ognizant Communication Corporation

CELL TRANSPLANTATION
The Regenerative Medicine Journal

ABSTRACTS
VOLUME 14, NUMBER 10, 2005

Cell Transplantation, Vol. 14, pp. 715-725, 2005
0963-6897/05 $20.00 + 00
E-ISSN 1555-3892
Copyright (c) 2005 Cognizant Comm. Corp.
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A Role of the Choroid Plexus in Transplantation Therapy

Dwaine F. Emerich,1 Stephen J. M. Skinner,2 Cesario V. Borlongan,3,4,5,6 and Christopher G. Thanos1

1LCT BioPharma, Inc., Providence, RI, USA
2Diatranz NZLtd/Living Cell Technologies, Auckland, New Zealand
3Department of Neurology, Medical College of Georgia, Augusta, GA, USA
4Institute of Molecular Medicine & Genetics, Medical College of Georgia, Augusta, GA, USA
5School of Graduate Studies, Medical College of Georgia, Augusta, GA, USA
6Research and Affiliations Service Line, Augusta VAMC, Augusta, GA, USA

The choroid plexuses (CPs) play pivotal roles in the most basic aspects of neural function. Some of the roles of the CP include maintaining the extracellular milieu of the brain by actively modulating chemical exchange between the CSF and brain parenchyma, surveying the chemical and immunological status of the brain, detoxifying the brain, secreting a nutritive "cocktail" of polypeptides, and participating in repair processes following trauma. This diversity of functions suggests that even modest changes in the CP can have far reaching effects. Indeed, changes in the anatomy and physiology of the CP have been linked to several CNS diseases. It is also possible that replacing diseased CP or transplanting healthy CP might be useful for treating acute and chronic brain diseases. Here we describe the wide-ranging functions of the CP, alterations of these functions in aging and neurodegeneration, and recent demonstrations of the therapeutic potential of transplanted CP for neural trauma.

Key words: Choroid plexus; CSF; Transplantation; Growth factor

Address correspondence to Dwaine F. Emerich, LCT BioPharma, 766 Laten Knight Rd., Cranston, RI 02921, USA. Tel: 401-785-2985; Fax: 401-823-0466; E-mail: ED3FJM@aol.com




Cell Transplantation, Vol. 14, pp. 727-733, 2005
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E-ISSN 1555-3892
Copyright (c) 2005 Cognizant Comm. Corp.
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Minimal Focal Steatosis of Liver After Islet Transplantation in Humans: A Long-Term Study

Paola Maffi,1 Enzo Angeli,2 Federico Bertuzzi,1 Carlo Paties,3 Carlo Socci,4 Carlo Fedeli,1 Francesca De Taddeo,1 Rita Nano,1 Valerio Di Carlo,4 Alessandro Del Maschio,2 and Antonio Secchi1

1Department of Medicine, Transplant Unit, Scientific Institute San Raffaele, Vita-Salute University, 20132 Milano, Italy
2Department of Radiology, Scientific Institute San Raffaele, Vita-Salute University, 20132 Milano, Italy
3Department of Pathology, Scientific Institute San Raffaele, Vita-Salute University, 20132 Milano, Italy
4Department of Surgery, Scientific Institute San Raffaele, Vita-Salute University, 20132 Milano, Italy

Several reports have been published on islet transplantation in humans, but few data are available on the effect of islet infusion on the hepatic structure. Our aim was to evaluate in a longitudinal study the impact on the liver of intrahepatic islet transplantation. Clinical outcome and liver imaging were evaluated in 31 cases of islet-kidney transplantation (follow-up 38 ± 4 months, range 12-96 months). Patients were divided into three groups: full function (FF, 9 cases: established insulin independence); partial function (PF, 16 cases: transient insulin independence, prolonged C-peptide secretion); no function (NF, 6 cases: exhaustion of C-peptide secretion within the first year). Upper abdomen sonogram was regularly performed during the whole follow-up period. Percutaneous liver biopsy was performed in case of echographic abnormalities. Multiple small areas of focal hyperechogenicity were observed in nine cases after 6-12 months. These findings were observed only in FF (two) and in PF (seven) patients Fasting C-peptide levels at the time of echography were higher in negative than in positive patients (2.42 ± 0.16 vs. 1.51 ± 0.10 ng/ml, p = 0,0001). Liver biopsies showed focal macrovesicular steatosis, surrounded by normal liver parenchyma. Normal liver function was maintained. In conclusion, our results indicate that islet transplantation can lead to structural changes of the liver parenchyma (focal steatosis). It is more often observed in patients with partial function. Sonogram can be considered a specific method to reveal liver changes after islet transplantation.

