ognizant Communication Corporation


VOLUME 11, NUMBER 6, 2002

Cell Transplantation, Vol. 11, pp. 499-505, 2002
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Identification of a Specific Sertoli Cell Marker, Sox9, for Use in Transplantation

R. A. Hemendinger,1 P. Gores,1 L. Blacksten,1 V. Harley,2 and C. Halberstadt1

1Department of General Surgery and The Transplant Center, Carolinas Medical Center, Charlotte, NC 28232
2Prince Henry's Institute, Monash Medical Centre, Clayton, Victoria 3168, Australia

The immunoprivileged environment of the testes was first described in the 1930s, and the Sertoli cell was later identified as the main cell type responsible for this phenomenon. Recent work has examined the possibility of recreating this immunoprivileged environment at heterotopic sites using isolated Sertoli cells. These studies have focused on protection of pancreatic islets and neuronal cells from immune destruction in the hopes of reversing type I diabetes and Parkinson's disease. The absence of a definitive marker for identifying Sertoli cells at the transplant site has been an obstacle to this research. The current study examines the presence of a nuclear transcription factor, Sox9, which is preferentially expressed in Sertoli cells. Syngeneic Lewis rat Sertoli cells were transplanted into the renal subcapsular space and a subcutaneous site in Lewis female rats and examined histologically 21 days later. In addition, porcine Sertoli cells were transplanted into the renal subcapsular space in female SCID mice. Control testes and the transplant sites were examined immunohistochemically using an antibody to Sox9. The results from the study demonstrate that Sox9 expression is restricted to the Sertoli cells of the neonatal rat and porcine testis, indicating high homology between species. In addition, Sox9 expression was also observed in the testicular-like tubules that formed in both syngeneic and xenogeneic heterotopic transplants in rats and SCID mice. The Sox9 expression was restricted to the regions where Sertoli cells would be found in the native testis. These results suggest that the Sox9 protein is a useful marker in identifying Sertoli cells in heterotopic transplants in a manner similar to insulin as a marker for pancreatic islets.

Key words: Sertoli cells; Sox9; Cell transplantation; Diabetes; Parkinson's disease

Address correspondence to Dr. Richelle A. Hemendinger, Department of General Surgery Research, Carolinas Medical Center, P.O. Box 32861, Charlotte, NC 28232. Tel: (704) 355-9786; Fax: (704) 355-7203; E-mail: rhemendinger@carolinas.org

Cell Transplantation, Vol. 11, pp. 507-512, 2002
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A Unique Cytoplasmic Marker for Extratesticular Sertoli Cells

D. F. Cameron,1,2 J. J. Hushen,2 T. Dejarlais,1,3 L. Colado,2 K. M. Wolski,1 P. R. Sanberg,2 and S. Saporta12

1Department of Anatomy and 2Department of Neurosurgery, University of South Florida College of Medicine, Tampa, FL
3Saneron CCEL Therapeutics, Inc., Tampa, FL

In the absence of a definitive cell marker for testis-derived Sertoli cells, their identification in cell culture or in Sertoli cell-facilitated cell transplantation protocols is difficult and limits the creditable evaluation of experimental results. However, the production by prepubertal Sertoli cells of Mullerian inhibiting substance (MIS) presents the possibility of specifically identifying extratesticular Sertoli cells as well as Sertoli cells in situ, by the immunodection of this unique glycoprotein. This study was designed to determine if isolated rat Sertoli cells could be identified by routine immunocytochemistry utilizing an antibody raised against MIS. Sertoli cells immunostained for MIS included Sertoli cells in situ and freshly isolated, cultured and cocultured Sertoli cells, and Sertoli cells structurally integrated with NT2 cells in simulated microgravity. Detection of MIS was also determined by Western blot analysis.

Key words: Sertoli cells; Cytoplasmic marker; Mullerian inhibiting substance (MIS)

Address correspondence to Don F. Cameron, Ph.D., Department of Anatomy, University of South Florida, P.O. Box MDC-06, 12901 Bruce B. Downs Blvd., Tampa, FL. Tel: (813) 974-9434; Fax: (813) 974-2058; E-mail: dcameron@hsc.usf.edu

Cell Transplantation, Vol. 11, pp. 513-518, 2002
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Successful Single Donor Islet Allotransplantation in the Streptozotocin Diabetes Rat Model

