ognizant Communication Corporation

Cell Transplantation, Vol. 9, pp. 1-10, 2000
0963-6897/00 $20.00 + 00
Copyright © 2000 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

¹Organogenesis, Inc., Canton, MA 02021
²Center for Engineering in Medicine, Massachusetts General Hospital and the Shriners Hospital for Children, Boston, MA 02114

The clinical consequences of acute liver failure are associated with high mortality. Intensive medical intervention is required to treat the symptoms of liver failure, including coagulopathy, metabolic instability, and encephalopathy. Providing temporary liver support with an extracorporeal liver assist device could stabilize the patient until a donor liver became available or the patient's own liver was able to recover. The use of human hepatocytes as the biologic component of the assist device is precluded by the scarcity of available tissue and the limited proliferative potential of adult hepatocytes in vitro. Consequently, porcine hepatocytes are being evaluated as a cell source for liver assist devices. Maintaining differentiated function in isolated hepatocytes, however, remains a challenge in the development of this technology and is complicated by the fact that the key therapeutic functions for short-term survival have not been well defined. Several approaches have been effective in prolonging rodent hepatocyte function in vitro, including manipulation of extracellular matrix. Here, we have investigated porcine hepatocyte function in vitro with a specific emphasis on the response to exogenous collagen matrix. In control cultures, albumin secretion increased during the first 7-10 days of culture to an average of 50 ± 17 mg/day/10⁶ cells and then decreased over the next 2 weeks. The pattern of urea synthesis was slightly different in that it was highest in the first 1-3 days postisolation (140 ± 19 mg/day/10⁶ cells) and then decreased by about 50% to a plateau level that was stable during the next 3-4 weeks of culture. Cytochrome P450-mediated activities were the most labile with time in culture and were undetectable after the first week in the absence of pharmacological inducers. In contrast to results reported for rat cells, porcine hepatocytes exhibited differentiated function in the absence of any modification of the culture dish surface and function was not increased or prolonged in the presence of exogenous collagen.

Key words: Porcine hepatocytes; Collagen; Matrix

Address correspondence to Susan J. Sullivan, Organogenesis, Inc., 150 Dan Road, Canton, MA 02021. Tel: (781) 575-0775; E-mail: ssullivan@organo.com

Laboratory of Diabetes and Experimental Endocrinology, Endocrine Unit, CSUB-Hospital de Bellvitge, University of Barcelona, Barcelona, Spain

Insulin-induced normoglycemia has shown to have a beneficial effect on the outcome of pancreatic islets transplanted to diabetic recipients. The aim of the study was to identify the insulin treatment that can maximize its beneficial effect on islet transplants. Six groups of streptozotocin diabetic C57Bl/6 mice were transplanted (Tx) with 100 syngeneic islets, an insufficient beta cell mass to restore normoglycemia, and were treated with insulin as follows: group 1 (n = 9): from day 10 before Tx to day 14 after Tx; group 2 (n = 11): from day 6 before Tx to Tx day; group 3 (n = 11): from Tx day to day 6 after Tx; group 4 (n = 7): from Tx day to day 14 after Tx; group 5 (n = 8): from day 10 to day 24 after Tx; group 6 (n = 18): Tx mice were not treated with insulin. Sixty days after Tx, normoglycemia was achieved in 100% of mice in groups 1, 4, and 5, in 73% of mice in group 2, and in only 45% and 33% of mice in groups 3 and 6, respectively (p < 0.01). Intraperitoneal glucose tolerance, determined only in normoglycemic mice, was similar in groups 1, 2, 4, and normal controls. In contrast, normoglycemic mice from groups 3, 5, and 6, exposed to more severe and prolonged hyperglycemia after Tx, showed higher glucose values after glucose injection, suggesting that hyperglycemia had a long-lasting deleterious effect on transplanted beta cell function. The initially transplanted beta cell mass was maintained in the grafts of normoglycemic mice, but was severely reduced in hyperglycemic mice. Transplanted beta cell mass was similar in normoglycemic groups with normal or impaired glucose tolerance, indicating that impaired glucose tolerance was not due to reduced beta cell mass. In summary, the beneficial effect of insulin-induced normoglycemia on transplanted islets was maximal when insulin treatment was maintained the initial 14 days after transplantation. Exposure to sustained hyperglycemia initially after transplantation had a long-lasting deleterious effect on transplanted islets.

Key words: Islet transplantation; Insulin; Glucose toxicity; Beta cell mass

Address correspondence to Eduard Montanya, Endocrine Unit (13-2), CSUB-Hospital Bellvitge, Feixa Llarga, s/n, 08907 L'Hospitalet de Llobregat, Barcelona. Spain. Tel: 34-93-4035804; Fax: 34-93-2607561.

¹Department of Surgery, Leicester General Hospital and the ²Department of Surgery, University of Leicester, Leicester, UK

The intraportal site is the most common site for islet transplantation. Many other sites have been tried experimentally, including the spleen, which has successfully lead to insulin independence in a number of animal models. Nevertheless, there are no detailed reports of total pancreatectomy and splenic islet autotransplantation in humans. Five patients underwent total pancreatectomy and splenic islet autotransplantation for chronic pancreatitis. Four patients had a pylorus-preserving total pancreatectomy and one patient a duodenal-preserving pancreatectomy. In three cases islets were embolized into both the portal vein and spleen. Two patients received splenic islet transplants alone. Islets were transplanted by retrograde venous infusion via the short gastric veins (n = 3), splenic vein stump (n = 1), and the left gastroepiploic vein (n = 1). The total volumes of transplanted pancreatic digest in those receiving combined intraportal and splenic autografts (n = 3) were 15.8, 13.0, and 13.5 ml. The volumes in those receiving a splenic-alone autograft (n = 2) were 12.0 and 5 ml. The mean rise in portal pressure was 18 cm of water. Complications related to the splenic autograft included a wedge splenic infarct, an emergency splenectomy, and a portal vein thrombosis in one patient having a combined intraportal and splenic autograft. Two patients developed insulin independence, two patients were still insulin independent at 1-year follow-up, and all had normal HbA1c levels (mean 5.6, range 5.2-6.3). Splenic islet autotransplantation, after total pancreatectomy, does lead to insulin independence. However, in our experience the combined procedure has a high morbidity because of splenic infarction and venous thrombosis.

Key words: Pancreactectomy; Splenic islet autograft; Intraportal autograft

Address correspondence to Mr. Steven A. White, FRCS, Department of Surgery, University of Leicester, Leicester General Hospital, Gwendolen Road, Leicester, LE5 4PW UK. Tel: 0116 249 0490; Fax: 0116 249 0064.

*Presented to the International Pancreas and Islet Transplant Congress, Milan, September 1997 and the Association of Surgeons of Great Britain and Ireland, Edinburgh, June 1998.