Key words: Islet transplantation; Sonography; Histology; Steatosis

Address correspondence to Paola Maffi, Department of Medicine, Transplant Unit, Via Olgettina 60, 20132 Milano, Italy. Tel: 0039-0226432575; Fax: 0039-0226433790; E-mail: paola.maffi@hsr.it




Cell Transplantation, Vol. 14, pp. 735-748, 2005
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E-ISSN 1555-3892
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Generation of Islet-Like Hormone-Producing Cells In Vitro From Adult Human Pancreas

Fouad Atouf,1 Yong Choi,1 Michael J. Fowler,2 Greg Poffenberger,2 Jan Vobecky,4 Malancha Ta,1 George B. Chapman,4 Alvin C. Powers,2,3 and Nadya L. Lumelsky1

1Islet and Autoimmunity Branch, National Institute of Diabetes & Digestive & Kidney Diseases, National Institutes of Health, Bethesda, MD, USA
2Department of Medicine, Division of Diabetes, Endocrinology and Metabolism, Vanderbilt University, Nashville, TN, USA
3VA Tennessee Valley Healthcare System, Nashville, TN, USA
4Department of Biology, Georgetown University, Washington, DC, USA

Transplantation of pancreatic islets can provide long-lasting insulin independence for diabetic patients, but the current islet supply is limited. Here we describe a new in vitro system that utilizes adult human pancreatic islet-enriched fractions to generate hormone-producing cells over 3-4 weeks of culture. By labeling proliferating cells with a retrovirus-expressing green fluorescent protein, we show that in this system hormone-producing cells are generated de novo. These hormone-producing cells aggregate to form islet-like cell clusters. The cell clusters, when tested in vitro, release insulin in response to glucose and other secretagogues. After transplantation into immunodeficient, nondiabetic mice, the islet-like cell clusters survive and release human insulin. We propose that this system will be useful as an experimental tool for investigating mechanisms for generating new islet cells from the postnatal pancreas, and for designing strategies to generate physiologically competent pancreatic islet cells ex vivo.

Key words: Pancreatic islet transplantation; Diabetes; Pancreatic stem cells; In vitro culture

Address correspondence to Nadya L. Lumelsky, Unit of Cellular Differentiation, Islet and Autoimmunity Branch, National Institute of Diabetes & Digestive & Kidney Diseases, National Institutes of Health, Bldg. 10/CRC, Room 5-5940, 10 Center Dr., Bethesda, MD 20892-1453, USA. Tel: 301-451-9834; Fax: 301-480-4518; E-mail: nadyal@intra.niddk.nih.gov




Cell Transplantation, Vol. 14, pp. 749-756, 2005
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AN69 Hollow Fiber Membrane Will Reduce But Not Abolish the Risk of Transmission of Porcine Endogenous Retroviruses

Oleg Pakhomov,1 Lionel Martignat,2 Jiri Honiger,3 Béatrice Clémenceau,2 Pierre Saï,2 and Sylviane Darquy1

1Biology of Nutrition, Paris 5 University Faculty of Pharmacy, Paris, France
2Cellular and Molecular Immuno-Endocrinology, INRA/ENVN/University, Nantes, France
3Bioengineering Laboratory, Saint-Antoine Hospital, France

As the risk of porcine endogenous retrovirus (PERV) infection is a major obstacle to the xenotransplantation of porcine tissue, we investigated whether an AN69 hollow fibre membrane, used for islets of Langerhans transplantation, could prevent the transfer of PERVs and thus reduce the risk of PERV infection. PK15 cells were used as a PERV source. A specific and highly sensitive RCR was used for detection of a PERV provirus DNA (gag region) and a porcine mtDNA. Human U293 cells were incubated in vitro with encapsulated PK15 cells, concentrated encapsulated PK15 supernatant, or concentrated PK15 supernatant as a control. CD1 mice were implanted in vivo with encapsulated PK15 cells or injected with PK15 supernatant. We found no infection in human cells incubated with either encapsulated PK15 supernatant or in 10 out of 11 samples after coincubation with encapsulated PK15 cells. Infection of human cells was, however, detected in 1 out of 11 samples after coincubation with encapsulated PK15 cells. The presence of PERV provirus DNA and porcine mtDNA was detected in all the investigated tissues of the mice injected with PK15 supernatant and in various tissues of the mice implanted with encapsulated PK15 cells. Four weeks after the last injection of PK15 supernatant or a fiber explantation, no mouse showed any presence of PERV provirus DNA or porcine mtDNA. Our results demonstrate that AN69 hollow fiber membrane will reduce but not abolish the risk of PERV infection. Because the real risk of PERV infection still remains unknown, it is necessary to investigate further the real protection that could be provided by hollow fibers to ensure the safety of clinical xenotransplantation.

