|ognizant Communication Corporation|
VOLUME 11, NUMBER 7, 2002
Cell Transplantation, Vol. 11, pp. 621-630, 2002
0963-6897/02 $20.00 + 00
Copyright © 2002 Cognizant Comm. Corp.
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Anoikis: Roadblock to Cell Transplantation?
Isabel Zvibel,1 Françoise Smets,2 and Humberto Soriano2
1Gastroenterology Institute, Tel Aviv Sourasky Medical Center,
Weizmann 6, Tel Aviv 64239, Israel
2Division of Gastroenterology and Hepatology, Department of Pediatrics, Northwestern University Medical School; Children's Memorial Institute for Education and Research. Siragusa Transplantation Center, Children's Memorial Hospital
Cell therapy, in particular liver cell transplantation, holds great therapeutic potential and is partially hindered by the high rate of apoptosis during cell isolation, cryopreservation, and engraftment. Apoptosis occurring due to cell detachment from the extracellular matrix is a phenomenon termed "anoikis." The purpose of this review is to describe signaling mechanisms pertinent to anoikis in both immortalized cell lines, but particularly in primary normal epithelial cells. The mechanisms described include integrin signaling and survival molecules, caspase activation, and the role of mitochondrial proteins in anoikis. Strategies to prevent anoikis during isolation and cryopreservation of hepatocytes are discussed.
Key words: Anoikis; Cell transplantation; Hepatocytes; Apoptosis; Transplantation
Address correspondence to Isabel Zvibel, Gastroenterology Institute, Tel Aviv Sourasky Medical Center, Weizmann 6, Tel Aviv 64239, Israel.
Monkey Embryonic Stem Cell Lines Expressing Green Fluorescent Protein
Tatsuyuki Takada,1* Yutaka Suzuki,3* Yasushi Kondo,3 Nae Kadota,3 Kinji Kobayashi,3 Shinji Nito,3 Hiroshi Kimura,1 and Ryuzo Torii2
1Department of Experimental Radiology and 2Research
Center for Animal Life Science, Shiga University of Medical Science, Ohtsu,
Shiga, 520-2192, Japan
3Discovery Research Laboratory, Tanabe Seiyaku Co., Ltd., Osaka 532-8505, Japan
The major limitation of nonhuman primate (NHP) embryonic stem (ES) cell research is inefficient genetic modification and limited knowledge of differentiation mechanisms. A genetically modified NHP-ES cell with biomarkers, such as green fluorescent protein (GFP), that allow noninvasive monitoring of transgenic cells, is a useful tool to study cell differentiation control during preimplantation and fetal development, which also plays a crucial role in the development of cell transplantation medicine. Here we report the establishment of transgenic NHP-ES cell lines that express GFP without jeopardizing their pluripotency, which was confirmed by in vitro and in vivo differentiation. These GFP-expressing ES cells reproducibly differentiated into embryoid bodies, neural cells, and cardiac myocytes. They formed teratoma composed of tissues derived from the three embryonic germ layers when transplanted into severe combined immunodeficient disease (SCID) mice. GFP expression was maintained in these differentiated cells, suggesting that these cells were useful for cell transplantation experiments. Furthermore, we showed that these ES cells have the ability to form chimeric blastocysts by introducing into the early preimplantation stage NHP embryo.
Key words: Nonhuman primate; Cynomolgus monkey; Embryonic stem cell; Green fluorescent protein; Pluripotency
Address correspondence to Ryuzo Torii, Research Center for Animal Life Science, Shiga University of Medical Science, Ohtsu, Shiga, 520-2192, Japan. Tel: +81-77-548-2334; Fax: +81-77-543-1990; E-mail: email@example.com
*These authors contributed equally to this work.