Henning Jahr, Birte Hußmann, Torsten Eckhardt, and Reinhard G. Bretzel

Third Medical Department, University of Giessen, D-35392 Giessen, Germany

The objective of this study was to define pretransplant islet culture conditions for optimum tissue engraftment in the rat islet allotransplantation model. Lewis rat islets were cultured in TCM 199/5% fetal calf serum for 1 day at 37°C, followed by 1 day of culture at 22°C. When islets from single donors were allotransplanted intraportally into single streptozotocin-diabetic Wistar-Furth rats, complete normoglycemia was restored within 1 day after transplantation in seven out of seven rats, and persisted up to immunological rejection about 1 week later. In five out of six rats receiving a posttransplant injection of antilymphocyte serum, plasma glucose was normalized for >100 days. These data demonstrate, for the first time, successful single-donor-to-single-recipient transplantation of allogeneic rat pancreatic islets. Because islet engraftment may still be regarded as a main problem for clinical islet transplantation, the pretransplant islet culture regimen outlined in this article may lead to a more efficient use of donor pancreatic islet tissue in the clinical setting, as well.

Key words: Islet allotransplantation; Single donor; Islet culture; Rat

Address correspondence to Dr. Henning Jahr, Third Medical Department, Justus-Liebig-University Giessen, Rodthohl 6, D-35392 Giessen, Germany. Tel: +49-641-9942 852; Fax: +49-641-9942 849; E-mail: Henning.Jahr@innere.med.uni-giessen.de

Cell Transplantation, Vol. 11, pp. 519-528, 2002
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Gene Transfection and Expression of Transforming Growth Factor-b1 in Nonobese Diabetic Mouse Islets Protects b-Cells in Syngeneic Islet Grafts From Autoimmune Destruction

Wilma L. Suarez-Pinzon,1 Yvonne Marcoux,2 Aziz Ghahary,2 and Alex Rabinovitch1

1Department of Medicine, and 2Department of Surgery, University of Alberta, Edmonton, Alberta T6G 2S2, Canada

Nonobese diabetic (NOD) mice develop diabetes and destroy syngeneic islet grafts through an autoimmune response. Because transforming growth factor (TGF)-b1 downregulates immune responses, we tested whether overexpression of TGF-b1 by gene transfection of NOD mouse islets could protect b-cells in islet grafts from autoimmune destruction. NOD mouse islet cells were transfected with an adenoviral DNA expression vector encoding porcine latent TGF-b1 (Ad TGF-b1) or the adenoviral vector alone (control Ad vector). The frequency of total islet cells expressing TGF-b1 protein was increased from 12 ± 1% in control Ad vector-transfected cells to 89 ± 4% in Ad TGF-b1-transfected islet cells, and the frequency of b-cells that expressed TGF-b1 was increased from 12 ± 1% to 60 ± 7%. Also, secretion of TGF-b1 was significantly increased in islets that overexpressed TGF-b1. Ad TGF-b1-transfected NOD mouse islets that overexpressed TGF-b1 prevented diabetes recurrence after transplantation into diabetic NOD mice for a median of 22 days compared with only 7 days for control Ad vector-transfected islets (p = 0.001). Immunohistochemical examination of the islet grafts revealed significantly more TGF-b1+ cells and insulin+ cells and significantly fewer CD45+ leukocytes in Ad TGF-b1-transfected islet grafts. Also, islet b-cell apoptosis was significantly decreased whereas apoptosis of graft-infiltrating leukocytes was significantly increased in Ad TGF-b1-transfected islet grafts. These observations demonstrate that overexpression of TGF-b1, by gene transfection of NOD mouse islets, protects islet b-cells from apoptosis and autoimmune destruction and delays diabetes recurrence after islet transplantation.

Key words: Type I diabetes; NOD mice; Islets; Gene therapy; TGF-b1

Address correspondence to Alex Rabinovitch, M.D., 430 Heritage Medical Research Centre, University of Alberta, Edmonton, Alberta T6G 2S2, Canada. Tel: (780) 492-6791; Fax: (780) 492-4666; E-mail: alex.rabinovitch@ualberta.ca

Cell Transplantation, Vol. 11, pp. 529-537, 2002
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Morphological and Functional Studies on Submucosal Islet Transplants in Normal and Diabetic Hamsters

Nikolay Tchervenivanov, Songyang Yuan, Mark Lipsett, Despina Agapitos, and Lawrence Rosenberg

Department of Surgery, McGill University and The Montreal General Hospital, Montreal, Quebec H3G 1A4, Canada