Key words: Xenotransplantation; Hollow fiber; PERV; Bioartificial pancreas

Address correspondence to Sylviane Darquy, Ph.D., Inserm, Biology of Nutrition, Faculty of Pharmacy, 4 avenue de l'Observatoire, 75270 Paris cedex 06, France. Tel/fax: (+33) 01 53 73 98 68; E-mail: sylviane.darquy@univ-paris5.fr




Cell Transplantation, Vol. 14, pp. 757-762, 2005
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Effective Islet Isolation Method With Extremely High Islet Yields From Adult Pigs

Yukihide Yonekawa,1 Shinichi Matsumoto,2 Teru Okitsu,1 Takashi Arata,1 Yasuhiro Iwanaga,1 Hirofumi Noguchi,1 Hideo Nagata,1 John J. O'Neil,3 and Koichi Tanaka1,2

1Department of Transplantation and Immunology, Graduate School of Medicine, Kyoto University, 54 Kawara-cho Shogoin, Sakyo-ku, Kyoto, 606-8507 Japan
2Kyoto University Hospital, Transplantation Unit, 54 Kawara-cho Shogoin, Sakyo-ku, Kyoto, 606-8507 Japan
3Johnson & Johnson, 199 Grandview Road, Skillman, NJ 08558, USA

Achieving good islet isolation is one of the most important factors for successful islet transplantation. Porcine pancreas is suitable for islet isolation research due to its anatomical and physiological similarities to human pancreas. In this study, we evaluated a new porcine islet isolation method designed to maximize islet yield and compared it with our previous open pan method and the standard method using a Ricordi chamber (Ricordi method). We performed 15 porcine islet isolations, five each with the new method, the open pan method, and the Ricordi method. The new method features several important improvements. Pancreata remain uncut and are kept intact during collagenase intraductal injection, a large filtration chamber to handle whole pancreata, low concentration of collagenase (Liberase(r)HI) for digestion, and large plastic containers for large-scale islet purification. All isolated islets were assessed for yield, purity, viability and in vitro function. Islets isolated with this new method were transplanted under the kidney capsules of SCID mice with chemically induced diabetes for in vivo functional assessment (n = 8). With the new method, we obtained on average more than 1,000,000 islet equivalents (IE) (1,236,266 ± 213,486 IE) (mean ± SE) before purification and 800,000 IE (879,815 ± 222,729 IE) after purification from one adult pig. Islet yield per pancreas was significantly higher compared with our previous open pan method (30,666 ± 11,532 IE, p < 0.01) and the Ricordi method (317,073 ± 86,093 IE, p < 0.05). All mice, transplanted with 1000 islets from the new method, returned to normoglycemia within 4 days after transplantation. Our new method makes it possible to obtain extremely high porcine islet yield with good function. It should produce useful information for human islet isolation and transplantation, and might be applied to single donor clinical xenogeneic transplantation.

Key words: Islet transplantation; Porcine islets; Whole pancreas; Intraductal injection; Xenotransplantation

Address correspondence to Shinichi Matsumoto, M.D., Ph.D., Kyoto University Hospital, Transplantation Unit, 54 Kawara-cho Shogoin, Sakyo-ku, Kyoto, Japan 606-8507. Tel: 81-75-751-4386; Fax: 81-75-751-4387; E-mail: shinichi@kuhp.kyoto-u.ac.jp




Cell Transplantation, Vol. 14, pp. 763-773, 2005
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The Effects of Bone Marrow-Derived Mesenchymal Stem Cells and Fascia Wrap Application to Anterior Cruciate Ligament Tissue Engineering

Zigang Ge,1 James Cho Hong Goh,1,2 and Eng Hin Lee1

1Department of Orthopaedic Surgery, National University of Singapore, 10 Kent Ridge Crescent, Singapore
2Division of Bioengineering, National University of Singapore, 10 Kent Ridge Crescent, Singapore