Tissue Engineering and Banking Laboratory, National Centre for Cell Science, Ganeshkhind, Pune 411 007, India

The success of immunoisolation devices for islet transplantation depends on the nature of semipermeable membranes, which permit the crossover of micronutrients, glucose, and insulin and prevent the entry of immunocytes and other transplant rejection mechanisms. In the present study we examined the properties of chitosan-polyvinyl pyrrolidone (PVP) hydrogels for possible application as an immunoisolation device. Hydrogels with two different proportions of chitosan-PVP (M1 1:1 and M2 2:1, v/v) were synthesized by cross-linking with glutaraldehyde. Hydrogels were characterized for their hydrophilic nature, protein adsorption, diffusion properties, cytotoxicity, and islet compatibility. Hydrogel membranes were found to be hydrophilic as determined by high octane contact angle value (M1: 142.9 ± 0.46; M2: 143.6 ± 0.49). Protein adsorption on the hydrogels was found to be low (0.0143 ± 0.0027 mg for M1 and 0.0136 ± 0.0049 mg for M2) compared to tissue culture polystyrene (TCPS) (0.0434 ± 0.001 mg) and pure chitosan (0.0214 ± 0.0025 mg) control. Hydrogel M1 was tested as a representative for diffusion studies. M1 allowed regulated transport of insulin and did not allow anti-insulin antibodies to pass through. In vitro biocompatibility of M1 and M2 was found to be excellent with no cytotoxic effects on the HeLa cells as determined by MTT and NR assay. Mouse islets cultured on the hydrogel membranes retained their integrity and intact morphology as assessed by image analysis study. Viability of islets cultured on hydrogels was comparable to that of controls (M1: 97%; M2: 90.4%) as assessed by trypan blue dye exclusion test. Islets retained their functionality when cultured on hydrogels, as judged by insulin secretion in response to glucose challenge (16.0 mM). Although in vivo experiments are awaited, the present study provides sufficient documentation to consider chitosan-PVP membranes as potential candidates for immunoisolation of islets.

Key words: Chitosan; Hydrogels; Biocompatibility; Islet; Immunoisolation

Address correspondence to Ramesh Bhonde, Tissue Engineering and Banking Laboratory, National Centre for Cell Science, Ganeshkhind, Pune 411 007, India. Tel: 91 20 5670922/31/41; Fax: 91 20 5672259; E-mail: mrisbud@hotmail.com

Department of Surgery, Duke University Medical Center, Durham, NC 27710

Microencapsulation is an effective means of immunoisolation for pancreatic islet transplants. However, the process of isolating, purifying, encapsulating, and transplanting islets in a single day is labor intensive and difficult for routine use. There is an apparent need for reliable methods of islet storage, and cryopreservation has emerged as an attractive system of islet banking. While studies have shown that cryopreserved islets are viable when tested unencapsulated after thawing, it is not clear if the combination of freezing and encapsulation would affect islet function. The purpose of the present study was to determine the in vitro function of cryopreserved islets following thawing and microencapsulation. Islets were isolated from the pancreata of Sprague-Dawley rats and cryopreserved under liquid nitrogen for either 1 week or 1 month, following an overnight culture at 37°C. Upon thawing, the islets were tested either unencapsulated or after encapsulation in polylysine-alginate membrane. In all experiments islets were preperifused for 1 h at 37°C with a modified Krebs-Ringer bicarbonate buffer containing 3.3 mM (60 mg/dl) glucose and maintained at pH 7.4 by continuous gassing with 95% air/5% CO₂. Following basal effluent sample collection on ice, the glucose concentration was raised to 16.7 mM (300 mg/dl). It was found that, within 10 min of high glucose stimulation, an average of twofold increase in insulin secretion (p < 0.01) was obtained in islets within or without microcapsules. We conclude that islets cryopreserved for 1 month prior to thawing and microencapsulation retained functional viability as determined in in vitro experiments.

Key words: Islets; Banking; Cryopreservation; Microencapsulation

Address correspondence to Dr. Emmanuel C. Opara, Box 3065, Duke University Medical Center, Durham, NC 27710. Tel: (919) 684-4120; Fax: (919) 681-7339; E-mail: opara001@mc.duke.edu

*This work, which was supported in part by a grant from the Lincy Foundation, Beverly Hills, CA, and MicroIslet, Inc., San Diego, CA, has been presented in part at a minisymposium of Experimental Biology '99 in Washington, DC and published as an abstract.

Laboratoire d'Endocrinologie Experimentale, UPRES-ULP 2106, Strasbourg I, CeeD, France

The present study concerns the influence of the transplantation of free and encapsulated (AN69 membrane, Hospal) islets on the chemotaxis of peritoneal macrophages. Fifty free or encapsulated rat islets, cultured for 24 h, were transplanted in the peritoneal cavity of mice (n = 12). Three days after transplantation, the chemotaxis of peritoneal murine macrophages was tested towards formyl-methionyl-leucyl-phenylalanine (fMLP) and a culture medium conditioned for 3 days by free rat islets isolated from the same rat donor. In response to fMLP, the chemotactic indexes of macrophages from mice transplanted with free or encapsulated islets were 8.09 ± 2.10 and 9.45 ± 2.76, respectively. These values were significantly higher than those obtained when macrophages from untreated mice were tested (2.42 ± 0.23; p < 0.01). In response to culture medium conditioned by free islets, the transplanted encapsulated islets failed to enhance macrophage chemotaxis (2.41 ± 0.53) compared to transplanted free islets (7.00 ± 2.63; p < 0.01). Thus, encapsulation decreased the specific chemotactic activity of peritoneal macrophages induced by free islet transplantation, probably by prohibiting the diffusion of chemoattractants.

Key words: Transplantation; Pancreatic islets; Bioartificial pancreas; Macrophages; Chemotaxis

Address correspondence to Dr. L. Kessler, Service d'Endocrinologie et de Diabétologie, Pavillon Leriche, 1, Place de l'Hôpital, 67091 Strasbourg, Cedex, France. Tel: 03 88 35 87 39; Fax: 03 88 11 62 63; E-mail: Laurence.Kessler@medecine.u-strasbg.fr

¹Unitat de Bioquímica, Departament de Ciències Fisiològiques II and ²Escola Universitària d'Infermeria, Campus de Bellvitge, Universitat de Barcelona, 08907-Hospitalet del Llobregat, Spain
³Department of Medical Biochemistry and Biophysics, Laboratory of Molecular Neurobiology, Karolinska Institute, Stockholm, S-171 77, Sweden

The transplantation of fetal mesencephalic cell suspensions into the brain striatal system is an emerging treatment for Parkinson's disease. However, one objection to this procedure is the relatively poor survival of implanted cells. The ability of neurotrophic factors to regulate developmental neuron survival and differentiation suggests they could be used to enhance the success of cerebral grafts. We studied the effects of neurotrophin-3 (NT-3) or glial cell line-derived neurotrophic factor (GDNF) on the survival of dopaminergic neurons from rat fetal ventral mesencephalic cells (FMCs) implanted into the rat striatum. Two conditions were tested: (a) incubation of FMCs in media containing NT-3 and GDNF, prior to grafting, and (b) co-grafting of FMCs with cells engineered to overexpress high levels of NT-3 or GDNF. One week after grafting into the rat striatum, the survival of TH+ neurons was significantly increased by pretreatment of ventral mesencephalic cells with NT-3 or GDNF. Similarly, co-graft of ventral mesencephalic cells with NT-3- or GDNF-overexpressing cells, but not the mock-transfected control cell line, increased the survival of graft-derived dopaminergic neurons. Interestingly, we also found that co-grafting of GDNF-overexpressing cells was less effective than NT-3 at improving the survival of fetal dopaminergic neurons in the grafts, and that only GDNF induced intense TH immunostaining in fibers and nerve endings of the host tissue surrounding the implant. Thus, our results suggest that NT-3, by strongly enhancing survival, and GDNF, by promoting both survival and sprouting, may improve the efficiency of fetal transplants in the treatment of Parkinson's disease.