The Release of Excitatory Amino Acids, Dopamine, and Potassium Following Transplantation of Embryonic Mesencephalic Dopaminergic Grafts to the Rat Striatum, and Their Effects on Dopaminergic Neuronal Survival In Vitro
Rike Zietlow,1,3 Simon R. Sinclair,1 Christof J. Schwiening,2 Stephen B. Dunnett,1,3 and James W. Fawcett1,2
1Cambridge University Centre for Brain Repair, Cambridge
CB2 2PY, UK
2Department of Physiology, Cambridge University, Cambridge CB2 3EG, UK
3Brain Repair Group, School of Biosciences, Cardiff University, Cardiff CF10 3US, UK
A major limitation to the effectiveness of grafts of fetal ventral mesencephalic tissue for parkinsonism is that about 90-95% of grafted dopaminergic neurones die. In rats, many of the cells are dead within 1 day and most cell death is complete within 1 week. Our previous results suggest that a major cause of this cell death is the release of toxins from the injured CNS tissue surrounding the graft, and that many of these toxins have dissipated within 1 h of inserting the grafting cannula. In the present experiments we measured the change over time in the concentration of several potential toxins around an acutely implanted grafting cannula. We also measured the additional effect of injecting suspensions of embryonic mesencephalon, latex microspheres, or vehicle on these concentrations. Measurements of glutamate, aspartate, and dopamine by microdialysis showed elevated levels during the first 20-60 min, which then declined to baseline. In the first 20 min glutamate levels were 10.7 times, aspartate levels 5 times, and dopamine levels 24.3 times baseline. Potassium levels increased to a peak of 33 ± 10.6 mM 4-5 min after cannula insertion, returning to baseline of <5 mM by 30 min. Injection of cell suspension, latex microspheres, or vehicle had no significant effect on these levels. We then assayed the effect of high concentrations of glutamate, aspartate, dopamine, and potassium on dopaminergic neuronal survival in E14 ventral mesencephalic cultures. In monolayer cultures only dopamine at 200 mM showed toxicity. In three-dimensional cultures only the combination of raised potassium, dopamine, glutamate, and aspartate together decreased dopaminergic neuronal survival. We conclude that toxins other than the ones measured are the main cause of dopaminergic cell death after transplantation, or the effects of the toxins measured are enhanced by anoxia and metabolic challenges affecting newly inserted grafts.
Key words: Substantia nigra; Transplantation; Glutamate; Dopamine; Aspartate; Potassium
Address correspondence to Rike Zietlow, Cardiff School of Biosciences, Museum Avenue, PO Box 911, Cardiff CF10 3US, UK. Tel: +44 2920 471288; Fax: +44 2920 876749; E-mail: firstname.lastname@example.org
Effects of Antioxidant Pretreatment on the Survival of Embryonic Dopaminergic Neurons In Vitro and Following Grafting in an Animal Model of Parkinson's Disease
R. M. Love,1 R. L. Branton,1 J. Karlsson,2 P. Brundin,2 and D. J. Clarke1
1Department of Human Anatomy and Genetics, University of
Oxford, Oxford, OX1 3QX, UK
2Wallenberg Neuroscience Center, Section for Neuronal Survival, BMC A10, S-221 84 Lund, Sweden
The effect of pretreating cell suspensions of embryonic rat ventral mesencephala (VM) with antioxidant combinations on the survival of dopaminergic (DA) neurons was studied in vitro and following transplantation into the unilateral 6-hydroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease. The in vitro experiments examined the effects of two thiol antioxidants, N-acetyl-L-cysteine (NAC) and reduced glutathione (GSH), and a member of the lazaroid family of 21-aminosteroids, U-83836E, singly and in combination, on survival of DA neurons derived from dissociated E14 rat VM tissue. For in vivo studies, cell suspensions were pretreated with combinations of NAC, GSH, and U-83836E prior to transplanting into 6-OHDA-lesioned rats to investigate whether DA neuron survival could be further improved. NAC, GSH, and U-83836E individually increased DA neuron survival in vitro and a combination of all three resulted in the greatest survival. In vivo, pretreatment with U-83836E alone resulted in a significantly greater reduction in amphetamine-induced rotation 6 weeks postgrafting compared with a control group receiving nontreated graft tissue. This functional effect correlated with a significant improvement in DA neuron survival 6 weeks postgrafting. The thiol combination pretreatment of NAC and GSH, and the triple combination of NAC, GSH, and U-83836E, however, failed to improve both functional recovery and DA neuron survival when compared with the nontreated control grafts.
