ognizant Communication Corporation


VOLUME 11, NUMBER 3, 2002

Cell Transplantation, Vol. 11, pp. 185-193, 2002
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A Sequential Intrastriatal Dopaminergic Graft Strategy in the Rodent Model for Parkinson's Disease: Implications for Graft Survival and Targeting

K. A. Baker,1,2 M. B. Purdy,1 D. Sadi,1 K. Mukhida,1 and I. Mendez1,2,3

1Neural Transplantation Laboratory, 2Department of Anatomy and Neurobiology and 3Department of Surgery (Division of Neurosurgery), Dalhousie University, Halifax, Nova Scotia, Canada, B3H 4H7

Optimal placement of intrastriatal dopaminergic grafts is likely crucial to optimize clinical recovery in Parkinson's disease (PD). The target sites of dopaminergic grafts vary among clinical trials and may partially explain the variable results in clinical efficacy reported thus far. In this study we hypothesized that a subsequent dopaminergic graft may promote functional recovery following a suboptimal initial graft. To test this hypothesis, rats with unilateral 6-hydroxydopamine lesions of the right nigrostriatal pathway were randomly divided into three groups. The first group received 900,000 fetal nigral cells in the medial striatum only (n = 6). The second group received 900,000 cells in both the medial and lateral striatum simultaneously (1.8 million total; n = 8). The final group received a second graft of 900,000 cells in the lateral striatum 6 weeks following initial transplantation of a medial graft (n = 6). Amphetamine-induced circling behavior was significantly reduced in both simultaneous and sequential graft groups at 9 and 12 weeks following transplantation of the initial graft. However, no recovery was noted in the single medial graft group at those time points. Furthermore, increased survival of dopaminergic cells was observed in the lateral graft of sequentially grafted animals compared with the medial graft. We conclude that a well-positioned subsequent graft can restore function in animals with a suboptimal initial graft and that the initial graft may improve survival of the second graft. These results are further discussed in relation to their important clinical implication for neural transplantation in PD.

Key words: Parkinson's disease; Sequential grafts; Graft survival

Address correspondence to Dr. I. Mendez, Neural Transplantation Laboratory, Rm 12H-1, Department of Anatomy & Neurobiology, Dalhousie University, Halifax, Nova Scotia, Canada, B3H 4H7. Tel: (902) 473-7046; Fax: (902) 473-3343; E-mail: mendez@is.dal.ca

Cell Transplantation, Vol. 11, pp. 195-205, 2002
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Tauroursodeoxycholic Acid Improves the Survival and Function of Nigral Transplants in a Rat Model of Parkinson's Disease

Wei-Ming Duan,1 Cecilia M. P. Rodrigures,2,3 Li-Ru Zhao,1 Clifford J. Steer,2 and Walter C. Low1

Departments of 1Neurosurgery and 2Medicine, University of Minnesota Medical School, Minneapolis, MN 55455
3Centro de Patogénese Molecular, Faculdade de Farmácia, University of Lisbon, Lisbon, Portugal

There is accumulating evidence showing that the majority of cell death in neural grafts results from apoptosis when cells are implanted into the brain. Tauroursodeoxycholic acid (TUDCA), a taurine-conjugated hydrophilic bile acid, has been found to possess antiapoptotic properties. In the present study we have examined whether the supplementation of TUDCA to cell suspensions prior to transplantation can lead to enhanced survival of nigral grafts. We first conducted an in vitro study to examine the effects of TUDCA on the survival of dopamine neurons in serum-free conditions. The number of tyrosine hydroxylase (TH)-positive neurons in the TUDCA-treated cultures was significantly greater than that of control cultures 7 days in vitro. In addition, a terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) assay showed that the number of apoptotic cells in the TUDCA-treated cultures was dramatically smaller than that in the control cultures. In the transplantation study, a 50 mM concentration of TUDCA was added to the media when nigral tissue from Sprague-Dawley (SD) rats was trypsinized and dissociated. Two microliters of cell suspension containing TUDCA was then stereotaxically injected into the striatum of adult SD rats subjected to an extensive unilateral 6-hydroxydopamine lesion of the nigrastriatal dopamine pathway. At 2 weeks after transplantation, the rats that received a cell suspension with TUDCA exhibited a significant reduction in amphetamine-induced rotation scores when compared with pretransplantation value. There was a significant increase (approximately threefold) in the number of TH-positive cells in the neural grafts for the TUDCA-treated group when compared with the controls 6 weeks postgrafting. The number of apoptotic cells was much smaller in the graft areas in the TUDCA-treated groups than in the control group 4 days after transplantation. These data demonstrate that pretreatment of the cell suspension with TUDCA can reduce apoptosis and increase the survival of grafted cells, resulting in an improvement of behavioral recovery.