Key words: Fetal ventral mesencephalic cells; Transplant; Neurotrophin-3; GDNF; Dopamine; Tyrosine hydroxylase; Parkinson's disease

Address correspondence to Santiago Ambrosio, Unitat de Bioquímica, Departament de Ciències Fisiològiques 11, Campus de Bellvitge, Universitat de Barcelona, c/.Feixa Llarga s/n, L'Hospitalet del Llobregat 08907, Barcelona, Spain. Tel: 343 93 402 90 94; Fax: 343 93 402 42 68; E-mail: ambrosio@bellvitge.bvg.ub.es

MRC Cambridge Centre for Brain Repair, University of Cambridge, Forvie Site, Robinson Way, Cambridge CB2 2PY, UK

Expanded neural precursor cells provide an attractive alternative to primary fetal tissue for cell replacement therapies in neurodegenerative diseases. In this study we transplanted epigenetically propagated human neural precursor cells into a rat model of Huntington's disease. Neural precursors survived transplantation and large numbers differentiated to express neuronal antigens, including some that expressed DARPP-32, indicating a mature striatal phenotype had been adopted. Neuronal fibers from the grafts projected diffusely throughout the host brain, although there was no evidence that outgrowth was specifically target directed. This study supports the contention that propagated human neural precursors may ultimately be of use in therapeutic neural transplantation paradigms for diseases such as Huntington's disease.

Key words: Neural stem cell, EGF, FGF-2, Striatal graft

Address correspondence to Richard J. E. Armstrong, MRC Cambridge Centre for Brain Repair, University of Cambridge, Forvie Site, Robinson Way, Cambridge CB2 2PY, UK. Tel: 00 44 1223 331160; Fax: 00 44 1223 331174; E-mail: rjea100@cam.ac.uk

¹MRC Cambridge Centre for Brain Repair, and the Departments of ²Neurosurgery and ³Experimental Psychology, University of Cambridge, Cambridge, UK

A growing body of evidence suggests that graft-mediated functional recovery in animal models of Huntington's disease is influenced by the morphology of the striatal grafts. Various parameters, including embryonic dissection, tissue preparation, and surgical delivery into the brain, have been investigated with the aim of increasing the proportion of the grafts comprising striatum-like tissue. While growing evidence suggests that implants derived from the selective dissection of the lateral ganglionic eminence (LGE) contain more striatal tissue, the relationship between the quantity of LGE tissue implanted and the striatum-like proportion of the resultant grafts has not been formally investigated. In this study the volume of striatum-like tissue within the grafts did not increase in a linear manner with increasing numbers of cells implanted. The proportion of the grafts that comprised the striatum-like patch compartment or P-zone remained constant after an initial rapid increase as the number of LGE cells implanted was increased. These results have important practical implications in determining the optimum number of LGE cells to implant and hence in the design of any surgical protocol for the clinical application of this technique.

Key words: Striatal grafts; Lateral ganglionic eminence; Optimum cell number; Graft morphology

Address correspondence to Colin Watts, MRC Cambridge Centre for Brain Repair, University of Cambridge, Forvie Site, Robinson Way, Cambridge CB2 2PY, UK. Tel: (+44) 1223 331160; Fax: (+44) 1223 331174; E-mail: cw209@cam.ac.uk

Section for Neuronal Survival, Department of Physiological Sciences, Wallenberg Neuroscience Center, Lund University, SE-223 62 Lund, Sweden

A large proportion of grafted striatal neurons die, and mechanisms by which they succumb may involve excitotoxicity and oxidative stress. We investigated the effects of pretreatment of the graft tissue with the \IC\N-methyl-D-aspartate (NMDA) receptor antagonist (+)dizocilpine hydrogen maleate (MK-801) and lipid peroxidation inhibitor lazaroid U-83836E on the survival of transplanted striatal neurons. Neither compound increased the survival of grafts, suggesting that NMDA-related excitotoxicity or oxidative stress may not be primary mediators of cell death in striatal grafts.

Key words: Embryonic striatum; Neural transplantation; Excitotoxicity; Oxidative stress

Address correspondence to Patrik Brundin, Section for Neuronal Survival, Department of Physiological Sciences, Wallenberg Neuroscience Center, Lund University, Solvegatan 17, SE-223 62 Lund, Sweden. Tel: +46 46 222 0524; Fax: +46 46 222 0531; E-mail: Patrik.Brundin@neural.lu.se

¹Immunology Department, ²Neurosurgery Department, and ³Cytology Department, Rangueil Hospital, F31403 Toulouse cedex 4, France
⁴Radioisotope Department, CHU Purpan, F31052 Toulouse cedex, France
⁵Group for Pain Study, and ⁶Pierre Fabre Research Institute, Cell Culture Department, Rangueil Medical School, Paul Sabatier University, 133, route de Narbonne, F31077 Toulouse, France
⁷Statistics and Epidemiology Department, Saint-Jacques Hospital, Medical School, Paul Sabatier University, 133, route de Narbonne, F31000 Toulouse, France

Adrenal medullary tissue including chromaffin cells was grafted intrathecally in cancer patients to relieve intractable pain. The central nervous system (CNS) is considered an immune privileged site. Therefore, non-HLA-matched and unencapsulated tissue was grafted in 15 patients and 1 sham control in a series of at least 20 grafts. We observed an increase in CSF lymphocyte counts in 15/20 allografts (75%). In contrast to peripheral blood, CD4 T cells predominated in the CSF, but failed to exhibit an activated phenotype (CD25+ CD45RO+ HLA-DR+). The positive effect of graft on pain, the high met-enkephalin levels, the absence of any increase in CSF cytokine levels particularly for IFN-g or IL-2 (but not IL-10 and IL-6), indirectly indicated that the graft was tolerated despite the presence of CSF lymphocytes. The single treatment failure and three of four cases of partial efficacy occurred in grafts where CSF lymphocytes were present. Moreover, when assayed (n = 7), the CD4+ CSF lymphocytes still retained the capacity to exhibit ex vivo a normal or enhanced frequency of T CD4 cells producing IFN-g and IL-2. Taken together, our observations indicate that impairment of the local immunosuppressive balance can lead to activation of those CSF CD4 T cells and drive a rejection process. This study suggests further work on the purification and/or the immunoisolation of tissues grafted in the CNS will be necessary, particularly when the possibility of long-term and repeated grafting is considered.

