ognizant Communication Corporation

CELL TRANSPLANTATION

ABSTRACTS
VOLUME 12, NUMBER 6, 2003

Cell Transplantation, Vol. 12, pp. 555-561, 2003
0963-6897/03 $20.00 + 00
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Human Cord Blood-Derived Mesenchymal Stem Cells Home and Survive in the Marrow of Immunodeficient Mice After Systemic Infusion

Alejandro A. Erices,1,2 Carolina I. Allers,1,3 Paulette A. Conget,1,3 Cecilia V. Rojas,1 and José J. Minguell1,2

1Programa Terapias Génicas y Celulares, INTA, Universidad de Chile, Santiago, Chile
2Laboratorio de Criopreservación, Clínica Las Condes, Santiago, Chile
3Facultad de Ciencias de la Salud, Universidad del Desarrollo, Santiago, Chile

Bone marrow is the residence site of mesenchymal stem cells (MSC), which upon commitment and maturation develop into several mesenchymal phenotypes. Recently, we have described the presence of MSC in human cord blood (cbMSC) and informed that their properties are the same as those for MSC obtained from adult bone marrow. In this study we have investigated the capability of transplanted cbMSC to home and survive in the marrow of unconditioned nude mice. cbMSC utilized for transplantation studies were characterized by morphology, differentiation potential, and immunophenotype. After transplantation by systemic infusion, human DNA (as detected by PCR amplification of human-specific b-globin gene) was detected in the marrow of recipients as well as in ex vivo-expanded stromal cells prepared from the marrow of transplanted animals. These results demonstrate homing and survival of cbMSC into the recipient marrow and also suggest a mesenchymal-orientated fate of engrafted cells, because human DNA was also detected in cells of other recipient tissues, like cardiac muscle, teeth, and spleen.

Key words: Mesenchymal stem cells; Cord blood; Transplantation; Homing

Address correspondence to Alejandro Erices, Ph.D., Programa de Terapias Génicas y Celulares, INTA, Universidad de Chile, Casilla 138, Santiago 11, Chile. Tel: (56)2-678-1510; Fax: (56)2-221-4030; E-mail: aerices@uec.inta.uchile.cl




Cell Transplantation, Vol. 12, pp. 563-568, 2003
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Bridging a Patient With Acute Liver Failure to Liver Transplantation by the AMC-Bioartificial Liver

Maarten-Paul van de Kerkhove,1 Ernesto Di Florio,3 Vincenzo Scuderi,3 Antonio Mancini,4 Antonello Belli,4 Adele Bracco,4 Daniela Scala,4 Simona Scala,4 Laura Zeuli,4 Giuseppe Di Nicuolo,5 Pietro Amoroso,6 Fulvio Calise,3 and Robert A. F. M. Chamuleau2

Surgical Laboratory (IWO-1), 1Department of Surgery, 2Department of Hepatology, Academic Medical Center, University of Amsterdam, The Netherlands
3Liver Transplantation Unit, Department of Surgery, Cardarelli Hospital, 4Centro di Biotecnologie A. O. Cardarelli, Naples, Italy
5Virology Laboratory, 6VI Division of Infectious Diseases, D. Cotugno Hospital, Naples, Italy

Recently a phase I clinical trial has been started in Italy to bridge patients with acute liver failure (ALF) to orthotopic liver transplantation (OLT) by the AMC-bioartificial liver (AMC-BAL). The AMC-BAL is charged with 10 x 109 viable primary porcine hepatocytes isolated from a specified pathogen-free (SPF) pig. Here we report a patient with ALF due to acute HBV infection. This patient was treated for 35 h by two AMC-BAL treatments and was bridged to OLT. There was improvement of biochemical and clinical parameters during the treatment. No severe adverse events were observed during treatment and follow-up of 15 months after hospital discharge. Possible porcine endogenous retrovirus (PERV) activity could not be detected in the patient's blood or blood cells up to 12 months after treatment.