The long-term outcome of human islet allotransplantation is poor, and it remains to be seen if the Edmonton Protocol will make a positive impact upon the extension of posttransplant islet function. Hence, establishing an implantation site capable of sustaining islet allografts for a prolonged duration needs to be explored. In this study we investigated the submucosal space of the duodenum in Syrian golden hamsters. Following transplantation of more than 800 islets into streptozotocin (STZ)-induced diabetic hamsters, basal nonfasted blood glucose levels decreased from 403 ± 14 to 143 ± 10 mg/dl within 5 weeks posttransplantation. In these animals, in vivo islet function, as determined by intravenous glucose tolerance test (IVGTT), was similar to nondiabetic controls (K values: 1.16 ± 0.12 vs. 0.95 ± 0.06, respectively) and was significantly greater than diabetic controls (K value: 0.47 ± 0.07). Islets transplanted into the submucosal space become richly vascularized within 2 weeks, and there is minimal host inflammatory infiltrate. The b-cells of the graft remain well granulated with insulin for at least 129 days. We conclude that the submucosal space is an effective engraftment site for islets that warrants further development in a large-animal model.

Key words: Diabetes mellitus; Immunocytochemistry; Islet cell isolation; Islet transplantation; Intravenous glucose tolerance test; Pancreatic digestion; Streptozotocin

Address correspondence to Dr. Lawrence Rosenberg, Montreal General Hospital, 1650 Cedar Ave., L9-313, Montreal, Quebec H3G 1A4, Canada. Tel: (514) 934-1934, ext. 44346; Fax: (514) 934-8438; E-mail: lawrence.rosenberg@mcgill.ca

Cell Transplantation, Vol. 11, pp. 539-545, 2002
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Comparison of Size, Viability, and Function of Fetal Pig Islet-Like Cell Clusters After Digestion Using Collagenase or Liberase

Pauline Georges, Roslyn P. Muirhead, Lindy Williams, Sara Holman, Muhammad Tani Tabiin, Sophia K. Dean, and Bernard E. Tuch

Diabetes Transplant Unit, Prince of Wales Hospital and The University of New South Wales, Sydney, New South Wales, 2031, Australia

Liberase is a highly purified blend of collagenases that has been specifically developed to eliminate the numerous problems associated with the conventional use of crude collagenase when isolating islet-like cell clusters (ICCs) from pancreases of different species. The influence of Liberase on yield, size, viability, and function of ICCs has been documented when this enzyme was used to digest adult but not fetal pancreases. In this study, we compared the effects of collagenase and Liberase on fetal pig ICCs. A total of eight fetal pig pancreas digestions were analyzed. Fetuses were obtained from Large White Landrace pigs of gestational age 80 ± 2.1 days. The pancreases were digested with either 3 mg/ml collagenase P or 1.2 mg/ml Liberase HI. The time taken to digest the pancreas was shorter for collagenase when compared with Liberase (22 ± 2 vs. 31 ± 2 min). The size of ICCs was similar for both collagenase (83 ± 0.5 mm) and Liberase (79 ± 0.4 mm) as was the number of ICCs produced per pancreas (7653 ± 1297 vs. 8101 ± 1177). Viability, as assessed using fluorescent markers, was slightly greater for Liberase (79 ± 1% vs. 76 ± 1%, p < 0.05). Responsiveness to b-cell stimulus (20 mM KCl) was similar for both methods of isolation, as was the insulin content of the ICCs, both in vitro and at 1 month after transplantation of 1500 ICCs beneath the renal capsule of immunoincompetent mice. Despite the high content of endotoxins in collagenase, the above results show that this enzyme was equally as efficient as Liberase in isolating functional ICCs from fetal pig pancreas.

Key words: Collagenase; Liberase; Fetal pig ICCs; Endotoxins

Address correspondence to Bernard Tuch, M.D., Ph.D., Diabetes Transplant Unit, Prince of Wales Hospital, High Street, Randwick, New South Wales, 2031, Australia. Tel: 61-2-9382 4814; Fax: 61-2-9382 4826; E-mail: b.tuch@unsw.edu.au

Cell Transplantation, Vol. 11, pp. 547-552, 2002
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Development of an Immunoprivileged Site to Prolong Islet Allograft Survival

Tatsuya Kin, Ray V. Rajotte, Jannette M. Dufour, and Gregory S. Korbutt

Surgical-Medical Research Institute, 1074 Dentistry/Pharmacy Building, University of Alberta, Edmonton, Alberta, Canada T6G 2N8