After an anterior cruciate ligament (ACL) injury, surgical reconstructions are necessary in most cases, either with autografts, allografts, or artificial ligaments. Potential tissue-engineered ligaments would circumvent the disadvantages apparent in these methods. While seeding of mesenchymal stem cells (MSCs) and fascia wrap could potentially improve tissue regeneration and mechanical properties, their exact roles were evaluated in the current study. Knitted biodegradable scaffolds of poly-L-lactic acid (PLLA) and poly-glycolic-lactic acid (PGLA) yarns were used to reconstruct ACL in 48 rabbits. These were divided into four equal groups: only knitted scaffolds were used in group I; knitted scaffolds and mesenchymal stem cells were used in group II; knitted scaffolds, MSCs, and fascia lata were used in group III; knitted scaffolds and fascia lata were used in group IV. Carboxyfluorescein diacetate (CFDA)-labeled MSCs were used to trace the fate of seeded cells in groups II and III. Histology, Western blot analysis, and mechanical properties of reconstructed ACL were analyzed after 20 weeks. Fibroblast ingrowths were seen in all four groups while CFDA-labeled MSCs could be found after 8 weeks of implantation in groups II and III. Both the amount of collagen type I and collagen type III in groups III and IV were significantly higher than in group II, which was much higher than in group I. Both maximal tensile loads and stiffness of the reconstructed ACLs in groups I, II, III, and IV were significantly lower than normal controls after 20 weeks of implantation. It is concluded that MSCs could promote synthesis of collagen type I and collagen type III in tissue-engineered ligaments, while fascia wraps have stronger effects. Both MSC seeding and fascia wrap could not enhance ultimate tensile load and stiffness.

Key words: Anterior cruciate ligament; Tissue engineering; Mesenchymal stem cell; Protein synthesis

Address correspondence to James C. H. Goh, Ph.D., Department of Orthopedic Surgery, National University of Singapore, 10 Kent Ridge Crescent, Singapore, 119260. Tel: (65)-67724423; Fax: (65)-67744082; E-mail: dosgohj@nus.edu.sg




Cell Transplantation, Vol. 14, pp. 775-786, 2005
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Analysis of Allogeneic and Syngeneic Bone Marrow Stromal Cell Graft Survival in the Spinal Cord

Sharon A. Swanger, Birgit Neuhuber, B. Timothy Himes, Ajay Bakshi, and Itzhak Fischer

Department of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia, PA, USA

Bone marrow stromal cells (MSC) are attractive candidates for developing cell therapies for central nervous system (CNS) disorders. They can be easily obtained, expanded in culture, and promote modest functional recovery following transplantation into animal models of injured or degenerative CNS. While syngeneic MSC grafts can be used efficiently, achieving long-term survival of allogeneic MSC grafts has been a challenge. We hypothesize that improved graft survival will enhance the functional recovery promoted by MSC. To improve MSC graft survival, we tested two dosages of the immune suppressant cyclosporin A (CsA) in an allogeneic model. Syngeneic transplantation of MSC where cells survive well without immune suppression was used as a control. Sprague-Dawley rats treated with standard dose (n = 12) or high-dose (n = 12) CsA served as allogeneic hosts; Fisher 344 rats (n = 12) served as syngeneic hosts. MSC were derived from transgenic Fisher 344 rats expressing human placental alkaline phosphatase and were grafted into cervical spinal cord. Animals treated with standard dose CsA showed significant decreases in allograft size 4 weeks posttransplantation; high CsA doses yielded significantly better graft survival 4 and 8 weeks posttransplantation compared to standard CsA. As expected, syngeneic MSC transplants showed good graft survival after 4 and 8 weeks. To investigate MSC graft elimination, we analyzed immune cell infiltration and cell death. Macrophage infiltration was high after 1 week in all groups. After 4 weeks, high-dose CsA and syngeneic animals showed significant reductions in macrophages at the graft site. Few T lymphocytes were detected in any group at each time point. Cell death occurred throughout the study; however, little apoptotic activity was detected. Histochemical analysis revealed no evidence of neural differentiation. These results indicate that allogeneic transplantation with appropriate immune suppression permits long-term survival of MSC; thus, both allogeneic and syngeneic strategies could be utilized in devising novel therapies for CNS injury.

Key words: Transplantation; Cell therapy; Autologous grafting; Allograft; Immune suppression; Immune response

Address correspondence to Itzhak Fischer, Ph.D., Department of Neurobiology and Anatomy, 2900 Queen Lane, Drexel University College of Medicine, Philadelphia, PA 19129, USA. Tel: 215 991 8400; Fax: 215 843 9802; E-mail: Itzhak.Fischer@drexel.edu




Cell Transplantation, Vol. 14, pp. 787-798, 2005
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Purified Human Bone Marrow Multipotent Mesenchymal Stem Cells Regenerate Infarcted Myocardium in Experimental Rats

Shaoheng Zhang,1,2,4* Zhuqing Jia,1,3* Junbo Ge,2 Lizhong Gong,1 Yanling Ma,1 Tao Li,3 Jingxuan Guo,4 Ping Chen,3 Qikuan Hu,1 Ping Zhang,1,4 Yonggang Liu,4 Zhaoping Li,4 Kangtao Ma,3 Linsong Li,1 and Chunyan Zhou1,3