Key words: Acute liver failure; Bioartificial liver; Hepatocyte; Transplantation; Human; Porcine; Clinical trial

Address correspondence to Maarten-Paul van de Kerkhove, Surgical Laboratory IWO-1-172, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. Tel: 0031-20-5666683; Fax: 0031-20-6976621; E-mail: m.p.vandekerkhove@amc.uva.nl




Cell Transplantation, Vol. 12, pp. 569-578, 2003
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Characterization of Long-Term Survival of Syngeneic Hepatocytes in Rat Peritoneum

Clare Selden, A. Casbard, M. Themis, and H. J. F. Hodgson

The Liver Group, Department of Gastroenterology, Division of Medicine, Imperial College School of Medicine, Hammersmith Hospital, London, W12 0NN, UK
Gene Therapy, Imperial College of Science Technology and Medicine, South Kensington campus, Exhibition Rd, London SW7, UK

Hepatocyte transplantation is a potential therapy for both acute and chronic hepatic insufficiency and also for treatment of inborn errors of metabolism affecting the liver. The peritoneum is one site for implantation and has several advantages: cells implanted there can be easily identified and observed, and it has a relatively large capacity. Long-term survival using "pure" hepatocytes in the peritoneum have been disappointing. We hypothesized that cotransplantation of hepatocytes with nonparenchymal cells would help maintain differentiated hepatocyte function. Rat liver cells transplanted intraperitoneally into August rats were sacrificed at 7 days, 1, 3, 6, 9, and 12 months and analyzed for presence, basal proliferation, and functionality of hepatocytes. To demonstrate that ectopic hepatocytes remained susceptible to exogenous growth factors affecting cell proliferation, rats 9 and 12 months after transplantation were stimulated with tri-iodothyronine and KGF. Hepatocytes were identified 7 days to >12 months, by H&E and immunohistochemically, as ectopic islands in the omental fat. Functionality was confirmed by glycogen deposition. Basal proliferation in 7-day rats was 28.0 ± 10/1000 hepatocytes in ectopic islands (cf. 5.70 ± 2.7/1000 in recipient liver). Proliferation in ectopic islands was greater than host liver. Growth factor-stimulated proliferation in ectopic islands induced a 70-fold increase in DNA synthesis. In conclusion, hepatocytes transplanted with nonparenchymal cells survive, proliferate, and function in the peritoneum of normal rats, and respond to exogenous growth stimuli. Their survival and proliferation in the presence of a normal functioning liver has implications for the potential use of the peritoneal site clinically for supplementation of liver function in metabolic disorders.

Key words: Hepatocytes; Nonparenchymal cells; Proliferation; T3; KGF; Intraperitoneal transplantation

Address correspondence to Dr. Clare Selden, The Liver Group, Department of Medicine-Centre for Hepatology, Royal Free and University College Medical School, Royal Free Campus, Rowland Hill Street, Hampstead, London NW3 2PF, UK. Tel: +44 (0)207 433 2854; Fax: +44 (0)207 433 2744; E-mail: c.selden@rfc.ucl.ac.uk




Cell Transplantation, Vol. 12, pp. 579-592, 2003
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Hypothermic Storage and Cryopreservation of Hepatocytes: The Protective Effect of Alginate Gel Against Cell Damages

Stephan Mahler,1,2* Mireille Desille,1* Benjamin Frémond,1 Christophe Chesné,2 André Guillouzo,1 Jean-Pierre Campion,1 and Bruno Clément1

1INSERM U456, Detoxication and Tissue Repair Unit, University of Rennes I, 2 avenue Pr Léon Bernard, 35043 Rennes, France
2Biopredic International, 14-18 rue Jean Pecker, 35000 Rennes, France