Key words: Brain immunology; T lymphocytes; Cytokines; Cell transplantation; Adrenal medulla; Immunoisolation

Address correspondence to Dr. Jean Tkaczuk, Laboratoire d'Immunologie, Hôpital Rangueil, F 31403 Toulouse cedex 4, France. Tel: (33) 5 61 32 28 53; Fax: (33) 5 61 32 22 82; E-mail: tkaczuk@cict.fr

¹Department of Bioengineering, National Cardiovascular Center Research Institute, 5-7-1, Fujishiro-dai, Suita, Osaka 565-8565, Japan
²Second Department of Surgery, Kyoto Prefectural University of Medicine, 465, Kawaramachi-Hirokoji, Kamikyo-ku, Kyoto 602-0841, Japan

This study was conducted to investigate how a continuous load of hydrodynamic stresses influences the tissue architecture of a branched hybrid vessel in vitro. Tubular hybrid medial tissue of small (3 mm) and large (6 mm) diameters, prepared by thermal gelation of a cold mixed solution of bovine smooth muscle cells (SMCs) and type I collagen in glass molds, was assembled into a branched hybrid medial tissue by end-to-side anastomosis. After a 2-week culture period, bovine endothelial cells (ECs) were seeded onto the luminal surface. The branched hybrid vessel was connected to a mock circulatory loop system and tested for two modes of flow: 1) low flow rate for 24 h, 2) high flow rate for 24 or 72 h. After exposure to a low flow rate for 24 h, cobblestone appearance of the ECs was dominant. After exposure to a high flow rate, EC alignment in the direction of flow was observed in the branch region, except at the region of predicted flow separation where ECs retained their polygonal configuration. Elongation of SMCs with no preferential orientation was observed in the case of vessels exposed to a high flow rate for 24 h, and circumferential orientation was prominent in those exposed to a high flow rate for 72 h. On the other hand, collagen fibrils exhibited no preferential orientation in either case. After injection of Evans blue-albumin conjugate into the circulating medium, the luminal surface of the hybrid vessel exposed to a high flow rate for 24 h was examined by confocal laser scanning microscopy. The fluorescence intensity was low at the high shear zone in the branch region, while at the flow separation region it was very high, indicating the increased albumin permeability at the latter region. These findings reflect region-specific tissue architecture in the branch region, in response to the local flow pattern, and may provide an in vitro atherosclerosis model as well as a fundamental basis for the development of functional branched hybrid grafts.

Key words: Branched hybrid vascular tissue; Hydrodynamic stress; Cellular orientation; Albumin permeability; Vascular wall architecture

Address correspondence to Takehisa Matsuda at his present address: Department of Biomedical Engineering, Kyushu University Graduate School of Medicine, 3-1-1, Maedashi, Higashi-ku, Fukuoka 812-8582, Japan. Tel: (+81) 92-642-6210; Fax: (+81) 92-642-6212; E-mail: matsuda@med.kyushu-u.ac.jp

Departments of ¹Transplantation Surgery, Huddinge Hospital, and ²Pharmacology, Karolinska Hospital, Karolinska Institute, Stockholm, Sweden

Encapsulation of cellular grafts in an immunoisolation membrane device may make it possible to perform transplantation without having to give immunosuppressive drugs. A common problem is the development of an avascular fibrotic zone around the implants, leading to impaired graft survival. The TheraCyte^TM macroencapsulation device has therefore been designed to facilitate neovascularization of the device's surface. In this study, we evaluated the microcirculation around empty TheraCyte^TM devices implanted SC in rats at various times after implantation, using a laser Doppler probe introduced via the device port. Studies were performed on day 1 or at 1, 2, and 4 weeks or at 2, 3, and 12 months after implantation. The mean flow was 158 ± 42, 148 ± 50, 133 ± 28, 72 ± 17, 138 ± 41, 165 ± 43, and 160 ± 29 perfusion units (PU), respectively. Thus, the microcirculation around the device was significantly reduced at 4 weeks after implantation (p < 0.01) while, from 2 months onwards the circulation had improved and did not differ significantly from that on day 1. The present study shows time-related changes in the microcirculatory flow around TheraCyte^TM macroencapsulation devices that agree with our previous microdialysis studies on in vivo exchange of insulin and glucose between the device and the circulation. Laser Doppler flowmetry seems to provide a reliable technique for screening blood perfusion around macroencapsulation devices.

Key words: Macroencapsulation device; Transplantation; Microcirculation; Blood flow measurements; Laser Doppler flowmetry

Address correspondence to Dr. Ehab Rafael, Department of Transplantation Surgery, Huddinge Hospital, S-141 86 Huddinge, Sweden. Tel: +46 8 58 58 0000; Fax: +46 8 774 31 91; E-mail: Ehab.Rafael@karo.ki.se

¹Research Division, Section of Islet Transplantation and Cell Biology, Joslin Diabetes Center, Boston, MA 02215
²Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139

Improving blood vessel formation around an immunobarrier device should improve the survival of the encapsulated tissue. In the present study we investigated the formation of new blood vessels around a planar membrane diffusion device (the Baxter Theracyte System^TM) undergoing a continuous infusion of vascular endothelial growth factor through the membranes and into the surrounding tissue. Each device (20 ml) had both an inner immunoisolation membrane and an outer vascularizing membrane. Human recombinant vascular endothelial growth factor-165 was infused at 100 ng/day (low dose: n = 6) and 500 ng/day (high dose: n = 7) for 10 days into devices implanted SC in Sprague-Dawley rats; noninfused devices transplanted for an identical period were used as controls (n = 5). Two days following the termination of VEGF infusion, devices were loaded with 20 ml of Lispro insulin (1 U/kg) and the kinetics of insulin release from the lumen of the device was assessed. Devices were then explanted and the number of blood vessels (capillary and noncapillary) was quantified using morphometry. High-dose vascular endothelial growth factor infusion resulted in two- to threefold more blood vessels around the device than that obtained with the noninfused devices and devices infused with low-dose vascular endothelial growth factor. This increase in the number of blood vessels was accompanied by a modest increase in insulin diffusion from the device in the high-dose vascular endothelial growth factor infusion group. We conclude that vascular endothelial growth factor can be used to improve blood vessel formation adjacent to planar membrane diffusion devices.

Key words: Transplantation; Immunobarrier; Lispro; Angiogenesis; Encapsulation

Address correspondence to Gordon C. Weir, M.D., Joslin Diabetes Center, One Joslin Place, Boston, MA 02215. Tel: (617) 732-2581; Fax: (617) 732-2650; E-mail: gordon.weir@joslin.harvard.edu

Department of Paediatrics, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, NT, Hong Kong

As an early acting growth factor, flt-3 ligand (FL) promotes the ex vivo expansion of hematopoietic stem and progenitor cells. The effect and mechanism of FL on the development of the megakaryocytic lineage remain unclear. In this study, we compared the effects of FL and stem cell factor (SCF) in combination with other megakaryocyte-promoting cytokines on the differentiation and proliferation of megakaryocytic progenitors and investigated the expression of flt-3 receptors on megakaryocytic cell lines. In liquid cultures of enriched CD34+ cells from human umbilical cord blood for 14 days, FL plus thrombopoietin (TPO), interleukin-3 (IL-3), and IL-6 promoted the expansion of nucleated cells, CD34+ cells, CD34+CD38- cells, and megakaryocyte colony-forming units (CFU-MK) by 300 ± 115-, 23.8 ± 11.3-, 33.9 ± 28.6-, and 584 ± 220-fold, respectively. Replacing FL with SCF significantly decreased the yield of all cell types. Using murine bone marrow (BM) cells, we demonstrated that FL at a range of 0-100 ng/ml had no significant mitogenic effect on CFU-MK formation. TPO increased CFU-MK (p < 0.001) but the effect was not significantly modified by FL. While one human acute lymphoblastic leukemia sample expressed high levels of flt-3 receptor, the four megakaryocytic cell lines (Meg-01, CHRF-288-11, M-07e, and Dami) did not show any positive expression. Our data suggest that the present cytokine combination and expansion conditions provide an effective and potentially useful system for the clinical expansion of cord blood for bone marrow transplantation (BMT). FL alone did not stimulate megakaryocytopoiesis, possibly due to the lack of receptor expression on megakaryocytes. The effect of FL in augmenting the expansion of CFU-MK in liquid culture might be due to the early action of FL at the pluripotent stem cell stage.