Key words: N-Acetyl-L-cysteine; Dopaminergic neurons; Glutathione; Lazaroid; Neural transplantation; Parkinson's disease
Address correspondence to Dr. Deborah J. Clarke, Department of Experimental Psychology, South Parks Road, Oxford, OX1 3UD, UK. Tel: 00 44 1865 281280; Fax: 00 44 1865 301447; E-mail: email@example.com
Intracerebral Transplantation and Successful Integration of Astrocytes Following Genetic Modification With a High-Capacity Adenoviral Vector
S. Arnhold,1 F. Kreppel,2 S. Kandirali,1 D. Lenartz,3 F.-J. Klinz,1 V. Sturm,3 S. Kochanek,2 C. Andressen,1 and K. Addicks1
1Department of Anatomy I, 2Center for Molecular Medicine (ZMMK), and 3Clinic for Stereotaxy and Functional Neurosurgery, University of Cologne, Joseph-Stelzmann Str. 9, 50931 Köln, Germany
To investigate the ability of genetically modified astrocytes to integrate into adult rat brain, two spontaneously immortalized cell lines and the allogenic nontumorigenic glioma cell line F98 were transduced with a high-capacity adenoviral vector (HC-Adv) expressing the EGFP gene from the hCMV promoter. In organotypic slice cultures the transduced astrocytes were shown to integrate into the brain tissue. Following transplantation of the transduced astrocytes into the striatum of adult rats, the transplanted cells survived at least for 6 weeks, continuously expressed the EGFP transgene, in close neighborhood with cells of the recipient tissue executing their differentiation capacity along the glial lineage. Thus, HC-Adv transduced astrocytes are promising vehicles to locally deliver therapeutic proteins for the treatment of neurodegenerative diseases.
Key words: Intracerebral transplantation; Neurotrophic factors; Parkinson's disease; Astrocytes; High-capacity adenoviral vector
Address correspondence to Dr. Stefan Arnhold, Department of Anatomy I, Joseph-Stelzmann Str. 9, 50931 Köln, Germany. Tel: ++49 221 4785438; Fax: ++49 221 4786711; E-mail: firstname.lastname@example.org
Dynamics of the Early Immune Cellular Reactions After Myogenic Cell Transplantation
Daniel Skuk, Nicolas Caron, Marlyne Goulet, Brigitte Roy, Francisco Espinosa, and Jacques P. Tremblay
Unité de recherche en Génétique humaine, Centre de Recherche du Centre Hospitalier de l'Universit, Laval, CHUL du CHUQ, Québec, Canada G1V 4G2
The role of immune cells in the early donor cell death/survival following myoblast transplantation is confusing, one of the reasons being the lack of data about the immune reactions following cell transplantation. We used outbred mice as hosts for transplantation of primary cultured muscle cells and T-antigen-immortalized myoblasts. The host muscles were analyzed 1 h to 7 days after cell injection. No net loss of the donor primary cultured cell population was observed in this period. The immune cellular reaction in this case was: 1) a brief (<48 h) neutrophil invasion; 2) macrophage infiltration from days 1 to 7; 3) a specific response involving CTL and few NK cells (days 6 and 7), preceded by a low CD4+ cell infiltration starting at day 3. In contrast, donor-immortalized myoblasts completely disappeared during the 7-day follow-up. In this case, an intense infiltration of CTL and macrophages, with moderate CD4+ infiltration and lower amounts of NK cells, was observed starting at day 2. The nonspecific immune response at days 0 and 1 was similar for both types of donor cells. The present observations set a basis to interpret the role of immune cells on the early death/survival of donor cells following myoblast transplantation.