Hepatocyte-based therapy has been proposed as an alternative to organ transplantation in the treatment of liver disorders. In the clinical context, a major issue is the constant supply of quality assurance-controlled hepatocytes, thereby requiring their cold storage in good conditions. We have analyzed the protective effects of alginate entrapment of rat hepatocytes after either 24 or 48 h of hypothermic storage or cryopreservation on the cell viability, cell yield, both mitochondrial and other cytoplasmic functional activities, and apoptosis. Decrease in viability, as evaluated by the MTT inclusion test, was 4% and 13% (24 h at 4°C), 15% and 33% (48 h at 4°C), and 9% and 19% (liquid nitrogen) for entrapped and free suspended hepatocytes, respectively. Viable cell yields were 86 ± 8% and 51 ± 6% for cryopreserved entrapped and free suspended hepatocytes, respectively. The mitochondrial (MTS assay), 7-ethoxyresorufin O-deethylase (EROD), and glutathione-S-transferase (GST) activities were better preserved in entrapped than in free suspended hepatocytes. Both hypothermic storage and cryopreservation were found to induce early caspase-3-like activities, being always much lower in entrapped hepatocytes, particularly after cryopreservation (98.4 ± 42.4 vs. 6.4 ± 4.0 fluorescence arbitrary units/hours/mg protein). Thus, cold-induced apoptosis in hepatocytes can be significantly reduced following their entrapment within alginate gel beads and this is associated with an improvement of both their viability and function.

Key words: Cell therapy; Cryopreservation; Hepatocytes; Hypothermia; Immobilized cells; Sodium alginate

Address correspondence to Dr. B. Clément INSERM U456, Detoxication and Tissue Repair Unit, Faculté de Pharmacie et de Médecine, 2 avenue Léon Bernard, 35043 Rennes, France. E-mail: bruno.clement@rennes.inserm.fr

*Stephan Mahler and Mireille Desille contributed equally to the study.




Cell Transplantation, Vol. 12, pp. 593-598, 2003
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Hepatocyte Isolation and Transplantation in the Pig

Masanobu Maruyama,1 Toshinori Totsugawa,1 Takemi Kunieda,2 Teru Okitsu,1 Norikuni Shibata,2 Michihiko Takesue,2 Yuzuru Kurabayashi,3 Mizuko Oshita,4 Shuhei Nakaji,4 Makoto Kodama,5 Noriaki Tanaka,1 and Naoya Kobayashi1

1Department of Surgery, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558, Japan
2Division of Gastroenterology I, Department of Medicine, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-0192 Japan
3Animal Center for Medical Research, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558, Japan
4Medical Products Development Department, Kuraray Medical Co., 1621 Sakazu, Kurashiki, Okayama 710-8622, Japan
5Tissue Engineering Research Center, The National Institute of Advanced Industrial Science and Technology. 1-1-4 Higashi, Tsukubashi, Ibaragi 305-8562, Japan

Hepatocyte transplantation (HTX) has received great expectation for the treatment of a wide spectrum of liver diseases. Considering the severe shortage of human livers for hepatocyte isolation, porcine hepatocytes are an attractive alternative to normal human hepatocytes. To develop such therapy, establishment of an efficient hepatocyte isolation and transplantation model that enables accurate assessment of safety and efficacy of HTX is extremely important. Porcine hepatocytes were isolated from a surgically removed liver segment with a four-step retrograde perfusion using dispase and collagenase. The resultant hepatocytes of >84% viability were used for transplantation experiment in a pig model of acute liver failure induced by intravenous administration of D-galactosamine (D-gal) (0.5 mg/kg). Twenty-four hours after D-gal injection, transplantation of freshly isolated porcine hepatocytes (1 x 109) was safely conducted and prolonged the survival of D-gal-treated pigs. We describe an efficient porcine hepatocyte isolation and subsequent cell transplantation in pigs with D-gal-induced liver failure.

Key words: Porcine hepatocytes; Hepatocyte isolation; D-Galactosamine; Acute liver failure

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) 86-221-8775; E-mail: immortal@md.okayama-u.ac.jp




Cell Transplantation, Vol. 12, pp. 599-606, 2003
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Maintenance of Cold-Preserved Porcine Hepatocyte Function With UW Solution and Ascorbic Acid-2 Glucoside

Michihiko Takesue,1 Masanobu Maruyama,2 Norikuni Shibata,1 Takemi Kunieda,1 Teru Okitsu,2 Masakiyo Sakaguchi,3 Toshinori Totsugawa,2 Yoshikazu Kosaka,2 Akira Arata,2 Hideaki Ikeda,2 Junji Matsuoka,2 Toshie Oyama,4 Makoto Kodama,4 Kenji Ohmoto,1 Shinichiro Yamamoto,1 Yuzuru Kurabayashi,5 Itaru Yamamoto,6 Noriaki Tanaka,2 and Naoya Kobayashi2