Key words: Neural transplantation; Dopamine; Apoptosis; Parkinson's disease; Tauroursodeoxycholic acid

Address correspondence to Walter C. Low, Ph.D., Department of Neurosurgery, University of Minnesota Medical School, Minneapolis, MN 55455. Tel: (612) 626-9200; Fax: (612) 626-9201; E-mail: lowwalt@tc.umn.edu

Cell Transplantation, Vol. 11, pp. 207-214, 2002
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Striatal Xenotransplantation of Human Retinal Pigment Epithelial Cells Attached to Microcarriers in Hemiparkinsonian Rats Ameliorates Behavioral Deficits Without Provoking a Host Immune Response

Thyagarajan Subramanian,1 Deanna Marchionini,1 Elizabeth M. Potter,2 and Michael L. Cornfeldt3

1Emory University, Atlanta, GA 30322
2Midatlantic Bio-Research, Rockville, MD 28051
3Titan Pharmaceuticals, Inc., Somerville, NJ 08876

Attachment of donor cells to microcarriers has been reported to make them more tolerable for transplantation into the brain. Human retinal pigment epithelial (hRPE) cells have been previously reported to contain enzymes for the production of dopa. Therefore, we examined the host immune response and behavioral effects of xenotransplantation of hRPE cells attached to microcarriers (hRPE-M) into the striatum of unilateral dopamine-depleted rats. Thirty-four adult rats were lesioned with 6-OHDA injections into the medial forebrain bundle on the right side. After 5 weeks of testing for apomorphine-induced rotations (AIR), animals were randomized for right striatal surgery into the following four groups: hRPE-M (group 1), hRPE alone (group 2), microcarriers alone (group 3), or needle tract alone (group 4). Following surgery, animals were tested for AIR every 4 weeks for a period of 12-18 weeks and thereafter euthanized. There was a significant reduction in AIR scores posttransplantation in all groups of animals in the initial observation points at 4 weeks and 8 weeks. However, there was a gradual return to baseline scores in groups 2, 3, and 4 animals at 12 weeks and at 18 weeks only group 1 animals had statistically significant (p = 0.001, repeated measures ANOVA, means comparison, predetermined contrasts) reduction in AIR scores. Brain tissue from representative animals from each group was cut into 30-mm coronal sections, stained for cresyl violet, tyrosine hydroxylase (TH), and markers for host immune activation. Sections through the striatum from group 1 animals revealed microcarriers with attached cells resembling RPE cells. No evidence of transplanted hRPE cells could be detected in sections from group 2 animals while those from groups 3 and 4 animals showed microcarriers and a needle tract alone, respectively. There was no host TH-immunoreactive sprouting response in the striatum in any of the groups and the host immune response was minimal. These results suggest that intrastriatal hRPE-M xenotransplantation into rats is well tolerated without systemic immunosuppression and that such transplants may provide behavioral benefit for parkinsonism.

