ognizant Communication Corporation


VOLUME 11, NUMBER 2, 2002

Cell Transplantation, Vol. 11, pp. 95-101, 2002
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Beneficial Effects of Hyperbaric Oxygen Therapy on Islet Transplantation

Jyuhn-Huarng Juang,1 Brend Ray-Sea Hsu,1 Chien-Hung Kuo,1 and Steve Wen-Neng Ueng2

1Division of Endocrinology and Metabolism, Department of Internal Medicine, and 2Department of Orthopedic Surgery, Chang Gung Memorial Hospital, Taoyuan, Taiwan

We envisage that hyperbaric oxygen (HBO) would ameliorate islet anoxia, preventing early graft failure. Thus, treatment of HBO to diabetic recipients should improve the outcome of islet transplantation. We tested this hypothesis by syngeneically transplanting insufficient number of islets (150 islets) into streptozotocin-diabetic C57BL/6 mice, each followed by HBO (2.4 ATA, 100% O2) therapy for 1.5 h from day 0 to 28, once daily (group A) or twice daily (group B), or from day 5 to 28, once daily (group C) or twice daily (group D), 6 days/week. Recipients without HBO treatment served as controls. At day 28 after transplantation, groups B, C, and D gained weight and had lower blood glucose compared with their baseline values. In addition, groups B and D had higher insulin content of the graft than the controls. To determine the optimal timing of HBO therapy, groups B and D were compared with recipients treated with HBO twice daily, 6 days/week, from day -14 to 0 (group E) and from day -14 to 28 (group F). At day 28 after transplantation, groups B, D, E, and F had significantly lower blood glucose than their individual baseline values and higher insulin content of the graft than controls. But only group F had more b-cell mass of the graft than controls. These findings lend credence to the expectation that peritransplant application of adequate frequency of HBO to diabetic recipients would enhance the performance and growth of the islet graft, resulting in an improvement of the outcome of the transplantation.

Key words: Hyperbaric oxygen; Pancreatic islet cells; Islet transplantation

Address correspondence to Dr. Jyuhn-Huarng Juang, Division of Endocrinology and Metabolism, Chang Gung Memorial Hospital, 5 Fu-Shin Street, Kweishan, Taoyuan, Taiwan. Tel: +886-3-3277976; Fax: +886-3-3277976; E-mail: jjuang@adm.cgmh.org.tw

Cell Transplantation, Vol. 11, pp. 103-112, 2002
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Islet Transplantation Under the Kidney Capsule Corrects the Defects in Glycogen Metabolism in Both Liver and Muscle of Streptozocin-Diabetic Rats

Margherita Matarazzo,1 Maria Grazia Giardina,1 Vincenzo Guardasole,1 Alberto M. Davalli,2,3 Edward S. Horton,3 Gordon C. Weir,3 Luigi Saccà,1 and Raffaele Napoli1,3

1Department of Internal Medicine and Cardiovascular Sciences, University Federico II School of Medicine, Napoli, Italy
2Scientific Institute S.Raffaele, Milano, Italy
3Joslin Diabetes Center, Harvard Medical School, Boston, MA

Insulin-deficient rats are characterized by multiple defects in the pathway of glycogen synthesis and breakdown in both liver and skeletal muscle. The aim of this study was to clarify whether islet transplantation under the kidney capsule, which is associated with delivery of insulin into the peripheral circulation, is able to normalize glycogen metabolism in liver and muscle of streptozotocin-diabetic rats. Three groups of male Lewis rats were studied under fasting condition: controls, untreated diabetics, and islet transplanted diabetics. Glycogen content, glucose-6-phosphate concentration, and glycogen synthase activity were measured in both liver and skeletal muscle. Untreated diabetic rats were characterized by an increase in glycogen content of 178% and a reduction of glucose-6-phosphate level of 50%. Both glycogen and glucose-6-phosphate contents were restored to normal in transplanted diabetic rats. Active glycogen synthase (0.35 ± 0.1 nmol/min/mg) and activity ratio (0.22 ± 0.04) were significantly impaired compared with controls (0.99 ± 0.2 nmol/min/mg and 0.43 ± 0.06, respectively) and were normalized by islet transplantation. In the skeletal muscle, glycogen content was similar in the three groups of animals, whereas muscle glucose-6-phosphate level was reduced by 28% and glycogen synthase was in a less active state in the untreated diabetic rats. Both the glucose-6-phosphate concentration and the kinetic profile of glycogen synthase were normalized by islet transplantation. In conclusion, islet transplantation under the kidney capsule corrects the diabetes-induced abnormalities in glycogen and glucose-6-phosphate content and glycogen synthase activity in both liver and skeletal muscle.

