ognizant Communication Corporation

Cell Transplantation, Vol. 13, pp. 1-6, 2004
0963-6897/04 $20.00 + 00
Copyright © 2004 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

¹Surgical-Medical Research Institute, Department of Surgery, University of Alberta, Edmonton T6G 2N8, Canada
²Department of General Surgery and The Transplant Center, Carolinas Medical Center, Charlotte, NC 28232
³Sertoli Technologies, Inc., Cranston, RI 02905

Gene therapy involves the manipulation of genetic material to replace defective or deficient proteins to restore function in disease states. These genes are introduced into cells by mechanical, chemical, and biological approaches. To date, cell-based gene therapy has been hampered by the lack of an abundant, safe, and immunologically acceptable source of tissue. As an alternative, transgenic animals designed to produce therapeutic proteins could overcome some of the issues facing gene therapy but the problem of immune rejection of the tissue remains. This article reports on recently published work indicating the potential to use transgenic Sertoli cells surviving in an allogeneic host by virtue of their ability to create a locally immunoprivileged environment, thereby providing for the continued delivery of a therapeutic protein to the systemic circulation.

Key words: Sertoli cell; Gene therapy; Transgenic; Immune privilege

Address correspondence to Craig Halberstadt, Ph.D., Director, Tissue Engineering, Senior Research Scientist, Carolinas Medical Center, Cannon Research Building, PO Box 32861, Charlotte, NC 28232-2861. Tel: (704) 355-2846; Fax: (704) 355-7203; E-mail: chalberstadt@carolinas.org

¹Department of Integrative Human Cardiovascular Physiology and Cardiac Surgery, ²Department of Cardiology, ³Division of Haematology, and ⁴Department of Surgery, University of Leicester, Glenfield Hospital, Leicester, LE3 9QP, UK

Stem cell transplants into damaged myocardium may have the potential to improve cardiac function. We investigated the safety of transplanting unmanipulated autologous bone marrow into infarcted myocardium of patients undergoing coronary bypass surgery and assessed its efficacy to improve cardiac function. Fourteen patients with one or more areas of transmural myocardial infarction were studied. Autologous bone marrow was obtained by sternal bone aspirate at the time of surgery, diluted in autologous serum at a ratio of 1:2, and then injected 1 cm apart into the mid-depth of the left ventricular scar. There were no deaths, no perioperative myocardial infarctions, and no significant ventricular arrhythmias. Dobutamine stress echocardiography demonstrated overall improvement in the global and regional left ventricular function 6 weeks and 10 months after surgery. Of 34 infarcted left ventricular segments, 11 were injected with bone marrow alone, 13 were revascularized with a bypass graft alone, and 10 received bone marrow transplantation and a bypass graft in combination. Only the left ventricle segmental wall motion score of the areas injected with bone marrow and receiving a bypass graft in combination improved at low dose and at peak dobutamine stress. These findings suggest that transplantation of unmanipulated autologous bone marrow into scar tissue of the human heart is safe and enhances cardiac function only when used in combination with myocardial revascularization. This benefit can be seen after 6 weeks of the bone marrow transplant and is maintained after 10 months of follow-up.

Key words: Human; Bone marrow; Transplantation; Infarcted myocardium

Address correspondence to Professor Manuel Galiñanes, Department of Integrative Human Cardiovascular Physiology and Cardiac Surgery, University of Leicester, Glenfield Hospital, Leicester LE3 9QP, UK. Tel: 0044 116 2563032; Fax: 0044 116 2502449; E-mail: mg50@le.ac.uk

¹School of Dentistry, The University of Queensland, Brisbane, Queensland, Australia
²Tissue BioRegeneration and Integration, Science Centre, Queensland University of Technology, Brisbane, Queensland, Australia
³Royal Children's Hospital, The University of Queensland, Brisbane, Queensland, Australia
⁴Colgate Australian Clinical Dental Research Centre, School of Dentistry, Adelaide University, Adelaide, South Australia