Key words: Parkinson's disease; Immunosuppression; Rodents; Brain; Cell culture

Address correspondence to Thyagarajan Subramanian, M.D., Department of Neurology/Mail code S90, Cleveland Clinic Foundation, Cleveland, OH 44195. Tel: (216) 444-4225; Fax: (216) 444-9401; E-mail: subarat@ccf.org

Cell Transplantation, Vol. 11, pp. 215-227, 2002
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Adeno-Associated Viral Delivery of GDNF Promotes Recovery of Dopaminergic Phenotype Following a Unilateral 6-Hydroxydopamine Lesion

John McGrath,1 Elishia Lintz,2 Barry J. Hoffer,3 Greg A. Gerhardt,4 E. Matthew Quintero,5 and Ann-Charlotte Granholm5

1Alkermes, Inc., 64 Sidney St., Cambridge, MA
2University of Colorado Health Science Center, Denver, CO
3Intramural Research Program National Institute on Drug Abuse, Baltimore, MD
4Departments of Anatomy & Neurobiology, and Neurology, and the Morris K. Udall Parkinson's Disease Research Center of Excellence, and the Center for Sensor Technology, University of Kentucky, Chandler Medical Center, Lexington, KY
5Department of Physiology and Neuroscience, Medical University of South Carolina, Charleston, SC

Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor for dopamine neurons that has been proposed for use in the treatment of Parkinson's disease (PD). Previous studies using viral vectors to deliver GDNF in rodent models of PD have entailed administering the virus either prior to or immediately after neurotoxin-induced lesions, when the nigrostriatal pathway is largely intact, a paradigm that does not accurately reflect the clinical situation encountered with Parkinson's patients. In this study, recombinant adeno-associated virus carrying the gene encoding GDNF (rAAV-GDNF) was administered to animals bearing a maximal lesion in the nigrostriatal system, more closely resembling fully developed PD. Rats were treated with 6-hydroxydopamine into the medial forebrain bundle and assessed by apomorphine-induced rotational behavior for 5 weeks prior to virus administration. Within 4 weeks of a single intrastriatal injection of rAAV-GDNF, unilaterally lesioned animals exhibited significant behavioral recovery, which correlated with increased expression of dopaminergic markers in the substantia nigra, the medial forebrain bundle, and the striatum. Our findings demonstrate that rAAV-GDNF is capable of rescuing adult dopaminergic neurons from near complete phenotypic loss following a neurotoxic lesion, effectively restoring a functional dopaminergic pathway and diminishing motoric deficits. These data provide further support for the therapeutic potential of rAAV-GDNF-based gene therapy in the treatment of PD.

Key words: Glial cell line-derived neurotrophic factor (GDNF); Parkinson's disease; Adeno-associated viral delivery

Address correspondence to Ann-Charlotte Granholm, Ph.D., Professor, Department of Physiology and Neuroscience, 173 Ashley Avenue, Medical University of South Carolina, Charleston, SC 29425. Tel: (843) 792-4527; E-mail: Granholm@musc.edu

Cell Transplantation, Vol. 11, pp. 229-239, 2002
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Upregulation of EphA Receptor expression in the injured adult rat spinal cord

Christopher A. Willson,1,2* Margarita Irizarry-Ramírez,5* Hope E. Gaskins,1,2 Lillian Cruz-Orengo,4 Johnny D. Figueroa,4 Scott R. Whittemore,1,2,3 and Jorge D. Miranda4

1Kentucky Spinal Cord Injury Research Center and Departments of 2Neurological Surgery and 3Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Louisville, KY 40202
4Departments of Physiology and 5Clinical Laboratory Science, University of Puerto Rico Medical Science Campus, San Juan, PR 00936

After spinal cord injury (SCI), the inability of supraspinal neurons to regenerate or reform functional connections is likely due to proteins in the surrounding microenvironment restricting regeneration. EphAs are a family of receptor tyrosine kinases that are involved in axonal guidance during development. These receptors and their ligands, the Ephrins, act via repulsive mechanisms to guide growing axons towards their appropriate targets and allow for the correct developmental connections to be made. In the present study, we investigated whether EphA receptor expression changed after a thoracic contusion SCI. Our results indicate that several EphA molecules are upregulated after SCI. Using semiquantitative RT-PCR to investigate mRNA expression after SCI, we found that EphA3, A4, and A7 mRNAs were upregulated. EphA3, A4, A6, and A8 receptor immunoreactivity increased in the ventrolateral white matter (VWM) at the injury epicenter. EphA7 had the highest level of immunoreactivity in both control and injured rat spinal cord. EphA receptor expression in the white matter originated from glial cells as coexpression in both astrocytes and oligodendrocytes was observed. In contrast, gray matter expression was localized to neurons of the ventral gray matter (motor neurons) and dorsal horn. After SCI, specific EphA receptor subtypes are upregulated and these increases may create an environment that is unfavorable for neurite outgrowth and functional regeneration.