Key words: Islet transplantation; Glycogen; Glucose-6-phosphate; Glycogen synthase; Liver; Muscle

Address correspondence to Luigi Saccà, Medicina Interna, Via Pansini, 5, 80131 Napoli, Italy. Tel: (+39) 0817463517; Fax: (+39) 0817463199; E-mail: sacca@unina.it

Cell Transplantation, Vol. 11, pp. 113-123, 2002
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Distinct Characteristics and Features of Allogeneic Chimerism in the NOD Mouse Model of Autoimmune Diabetes

Tatiana D. Zorina,1 Vladimir M. Subbotin,2 Suzanne Bertera,1 Angela M. Alexander,1 Catherine Haluszczak,1 Alexis J. Styche,1 and Massimo Trucco1

1Division of Immunogenetics, Department of Pediatrics, University of Pittsburgh, School of Medicine, Pittsburgh, PA 15213
2Mirus Corporation, Madison, WI 53719

The adaptation of allogeneic chimerism in treatment of autoimmune diabetes has been shown as a promising approach in numerous studies in both experimental and clinical settings. Establishment of hemopoietic chimerism in NOD mice is the most adequate animal model to study mechanisms involved in the multiple aspects of the curative effects of chimerism in autoimmunity-prone individuals. However, there are some discrepancies in the current literature for parameters and criteria used to characterize chimerism in the NOD model. This study was aimed to standardize the criteria for the different pathological stages of diabetogenesis in chimeric versus unmanipulated NOD mice. We report two well-defined scoring systems and a new Index N for the assessment of the pathological characteristics of diabetogenesis and GVHD in chimeric NOD mice. Also, we have demonstrated that, in the NOD model, recipient conditioning resulting in as low as 1% of chimerism is sufficient to promote engraftment of the BM donor-specific islets of Langerhans.

Key words: Autoimmune diabetes; Allogeneic chimerism; NOD mouse

Address correspondence to Tatiana D. Zorina, M.D., Ph.D., Division of Immunogenetics, Department of Pediatrics, University of Pittsburgh, School of Medicine, Pittsburgh, PA 15213. Tel: (412) 692-5238; Fax: (412) 692-5809; E-mail: tatiana@pitt.edu

Cell Transplantation, Vol. 11, pp. 125-138, 2002
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Induction of Ectopic Bone Formation by Using Human Periosteal Cells in Combination With a Novel Scaffold Technology

Jan-Thorsten Schantz,1,6 Dietmar Werner Hutmacher,2,3 Harvey Chim,4 Kee Woei Ng,1,5 Thiam Chye Lim,6 and Swee Hin Teoh1,5

1Laboratory for Biomedical Engineering, 2Department of Bioengineering, 3Department of Orthopaedic Surgery, 4Faculty of Medicine, 5Department of Mechanical Engineering, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260
6Department of Plastic Surgery, National University Hospital, 5 Lower Kent Ridge Road, Singapore 119074