Key words: Myoblast; Neutrophil; Macrophage; CD8+ lymphocyte; CD4+ lymphocyte; Natural killer cell
Address correspondence to Jacques P. Tremblay, Ph.D., Unité de recherche en Génétique humaine, Centre Hospitalier de l'Universit, Laval, 2705, boulevard Laurier, Ste-Foy, Québec, Canada G1V 4G2. Tel: (418) 654-2186; Fax: (418) 654-2207; E-mail: Jacques-P.Tremblay@crchul.ulaval.ca
Mechanisms of Alloimmune Tolerance Associated With Mixed Chimerism Induced by Vascularized Bone Marrow Transplants
Rajen Ramsamooj,1 Ramon Llull,2 Mayuri P. Patel,3 Kenneth R. Beko, II,4 Kirby S. Black,5 and Charles W. Hewitt3
1University of California Davis Medical Center, Division
of Surgical & Transplant Pathology, Sacramento, CA 95817
2University of Pittsburgh, Department of Surgery, Pittsburgh, PA 15621
3Cooper Hospital/University Medical Center, UMDNJ-Robert Wood Johnson Medical School, Division of Surgical Research, Camden, NJ, 08103
4University of California, Department of Surgery, Irvine, CA 92717
5CryoLife, Inc., Department of Research and Development, 1655 Roberts Boulevard, Kennesaw, GA 30144
Rat limb allograft recipients represent surgically induced, immediately vascularized bone marrow transplant (VBMT) chimeras. The majority of these chimeras undergo tolerance while a minority develop graft versus host disease (GVHD). T-cell chimerism and associated mechanisms of cellular immune nonresponsiveness were investigated in tolerant VBMT chimeras. A strong correlation (p < 0.01) was observed between the clinical onset of GVHD and levels of donor T-cell chimerism approximating or greater than 50%. However, stable mixed chimerism was associated with tolerance. In conclusion, three major sequential mechanisms of immune nonresponsiveness were elucidated in tolerant VBMT chimeras over time and included development of nonspecific suppressor cells (which potentially represent natural suppressor cells), maturation of antigen-specific suppressor cell circuits, and eventually putative clonal inactivation.
Key words: Extremity transplant; Composite tissue limb allograft
Address correspondence to Charles W. Hewitt, Ph.D., Director of Surgical Research, Cooper Hospital/University Medical Center, UMDNJ-RWJMS, Three Cooper Plaza, Suite 411, Camden, NJ 08103. Tel: (856) 757-7854; Fax: (856) 757-9647; E-mail: email@example.com
Proliferative and Cytokine Responses in CTLA4-Ig-Treated Diabetic NOD Mice Transplanted With Microencapsulated Neonatal Porcine ICCs
Susan A. Safley,1 Judith A. Kapp,2 and Collin J. Weber1
Departments of 1Surgery and 2Ophthalmology, Emory University School of Medicine, Atlanta, GA 30322
Our goal is to develop effective islet xenografts for treating human diabetes. We have studied microencapsulated neonatal porcine islet cell clusters (ICCs) transplanted intraperitoneally in spontaneously diabetic NOD mice, where they function to maintain normoglycemia in the autoimmune host. Nonencapsulated neonatal porcine ICCs functioned for 4.5 ± 0.5 days before being rejected; encapsulation prolonged graft function to 17 ± 2 days. CTLA4-Ig treatment did not enhance the survival of nonencapsulated ICCs. However, CTLA4-Ig treatment significantly extended the function of encapsulated ICCs to 73 ± 5 days. Histological analyses demonstrated a profuse pericapsular cellular reaction associated with rejection of encapsulated islet xenografts in untreated mice, while this reaction was significantly reduced in CTLA4-Ig-treated mice. To study mechanisms of xenograft rejection in this model, we analyzed proliferative responses to neonatal porcine ICCs and cytokines present in the peritoneal cavities of transplanted mice. Spleen cells from both CTLA4-Ig-treated and untreated rejecting NODs exhibited vigorous proliferation in the absence of antigenic stimulation, suggesting prior activation in vivo, while splenocytes from CTLA4-Ig-treated NODs with functioning grafts had low proliferative levels, equal to controls. Islet-specific proliferation was not detected in islet-rejecting mice, perhaps due to their high background levels. With the exception of elevated IL-6 levels, empty capsules did not provoke a significant peritoneal cytokine response compared with sham surgery or untransplanted control mice. However, IL-5, IL-12, TGF-b, and IL-1b were significantly elevated in NODs receiving encapsulated neonatal porcine ICCs compared with untransplanted controls. There were no significant differences between peritoneal cytokine concentrations in CTLA4-Ig-treated mice with long-term functioning grafts compared to mice that rejected grafts at earlier time points. We conclude that the combination of donor islet microencapsulation and brief treatment of the recipient with co-stimulatory blockade delays sensitization of the host, possibly by altering mechanism(s) for recruitment and/or activation of host effector cells.