The use of extracellular matrix materials as scaffolds for the repair and regeneration of tissues is receiving increased attention. The current study was undertaken to test whether extracellular matrix formed by osteoblasts in vitro could be used as a scaffold for osteoblast transplantation and induce new bone formation in critical size osseous defects in vivo. Human osteoblasts derived from alveolar bone were cultured in six-well plates until confluent and then in mineralization media for a further period of 3 weeks to form an osteoblast-mineralized matrix complex. Histologically, at this time point a tissue structure with a "connective tissue"-like morphology was formed. Type I collagen was the major extracellular component present and appeared to determine the matrix macrostructure. Other bone-related proteins such as alkaline phosphatase (ALP), bone morphogenetic protein (BMP)-2 and -4, bone sialoprotein (BSP), osteopontin (OPN), and osteocalcin (OCN) also accumulated in the matrix. The osteoblasts embedded in this matrix expressed mRNAs for these bone-related proteins very strongly. Nodules of calcification were detected in the matrix and there was a correlation between calcification and the distribution of BSP and OPN. When this matrix was transplanted into a critical size bone defect in skulls of immunodeficient mice (SCID), new bone formation occurred. Furthermore, the cells inside the matrix survived and proliferated in the recipient sites, and were traceable by the human-specific Alu gene sequence using in situ hybridization. It was found that bone-forming cells differentiated from both transplanted human osteoblasts and activated endogenous mesenchymal cells. This study indicates that a mineralized matrix, formed by human osteoblasts in vitro, can be used as a scaffold for osteoblast transplantation, which subsequently can induce new bone formation.

Key words: Extracellular matrix; Mineralized matrix; Osteoblasts; Bone formation

Address correspondence to P. M. Bartold, University of Adelaide, CACDRC Dental School, Frome Road, Adelaide, South Australia 5005, Australia. E-mail: mark.bartold@adelaide.edu.au

Departments of ¹Otolaryngology, ²Pathology, and ³Surgery, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501, Japan

Transplantation of rat hepatocytes into the syngeneic rat spleen results in the appearance of cytokeration (CK)-19-positive biliary cells that form ductules. The exact origin of CK-19-positive cells is not known and the possibility that they are derived from biliary cells or precursors of oval cells in transplanted hepatocyte preparations has been raised. In the present study, we found that the number of CK-19-positive biliary cells increased rapidly after transplantation of hepatocytes, reached the maximum at 4 weeks, and then gradually decreased. However, a Ki-67 labeling index of CK-19-positive biliary cells was low and showed no significant changes throughout the experimental period. In addition, no or few CK-19-positive cells appeared in the spleen after transplantation of nonparenchymal liver cells enriched with biliary cells. These results showed that biliary cells were not the source of CK-19-positive cells in the spleen. Impairment of precursors of oval cells in the liver by administration of 4,4´-diaminodiphenylmethane 24 h before transplantation of hepatocytes did not prevent the appearance of CK-19-positive biliary cells in the spleen. Moreover, transplantation of nonparenchymal cells carrying an increased number of oval cells by means of treatment with 2-acetylaminofluorene and partial hepatectomy resulted in no appearance of CK-19-positive biliary cells in the spleen. These results ruled out oval cells as the origin of CK-19-positive biliary cells in the spleen. Because CK-19-positive biliary cells appeared in the spleen only when hepatocyte fractions were transplanted, we suggest transdifferentiation of heptocytes may be the mechanism by which CK-19-positive biliary cells are generated.

Key words: Cytokeratin-19; Transplantation; Spleen; Hepatocyte; Biliary cell; Rat

Address correspondence to Nobuyuki Terada, M.D., Department of Pathology, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501, Japan. Fax: 0798-45-6431; E-mail: terada@hyo-med.ac.jp

¹NORLUX Neuro-Oncology, Department of Anatomy and Cell Biology, University of Bergen, Jonas Lies vei 91, 5009 Bergen, Norway
²Department of Oncology, Haukeland University Hospital, 5021 Bergen, Norway
³NORLUX Neuro-Oncology, Centre de Recherche Public Santé, Luxembourg