1Division of Gastroenterology, Department of Internal Medicine, Kawasaki Medical School, 577 Matsushima, Kurashiki 701-0192 Japan
2Department of Surgery and 3Department of Cell Biology, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558, Japan
4Tissue Engineering Research Center, The National Institute of Advanced Industrial Science and Technology, 1-1-4 Higashi, Tsukubashi, Ibaragi 305-8562, Japan
5Animal Center for Medical Research, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558, Japan
6Department of Immunochemistry, Faculty of Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima-naka, Okayama 700-8530, Japan

Normal human hepatocytes are an ideal source of liver-targeted cell therapies, such as hepatocyte transplantation and bioartificial livers, but availability of human donor livers for liver cell isolation is severely limited. To effectively utilize scarce donor organs for cell therapies, it is of extreme importance to establish an efficient isolation technique and an effective cold preservation solution for transportation of isolated cells. A lateral segment of the liver was surgically resected from pigs weighing 10 kg and a four-step collagenase and dispase digestion was conducted. Isolated hepatocytes were subjected to 8-h cold storage on ice. The following preservation solutions were tested: 1) University of Wisconsin (UW) solution, 2) UW with 100 mmg/ml of ascorbic acid-2 glucoside (AA2G), 3) 100% fetal bovine serum (FBS), and 4) Dulbecco's modified Eagle's medium (DMEM) supplemented with 100% FBS. The mean viability of porcine hepatocytes was 95.5 ± 2.5% when isolated in three independent experiments. Viability, plating efficiency, membrane stability, and ammonia metabolic capacity of cold-preserved hepatocytes were significantly better maintained by the use of UW solution. When AA2G (100 mg/ml) was combined with UW solution, such parameters were further improved. It was explained by inhibition of caspase-3 activation and retention of ATP at high levels of hepatocytes preserved with UW solution containing AA2G. The present work demonstrates that a combination of UW solution with AA2G (100 mg/ml) would be a useful cold preservation means for the development of cell therapies.

Key words: Hepatocytes; University of Wisconsin solution; Ascorbic acid-2 glucoside; Cold preservation

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) 86-235-7259; Fax: (+81) 86-234-9328; E-mail: immortal@md.okayama-u.ac.jp




Cell Transplantation, Vol. 12, pp. 607-616, 2003
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Cryopreservation of Primarily Isolated Porcine Hepatocytes With UW Solution

Takemi Kunieda,1 Masanobu Maruyama,2 Teru Okitsu,2 Norikuni Shibata,1 Michihiko Takesue,1 Toshinori Totsugawa,2 Yoshikazu Kosaka,2 Takashi Arata,2 Kazuya Kobayashi,2 Hideaki Ikeda,2 Mizuko Oshita,3 Shuhei Nakaji,3 Kenji Ohmoto,1 Shinichiro Yamamoto,1 Yuzuru Kurabayashi,4 Makoto Kodama,5 Noriaki Tanaka,2 and Naoya Kobayashi2

1Division of Gastroenterology I, Department of Medicine, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-0192 Japan
2Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan
3Medical Products Development Department, Kuraray Medical Co., 1621 Sakazu, Kurashiki, Okayama 710-8622, Japan
4Animal Center for Medical Research, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558, Japan
5Tissue Engineering Research Center, The National Institute of Advanced Industrial Science and Technology. 1-1-4 Higashi, Tsukubashi, Ibaragi 305-8562, Japan