Key words: Diabetes; Xenograft; Porcine islet cell clusters; Microencapsulation; NOD mice; CTLA4-Ig
Address correspondence to Dr. Susan A. Safley, the Elizabeth Brooke Gottlich Laboratory for Diabetes and Islet Transplant Research, Department of Surgery, 5207 Woodruff Memorial Building, 1639 Pierce Drive, Atlanta, GA 30322. Tel: (404) 712-9676; Fax: (404) 727-3660; E-mail: firstname.lastname@example.org
Decreased Survival of Islet Allografts in Rats With Advanced Chronic Complications of Diabetes
Wayne V. Moore, Karen Bieser, Zhoahui Geng, Pei Y. Tong, and Karen Kover
Department of Endocrinology at The Children's Mercy Hospital and University of Missouri-Kansas City, Kansas City, MO
Successful islet transplantation has been possible in experimental animals in contrast to humans. One difference between animal models of diabetes and human islet transplantation is the presence of advanced chronic complications in humans. Even longer-term follow-up of islet transplantation in humans according to the Edmonton protocol suggests that advanced chronic complications may adversely affect allograft survival with the glucocorticoid-free immunosuppressive regimen as well. We developed a rat model of chronic complications of diabetes and compared islet allograft survival in rats with advanced chronic complications to age-matched control rats with acute onset diabetes. Islets were transplanted at either the renal supcapsular, intrahepatic, or intramuscular location. The survival of islet allografts in rats with chronic complications was decreased at all sites compared with the age-matched controls. The best survival in the rats with advanced chronic complications occurred at the renal subcapsular site. Blood sugar measurements indicated impaired glucose tolerance in most of the rats with chronic complications and surviving renal subcapsular islet allograft. Histological and gross examination of the surviving renal subcapsular islet allografts indicated disordered angiogenesis in the rats with chronic complications. Rats with successful intrahepatic islet allografts and the respective age-matched controls had comparable blood sugars. Survival of islet allografts at the intramuscular site was poor in rats with chronic complications or acute onset diabetes. We conclude that the structural or metabolic abnormalities associated with chronic poor control of diabetes impair islet allograft survival and function. This should be considered as a possible explanation for failure of islet allograft survival in human islet transplantation.