Key words: Spinal cord injury; Receptor tyrosine kinase; Immunohistochemistry; RT-PCR; Trauma

*These two authors contributed equally to this study.

Address correspondence to Jorge D. Miranda, Ph.D., University of Puerto Rico, School of Medicine Physiology Department, PO Box 365067, San Juan, PR 00936-5067. Tel: (787) 758-2525, ext. 1631; Fax: (787) 753-0120; E-mail: jdmiranda@rcm.upr.edu

Cell Transplantation, Vol. 11, pp. 241-250, 2002
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Muscle Reinnervation With Delayed or Immediate Transplant of Embryonic Ventral Spinal Cord Cells Into Adult Rat Peripheral Nerve

Robert M. Grumbles,1 Patrick Wood,1,2 Michelle Rudinsky,1 Anna M. Gomez,1 and Christine K. Thomas1,2

1The Miami Project to Cure Paralysis, Department of Neurological Surgery and 2Department of Physiology and Biophysics, University of Miami School of Medicine, P.O. Box 016960, R-48, Miami, FL 33101

Muscle denervation is common in various neuromuscular diseases and after trauma. It induces skeletal muscle atrophy. Only muscle reinnervation leads to functional recovery. In previous studies, denervated adult rat muscles were rescued by transplantation of embryonic day 14-15 (E14-15) ventral spinal cord cells into a nearby peripheral nerve. In the present study, changes were made in the environment into which the cells were placed to test whether reinnervation was improved by: 1) prior nerve degeneration, induced by sciatic nerve transection 1 week before cell transplantation; 2) transplantation of 1 million versus 5 million cells; 3) addition of nerve growth factor (NGF) to the transplant. Ten weeks after cell transplantation, axons had grown from all of the transplants. The numbers of myelinated axons that regenerated into the tibial, medial (MG), and lateral gastrocnemius-soleus (LGS) nerves were similar across treatments. The mean diameters of large LGS axons (>6 mm) were significantly larger with nerve degeneration before transplantation. The mean diameters of MG and LGS axons were significantly larger with transplantation of 1 million versus 5 million cells. Silver-stained experimental and control lateral gastronemius (LG) muscles showed axons that terminated at motor end plates. Nodal and terminal sprouts were more common in reinnervated muscles (45-63% of all end plates) than in control muscles (10%). Electrical stimulation of the transplants induced weak contractions in 39 of 47 MG muscles (83%) and 33 of 46 LG muscles (72%) but at higher voltages than needed to excite control muscles. The threshold for MG contraction was lower with transplantation of 1 million cells, while LG thresholds were lower without NGF. The cross-sectional area of whole LG muscles was significantly larger with cell transplantation (immediate or delayed) than with media alone, but all of these muscle areas were reduced significantly compared with control muscle areas. These data suggest that delayed transplantation of fewer cells without NGF assists regeneration of larger diameter axons and prevents some muscle atrophy.

Key words: Muscle denervation; Cell transplantation; Motor axons; Motor units; Neuromuscular junctions

Address correspondence to Christine K. Thomas, Ph.D., The Miami Project to Cure Paralysis, University of Miami School of Medicine, PO Box 016960 (R-48), Miami, FL 33101-9844. Tel: (305) 243-7109; Fax: (305) 243-3913; E-mail: cthomas@miamiproject.med.miami.edu

Cell Transplantation, Vol. 11, pp. 251-259, 2002
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T155g-Immortalized Kidney Cells Produce Growth Factors and Reduce Sequelae of Cerebral Ischemia