Key words: Flt-3 ligand; Flt-3 receptor; Megakaryocytes; Ex vivo expansion

Address correspondence to Karen Li, Ph.D., Associate Professor, Department of Paediatrics, 6th fl, Prince of Wales Hospital, Shatin, NT, Hong Kong. Tel: (852) 2632 2859; Fax: (852) 2636 0020; E-mail: lipang@cuhk.edu.hk

Centre for Brain Repair, University of Cambridge, Cambridge, UK

A large number of crippling neurological conditions result from the loss of certain cell populations from the nervous system through disease or injury, and these cells are not intrinsically replaced. Mounting evidence now suggests that replacement of depleted cell populations by transplantation may be of functional benefit in many such diseases. A diverse range of cell populations is vulnerable, and the loss of specific populations results in circumscribed deficits in different conditions. This diversity presents a considerable challenge if cell replacement therapy is to become widely applicable in the clinical domain, because each condition has specific requirements for the phenotype, developmental stage, and number of cells required. An ideal cell for universal application in cell replacement therapy would possess several key properties: it would be highly proliferative, allowing the ex vivo production of large numbers of cells from minimal donor material; it would also remain immature and phenotypically plastic such that it could differentiate into appropriate neural and glial cell types on, or prior to, transplantation. Critically, both proliferation and differentiation would be controllable. This review considers some of the evidence that stem cells exist in the central nervous system and that they may possess characteristics that make them ideal for broad application in cell replacement therapy.

Key words: Stem cell; Neural Transplantation; EGF; FGF-2

Address correspondence to Richard J. E. Armstrong, Cambridge Centre for Brain Repair, Forvie Site, Robinson Way, Cambridge CB2 2PY UK. Tel: +44-1223-331160; Fax: +44-1223-331174; E-mail: rjea100@cam.ac.uk

¹Department of Psychology, The Institute of Psychiatry, De Crespigny Park, London SE5 8AF, UK
²ReNeuron Ltd, London, UK

Experiments are described using rats with two kinds of brain damage and consequent cognitive deficit (in the Morris water maze, three-door runway, and radial maze): 1) ischemic damage to the CA1 hippocampal cell field after four-vessel occlusion (4VO), and 2) damage to the forebrain cholinergic projection system by local injection of excitotoxins to the nuclei of origin or prolonged ethanol administration. Cell suspension grafts derived from primary fetal brain tissue display a stringent requirement for homotypical cell replacement in the 4VO model: cells from the embryonic day (E)18-19 CA1 hippocampal subfield, but not from CA3 or dentate gyrus or from E16 basal forebrain (cholinergic rich) led to recovery of cognitive function. After damage to the cholinergic system, conversely, recovery of function was seen with cell suspension grafts from E16 basal forebrain or cholinergic-rich E14 ventral mesencephalon, but not with implants of hippocampal tissue. These two models therefore provided a test of multifunctionality for a clonal line of conditionally immortalized neural stem cells, MHP36, derived from the E14 "immortomouse" hippocampal anlage. Implanted above the damaged CA1 cell field in 4VO-treated adult rats, these cells (multipotential in vitro) migrated to the damaged area, reconstituted the gross morphology of the CA1 pyramidal layer, took up both neuronal and glial phenotypes, and gave rise to cognitive recovery. Similar recovery of function and restoration of species-typical morphology was observed when MHP36 cells were implanted into marmosets with excitotoxic CA1 damage. MHP36 implants led to recovery of cognitive function also in two experiments with rats with excitotoxic damage to the cholinergic system damage, either unilaterally in the nucleus basalis or bilaterally in both the nucleus basalis and the medial septal area. Thus, MHP36 cells are both multipotent (able to take up multiple cellular phenotypes) and multifunctional (able to repair diverse types of brain damage).

Key words: Neural stem cells; Conditional immortalization; Cognitive function; Multifunctional repair

Address correspondence to Professor Jeffrey Gray, Department of Psychology, The Institute of Psychiatry, De Crespigny Park, London SE5 8AF, UK. Tel: 44 (0) 207-848 0245; Fax: 44 (0) 171-708 3497; E-mail: J.Gray@iop.kcl.ac.uk

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

Regeneration of injured axons following injury depends on a delicate balance between growth-promoting and growth-inhibiting factors. Overexpression of neurotrophin genes seems a promising strategy to promote regeneration. Trophic genes can be overexpressed at the site of injury at the axonal stumps, or at the perikaryal level of the injured neuron. Transduction of the neural cells can be achieved by applying adenoviral vectors, either directly in vivo or--in the case of neurotransplantation--as an ex vivo approach. In both cases it would create a more permissive environment for axonal growth and therefore in functional regeneration. In this article, the feasibility of the use of adenoviral vectors in several neuroregeneration models--in particularly in spinal cord lesion models and the biological clock transplantation model--is illustrated. The results show that the adenoviral vectors can be a powerful tool to study the effects of overexpression of genes in an in vivo paradigm of nerve regeneration or nerve outgrowth. The potential use of adenoviral vectors and ex vivo transduced neurotransplants is discussed.

Key words: Adenoviral vector; Nerve injury; Neurotrophic factors; Regeneration; Transplantation; Suprachiasmatic nucleus; Peripheral nerve; Spinal cord; Ex vivo transduction

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

Section for Neuronal Survival, Wallenberg Neuroscience Center, Department of Physiological Sciences, Lund University, Sölvegatan 17, S-223 62 Lund, Sweden

Neural transplantation is developing into a therapeutic alternative in Parkinson's disease. A major limiting factor is that only 3-20% of grafted dopamine neurons survive the procedure. Recent advances regarding how and when the neurons die indicate that events preceding actual tissue implantation and during the first week thereafter are crucial, and that apoptosis plays a pivotal role. Triggers that may initiate neuronal death in grafts include donor tissue hypoxia and hypoglycemia, mechanical trauma, free radicals, growth factor deprivation, and excessive extracellular concentrations of excitatory amino acids in the host brain. Four distinct phases during grafting that can involve cell death have been identified: retrieval of the embryo; dissection and preparation of the donor tissue; implantation procedure followed by the immediate period after graft injection; and later stages of graft maturation. During these phases, cell death processes involving free radicals and caspase activation (leading to apoptosis) may be triggered, possibly involving an increase in intracellular calcium. We review different approaches that reduce cell death and increase survival of grafted neurons, typically by a factor of 2-4. For example, changes in transplantation procedure such as improved media and implantation technique can be beneficial. Calcium channel antagonists such as nimodipine and flunarizine improve nigral graft survival. Agents that counteract oxidative stress and its consequences, such as superoxide dismutase overexpression and lazaroids, can significantly increase the survival of transplanted dopamine neurons. Also, the inhibition of apoptosis by a caspase inhibitor has marked positive effects. Finally, basic fibroblast growth factor and members of the transforming growth factor-beta superfamily, such as glial cell line-derived neurotrophic factor, significantly improve the outcome of nigral transplants. These recent advances provide hope for improved survival of transplanted neurons in patients with Parkinson's disease, reducing the need for human embryonic donor tissue and increasing the likelihood of a successful outcome.