Due to their osteogenic germination potential, periosteum-derived osteoprogenitor cells are a potential source for tissue engineering a bone graft that could be used to regenerate skeletal defects. In this study we evaluated if ectopic bone formation could be induced by a construct made of human periosteal cells and a novel scaffold architecture whose mechanical properties are in the range of cancellous bone. Biopsies from human calvarial periosteum were harvested and cells were isolated from the inner cambial layer. Fifty thousand periosteal cells were seeded into the scaffolds measuring 6 x 6 x 2 mm. The cell-scaffold constructs were cultured for a period of 3 weeks prior to implantation into balb C nude mice. Mice were sacrificed and implants were analyzed 6 and 17 weeks postoperatively. Immunohistochemical analysis confirmed the osteoblastic phenotype of the seeded cells. Formation of focal adhesions and stress fibers could be observed in both scaffold architectures. Three-dimensional cell proliferation was observed after 2 weeks of culturing with centripetal growth pattern inside the pore network. The deposition of calcified extracellular matrix was observed after 3 weeks of culturing. In vivo, endochondral bone formation with osteoid production was detectable via von Kossa and Osteocalcin staining after 6 and 17 weeks. Histology and SEM revealed that the entire scaffold/bone grafts were penetrated by a vascular network. This study showed the potential of bone tissue engineering by using human periosteal cells in combination with a novel scaffold technology.

Key words: Bone tissue engineering; Periosteal cells; Scaffolds; Polycaprolactone; Rapid prototyping

Address correspondence to Assistant Professor Dietmar W. Hutmacher M.S., Ph.D., M.B.A., Department of Bioengineering, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260. Tel: 65 874 5101; Fax: 65 777 3537

Cell Transplantation, Vol. 11, pp. 139-145, 2002
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Intradermal Cell Transplantation in Soluble Collagen

Hao Zhang and Peter J. Hornsby

Huffington Center on Aging and Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030

Intradermal, as opposed to subcutaneous, cell transplantation, was previously shown to be advantageous for tumor cell growth, but this site has not been used for transplantation of normal nonneoplastic cells. In preliminary experiments we found that it was difficult to control the size and shape of transplants when we injected dissociated cells intradermally. This problem was solved by placing cells in nongelled, pepsin-solubilized collagen prior to injection. This technique permitted the successful transplantation of normal bovine adrenocortical cells and of neoplastic cells (3T3 cells secreting FGF) in scid mice. Primary bovine adrenocortical cells formed functional vascularized tissue and the transplants rescued the animals from the lethal effects of adrenalectomy. The histological structure of transplant tissues resembled that previously observed when cells were transplanted in the subrenal capsule space. We also used a line of 3T3 cells that has been genetically modified to secrete a form of acidic FGF. When transplanted intradermally in collagen, they formed rapidly enlarging masses of cells that could easily be palpated beneath the skin of the animal. Intradermal injection of cells in pepsin-solubilized collagen is a simple and reliable technique for transplanting normal primary cells and preneoplastic cells. The ability to grow both types of cells in an easily accessible site allows less invasive monitoring of growth, angiogenesis, and other features of the transplant.

Key words: Cell transplantation; Intradermal transplants; Collagen; Adrenocortical cells; 3T3 cells

Address correspondence to Peter J. Hornsby, Ph.D., Professor, Department of Physiology, Sam and Ann Barshop Center for Longevity and Aging Studies, University of Texas Health Science Center, 15355 Lambda Drive STCBM 2.200, San Antonio, TX 78245. Tel: (210) 562-5080; Fax: (281) 582-3538; E-mail: hornsby@uthscsa.edu

Cell Transplantation, Vol. 11, pp. 147-159, 2002
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Flt3-L Augments the Engraftment of Donor-Derived Bone Marrow Cells When Combined With Sublethal Irradiation and Costimulatory (CD28/B7 and CD40/CD40L) Blockade

Jennifer E. Woodward,1 Abdus Salam,1 Alison J. Logar,1 Adam T. Schaefer,1 and Abdul S. Rao1,2*

Section of Cellular Transplantation, Thomas E. Starzl Transplantation Institute, and the Departments of 1Surgery and 2Pathology, University of Pittsburgh Medical Center, Pittsburgh PA 15261