1Stem Cell Research Center, Peking University, 38 Xue Yuan Road, Hai Dian District, Beijing, 100083 China
2Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Fudan University, 180 Feng Ling Road, Shanghai, 20032 China
3Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Peking University, 38 Xue Yuan Road, Hai Dian District, Beijing, 10083 China
4Department of Cardiology, Third Hospital of Peking University, 49 North Garden Road, Hai Dian District, Beijing, 10083 China

Recent findings suggest the feasibility of cardiac repair by transplantation of bone marrow mesenchymal stem cell (MSCs). However, it remains controversial regarding which cell type is the best source for transplanting into the ischemic heart because of lack of well-defined cell markers. In this study, we investigated the in vitro and in vivo effects of the novel multipotent marrow mesenchymal stem cells (MMSCs) from human bone marrow Pluripotent markers (Oct4, Bmi1, and Abcg2) and vascular endothelial growth factor (VEGF) were detected by RT-PCR and immunofluorescence in MMSCs. Myocardial differentiation was induced in the expanded MMSC cultures by treatment with 5-azacyline. Expressions of VEGF in the animals transplanted with MMSCs were markedly increased in comparison with the animals injected with fibroblasts or saline at both mRNA and protein levels. VEGF expression was observed in both transplanted MMSCs and recipient cardiomyocytes by immunofluorescence. Confocal immunofluorescence microscopy revealed the specific markers for cardiomyocytes and endothelial cells in transplanted MMSCs 14 days after transplantation. Vessel count was increased and left ventricular function improved post-MMSC transplantation. These results indicate that transplantation of purified MMSCs from human bone marrow upregulated VEGF expression, enhanced angiogenesis, and improved the functional recovery following myocardial infarction in rats.

Key words: Mesenchymal stem cell; Transplantation; Differentiation; Myocardial infarction; Angiogenesis

Address correspondence to Chunyan Zhou, Department of Biochemistry and Molecular Biology, the School of Basic Medical Sciences, Peking University, 38 Xue Yuan Road, Hai Dian District, Beijing, China, 100083. Tel/Fax: 86 10 82802417; E-mail: chunyanzhou@bjmu.edu.cn

*These authors contributed equally to this work.




Cell Transplantation, Vol. 14, pp. 799-808, 2005
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Protection of Photoreceptor Cells From Phototoxicity by Transplanted Retinal Pigment Epithelial Cells Expressing Different Neurotrophic Factors

Toshiaki Abe,1 Yoko Saigo,2 Masayoshi Hojo,2 Tetsuya Kano,2 Ryosuke Wakusawa,2 Yumi Tokita,1 and Makoto Tamai2

1Division of Clinical Cell Therapy, School of Medicine, Tohoku University, 1-1 Seiryomachi, Aobaku, Sendai, Miyagi, 980-8574 Japan
2Department of Ophthalmology and Visual Science, School of Medicine, Tohoku University, 1-1 Seiryomachi, Aobaku, Sendai, Miyagi, 980-8574 Japan

Transplantation of cells or tissues and the intravitreal injection of neurotrophic factors are two methods that have been used to treat retinal diseases. The purpose of this study was to examine the effects of combining both methods: the transplantation of retinal pigment epithelial (RPE) cells expressing different neurotrophic factors. The neutrophic factors were Axokine, brain derived-neurotrophic factor (BDNF), and basic fibroblast growth factor (bFGF). The enhanced green fluorescence protein (eGFP) gene was used as a reporter gene. These genes were transduced into RPE cells by lipofection, selected by antibiotics, and transplanted into the subretinal space of 108 rats. The rats were examined at 1 week and 3 months after the transplantation to determine whether the transduced cells were present, were expressing the protein, and were able to protect photoreceptors against phototoxicity. The survival of the transplanted cells was monitored by the presence of eGFP. The degree of protection was determined by the thickness of the outer nuclear layer. Our results showed that the degree of photoreceptor protection was different for the different types of neurotrophic factors at 1 week. After 3 months, the number of surviving transplanted cell was markedly reduced, and protection was observed only with the BDNF-transduced RPE cells. A significant degree of rescue was also observed by BDNF-transduced RPE cells in the nontransplanted area of the retina at both the early and late times. Lymphocytic infiltration was not detected in the vitreous, retina, and choroid at any time. We conclude that the transplantation of BDNF-transduced RPE cells can reduce the photoreceptor damage induced by phototoxicity in the transplanted area and weakly in the nontransplanted area.