Key words: Transplantation; Parkinson's disease; Neuroprotection; Growth factor; Cell death.

Address correspondence to Patrik Brundin, Section for Neuronal Survival, Wallenberg Neuroscience Center, Slövegatan 17, SE-223 62 Lund, Sweden. Tel: +46-46-222 05 29; Fax: +46-46-222 05 31; E-mail: Patrik.Brundin@neurol.lu.se

Neurosurgical Clinic, Nordstadt Hospital, Haltenhoffstrasse 41, 30167 Hannover, Germany

In this study sensorimotor behavioral changes were monitored in rats following bilateral 6-hydroxydopamine (6-OHDA) axon terminal lesion and uni- or bilateral implantation of embryonic dopaminergic (DA) micrografts. A total of 28 mg of 6-OHDA was distributed over four injection tracts in the dorsolateral part of the caudate-putamen (CPU) bilaterally followed 4 months later by the implantation of DA micrografts spread over seven implantation tracts placed within the denervated area. Bilaterally 6-OHDA-lesioned animals exhibited significantly reduced behavioral performance scores in tests of explorational and stepping behavior as well as in skilled forelimb use. However, in contrast to the established medial forebrain bundle (MFB) lesion model of PD, these animals showed a spontaneous recovery in the side falling and skilled forelimb behavior and no deficits in overnight locomotor activity at 6 months after the lesion. Unilateral DA micrografts elicited a substantial amphetamine-induced rotational bias contralateral to the graft, but led to a significant impairment of contralateral skilled forelimb use and reduced scores in overnight locomotor activity. Bilateral DA micrografts caused a significant, though partial, increase in explorational and backhand stepping behavior, but resulted also in a significant decrease in performance levels in overnight locomotor activity and skilled forelimb use on both paws. In conclusion, DA grafts placed ectopically in the CPU in the partial lesion model of PD result in a double innervation of the GABAergic striatal neurons, arising from the residual nigrostriatal DA projections of the host and from the graft-derived DA efferent fibers. These two DA fiber systems may indeed function in a cooperative and competitive manner depending on their respective and different afferent and efferent connections, which, in turn, may lead to positive or negative influences on basal ganglia function and behavioral performances. The different patterns of 6-OHDA lesion and transplant-induced behavioral changes demonstrated in the present study compared to the "classical" MFB lesion model of PD may thus provide further insights in the complex functional organization of the basal ganglia and, thereby, may help to further optimize restorative strategies for neurodegenerative diseases, such as Parkinson's disease.

Key words: Neurotransplantation; Parkinson's disease; Neurodegeneration; Basal ganglia

Address correspondence to Guido Nikkhah, Neurosurgical Clinic, Nordstadt Hospital, Haltenhoffstrasse 41, 30167 Hannover, Germany. Tel: +49 511 9700; Fax: +49 511 9701606; E-mail: GNikkhah@compuserve.com

Laboratoire de Neurobiologie Préclinique, INSERM U.318, CHU-Pavillon B, BP 217, 38043 Grenoble Cedex 09, France

The subthalamic nucleus (STN) has been shown to play an important role in the control of movement and has been considered as a key structure in the functional organization of the basal ganglia. Several studies postulated that the STN plays a critical role in the pathophysiology of Parkinson's disease and that its inhibition or its lesioning can reverse the cardinal motor symptoms. Nevertheless, the beneficial effect was accompanied by dyskinetic abnormal movements. In order to avoid unpleasant and irreversible side effects we used high-frequency stimulation (HFS) of the STN instead of lesions. We have shown that parkinsonian motor symptoms, akinesia, rigidity, and tremor can be alleviated by HFS of the STN in the nonhuman primate model. Side effects were controllable and appeared only at intensities higher than that inducing the improvement of motor symptoms. In severe parkinsonian patients, bilateral STN-HFS greatly improved parkinsonian motor symptoms. Motor fluctuations were attenuated and patients became independent in most activities of daily living. It appears that STN-HFS mimics the effects of lesions by inhibiting its neuronal activity. In a rat model of parkinsonism, we studied the implication of the STN in the excitotoxicity of nigral dopamine cells. We showed that kainic acid lesioning of the STN can protect nigral dopaminergic cells against 6-hydroxydopamine-induced toxicity. The evidence reviewed in the present article clearly demonstrates that the STN is implicated in the pathophysiology and pathogenesis of Parkinson's disease.

Key words: Parkinson's disease; Subthalamic nucleus; Neuroprotection; High-frequency stimulation

Address correspondence to Dr. A. Benazzouz, Laboratoire de Neurobiologie Préclinique, INSERM U.318, CHU-Pavillon B, BP 217, 38043 Grenoble Cedex 09, France. Tel: (+33) 476 76 56 34; Fax: (+33) 476 76 56 19; E-mail: Abdelhamid.Benazzouz@ujf-grenoble.fr

¹MRC Cambridge Centre for Brain Repair, and the Departments of ²Neurosurgery and ³Experimental Psychology, University of Cambridge, Cambridge, UK

There is a growing body of scientific evidence contributing to the development of clinical transplantation programs in patients with Huntington's disease. Phase I clinical trials have already commenced in France and North America and are starting in the near future in Sweden and the UK. Protocols for patient selection, surgical implantation, and pre- and postoperative follow-up are well defined. However, considerable variability exists with respect to the harvesting, preparation, and timing of implantation of the donor material. In this article we review the scientific evidence on which a rational protocol for donor tissue preparation and delivery may be based. Strategies aimed at minimizing the variability of tissue preparation should reduce the variability of functional outcome of striatal transplantation observed in animal models of Huntington's disease.

Key words: Huntington's disease; Donor tissue preparation; Clinical trial protocol

Address correspondence to Colin Watts, MRC Cambridge Centre for Brain Repair, University of Cambridge, Forvie Site, Robinson Way, Cambridge CB2 2PY, UK. Tel: (+44) 1223 331160; Fax: (+44) 1223 331174; E-mail: cw209@cam.ac.uk

¹Department of Experimental Psychology and Centre for Brain Repair, University of Cambridge, UK
²Neuronal Survival Unit, Wallenberg Neuroscience Center, University of Lund, Sweden
³Member Groups of the European Biomed II Programme "Development of Xenogeneic Donor Tissue for Transplantation in Neurodegenerative Disorders"**

Embryonic allografted human tissue in patients with Parkinson's disease has been shown to survive and ameliorate many of the symptoms of this disease. Despite this success, the practical problems of using this tissue coupled to the ethical restrictions of using aborted human fetal tissue have lead to an exploration for alternative sources of suitable material for grafting, including xenogeneic embryonic dopaminergic-rich neural tissue. Nevertheless, xenografted neural tissue itself generates a number of practical, ethical, safety, and immunological issues that have to be addressed prior to any clinical xenotransplant program. In this article we review these critical issues and set out the criteria that we consider need to be met in the development of our clinical xenotransplantation research programs. We advocate that these, or similar, criteria should be adopted and made explicit by other centers contemplating similar clinical trials.