Key words: Islet allograft; Survival; Chronic complications
Address correspondence to Wayne V. Moore, M.D., Ph.D., Children's Mercy Hospital, 2401 Gilham Rd., Kansas City, MO 64108. Fax: (816) 855-1919; E-mail: email@example.com
Co-Stimulatory Molecules in Islet Xenotransplantation: CTLA4Ig Treatment in CD40 Ligand-Deficient Mice
Birgitta Benda,1 Hans-Gustaf Ljunggren,2 Robert Peach,3 Jan-Olov Sandberg,4 and Olle Korsgren1
1Section of Clinical Immunology, Department of Oncology,
Radiology, and Clinical Immunology, The Rudbeck Laboratory, Uppsala University,
SE-751 85 Uppsala, Sweden
2Microbiology and Tumor Biology Center, Karolinska Institute, SE-171 77 Stockholm, Sweden
3Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543, USA
4Roche AB, SE-100 74 Stockholm, Sweden
Previous work has demonstrated that short-term systemic administration of cytotoxic T lymphocyte antigen-4 (CTLA-4) Ig blocks human pancreatic islet xenograft rejection in mice and induces long-term, donor-specific tolerance, whereas studies on pig pancreatic islet rejection in mice have failed to demonstrate a role for CTLA4Ig in preventing rejection. Treatment with anti-CD40 ligand (L) monoclonal antibodies alone is somewhat effective in prolonging the survival of islet xenografts, but ineffective when applied to skin xenografts. However, simultaneous blockade of the CD28 and CD40 co-stimulatory pathways prolongs the survival of pig skin on recipient mice. To evaluate the role of CD28 and CD40 co-stimulatory pathways in pig islet-like cell cluster (ICC) xenograft rejection in mice, CD40L-deficient mice transplanted with fetal porcine ICCs were given posttransplant treatment with human (h) CTLA4Ig or a human IgG1 chimeric mAb (hL6). Xenografts were evaluated 6 or 12 days after transplantation. Fetal porcine ICC xenografts were protected from rejection in hCTLA4Ig-treated CD40L-deficient mice, whereas xenograft rejection persisted in untreated CD40L-deficient mice. Simultaneous blockade of the CD28 and CD40 co-stimulatory pathways is mandatory to inhibit ICC xenograft rejection in the pig-to-mouse model, because the CD28 and CD40 co-stimulatory pathways seem capable of efficiently substituting for one another.
Key words: Xenotransplantation; Islet; CTLA4Ig; CD40L; Co-stimulation; In vivo animal model
Address correspondence to Dr. Olle Korsgren, Section of Clinical Immunology, Department of Oncology, Radiology, and Clinical Immunology, The Rudbeck Laboratory, Uppsala University, SE-751 85 Uppsala, Sweden. Tel: +46 18 6114187; Fax: +46 18 6110222; E-mail: Olle.Korsgren@klinimm.uu.se
Insulin Treatment of Mice Recipients Preserves b-Cell Function in Porcine Islet Transplantation
Oleg Pakhomov,1 Jiry Honiger,2 Edouard Gouin,3 Roland Cariolet,4 Gerard Reach,1 and Sylviane Darquy1
1INSERM U 341, Diabetes Department, Hôtel-Dieu Hospital,
2Bioengineering Laboratory, Saint-Antoine Hospital, France
3Zoopole Development, Ploufragan, France
4CNEVA, Ploufragan, France
Encapsulation of islets of Langerhans confers protection against cell-mediated immune destruction and so should allow the transplantation of islets without immunosuppression. Xenotransplantation of encapsulated islets of Langerhans might therefore help overcome problems of human organ donor shortage. Given that islets exposed to sustained hyperglycemia show impaired b-cell function, we set out to determine whether recipient treatment with insulin could improve transplantation success rate. Islets of Langerhans were obtained from Specific Germ-Free (SPF) pig pancreas and cultured overnight. Islets were encapsulated in AN69 fibers and implanted into the peritoneal cavity of diabetic mice. A group of implanted mice was treated with exogenous insulin from day 3 to day 7 after grafting. Islet implantation depressed plasma glucose in all the mice, both insulin treated and untreated. Glycemia slowly increased in the non-insulin-treated mice, whereas the decrease observed in the insulin-treated mice was maintained until day 29 of follow-up. We found significant differences between the two groups (p < 0.05 at day 18 and day 20, p < 0.001 at day 23 and day 29). No improvement of hyperglycemia was observed in diabetic mice implanted with empty fibers. When islet-containing fibers were removed from the peritoneal cavity of mice 1 month after the graft plasma glucose increased markedly. We demonstrate that treatment of recipients with exogenous insulin in the immediate posttransplantation period has a positive effect on \GK\b-cell function in transplanted macroencapsulated porcine islets.
Key words: Porcine islet of Langerhans; Xenotransplantation; AN69 hollow fibers; Bioartificial pancreas
Address correspondence to Sylviane Darquy, Ph.D., Inserm U 341, Diabetes Department, 1 place du Parvis Notre-Dame, 75004 Paris, France. E-mail: firstname.lastname@example.org