The potential benefit of continuous local administration of antiangiogenic proteins to CNS tumors in vivo has recently been demonstrated using endostatin-producing recombinant cells encapsulated in alginate beads. Due to the treatment potential of transplanted alginate-encapsulated cells producing therapeutic proteins, we describe a successful method of cryopreservation (CP) of such beads, in which cellular viability, alginate structure, and protein secretion were maintained. Alginate beads containing human embryonic kidney cells (HEK 293 cells) stably transfected with the gene encoding for endostatin were cryopreserved in dimethyl sulfoxide (DMSO) using a slow freezing procedure. Briefly, the DMSO concentration was gradually increased prior to the freezing procedure. The cryotubes were further supercooled to -7.5°C and nucleated. Thereafter, the samples were cooled at a rate of 0.25°C/min and stored in liquid nitrogen. The viability of the encapsulated cells was assessed using confocal microscopy quantification (CLSM) technique and a MTS assay. The cell cycle distribution inside the beads was assessed by DNA flow cytometry and endostatin production was determined by an endostatin-specific ELISA assay, both prior to and after CP. CLSM measurements showed sustained esterase activity in the beads after thawing, with only a slight transient decrease 24 h after CP. The MTS assay verified these findings by displaying similar variations of intracellular dehydrogenase activity. Flow cytometric analyses revealed no cryorelated disturbances in cellular ploidy. Furthermore, ELISA measurements showed a well-preserved endostatin production after CP. In conclusion, this work describes the successful CP of alginate-encapsulated recombinant cells secreting a therapeutic protein. Together with previous published reports, these results further substantiate the feasibility and potential of cell encapsulation therapy in the treatment of malignant tumors.

Key words: Cryopreservation; Alginate encapsulation; Recombinant cells; Endostatin; Antiangiogenic therapy

Address correspondence to Rolf Bjerkvig, Ph.D., NORLUX Neuro-Oncology, Department of Anatomy and Cell Biology, University of Bergen, Jonas Lies vei 91, 5009 Bergen, Norway. Tel: +47 55 58 63 52; Fax: +47 55 58 63 60; E-mail: rolf.bjerkvig@pki.uib.no

Department of Plastic and Hand Surgery, Tissue Engineering Laboratory, University of Freiburg Medical Center, Freiburg, Germany

Surgical reconstruction of muscle tissue lost by trauma or tumor ablation is limited by the lack of availability of functional native tissue substitution. Moreover, so far most inherited or acquired muscle diseases are lacking sufficient treatment, because only few alternatives exist to provide functional restoration of lost muscle tissues. Engineering those tissues and transplantation into sites of dysfunction may be an alternative approach and may allow replacement of such damaged or failing skeletal muscle tissues. Techniques attempting reconstruction of some human tissues and organs (tissue engineering) have been introduced into clinical practice recently. One major problem that previous transplantation studies were facing is the ability of detection of transplanted cells after integration. Using the Y chromosome in situ hybridization technique in a syngeneic rat model allows transplantation of cell constructs orthotopically, without manipulation of the cells, with no rejection or immunosuppression being implied, but providing a nondilutable genetic marker to identify transplanted cells. The purpose of our study was to create functional skeletal muscle tissue in vivo using the transplantation of primary myoblasts precultivated within a three-dimensional (3D) fibrin matrix and to determine the fate of the transplanted cells using the Y chromosome detection technique. 3D myoblast cultures were established derived from male donor rats and after 7 days of cultivation we performed an orthotopic transplantation of 3D cell constructs into a created muscle defect within the gracilis muscle of syngeneic female rats. Anti-desmin immunostaining and Y chromosome in situ hybridization indicated the survival and integration of transplanted male myoblasts into the female recipient animal, thus demonstrating the feasibility of this approach in tissue engineering and the research of cell transplantation in general.

Key words: Muscle transplantation; Three-dimensional culture; In situ hybridization; Skeletal muscle; Tissue engineering; Y chromosome

Address correspondence to Justus P. Beier, Department of Plastic and Hand Surgery, Tissue Engineering Laboratory, University of Freiburg Medical Centre, Hugstetter Str. 55, D-79106 Freiburg I Br., Germany. Tel: 0049-761-270-6367; Fax: 0049-761-270-6368; E-mail: beier@chir11.ukl.uni-freiburg.de

University of Miami, ¹Cell Transplant Division of Surgery, ²Department of Radiology, and ³Department of Medicine, Miami, FL 33136