Key words: Neural transplantation; Parkinson's disease; Xenograft; Porcine; Cell transplantation

Address correspondence to Dr. Roger Barker, MRC Cambridge Centre for Brain Repair, Forvie Site, Robinson Way, Cambridge CB2 2PY, UK. Tel: +44-233-33 11 60; Fax: +44-223 33 11 74; E-mail: rab46@cus.cam.ac.uk

*All three lead authors have made an equal contribution.

**Contract number BMH4-CT-97-2596, include the following contractors: Neuronal Survival Unit, Wallenberg Neuroscience Center, Lund University, Sweden (H. Widner, L. Larsson, K. A. Czech, P. Anderson, B. Mirza); Centre for Brain Repair, University of Cambridge, UK (S. B. Dunnett, A. L. Kendall, L .E. Annett, R. Barker, E. Torres, A. E.Rosser, C. Watts); Department of Anatomy and Cell Biology, Odense University, Denmark (J. Zimmer, B. R. Finsen, A. Dahl-Jørgensen, E. B. Pedersen, T. Brevig, M. Meyer); and associated contractors: Division of Clinical Immunology at Karolinska Institute at Huddinge Hospital, Sweden (J. Holgersson); Department of Clinical Laboratory Sciences, Division of Clinical Immunology and Transfusion Medicine, Uppsala University Hospital, Sweden (O. Korsgren); Department of Clinical Chemistry and Transfusion Medicine, Sahlgren University Hospital Gothenburg, Sweden (M. Breimer); Positron Emission Tomography Center, Aarhus University Hospital, Denmark (A. Gjedde, E. Danielsen); Neurosearch A/S, Glostrup, Denmark (A. Møller).

¹PET-Center, ²Department of Neuroradiology, ³Department of Neurosurgery, and ⁴Department of Neuroanaesthesia at Aarhus University Hospital, Denmark
⁵Department of Biological Psychiatry, Aarhus University Psychiatric Hospital, Denmark
⁶Department Anatomy and Neurobiology, University of Southern Denmark-University of Odense, Denmark
⁷Neuro Search, Glostrup, Denmark

A multicenter study is under way to investigate the efficacy of allografting of embryonic mesencephalic neurons in a pig model of Parkinson's disease. We have first established that a stable parkinsonian syndrome can be established by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication of adult male Göttingen minipigs. We are now using positron emission tomography (PET) methods for testing the physiological responses to MPTP intoxication and the time course of the response to several treatment strategies. We now report preliminary results obtained in 11 pigs employed in the initial phase of the study; the completed study shall ultimately include 30 pigs. Animals were randomly assigned to one of five groups: 1) Control, 2) MPTP intoxication, 3) MPTP intoxication followed by allograft, 4) MPTP intoxication followed by allograft with immunosuppression, and 5) MPTP intoxication followed by allograft with immunosuppression and co-grafting of immortalized HiB5 cells, which had been manipulated to secrete glia cell line-derived neurotrophic factor (GDNF) ( approx. 2 ng GDNF/h/10⁵ cells). MPTP was administered (1 mg/kg/day, SC) for 7-10 days until the pigs had developed mild parkinsonian symptoms of muscle rigidity, hypokinesia, and impaired coordination, especially of the hind limbs. Approximately 2 weeks after the last MPTP dose, animals received a T1-weighted magnetic resonance imaging (MRI) scan, and a series of dynamic PET recordings. After the first series of PET scans, four grafts of porcine embryonic mesencephalic tissue (E28 days) were placed in each striatum of some MPTP-intoxicated pigs, using MRI-based stereotactic techniques. Immunosuppression of some animals with cyclosporin and prednisolone began just prior to surgery. Two more series of PET scans were performed at 4-month intervals after surgery. After the last scans, pigs were killed and the brains were perfused for unbiased stereological examination of cytological and histochemical markers in striatum and substantial nigra. The behavioral impairment of the animals (the "Parkinson's score") had been evaluated throughout the 8-month period. Kinetic analysis of the first set of PET scans has indicated that the rate constant for the decarboxylation of FDOPA in catecholamine fibers was reduced by 33% in striatum of the mildly parkinsonian pigs. The rate of association of [¹¹C]NS-2214 to catecholamine uptake sites was reduced by 62% in the same groups of pigs. No significant difference was found in the binding potential of [¹¹C]raclopride to the dopamine D₂-like receptors in striatum of the MPTP-intoxicated versus control pigs. These preliminary results are suggestive that the activity of DOPA decarboxylase may be upregulated in the partially denervated pig striatum.

Key words: Parkinson's disease; Animal models; Allografts; Positron emission tomography (PET)

Address correspondence to Erik Hvid Danielsen, M.D., Ph.D., PET-Center, Aarhus University Hospital, Norrebrogade 44. DK-8000 Aarhus C, Denmark. Tel: +45 89493299; Fax: +45 89493020; E-mail: erik@pet.auh.dk

*Contributing authors are from the Danish Neuronal Xenografting (DaNeX) Group (see the Appendix).

Section for Neuronal Survival, Department of Physiological Sciences,
Wallenberg Neuroscience Center, Lund University, Sweden

Transplantation of neural tissue from other species has the potential to improve function in patients with neurodegenerative disorders. We investigated the functional effects of embryonic porcine dopaminergic neurons transplanted in a rat model of Parkinson's disease and the immune responses to the grafts in immunosuppressed and nonimmunosuppressed hosts. Twenty-three rats with unilateral 6-hydroxydopamine lesions received dissociated, 27-day-old embryonic porcine ventral mesencephalic tissue in the right striatum. Eighteen rats received cyclosporine (10 mg/kg, IP, daily) during the whole period of 14 weeks, in combination with prednisolone (20 mg/kg, IP, daily) the first 4 days. Five rats served as nonimmunosuppressed controls. All rats were tested for amphetamine-induced rotational behavior at 3-week intervals. Two immunosuppressed rats were excluded due to severe side effects of the treatment. Functional recovery was seen in 9 of 16 immunosuppressed rats at 12 weeks. Six animals remained functionally recovered at 14 weeks and contained an average of 5750 ± 1450 (SEM) dopaminergic neurons. Between 9 and 14 weeks, three immunosuppressed rats rejected their grafts, based on rotation scores and immunohistochemical demonstration of cell infiltrates. One additional immunosuppressed rat showed evidence of ongoing rejection at 14 weeks. The striata in animals with ongoing or recent rejection contained large numbers of CD4- and CD8-positive lymphocytes, NK cells, macrophages, and microglia cells, whereas scar tissue was found in rats with grafts rejected at earlier time points (n = 11). Embryonic porcine ventral mesencephalic tissue matures in the adult rat striatum, reinnervates the host brain, and restores behavioral defects. Immunosuppressive treatment was necessary for long-term graft survival and functional recovery, but did not sufficiently protect from rejection mechanisms. Porcine neural tissue is an interesting alternative to embryonic human tissue for intracerebral transplantation in neurodegenerative diseases. However, to achieve stable graft survival in discordant xenogeneic combinations, an appropriate immunosuppressive treatment or donor tissue modifications are needed.