T-cell costimulatory blockade as a constituent for recipient conditioning prior to bone marrow transplantation has led to the development of less toxic protocols for the establishment of donor cell chimerism. We therefore hypothesized that the addition of the hematopoietic growth factor, Flt3-ligand (Flt3-L), to the perioperative inhibition of the CD28/B7 and CD40/CD40 ligand costimulatory pathways would enhance the engraftment of allogeneic bone marrow. Recipient BALB/c ByJ (H-2d, Mlsc, Vb6+/Vb8+ TCR) received a single sublethal dose of total body irradiation (300 rad) 6 h prior to transplantation IV with unfractionated donor CBA/J (H-2k, Mlsd, Vb6-/Vb8+ TCR) bone marrow cells. CTLA4-Ig and/or MR1 were administered at 500 mg IP on days 0, 2, 4, and 6 posttransplantation. Flt3-L was administered at 10 mg IP on days 0-6. Donor cell chimerism was determined on days 30-90 by flow cytometric analysis. Donor-specific tolerance was assessed by skin grafting. In vitro TCR cross-linking assays and flow cytometry were utilized to explore the deletion of donor-reactive T cells. Recipients receiving CTLA4-Ig and MR1 engrafted allogeneic bone marrow cells in the peripheral blood (3/6; 50%) with chimerism being detected at 2-31%. Addition of Flt3-L to this preconditioning regimen enhanced the incidence of engraftment of donor bone marrow cells (10/13; 3-70%). Long-term survival of donor but not third-party-specific skin grafts demonstrated that donor-specific tolerance had been achieved in the chimeric recipients. Deletion of the donor-reactive T cells within the chimeric recipients was also observed. The addition of hematopoietic growth factors and cytokines to the nonmyeloablative regimen of sublethal irradiation and T-cell costimulatory blockade provides a novel strategy for the establishment of donor cell chimerism and for the induction of stable and robust donor-specific tolerance. The deletion of donor-reactive T cells using this protocol suggests the reliability and feasibility of this protocol for clinical transplantation.

Key words: Flt3-ligand; Costimulation; Chimerism; Bone marrow; Transplantation

Address correspondence to Jennifer E. Woodward, Ph.D., Thomas E. Starzl Transplantation Institute, University of Pittsburgh, E1545 Biomedical Science Tower, 200 Lothrop Street, Pittsburgh, PA 15261. Tel: (412) 383-8884; Fax: (412) 624-9493; E-mail woodwardje@msx.upmc.edu

*Present address: Office of the Dean, MCP Hahnemann University, School of Medicine, Philadelphia, PA 19102.

Cell Transplantation, Vol. 11, pp. 161-168, 2002
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Engraftment and Function of Intrasplenically Transplanted Cold Stored Rat Hepatocytes

Silvana V. Spinelli,1 Joaquín V. Rodríguez,2 Alejandra B. Quintana,3 María G. Mediavilla,1 and Edgardo E. Guibert1

1Biología Molecular, Dto. Cs. Biológicas, Facultad de Cs. Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario
2Farmacología, Dto. Cs. Fisiológicas, Facultad de Cs. Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario
3Morfología, Dto. Cs. Biológicas, Facultad de Cs. Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario

Hepatocellular transplant may potentially be efficacious for the treatment of selected liver metabolic disorders and acute hepatic failure. On the other hand, the use of hepatocyte cold preservation techniques in these transplantation protocols would allow to have available cells at the right time and place and, consequently, make an optimal use of scarce human hepatocytes. In our experiments we evaluated the biodistribution and functionality of cold preserved hepatocytes transplanted in the spleen of syngeneic rats. Isolated hepatocytes were labeled with the fluorescent dye 5(6)-carboxyfluorescein diacetate succinimidyl-ester, cold-preserved in modified University of Wisconsin (UW) solution for 48 or 96 h, and then transplanted into the spleen. Recipient animals were euthanized at 0 and 3 h, and at 1, 2, 3, 5, 10, and 14 days after transplantation for tissue analysis. Labeled hepatocytes were clearly identifiable in the recipient tissues up to 14 days later. Fluorescence microscopy also showed no significant differences in biodistribution when either cold stored or freshly isolated hepatocytes were transplanted. In addition, functional activity of transplanted cells was demonstrated by immunohistochemical detection of albumin at levels comparable to those found in normal hepatocytes. Our findings establish that cold preserved hepatocytes appear morphologically and biochemically normal after intrasplenic transplantation. Consequently, it indicates that modified UW solution makes it possible to safety preserve hepatocytes for up to 96 h before transplantation, perhaps providing sufficient time for hepatocyte allocation and potential recipient preparation, if applicable clinically.