Key words: Brain-derived neurotrophic factor; Transplantation; Phototoxicity; Retinal pigment epithelium; Plasmid vector

Address correspondence to Toshiaki Abe, Division of Clinical Cell Therapy Tohoku University, School of Medicine, 1-1 Seiryoumachi Aobaku Sendai, Miyagi, 980-8574 Japan. Tel: 81-22-717-7294; Fax: 81-22-717-7298; E-mail: toshi@oph.med.tohoku.ac.jp




Cell Transplantation, Vol. 14, pp. 809-817, 2005
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Synergistic Effect of Keratinocyte Transplantation and Epidermal Growth Factor Delivery on Epidermal Regeneration

So-Jung Gwak,1 Sang-Soo Kim,2 Kyungeun Sung,1 Joungho Han,3 Cha Yong Choi,2 and Byung-Soo Kim4

1Department of Chemical Engineering, Hanyang University, Seoul, Korea
2Interdisciplinary Program for Biochemical Engineering and Biotechnology, Seoul National University, Seoul, Korea
3Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
4Department of Bioengineering, Hanyang University, Seoul, Korea

Both keratinocyte transplantation and epidermal growth factor (EGF) delivery stimulate epidermal regeneration. In this study, we hypothesized that the combined therapy of keratinocyte transplantation and EGF delivery accelerates epidermal regeneration compared to the single therapy of either keratinocyte transplantation or EGF delivery. To test this hypothesis, we utilized fibrin matrix as a keratinocyte/EGF delivery vehicle for epidermal regeneration. Full-thickness wounds were created on the dorsum of athymic mice, and human keratinocytes and EGF in fibrin matrix were sprayed onto the wounds to regenerate epidermal layers (group 1). As controls, human keratinocytes in fibrin matrix (group 2), EGF in fibrin matrix (group 3), or fibrin matrix alone (group 4) was sprayed onto the wounds. Spraying keratinocytes suspended in fibrin matrix did not affect the keratinocyte viability, as the cell viabilities before and after spraying were not different. EGF was released from fibrin matrix for 3 days. The wounds were analyzed with histology and immunohistochemistry at 1 and 3 weeks after treatments. Compared with the control groups, initial wound closure rate was highest in group 1. Histological analyses indicated that group 1 exhibited faster and better epidermal regeneration than the other groups. Immunohistochemical analyses showed that regenerated epithelium in groups 1 and 2 stained positively for human involucrin at 3 weeks, whereas the tissue sections of the groups 3 and 4 stained negatively. Human laminin was detected at the dermal-epidermal junction of the regenerated tissues in groups 1 and 2 at 3 weeks and was not detected in groups 3 and 4. The epidermal thickness of the regenerated tissues in group 1 was significantly thicker than that of the other groups at all time points. These results suggest that the combined therapy of keratinocyte transplantation and EGF delivery is more efficacious for epidermal regeneration than each separate therapy alone.

Key words: Epidermal growth factor; Epidermal regeneration; Fibrin matrix; Keratinocyte

Address correspondence to Byung-Soo Kim, Department of Bioengineering, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul 133-791, Korea. Tel: +82-2-2290-0491; Fax: +82-2-2298-4101; E-mail: bskim@hanyang.ac.kr




Cell Transplantation, Vol. 14, pp. 819-827, 2005
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Differentiation Effects by the Combination of Spheroid Formation and Sodium Butyrate Treatment in Human Hepatoblastoma Cell Line (Hep G2): A Possible Cell Source for Hybrid Artificial Liver

J. Fukuda,1 K. Okamura,1 K. Ishihara,1 H. Mizumoto,1 K. Nakazawa,2 H. Ijima,1 T. Kajiwara,1 and K. Funatsu1

1Department of Chemical Engineering, Faculty of Engineering, Kyushu University, Fukuoka, Japan
2Department of Chemical Processes and Environments, University of Kitakyushu, Kitakyushu, Japan

The aim of this study was to investigate the feasibility of human hepatoblastoma cell line (Hep G2), which differentiates by spheroid formation, and treatment with sodium butyrate (SB) as a cell source for hybrid artificial liver (HAL). Hep G2 spontaneously formed spheroids in polyurethane foam (PUF) within 3 days of culture and restored weak ammonia removal activity. Treatment with SB, which is a histone deacetylase inhibitor, further increased the ammonia removal activity of Hep G2 spheroids in a concentration-dependent manner. The activation of ornithine transcarbamylase - a urea cycle enzyme - was significantly related to the upregulation of ammonia removal by spheroid formation, but scarcely contributed to the further upregulation following SB treatment. In contrast with ammonia removal, treatment with SB reduced the albumin secretion of Hep G2 spheroids in a concentration-dependent manner. In the PUF-HAL module in a circulation culture, the ammonia removal rate and albumin secretion rate (per unit volume of the module) of Hep G2 spheroids treated with 5 mM SB were almost the same as those of primary porcine hepatocyte spheroids. These results suggest that simultaneous use of spheroid formation and SB treatment in Hep G2 is beneficial in enhancing the functions of human hepatocytes with potential applications in regenerative medicine and drug screening.