Development of liver-targeted cell therapies, such as hepatocyte transplantation and bioartificial livers, requires a large amount of functional hepatocytes as needed. To achieve this development, establishing an excellent cryopreservation method of hepatocytes is an extremely important issue. Therefore, we performed a comparative review of cryoprotective effects of various cryopreservation solutions using primarily isolated porcine hepatocytes. Porcine hepatocytes were isolated with a four-step dispase and collagenase perfusion method. The obtained hepatocytes with the initial viabilities of 76%, 84%, and 96% were assigned to the following four groups for cryopreservation at -80°C: Dulbecco's modified Eagle's medium (DMEM) + 10% fetal bovine serum (FBS) + 12% dimethyl sulfoxide (DMSO) (group A), University of Wisconsin (UW) solution + 12% DMSO (group B), Cell Banker 1 (group C), and Cell Banker 2 (group D). The hepatocytes in each group were thawed at 3 days, 10 days, and 5 months of cryopreservation and subjected to comparative analyses, including viability, plating efficiency, LDH release, ammonia removal test, and lentiviral gene transfer. These parameters were the most favorable in the hepatocytes cryopreserved with UW solution. Approximately 5% of thawed cryopreserved porcine hepatocytes expressed LacZ activity after lentiviral transduction. Intrasplenic transplantation of UW solution-cryopreserved hepatocytes improved the survival of rats treated with D-galactosamine. UW solution maintained the functions of cryopreserved porcine hepatocytes.

Key words: Cryopreservation; Hepatocyte; UW solution

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) 86-221-8775; E-mail: immortal@md.okayama-u.ac.jp




Cell Transplantation, Vol. 12, pp. 617-625, 2003
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Factors Influencing Insulin Secretion From Encapsulated Islets

Bart J. de Haan, Marijke M. Faas, and Paul de Vos

Immunoendocrinology, Department of Pathology and Laboratory Medicine, Section of Medical Biology, University of Groningen, Hanzeplein 1, 9700 RB Groningen, The Netherlands

Adequate regulation of glucose levels by a microencapsulated pancreatic islet graft requires a minute-to-minute regulation of blood glucose. To design such a transplant, it is mandatory to have sufficient insight in factors influencing the kinetics of insulin secretion by encapsulated islets. The present study investigates factors influencing the glucose-induced insulin response of encapsulated islets in vitro. We applied static incubations and did the following observations. (i) Small islets (90-120 mm) showed a similar instead of a lower glucose-induced insulin response, suggesting that inclusion of only small islets, which are associated with lower protrusion and failing rates, has no consequences for the functional performance of the graft. (ii) A capsule diameter of 800 mm showed identical rather than lower glucose-induced insulin responses as smaller, 500-mm capsules. (iii) Capsule membranes constructed with a conventional permeability interfered with diffusion of insulin, as illustrated by a lower response of islets in capsules with a 10-min poly-L-lysine (PLL) membrane than islets in capsules with a 5-min PLL membrane. (iv) Irrespective of the tested porosity, the capsules provided sufficient immunoprotection because the 10-min PLL membranes did block diffusion of the cytokines IL-1b (17 kDa) and TNF-a (70 kDa) while the 5-min PLL membranes interfered with the diffusion of the vast majority of the cytokines. We conclude that capsules containing small islets (90-120 mm) and a membrane with a lower permeability than routinely applied is preferred in order to obtain a graft with adequate glucose-induced insulin responses.

Key words: Alginate; Islets; Transplantation; Poly-L-lysine; Encapsulation

Address correspondence to B. J. De Haan, Ph.D., Pathology and Laboratory Medicine, Section of Medical Biology, University of Groningen, Hanzeplein 1, 9700 RB Groningen, The Netherlands. Tel: (31) (50) 3619826; Fax: (31) (50) 3619911; E-mail: B.J.DE.HAAN@MED.RUG.NL




Cell Transplantation, Vol. 12, pp. 627-635, 2003
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Influence of VEGF on the Viability of Encapsulated Pancreatic Rat Islets After Transplantation in Diabetic Mice

S. Sigrist,1 A. Mechine-Neuville,2 K. Mandes,1 V. Calenda,3 S. Braun,3 G. Legeay,4 J-P. Bellocq,2 M. Pinget,1 and L. Kessler1



1Centre européen d'étude du Diabète, Faculté de Médecine, 11, rue Humann, 67 000, Strasbourg, France
2Laboratoire d'Anatomie Pathologique, Hôpital de Hautepierre, 67 000 Strasbourg, France
3Transgène S.A., 11, rue de Molsheim, 67 000 Strasbourg, France
4Centre de Transfert et de Technologie du Mans, 20, rue Thalès de Milet, 72 000 Le Mans, France