Key words: Parkinson's disease; Pig; Xenograft; Immune response

Address correspondence to Lena C. Larsson, Section for Neuronal Survival, Department of Physiological Sciences, Wallenberg Neuroscience Center, Lund University, Sölvegatan 17, SE-223 62 Lund, Sweden. Tel: +46-46-222 05 63; Fax: +46-46-222 05 31; E-mail: Lena.Larsson@mphy.lu.se

¹Genzyme Corporation, Cambridge, MA 02139
²Massachusetts General Hospital/Harvard Medical School, Boston, MA 02114
³Lahey Medical Center, Burlington, MA 01805
⁴Boston Medical Center/Boston University School of Medicine, Boston, MA 02118
⁵Rush-Presbyterian Hospital, Chicago, IL
⁶Brigham and Women's Hospital/Harvard Medical School, Boston, MA 02115
⁷Primedica Corporation, Rockville, MD 20850
⁸Diacrin, Inc., Charlestown, MA 02129
⁹McLean Hospital/Harvard Medical School, Belmont, MA 02178

The observation that fetal neurons are able to survive and function when transplanted into the adult brain fostered the development of cellular therapy as a promising approach to achieve neuronal replacement for treatment of diseases of the adult central nervous system. This approach has been demonstrated to be efficacious in patients with Parkinson's disease after transplantation of human fetal neurons. The use of human fetal tissue is limited by ethical, infectious, regulatory, and practical concerns. Other mammalian fetal neural tissue could serve as an alternative cell source. Pigs are a reasonable source of fetal neuronal tissue because of their brain size, large litters, and the extensive experience in rearing them in captivity under controlled conditions. In Phase I studies porcine fetal neural cells grafted unilaterally into Parkinson's disease (PD) and Huntington's disease (HD) patients are being evaluated for safety and efficacy. Clinical improvement of 19% has been observed in the Unified Parkinson's Disease Rating Scale "off" state scores in 10 PD patients assessed 12 months after unilateral striatal transplantation of 12 million fetal porcine ventral mesencephalic (VM) cells. Several patients have improved more than 30%. In a single autopsied PD patient some porcine fetal VM cells were observed to survive 7 months after transplantation. Twelve HD patients have shown a favorable safety profile and no change in total functional capacity score 1 year after unilateral striatal placement of up to 24 million fetal porcine striatal cells. Xenotransplantation of fetal porcine neurons is a promising approach to delivery of healthy neurons to the CNS. The major challenges to the successful use of xenogeneic fetal neuronal cells in neurodegenerative diseases appear to be minimizing immune-mediated rejection, management of the risk of xenotic (cross-species) infections, and the accurate assessment of clinical outcome of diseases that are slowly progressive.

Key words: Parkinson's disease; Huntington's disease; Porcine; Xenotransplantation

Address correspondence to J. Stephen Fink, M.D., Ph.D., Department of Neurology, Boston University School of Medicine, C-314, One Boston Medical Center Place, Boston, MA 02118. Tel: (617) 638-5350; Fax: (617) 638-5354; E-mail: jsfink@bu.edu

¹Department of Neurology, University Hospital, A-6020 Innsbruck, Austria
²CNRS-UMR 5543, Universit‚ de Bordeaux 2, Bordeaux, France

Multiple system atrophy (MSA) is a neurodegenerative disorder that occurs sporadically and causes parkinsonism, cerebellar, autonomic, urinary, and pyramidal dysfunction in many combinations. Progressive L-dopa-unresponsive parkinsonism due to underlying striatonigral degeneration dominates the clinical syndrome in the majority of cases (MSA-P subtype). MSA-P is characterized pathologically by degenerative changes in somatotopically related areas of the substantia nigra pars compacta and of the putamen. Furthermore, oligodendroglial cytoplasmic inclusions (GCIs) are observed throughout the cortico-striato-pallido-cortical loops and may contribute to the basal ganglia dysfunction. Neurotransplantation strategies are of potential interest in this disease, which causes marked and early disability and dramatically reduces life expectancy. A number of experimental MSA-P models have been employed to evaluate neurotransplantation approaches. Sequential nigral and striatal lesions using 6-hydroxydopamine and quinolinic acid (double toxin-double lesion approach) indicate that apomorphine-induced contralateral rotation is abolished by a secondary striatal lesion. Intrastriatal injection of mitochondrial respiratory chain toxins produces secondary excitotoxic striatal lesions combined with retrograde nigral degeneration and therefore provides an alternative single toxin-double lesion approach. Neurotransplantation in MSA-P animal models has been used to improve functional deficits by replacing lost nigral and/or striatal circuitry (neuroregenerative approach). The available data indicate that embryonic mesencephalic grafts alone or combined with striatal grafts partially reverse drug-induced rotation asymmetries without improving deficits of complex motor function. The potential neuroprotective efficacy of embryonic striatal grafts against striatal excitotoxicity is presently under investigation in the double toxin-double lesion MSA-P rat model. Anecdotal clinical evidence in one MSA-P patient misdiagnosed as Parkinson`s disease indicates that embryonic mesencephalic grafts produce incomplete clinical benefit. Striatal co-grafts may increase functional improvement. Further experimental studies are required prior to the clinical application of embryonic neurotransplantation in MSA-P. Future research strategies should explore the effect of neurotransplantation in partial MSA-P rat models with less severe nigral and striatal degeneration, the feasibility of a primate model closely mimicking the human disease, and the replication of oligodendroglial dysfunction.

Key words: Multiple system atrophy; Neurotransplantation

Address correspondence to Dr. Gregor K. Wenning, Neurological Research Laboratory, Department of Neurology, University Hospital, Anichstrasse 35, A-6020 Innsbruck, Austria. Tel: 0043 512 504 4292; Fax: 0043 512 504 3852; E-mail: gregor.wenning@uibk.ac.at

Department of Clinical Veterinary Medicine, Madingley Road, Cambridge CB3 OES, UK

Persistent demyelination, in addition to being the major pathology of multiple sclerosis and the leucodystrophies, is also a feature of spinal cord trauma where there is evidence that it contributes to the functional deficit. In experimental animals it is possible to remyelinate demyelinated CNS axons by transplanting cultures containing central or peripheral myelinogenic cells. Using functional testing we have been able to show that transplant-mediated remyelination results in restoration of function lost as a consequence of demyelination. Glial cell transplantation may therefore provide a therapeutic strategy for remyelinating areas of chronic demyelination. This article reviews issues that have to be addressed before glial transplantation can be undertaken in humans. These include: what cells to use, where would the cells come from, and can we predict how much remyelination will be achieved? It concludes that the most promising approach will be to use neural multipotential stem cells isolated from embryonic CNS, expanded in vitro as neurospheres and then committed to oligodendrocyte lineage differentiation prior to implantation. However, even with such preparations, which have considerable myelinating potential, the extent of remyelination that will be achieved cannot currently be predicted with any degree of certainty.

Key words: Oligodendrocytes; Schwann cells; Transplantation; Demyelination; Remyelination

Address correspondence to W. F. Blakemore, Department of Clinical Veterinary Medicine, Madingley Rd., Cambridge CB3 OES, UK. Tel: +44 (0)1223 337639; Fax: +44 (0)1223 337610.