Key words: Hybrid artificial liver; Sodium butyrate; Spheroid; Polyurethane foam; Hep G2

Address correspondence to Kazumori Funatsu, Ph.D., 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan. Tel: 81-92-802-2759; Fax: 81-92-802-2796; E-mail: junjif@mit.edu




Cell Transplantation, Vol. 14, pp. 829-835, 2005
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The Effect of Simulated Microgravity by Three-Dimensional Clinostat on Bone Tissue Engineering

Masataka Nishikawa,1 Hajime Ohgushi,2 Noriyuki Tamai,1 Koichi Osuga,3 Masaru Uemura,3 Hideki Yoshikawa,1 and Akira Myoui1

1Department of Orthopaedics, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita City, Osaka 565-0871, Japan
2Tissue Engineering Research Group, Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology, 3-11-46 Nakouji, Amagasaki City, Hyogo 661-0974, Japan
3Mitsubishi Heavy Industries, Ltd, Japan, 1-1-1 Wadasaki-cho, Hyogo-ku, Kobe City, Hyogo 652-8585, Japan

Evidence suggests that mechanical stress, including gravity, is associated with osteoblast differentiation and function. To examine effects of microgravity on bone tissue engineering, we used a three-dimensional (3D) clinostat manufactured by Mitsubishi Heavy Industries (Kobe, Japan). A 3D clinostat is a device that generates multidirectional G force. By controlled rotation on two axes, it cancels the cumulative gravity vector at the center of the device. We cultured rat marrow mesenchymal cells (MMCs) in the pores of interconnected porous calcium hydroxyapatite (IP-CHA) for 2 weeks in the presence of dexamethasone using the 3D clinostat (clinostat group). MMCs cultured using the 3D clinostat exhibited a 40% decrease in alkaline phosphatase activity (a marker of osteoblastic differentiation), compared with control static cultures (control group). SEM analysis revealed that although there was no difference between the two groups in number or distribution of cells in the pores, the clinostat group exhibited less extensive extracellular matrix formation than the control group. Cultured IP-CHA/MMC composites were then implanted into subcutaneous sites of syngeneic rats and harvested 8 weeks after implantation. All implants showed bone formation inside the pores, as indicated by decalcified histological sections and microfocus computed tomography. However, the volume of newly formed bone was significantly lower for the clinostat group than for the control group, especially in the superficial pores close to the implant surface. These results indicate that new bone formation in culture was inhibited by use of the 3D clinostat, and that this inhibition was mainly due to suppression of osteoblastic differentiation of MMCs.

Key words: Simulated microgravity; Bone tissue engineering; 3D clinostat; Microfocus CT; Osteoblast differentiation; Marrow mesenchymal cell

Address correspondence to Akira Myoui, M.D., Ph.D., Department of Orthopaedics, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. Tel: (+81) 6-6879-3552; Fax: (+81) 6-6879-3559; E-mail: myoi@ort.med.osaka-u.ac.jp




Cell Transplantation, Vol. 14, pp. 837-843, 2005
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Influence of Preservation Solution on the Isolation and Culture of Human Hepatocytes From Liver Grafts

Alfonso Serralta,1,2 Maria Teresa Donato,3 Amparo Martinez,1 Eugenia Pareja,1 Francisco Orbis,1 Jose Vicente Castell,3 Jose Mir,1 and Maria Jose Gómez-Lechón3

1Division of Liver Surgery and Transplantation, University Hospital La FE, Avda Campanar 21, 46009, Valencia, Spain
2Regenerative Medicine & Cell Transplantation Unit, University Hospital La FE, Avda Campanar 21, 46009, Valencia, Spain
3Unidad de Hepatología Experimental, Centro de Investigación, University Hospital La FE, Avda Campanar 21, 46009, Valencia, Spain