After pancreatic islet transplantation, insufficient blood supply is responsible for the loss of islet viability. The aim of our study was: 1) to determine the influence of vascular endothelial growth factor (VEGF) on the survival of encapsulated rat islets transplanted into healthy and diabetic mice and 2) to evaluate the metabolic efficiency of the VEGF-supplemented grafts. Twenty-four hours after culture, 50 rat islets immobilized into collagen in the presence of VEGF (100 ng/ml) and encapsulated (AN69 membrane, HOSPAL) were grafted in the peritoneal cavity of healthy or streptozotocin-induced diabetic mice (n = 6). Seven, 14, and 28 days after implantation, the encapsulation device and tissue surrounding the device were removed and the following parameters were analyzed: the number and the diameter of buds, the distance between devices and buds, the amount of cellular adhesion on the capsule surface, and the level of insulin secreted by encapsulated islet. For reversal of diabetes, 1000 rat islets encapsulated in the presence of VEGF were implanted in the peritoneal cavity of diabetic mice and fasting glycemia was analyzed. After 7 days of islet implantation in the absence of VEGF, the bud diameter was 16.1 ± 6.9 mm in diabetic mice and 34.4 ± 3.9 mm in healthy mice. However, the number of buds increased by a factor 2.5 in the presence of VEGF in both types of mice. Furthermore, when islets were transplanted in the presence of VEGF, the distance between the device and the buds was significantly decreased in both types of mice (p < 0.001) after 7, 14, and 28 days of islet implantation. Capsule analysis showed a decrease in cellular adhesion when the islets were encapsulated in the presence of VEGF. Insulin secretion of the islets was higher in the presence of VEGF compared with islets alone at all steps of the study. When 1000 rat islets were transplanted in the presence of VEGF, the glycemia level decreased to 6.2 ± 0.8 mmol/L after 3 days and remained stable until at least 28 days. In contrast, in the absence of VEGF, the initial decrease in the glucose level was rapidly followed by a relapse in hyperglycemia. In summary, VEGF increased the viability of engrafted encapsulated islets, increasing the duration of a normalized glycemia in diabetic mice following transplantation. Local adjunction of VEGF may therefore improve the clinical outcome of islet transplantation.

Key words: Islets; Transplantation; Encapsulation; VEGF; Angiogenesis; Diabetes

Address correspondence to Laurence Kessler, M.D., Ph.D., Centre européen d'étude du Diabète, Boulevard René Leriche, 67 200 Strasbourg, France. Tel: (+33) 3 90 20 12 12; Fax: (+33) 3 90 20 12 19; E-mail: Laurence.Kessler@medecine.u-strasbg.fr




Cell Transplantation, Vol. 12, pp. 637-646, 2003
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Activated Porcine Embryonic Brain Endothelial Cells Induce a Proliferative Human T-Lymphocyte Response

Suchitra Sumitran-Holgersson,1 Thomas Brevig,2 Håkan Widner,3 and Jan Holgersson1

1Division of Clinical Immunology, Karolinska Institutet, Huddinge University Hospital AB, S-141 86 Stockholm, Sweden
2Department of Molecular Characterization, Biotechnological Institute, DK-2970 Horsholm, Denmark
3Departments of Physiological Sciences and Clinical Neuroscience, Lund University, BMC-A10, S-223 62 Lund, Sweden