Sertoli cells (SC) play a critical role in the maintenance of the immunoprivileged environment of the testis. We hypothesized that preengrafting SC would allow one to develop a vascularized immunoprivileged ectopic site that provides protection for mouse islet allografts. SC, prepared from 9-day Balb/c mice, were transplanted under the kidney capsule in adult Balb/c mice. After SC engraftment (~30 days), mice were rendered diabetic and subsequently implanted with Balb/c or CBA/J islets directly adjacent to the established SC grafts. Preengrafted SC (5.7 ± 0.2 x 106) had no adverse effect on syngeneic islet graft function. When allogeneic islets were transplanted into the immunoprivileged ectopic site created by preengrafting 6.4 ± 0.3 x 106 SC, mean graft survival was slightly prolonged (32.4 ± 6.0 days) compared with control mice that received allogeneic islets alone (16.3 ± 1.5 days; p = 0.329). In contrast, when 4.8 ± 0.4 x 106 SC were preengrafted, islet allograft survival was significantly prolonged (66.1 ± 9.8 days; p = 0.001). Four of eight mice, preimplanted with 4.8 ± 0.4 x 106 SC, remained normoglycemic throughout the follow-up period (83.8 ± 8.6 days) and returned to a diabetic state only when the kidneys bearing the composite grafts were removed. Transplantation of islets into an immunoprivileged ectopic site created by preengrafting SC did not affect islet function and, moreover, provided a means of developing an immunopriveliged ectopic site that permits prolonged islet allograft survival without systemic immunosuppression.

Key words: Sertoli cells; Islet transplantation; Diabetes

Address correspondence to Gregory S. Korbutt, Surgical-Medical Research Institute, 1074 Dentistry/Pharmacy Building, University of Alberta, Edmonton, Alberta, Canada T6G 2N8. E-mail: korbutt@ualberta.ca

Cell Transplantation, Vol. 11, pp. 553-561, 2002
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Intrasplenic Transplantation of Encapsulated Cells: A Novel Approach to Cell Therapy

Takeshi Aoki, Yutaka Umehara, Chiara Ferraresso, Nozomu Sugiyama, Yvette Middleton, Itzhak Avital, Daniel Inderbitzin, Achilles A. Demetriou, and Jacek Rozga

Liver Support Research Laboratory, Department of Surgery, Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles, CA 90048

Cell therapy is likely to succeed clinically if cells survive at the transplantation site and are protected against immune rejection. We hypothesized that this could be achieved with intrasplenic transplantation of encapsulated cells because the cells would have instant access to oxygen and nutrients while being separated from the host immune system. In order to provide proof of the concept, primary rat hepatocytes and human hepatoblastoma-derived HepG2 cells were used as model cells. Rat hepatocytes were encapsulated in 100-kDa hollow fibers and cultured for up to 28 days. Rat spleens were implanted with hollow fibers that were either empty or contained 1 x 107 rat hepatocytes. Human HepG2 cells were encapsulated using alginate/poly-L-lysine (ALP) and also transplanted into the spleen; control rats were transplanted with free HepG2 cells. Blood human albumin levels were measured using Western blotting and spleen sections were immunostained for albumin. Hepatocytes in monolayer cultures remained viable for only 6-10 days, whereas the cells cultured in hollow fibers remained viable and produced albumin throughout the study period. Allogeneic hepatocytes transplanted in hollow fibers remained viable for 4 weeks (end of study). Free HepG2 transplants lost viability and function after 7 days, whereas encapsulated HepG2 cells remained viable and secreted human albumin at all time points studied. ALP capsules, with or without xenogeneic HepG2 cells, produced no local fibrotic response. These data indicate that intrasplenic transplantation of encapsulated cells results in excellent survival and function of the transplanted cells and that the proposed technique has the potential to allow transplantation of allo- and xenogeneic cells (e.g., pancreatic islets) without immunosuppression.

