagoya University Graduate School of Medicine, Nagoya, Japan
2Baylor All Saints Medical Center and Baylor Research Institute, Fort Worth, TX, USA
3Nagoya Research Laboratory, MEITO Sangyo Co., Ltd., Kiyosu, Japan

Cell therapy is a proven and efficient method for treating multiple diseases. For both basic research and clinical practice, the development of noninvasive in vivo imaging methods is essential for monitoring the trafficking or homing of transplanted cells. One attractive approach for the effective imaging of transplanted cells is the efficient labeling of cells with a contrast agent. In this study, we developed a novel contrast agent of magnetic resonance imaging (MRI), TMADM-02. TMADM-02 was efficiently transduced into cells without toxicity. However, the aggregation of TMADM-02 was observed because of its low stability in culture medium. Therefore, TMADM-02 may have led to a false-positive test result. In future studies, we should verify not only the efficiency of labeling cells but also the stability of the contrast agent of MRI for clinical applications.

Key words: Cationic nanoparticles; Stability; Cell labeling; In vivo imaging; Cell therapy

Address correspondence to Hirofumi Noguchi, M.D., Ph.D., Baylor All Saints Medical Center, Baylor Research Institute, 1400 8th Avenue, Fort Worth, TX 76104, USA. Tel: (214) 820-9016; Fax: (214) 820-4952; E-mail: hirofumn@baylorhealth.edu




Cell Transplantation, Vol. 19, pp. 893-900, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X509022
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Efficient Transfection of Sendai Virus Vector to Mouse Pancreatic Stem Cells in the Floating State

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

1Department of Advanced Medicine in Biotechnology and Robotics, Nagoya University Graduate School of Medicine, Nagoya, Japan
2Baylor All Saints Medical Center and Baylor Research Institute, Fort Worth, TX, USA
3DNAVEC Corporation, Ibaraki, Japan
4Department of Biomedical Sciences, Chubu University College of Life and Health Sciences, Aichi, Japan

Sendai virus (SeV) vectors can efficiently introduce foreign genes without toxicity into various organs and are expected to be clinically applicable. We previously compared the transfectional efficiency of SeV and adenovirus (AdV) vectors by assessing the transfer of the green fluorescent protein (GFP) gene to pancreatic stem cells. Although the gene transfer efficiency was similar between these vectors, SeV vector had a lower toxicity in comparison to the AdV vector. In this study, we assessed the gene transfer efficiency of SeV vector in the floating state to pancreatic stem cells. The efficiency of gene transfer was much higher at all time points and at all concentrations in the floating state versus in the adhesion state. In addition, the pancreatic stem cells transfected with SeV in the floating state maintained their differentiation ability. These data suggest that SeV transfection to pancreatic stem cells in the floating state may be useful in gene transfer technology.

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

Address correspondence to Hirofumi Noguchi, M.D., Ph.D., Baylor All Saints Medical Center, Baylor Research Institute, 1400 8th Avenue, Fort Worth, TX 76104, USA. Tel: (214) 820-9016; Fax: (214) 820-4952; E-mail: hirofumn@baylorhealth.edu




Cell Transplantation, Vol. 19, pp. 901-909, 2010
0963-6897/10 $90.00 + 00
DOI: 10.3727/096368910X509031
E-ISSN 1555-3892
Copyright © 2010 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Transduction of Cell-Penetrating Peptides Into Induced Pluripotent Stem Cells

Hiroshi Yukawa,1 Hirofumi Noguchi,2 Ikuhiko Nakase,3 Yoshitaka Miyamoto,1 Koichi Oishi,1 Nobuyuki Hamajima,4 Shiroh Futaki,3 and Shuji Hayashi1

1Department of Advanced Medicine in Biotechnology and Robotics, Nagoya University Graduate School of Medicine, Nagoya, Japan
2Baylor All Saints Medical Center and Baylor Research Institute, Dallas, TX, USA
3Institute for Chemical Research, Kyoto University, Kyoto, Japan
4Department of Preventive Medicine, Biostatistics and Medical Decision Making, Nagoya University School of Medicine, Nagoya, Japan

Induced pluripotent stem (iPS) cells have recently been generated by Yamanaka's group, and then followed by others. iPS cells are expected to have clinical applications including an important role in regenerative medicine. This study focused on the cell-penetrating peptides (CPPs) for differentiation or functional application of iPS cells, because several transduction domains can deliver a large size-independent variety of molecules into cells. Two CPPs, Texas Red-R8 and Rhodamine-TAT, were generated as representative CPPs and these CPPs were tested to determine their ability to penetrate the membrane of iPS cells. Both CPPs were transduced in iPS cells through macropinocytosis classified in endocytosis within 2 h in a manner consistent with many other cells, and no cytotoxicity and influence on their undifferentiated state was observed. In conclusion, CPPs can be utilized for their differentiation or functional application in iPS cells.

Key words: Cell-penetrating peptides (CPPs); Protein transduction domains (PTDs); Induced pluripotent stem (iPS) cells; Endocytosis; Macropinocytosis

Address correspondence to Hirofumi Noguchi, Baylor All Saints Medical Center, Baylor Research Institute, 1400 8th Avenue, Fort Worth, TX 76104, USA. Tel: (214) 820-9016; Fax: (214) 820-4952; E-mail: hirofumn@baylorhealth.edu