Transplantation of allogeneic embryonic neural tissue is a potential treatment for patients with Parkinson's and Huntington's diseases. The supply of human donor tissue is limited, and alternatives such as the use of animal (e.g., porcine) donor tissue are currently being evaluated. Before porcine grafts can be used clinically, strategies to prevent neural xenograft rejection must be developed. Knowledge on how human T lymphocytes recognize porcine embryonic neural tissue would facilitate the development of such strategies. To investigate the ability of porcine embryonic brain microvascular endothelial cells (PBMEC) to stimulate human T-cell proliferation, PBMEC were immuno-magnetically isolated and cocultured with purified human CD4 or CD8 single-positive T cells. PBMEC had a cobblestone-like growth pattern and expressed the endothelial cell markers CD31 and CD106. PBMEC stimulated with the supernatant of phytohemagglutinin-activated porcine peripheral blood mononuclear cells or porcine IFN-g, but not nonstimulated PBMEC, induced proliferation of both CD8 and CD4 T cells as assessed by [3H]thymidine incorporation. Flow cytometric analyses showed that the degree of CD8 and CD4 T cell proliferation correlated with the expression levels of class I and II major histocompatibility complex (MHC) antigens, respectively. PBMEC expressed a CTLA-4/Fc-reactive molecule, most likely CD86, suggesting that these cells are able to deliver a costimulatory signal to the T cells. Human TNF-a, but not human IFN-g, induced class I, but not class II, MHC expression on PBMEC. Within a neural graft or the regional lymph nodes, PBMEC might stimulate human T cells via the direct pathway, and should therefore be removed from the donor tissue prior to transplantation.

Key words: Endothelial cell; Antigen presentation; T lymphocyte; Xenotransplantation; Neural transplantation; Parkinson's disease; Pig

Address correspondence to Jan Holgersson, M.D., Ph.D., Division of Clinical Immunology, F79, Huddinge University Hospital AB, S-141 86 Stockholm, Sweden. Tel: +46 8 585 81384; Fax: +46 8 585 81390; E-mail: jan.holgersson@impi.ki.se




Cell Transplantation, Vol. 12, pp. 647-657, 2003
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Transplanted Endothelial Progenitor Cells Augment the Survival Areas of Rat Dorsal Flaps

Yoshiaki Kubota,1 Kazuo Kishi,1 Hiroko Satoh,2 Takara Tanaka,1 Hideo Nakajima,1 and Tatsuo Nakajima1

1Department of Plastic and Reconstructive Surgery, School of Medicine, Keio University, 35, Shinanomachi Shinjuku-ku, Tokyo 160-8582, Japan
2Department of Plastic and Reconstructive Surgery, National Tokyo Medical Center, 2-5-1, Higashigaoka, Meguro-ku, Tokyo 152-8902, Japan

Endothelial progenitor cells (EPCs) have been identified in peripheral blood, and have been reported to be incorporated into ischemic regions such as the ischemic hindlimb. In this study, we examined whether or not transplantation of EPCs is useful for salvaging surgical flaps in vivo. At the same time, we quantitatively compared the neovascularization ability of transplanted EPCs and that of mature endothelial cells (ECs). ECs obtained from the aorta of rats by explantation and passaged several times were used in the present study. EPCs were obtained from the blood of rat hearts. The blood samples were separated by density gradient centrifugation. Light-density mononuclear cells (MNCs) were collected and cultured on plastic plates coated with rat plasma vitronectin. Cells attached at day 7 of culture were deemed to be EPCs. Then PBS (control), ECs, or EPCs (3.0 x 105 suspended in 1.0 ml PBS) were injected at the middle of a flap. Seven days after surgery, the survival lengths of the flaps were evaluated. EPC-transplanted groups revealed statistically significant augmentation of survival length compared with the other two groups (p < 0.003). EPC-transplanted groups had significantly more angiographically detectable blood vessels (p < 0.003) and significantly higher capillary density (p < 0.03) than the other two groups. Confocal microscopy revealed that EPCs were incorporated into enhanced neovascularization. These results suggest that transplantation of EPCs may be useful for salvaging surgical flaps, and EPCs are superior to ECs in neovascularization ability.

Key words: Endothelial cell; Endothelial progenitor cell (EPC); Flap; Neovascularization; Rat

Address correspondence to Kazuo Kishi, Department of Plastic and Reconstructive Surgery, School of Medicine, Keio University, 35 Shinanomachi Shinjuku-ku Tokyo 160-8582, Japan. Tel: +81-(0)3-5363-3814; Fax: +81-(0)3-3352-1054; E-mail: kkishi@sc.itc.keio.ac.jp




Cell Transplantation, Vol. 12, pp. 659-666, 2003
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Decellularized Native and Engineered Arterial Scaffolds for Transplantation