Key words: Cell transplantation; Cell therapy; Cell encapsulation; Hepatocyte

Address correspondence to Jacek Rozga, M.D., Ph.D., Cedars-Sinai Medical Center, 8700 Beverly Blvd., D-4018, Los Angeles, CA 90048. Tel: (310) 423-7702; Fax: (310) 423-0224; E-mail: Jacek.Rozga@cshs.org

Cell Transplantation, Vol. 11, pp. 563-571, 2002
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Innocuous Intracellular Ice Improves Survival of Frozen Cells

Jason P. Acker and Locksley E. McGann

Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, T6G 2R8 Canada

Extensive efforts to avoid intracellular ice formation (IIF) during freezing have been central to current methods used for the preservation and long-term storage of cells and tissues. In this study, we examined the effect of intracellular ice formation on the postthaw survival of V-79W fibroblast and MDCK epithelial cells using convection cryomicroscopy and controlled-rate freezing. V-79W and MDCK cells were cultured as single attached cells or as confluent cell monolayers. Postthaw cell survival was assessed using three different indices: the presence of an intact plasma membrane, the ability to reduce alamarBlue, and the capacity to form colonies in culture. Regulating the isothermal nucleation temperature was used to control the incidence of IIF in the model systems. We report that the presence of intracellular ice in confluent monolayers at high subzero temperatures does not adversely affect postthaw cell survival. Further, we show that in the absence of chemical cryoprotectants, the formation of intracellular ice alone improves the postthaw survival of cultured V-79W fibroblast and MDCK epithelial cells. Improved long-term storage of cells and tissues will result by incorporating innocuous intracellular ice formation into current strategies for cryopreservation.

Key words: Cryopreservation; Intracellular ice formation; Cryoprotectant; Cell viability; Dimethyl sulfoxide

Address correspondence to Dr. Jason Acker, Ph.D., Canadian Blood Services, 3rd Floor Canadian Blood Services Building, 8249-114 Street, Edmonton, Alberta T6G 2R8 Canada. Tel: (780) 702-2533; Fax: (780) 702-2501; E-mail: jason.acker@bloodservices.ca

Cell Transplantation, Vol. 11, pp. 573-582, 2002
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Gene Therapy: A Lipofection Approach for Gene Transfer Into Primary Endothelial Cells

A. T. L. Young,1 J. R. T. Lakey,1 A. G. Murray,2 and R. B. Moore1

1Department of Surgery, Surgical-Medical Research Institute and 2Department of Medicine, University of Alberta, Edmonton, Canada T6G 2N8

Despite the great potential of gene therapy to become a new treatment modality in future medicine, there are still many limitations to overcome before this gene approach can pass to the stage of human trial. The foremost obstacle is the development of a safe, efficient, and efficacious vector system for in vivo gene application. This study evaluated the efficacy of lipofection as a gene delivery vehicle into primary endothelial cells. Transfection efficiency of several lipid-based reagents (Effectene, Fugene 6, DOTAP) was examined at experimental temperatures of 37°C, 24°C, and 6°C. Human umbilical vein endothelial cells (HUVECs) were transfected with the enhanced green fluorescent protein (EGFP) using precise amounts of DNA (Effectene, 0.2 mg; Fugene 6, 0.5 mg; DOTAP, 2.5 mg) and lipids (Effectene, 10 ml; Fugene 6, 6 ml; DOTAP, 15 ml) optimized in our laboratory. Duration of incubation in the DNA/lipid transfection mixture varied for each lipid transfectant as follows: 5 h for both Fugene 6 and DOTAP and 3 h for Effectene. Efficiency of transfection was quantified by microscopic evaluation of EFGP expression in a minimum of 100 cells per group. Transfection efficiencies achieved with these lipofection agents were 34 ± 1.3% (mean ± SEM), 33 ± 1.4%, and 18 ± 1.5% for Effectene, Fugene 6, and DOTAP, respectively, at 37°C. Transfection results were lower at 24°C with mean efficiencies of 26 ± 2.4% for Effectene, 14 ± 2.9% for Fugene 6, and 15 ± 3.2% for DOTAP. Furthermore, mean efficiencies at 6°C were 6 ± 0.5%, 8 ± 1.5%, and 6 ± 0.0% for Effectene, Fugene 6, and DOTAP, respectively. Efficiency of transfection appeared to be temperature dependent (ANOVA; p < 0.0001). In spite of a significant decrease (37°C vs. 24°C: p < 0.0001; 37°C vs. 6°C: p < 0.0001; 24°C vs. 6°C: p < 0.0115) in transfection efficiency at low temperatures, the successful in vitro gene manipulation renders lipofection a potential gene delivery strategy for in vivo gene therapy.