An infrequent but nevertheless concerning complication associated with percutaneous transhepatic islet transplantation is bleeding. Historically in 61 procedures at this institution, we experienced four bleeding complications in three patients (6.6%), two requiring blood transfusion (3.3%) and two asymptomatic intraperitoneal bleeds detected sonographically at 24 h postprocedure (3.3%). It is suggested that the source of the majority of these bleeds is the liver parenchymal tract following removal of the infusion catheter combined with a significant dose of heparin administered to prevent portal vein thrombosis. Various techniques have been used to reduce the risk of tract bleeding, including gelfoam, intravascular coils, and cautery. In our experience gelfoam alone has been used to plug the catheter tract (n = 47); however, in the aforementioned three patients, this technique failed, either due to dislodgement of, or bleeding peripheral to, the plug. This article describes the use of D-Stat^TM, a collagen/thrombin paste that is injected into the peripheral tract. In five consecutive cases performed using D-Stat^TM, there has been no bleeding or thromboses detected. D-Stat^TM combined with a single gelfoam plug offers a quick, easy, efficacious way of sealing the entire catheter tract without leaving any permanent hardware in the liver. This new method may simplify tract closure and reduce bleeding complications in islet transplantation.

Key words: D-Stat^TM; Percutaneous transhepatic islet transplantation; Bleeding; Gelfoam plugs

Address correspondence to Tatiana Froud, M.D., Diabetes Research Institute, R-134, 1450 NW 10th Avenue, Miami, FL 33136. Tel: (305) 534-0195; Fax: (305) 243-1058; E-mail: tfroud@med.miami.edu

Kidney and Pancreas Transplantation Program, Hospital Italiano de Buenos Aires, Gascon 450, Buenos Aires (1181), Argentina

Here we report on the impact of completely unpurified islet transplantation on the portal vein pressure (PVP) and the hepatic biochemistry in the peritransplant period and on follow-up. Type I diabetic patients underwent simultaneous kidney and islet transplantation. Islets were not purified from the acinar tissue to prevent loss of endocrine mass. Each patient received a mean 521,846 ± 201,539.4 islet equivalents (7812.1 islet equivalents/kg/recipient). Immunosuppression and peritransplant medication were given according to the Giessen protocol. The islets were injected into the left hepatic lobe through the umbilical vein. PVP was recorded at time 0 and every 5 min throughout cell infusion. Liver function was assessed daily for the first 10 days, and on follow-up. Basal, peak, and final PVP were 12 ± 3.8, 25.1 ± 7.9, and 19.5 ± 6.2 mmHg, respectively (basal vs. final, p < 0.05). Bilirubin, alkaline phosphatase, prothrombin time, and APTT stayed within normal range. Peak aspartate aminotransferase (AST), alanine aminotransferase (ALT), and serum amylase were 109.4 ± 61.2 IU/L (basal vs. peak, not significant), 79.5 ± 56.9 IU/L (basal vs. peak, not significant), and 887.5 ± 153.6 IU/L (basal vs. peak, p = 0.02), respectively. In all cases AST, ALT, and amylase normalized within 6 days posttransplant and remained so on follow-up (longest control, 33 months posttransplant). Although the intrahepatic infusion of unpurified pancreatic islets affects both the portal vein pressure and the hepatic biochemical profile, this effect is transient and does not compromise the safety of the procedure.

Key words: Human islet transplantation; Unpurified islets; Intraportal islet injection; Portal vein pressure; Hepatic biochemistry

Address correspondence to Pablo F. Argibay, M.D., Ph.D., Kidney and Pancreas Transplantation Program, Hospital Italiano de Buenos Aires, Potosi 4240, 8th Floor, Buenos Aires C1199ACL, Argentina. Tel/Fax: (+54-11) 4959-0200, ext. 8919; E-mail: pablo.argibay@hospitalitaliano.org.ar

¹Surgical-Medical Research Institute, Department of Surgery, University of Alberta, Edmonton, Alberta, T6G2N8, Canada
²Department of Gastroenterological Surgery, Graduate School of Medical Sciences, Kobe University, Kobe, Hyogo, 650-0017, Japan