Shannon L. M. Dahl,1* Jennifer Koh,3* Vikas Prabhakar,4 and Laura E. Niklason1,2

Departments of 1Biomedical Engineering and 2Anesthesiology, Duke University, Durham, NC 27708
3Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093
4Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, MA 02139

More than 570,000 coronary artery bypass grafts are implanted each year, creating an important demand for small-diameter vascular grafts. For patients who lack adequate internal mammary artery or saphenous vein, tissue-engineered arteries may prove useful. However, the time needed to tissue engineer arteries (7 weeks or more) is too long for many patients. Decellularized cadaveric human arteries are another possible source of vascular conduit, but limited availability and the potential for disease transmission limit their widespread use. In contrast, decellularized tissue-engineered arteries could serve as grafts for immediate implantation, as scaffolds onto which patients' cells could be seeded, or as carriers for genetically engineered cells to aid cell transplantation. The goal of this study was to quantify the effects of decellularization on vascular matrix and mechanical properties. Specifically, we compared cellular elimination, extracellular matrix retention, and mechanical characteristics of porcine carotid arteries before and after treatment with three decellularization methods. In addition, for the first time, tissue-engineered arteries were decellularized. Decellularized native arteries were also used as a scaffold onto which vascular cells were seeded. These studies identified a decellularization method for native and engineered arteries that maximized cellular elimination, without greatly compromising mechanical integrity. We showed that engineered tissues could be decellularized, and demonstrated the feasibility of reseeding decellularized vessels with vascular cells.

Key words: Arteries; Decellularization; Extracellular matrix; Scaffolds; Tissue engineering

Address correspondence to Laura E. Niklason, M.D., Ph.D., Department of Biomedical Engineering, Box 90281, 136 Hudson Hall, Duke University, Durham, NC 27708. Tel: (919) 660-5149; Fax: (919) 684-5777; E-mail: nikla001@mc.duke.edu

*These authors contributed equally to this work.




Cell Transplantation, Vol. 12, pp. 667-676, 2003
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Novel Technique for Suspension Culture of Autologous Chondrocytes Improves Cell Proliferation and Tissue Architecture

Toshiaki Takahashi,1 Takamasa Nieda,1 Eriko Miyazaki,2 and Hideaki Enzan2

1Department of Orthopaedic Surgery, 2First Department of Pathology, Kochi Medical School, Oko-cho, Nankoku, Kochi, 783-8505, Japan

We have developed a new and simple method of chondrocyte suspension culture using a spinner bottle with rotation of the matrices. We compared the characteristics of chondrocytes cultured by this method with those grown in standard monolayer cultures. We also determined the optimal nutritional medium for suspension cultures. Periosteum explants seeded with chondrocytes were grown in monolayer and suspension cultures under three conditions: in medium with no additive (control), with 10% fetal bovine serum (FBS), or with 10% autologous serum (AS). After culturing, the explants were harvested, processed for histology, and stained with hematoxylin-eosin or TUNEL, or immunostained for type I, II, and III collagen, and Ki-67 antigen. In monolayer cultures, the attachment of the chondrocytes to the periosteum was weak and the superficial layer consisted of fibrotic tissue and few nucleated cells. Collagen type II staining was strong, but types I and III were weak. Among the suspension cultures the AS group produced the thickest layer of chondrocytes with the fewest apoptotic cells. The superficial layer of cartilage in these cultures stained positive for type I and III collagen and Ki-67 antigen. Among the suspension cultures, total chondroitin and chondroitin-4 sulfate (C-4S) concentration was highest in the AS group, while prostaglandin E2 (PGE2) was highest in the FBS group. In summary, our new method of suspension culture of periosteal explants using rotational matrices combined with AS nutritional media was the most effective method for maintaining the bond between the chondrocyte layer and periosteum, as well as the production of type I and III collagen in the superficial layer.

Key words: Suspension culture; Chondrocytes; Periosteum; Collagen; Autologous serum; Superficial layer

Address correspondence to Toshiali Takahashi, Department of Orthopaedics, Kochi Medical School, Oko-cho, Nankoku, Kochi, 783-8505, Japan. Tel: 81-88-880-2387; Fax: 81-88-880-2388; E-mail: takahast@kochi-ms.ac.jp