A major problem for the isolation and transplantation of hepatocytes is the lack of resources for obtaining viable hepatocytes. Improving this situation would enhance hepatic cell transplantation programs. Our objective was to evaluate the influence of the preservation solutions used during organ retrieval on the quality of hepatocytes isolated from liver tissue. We compared the results of the collagenase perfusion technique for isolation of hepatocytes in human livers flushed with University of Wisconsin (UW) and Celsior preservation solutions. Yield (number of viable cells per gram of tissue), cellular viability, efficiency of cells to attach to culture plates and form a monolayer, and drug metabolizing competence of the hepatocytes were measured. Successful isolation was achieved in 63% of the procedures using the UW solution and 100% of the procedures using the Celsior solution. In the UW group, significantly lower cell viability (38 ± 41% vs. 79 ± 14%, p < 0.05), yield of cells (4.0 ± 5.2 x106 vs. 8.2 ± 5.6 x106 cells/g, p < 0.05), and protein content at 24 h of culture (0.6 ± 0.6 vs. 1.2 ± 0.3 mg protein per plate, p < 0.05) than in Celsior solution were found. However, similar values of P450 activities were found in both groups. The more successful isolation, better yield, and higher cell viability obtained from human liver grafts preserved in Celsior solution, in comparison to UW solution, suggest Celsior solution as the most appropriate for preserving cadaveric hepatic tissue to be used for hepatocyte harvesting.

Key words: Human hepatocytes; Isolation; Preservation solution

Address correspondence to Alfonso S. Serralta, M.D., Ph.D., Department of Surgery, Division of Liver Surgery and Transplantation, Regenerative Medicine & Cell Transplantation Unit, 2° Planta Pabellón de Gobierno, Hospital Universitario LA FE, Avda Campanar 21, 46009, Valencia, Spain. Tel: ++34 963862754; Fax: ++34 961973286; E-mail: serralta_alf@gva.es




Cell Transplantation, Vol. 14, pp. 845-853, 2005
0963-6897/05 $20.00 + 00
E-ISSN 1555-3892
Copyright (c) 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Large-Scale Isolation of Human Hepatocytes for Therapeutic Application

Krassimira Alexandrova,1* Carsten Griesel,1* Marc Barthold,1 Hans-Gert Heuft,1,2 Michae Ott,1,3 Michae Winkler,1,4 Harald Schrem,1 Michael P. Manns,3 Timm Bredehorn,5 Marc Net,6 Martí Manyalich I Vidal,6 Sabine Kafert-Kasting,1 and Lubomir Arseniev1,7

1Cytonet GmbH & Co. KG, Branch Hannover, Feodor-Lynen-Str. 21, D-30625 Hannover, Germany
2Department of Transfusion Medicine, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany
3Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany
4Department of Visceral and Transplantation Surgery, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany
5DSO-G, Feodor-Lynen-Str. 21, D-30625 Hannover, Germany
6Fundació Clínic per a la Recerca Biomédica, Hospital Clínic Barcelona, Villarroel, 170, E-08036 Barcelona, Spain
7Department of Hematology and Oncology, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany

During the last decade, hepatocyte transplantation has been suggested as a safe and potentially effective clinical option for the treatment of acute or decompensating chronic liver failure as well as for hereditary liver disease. Currently, one of the major limiting factors for clinical application is the insufficient access to suitable liver cell preparations. In cooperation with the German and Catalane organ procurement organizations, a routine procedure for the isolation of hepatocytes from donor organs rejected for transplantation (n = 117) has been established. The process is performed according to the current EC Guidelines for Good Manufacturing Practice (cGMP) and all corresponding national laws and regulations concerning donor organ and tissue procurement. In about 50% of the cases (n = 58) the three-step perfusion procedure has been completed with an average total cell yield of 5.9 x109 cells per organ, the cell preparations displaying a mean viability of 64%. The mean specific yield was 3.6 x106 total and 2.6 x106 viable cells per gram liver tissue, respectively. Specific cell yields from three infantile donor livers were considerably higher. No correlation between isolation efficiency and cold ischemia time or donor age was found within the adult organ donors. In contrast, organs with a severe steatosis generally did not result in successful cell isolation. Results of sterility and endotoxin determination are also presented. In summary, a standardized and cGMP conform method of hepatocyte isolation from nontransplantable liver organs was established, which reproducibly yields large amounts of hepatocytes suitable for therapeutic application.

Key words: Hepatocyte isolation; Cell transplantation; GMP; Steatosis

Address correspondence to Dr. Sabine Kafert-Kasting, Cytonet Hannover, Feodor-Lynen-Str. 21, D-30625 Hannover, Germany. Tel: +49-511-55 47 43 23; Fax: +49-511-55 47 43 10; E-mail: sabine.kafert-kasting@cytonet.de

*Both authors contributed equally to this publication.