A two-layer cold storage method (TLM) allows sufficient oxygen delivery to pancreata during preservation and resuscitates the viability of ischemically damaged pancreata in the canine pancreas transplant model. In this study, we applied a short-term preservation of the TLM to human pancreata after prolonged cold ischemia prior to islet isolation, and investigated the mechanisms of resuscitation of the ischemically damaged human pancreas by the TLM. Human pancreata were procured from cadaveric donors and preserved by the TLM for 3.2 ± 0.5 h after 11.1 ± 0.9 h of cold storage in UW (TLM group), or by cold UW alone for 11.0 ± 0.3 h (UW group). Islet isolations of all pancreata were performed using the Edmonton protocol. Islet recovery and in vitro functional viability of isolated islets were significantly increased in the TLM group compared with the UW group. According to the criteria of the Edmonton protocol, 10/14 cases (71%) in the TLM group were transplanted to patients with type I diabetes mellitus compared with only 5/21 cases (24%) in the UW group. In the metabolic assessment of human pancreata, levels of energetic parameters (ATP, total adenylates, and energy charge) were significantly increased, and malondialdehyde (MDA) levels were significantly decreased after the TLM preservation. There was no observable change in the incidence or degree of mitochondrial injury after the TLM preservation. Additional short-term storage by the TLM resuscitates the ischemically damaged human pancreas by regenerating the energetic status and prevents further damage by oxidative stress, ultimately leading to improvements of islet recovery and in vitro function. Use of the TLM following prolonged storage in UW provides an excellent adjunctive protocol for treating human pancreata for the rigors of the islet isolation process.

Key words: Human islet isolation; Pancreas preservation; Two-layer method; Perfluorochemical; Oxygenation

Address correspondence to Jonathan R. T. Lakey, Ph.D., Director, Clinical Islet Laboratory, Assistant Professor of Surgery, Surgical-Medical Research Institute, University of Alberta, 1074 Dentistry/Pharmacy, Edmonton, Alberta, T6G2N8, Canada. Tel: (780) 492-3077; Fax: (780) 492-6335; E-mail: jonathan.lakey@ualberta.ca

¹Departments of Molecular and Preclinical Sciences, Alexion Pharmaceuticals Inc., 352 Knotter Drive, Cheshire, CT 06410
²Department of Molecular and Cellular Biology, CT Center for Regenerative Biology, University of Connecticut, Storrs, CT 06269

Porcine xenografts transplanted into primates are rejected in spite of immunosuppression. Identification of the triggering mechanisms and the strategies to overcome them is crucial to achieve long-term graft survival. We hypothesized that porcine CD86 (pCD86) contributes to xenograft rejection by direct activation of host T cells and NK cells. Formerly, we designed the human chimeric molecule hCD152-hCD59 to block pCD86 in cis. To test the efficacy in vivo, we have utilized a pig-to-mouse xenotransplant model. First, we showed that hCD152-hCD59 expression prevents the binding of murine CD28Ig to pCD86 on porcine aortic endothelial cells (PAEC) and dramatically reduces IL-2 secretion by Con A-stimulated mouse splenocytes in coculture. Moreover, IFN-g secretion by IL-12-stimulated mouse NK cells was averted after coculture with hCD152-hCD59 PAEC. In vivo, control PAEC implanted under the kidney capsule were rapidly rejected (2-4 weeks) in BALB/c and BALB/c SCID mice. Rejection of hCD152-hCD59 PAEC was significantly delayed in both cases. Signs of immune modulation in the hCD152-hCD59-PAEC BALB/c recipients were identified such as early hyporesponsiveness and diminished antibody response. Thus, simply modifying the donor xenogeneic cell can diminish both T cell and NK cell immune responses. We specifically demonstrate that pCD86 contributes to rejection of porcine xenografts.

Key words: CD86 antigen; CD152 antigen; T cells; NK cells; Antibody response; Xenotransplantation

Address correspondence to Cristina Costa, Alexion Pharmaceuticals Inc., 352 Knotter Drive, Cheshire, CT 06410. Tel: (203) 271-8252; Fax: (203) 271-8195; E-mail: costac@alxn.com or to William L. Fodor, Department of Molecular and Cellular Biology, CT Center for Regenerative Biology, University of Connecticut, 1390 Storrs Road, Storrs, CT 06269-4243. Tel: (860) 486-8336; Fax: (860) 486-0534; E-mail: william.fodor@uconn.edu