Key words: Lipofection; Endothelial cells; Gene therapy; Transplantation

Address correspondence to Jonathan R.T. Lakey, Ph.D., Assistant Professor of Surgery, Surgical-Medical Research Institute, University of Alberta, 1074 Dentistry/Pharmacy Building, Edmonton, Alberta T6G 2N8 Canada. Tel: (780) 492-3077; Fax: (780) 492-6335; E-mail: jonathan.lakey@ualberta.ca

Cell Transplantation, Vol. 11, pp. 583-592, 2002
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Vascular Adventitia Is a Suitable Compartment to Transplant Transduced Vascular Smooth Muscle Cells for Ex Vivo Gene Expression

Patricia C. B. Beltrão-Braga,1 Ivan H. J. Koh,2 Maria R. R. Silva,3 Paulo S. Gutierrez,4 and Sang W. Han1

1Department of Biophysics, 2Department of Pediatrics, and 3Department of Pathology, UNIFESP-EPM, São Paulo, 04023-062, Brazil
4Laboratory of Pathology, INCOR-USP, São Paulo, 05403-000, Brazil

Vascular smooth muscle cells (VSMC) are ideal for systemic gene therapy because of their proximity to blood vessels and they have demonstrated long-term exogenous gene expression in vivo. However, the procedure generally followed to seed the transduced VSMC onto arteries denuded of endothelial cells usually induces stenosis and thrombosis, with a consequent high risk for use in humans. We demonstrate here that the vascular adventitia is a suitable place to introduce transduced VSMC and to secrete therapeutic proteins into the blood stream by a simple procedure, avoiding postoperative vascular complications. Transduced VSMC, with the retroviral vectors carrying the human growth hormone gene (hGH), were seeded into the adventitia of the rat abdominal aorta by single injection of a cell suspension. Based on the hGH and anti-hGH production in serum and on histological analysis of the removed aortas, we demonstrated hGH production over the 2-month experimental period. None of the animals used in the experiment showed stenosis, thrombosis, or other vascular or visible physiological abnormalities.

Key words: Smooth muscle cells; Adventitia; Retroviral vector; Gene therapy; hGH

Address correspondence to Sang Won Han, UNIFESP-EPM, Department of Biophysics, Rua Botucatu, 862 São Paulo-SP, Brazil 04023-062. Tel: 55-11-5576 4530 or 5576 4555, ext. 202; Fax: 55-11-5571 5780; E-mail: sang@biofis.epm.br

Cell Transplantation, Vol. 11, pp. 593-613, 2002
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Pharmacological, Cell, and Gene Therapy Strategies to Promote Spinal Cord Regeneration

Bas Blits, Gerard J. Boer, and Joost Verhaagen

Graduate School Neurosciences Amsterdam, Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ Amsterdam-ZO, The Netherlands

In this review, recent studies using pharmacological treatment, cell transplantation, and gene therapy to promote regeneration of the injured spinal cord in animal models will be summarized. Pharmacological and cell transplantation treatments generally revealed some degree of effect on the regeneration of the injured ascending and descending tracts, but further improvements to achieve a more significant functional recovery are necessary. The use of gene therapy to promote repair of the injured nervous system is a relatively new concept. It is based on the development of methods for delivering therapeutic genes to neurons, glia cells, or nonneural cells. Direct in vivo gene transfer or gene transfer in combination with (neuro)transplantation (ex vivo gene transfer) appeared powerful strategies to promote neuronal survival and axonal regrowth following traumatic injury to the central nervous system. Recent advances in understanding the cellular and molecular mechanisms that govern neuronal survival and neurite outgrowth have enabled the design of experiments aimed at viral vector-mediated transfer of genes encoding neurotrophic factors, growth-associated proteins, cell adhesion molecules, and antiapoptotic genes. Central to the success of these approaches was the development of efficient, nontoxic vectors for gene delivery and the acquirement of the appropriate (genetically modified) cells for neurotransplantation. Direct gene transfer in the nervous system was first achieved with herpes viral and E1-deleted adenoviral vectors. Both vector systems are problematic in that these vectors elicit immunogenic and cytotoxic responses. Adeno-associated viral vectors and lentiviral vectors constitute improved gene delivery systems and are beginning to be applied in neuroregeneration research of the spinal cord. Ex vivo approaches were initially based on the implantation of genetically modified fibroblasts. More recently, transduced Schwann cells, genetically modified pieces of peripheral nerve, and olfactory ensheathing glia have been used as implants into the injured spinal cord.

Key words: Injury; Neurotrophins; Regeneration; Spinal cord; Transplantation; Viral vector

Address correspondence to Joost Verhaagen, Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ Amsterdam-ZO, The Netherlands. Tel: +31 20 5665500; Fax: +31 20 6961006; E-mail: j.verhaagen@nih.knaw.nl