Key words: University of Wisconsin solution; Hepatocellular transplantation; CFSE; Albumin distribution; Cold preservation

Address correspondence to Edgardo E. Guibert, Biología Molecular, Dto. Cs. Biológicas, Facultad de Cs. Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, S2002LRK Rosario, Argentina. Tel/Fax: 054-341-4804601; E-mail: eguibert@fbioyf.unr.edu.ar

Cell Transplantation, Vol. 11, pp. 169-180, 2002
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Changes in Mass and Performance in Rabbit Muscles After Muscle Damage With or Without Transplantation of Primary Satellite Cells

Ramzi Boubaker el Andalousi,1 Paul-André Daussin,1,2 Jean-Paul Micallef,3 Colette Roux,4 Jean Nougues,1 Michel Chammas,2 Yves Reyne,1 and Francis Bacou1

1UMR Différenciation cellulaire et Croissance, INRA, 2 Place Pierre Viala, 34060 Montpellier Cedex 1, France
2Service de Chirurgie Orthopédique 2 et Chirurgie de la Main, Hôpital Lapeyronie, CHU Montpellier, 34295 Montpellier Cedex 5, France
3INSERM ADR 08, Parc Euromédecine, 99 rue Puech Villa, 34197 Montpellier Cedex 5, France
4Laboratoire de Biométrie, INRA, 2 Place Pierre Viala, 34060 Montpellier Cedex 1, France

Changes in morphology, metabolism, myosin heavy chain gene expression, and functional performances in damaged rabbit muscles with or without transplantation of primary satellite cells were investigated. For this purpose, we damaged bilaterally the fast muscle tibialis anterior (TA) with either 1.5 or 2.6 ml cardiotoxin 10-5 M injections. Primary cultures of satellite cells were autotransplanted unilaterally 5 days after muscle degeneration. Two months postoperation, the masses of damaged TAs, with or without transplantation, were significantly larger than those of the controls. Furthermore, damaged transplanted muscles weighed significantly more than damaged muscles only. The increase in muscle mass was essentially due to increased fiber size. These results were independent of the quantity of cardiotoxin injected into the muscles. Maximal forces were similar in control and 2.6 ml damaged TAs with or without satellite cell transfer. In contrast, 1.5 ml damaged TAs showed a significant decrease in maximal forces that reaches the level of controls after transplantation of satellite cells. Fatigue resistance was similar in control and 1.5 ml damaged TAs independently of satellite cell transfer. Fatigue index was significantly higher in 2.6 ml damaged muscles with or without cell transplantation. These changes could be explained in part by muscle metabolism, which shifted towards oxidative activities, and by gene expression of myosin heavy chain isoforms, which presented an increase in type IIa and a decrease in type I and IIb in all damaged muscles with or without cell transfer. Under our experimental conditions, these results show that muscle damage rather than satellite cell transplantation changes muscle metabolism, myosin heavy chain isoform gene expression, and, to a lesser extent, muscle contractile properties. In contrast, muscle weight and fiber size are increased both by muscle damage and by satellite cell transfer.

Key words: Muscle regeneration; Myoblast transfer; Satellite cells; Skeletal muscle; Functional and morphological improvement

Address correspondence to Francis Bacou, UMR 866 Différenciation cellulaire et Croissance, INRA, 2 Place Pierre Viala, 34060 Montpellier Cedex 1, France. Tel: (33) 04 99 61 24 07; Fax: (33) 04 67 54 56 94; E-mail: francis.bacou@ensam.inra.fr