Ora Dillon-Carter,* Rowena E. Johnston,* Cesario V. Borlongan,* Mary Ellen Truckenmiller, Mark Coggiano, and William J. Freed

Cellular Neurobiology Branch, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, 5500 Nathan Shock Drive, Baltimore, MD 21224

Fetal rat kidney cells produce high levels of glial-derived neurotrophic factor (GDNF) and exert neuroprotective effects when transplanted into the brain in animal models of Parkinson's disease and stroke. The purpose of the present experiment was to produce kidney cell lines that secrete GDNF. Genes encoding two truncated N-terminal fragments of SV40 large T antigen, T155g and T155c, which does not code for small t antigen, were used. T155g was transduced into E17 cultured fetal Sprague-Dawley rat kidney cortex cells using a plasmid vector, and T155c was transduced with a plasmid and a retroviral vector. Sixteen clones were isolated from cultures transfected with the T155g-expressing plasmid. No cell lines were obtained with T155c. Four clones produced GDNF at physiological concentrations ranging from 55 to 93 pg/ml of medium. These four clones were transplanted into the ischemic core or penumbra of rats that had undergone middle cerebral artery occlusion (MCAO). Three of the four clones reduced the volume of infarction and the behavioral abnormalities normally resulting from MCAO. Blocking experiments with antibodies to GDNF and platelet-derived growth factor (PDGF) suggested that these growth factors contributed only minimally to the reduction in infarct volume and behavioral abnormality. These cell lines may be useful for intracerebral transplantation in animal models of brain injury, stroke, or Parkinson's disease.

Key words: Cell line; Transplantation; Stroke; Neurotrophic factors; Behavioral recovery

*These authors contributed equally to this study.

Address correspondence to Cesario V. Borlongan, Ph.D., at his present address: Department of Neurology, Medical College of Georgia, 1120 15th Street, Augusta, GA 30912. Tel: (706) 733-0188, Ext. 2484; Fax: (706) 721-7619; E-mail: cborlong@neuro.mcg.edu

Cell Transplantation, Vol. 11, pp. 261-264, 2002
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Human Umbilical Cord Blood Cells Can Be Induced to Express Markers for Neurons and Glia

Alma R. Bicknese,123 Holly S. Goodwin,3 Cheryl O. Quinn,23 Verneake C. D. Henderson,1 Shin-Nan Chien,3 and Donna A. Wall23

Departments of 1Neurology and 2Pediatrics of Saint Louis University, and 3The Pediatric Research Institute, Cardinal Glennon Children's Hospital, St. Louis, MO 63110

Rare cells are present in human umbilical cord blood that do not express the hematopoietic marker CD45 and in culture do not produce cells of hematopoietic lineage. These umbilical cord multipotent stem cells (UC-MC) behave as multilineage progenitor cells (stem cells) and can be expanded in tissue culture. Exposure to basic fibroblast growth factor (bFGF) and human epidermal growth factor (hEGF) for a minimum of 7 days in culture induces expression of neural and glial markers. Western immunoblots demonstrate expression of both \GK\b-tubulin III and glial fibrillary acidic protein (GFAP). Immunocytochemistry of the cells showed intense labeling to both compounds on the intracellular cytoskeleton. The oligodendrocyte cell surface marker galactocerebroside (Gal-C) was present on most cells. Many cells show dual labeling, expressing both neuronal and glial markers.

Key words: Stem cell; Umbilical cord blood; Neuron; Astrocyte; Glia; Lineage

Address correspondence to Alma R. Bicknese, M.D., Department of Neurology, Saint Louis University, 3635 Vista Avenue, St. Louis, MO 63110. Tel: (314) 577-5338; Fax: (314) 268-6411; E-mail: bicknese@slu.edu

Cell Transplantation, Vol. 11, pp. 265-274, 2002
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Human Umbilical Cord Blood Cells Express Neural Antigens After Transplantation Into the Developing Rat Brain

Tanja Zigova,1,2,4 Shijie Song,1,3,5 Alison E. Willing,1,2,4 Jennifer E. Hudson,1,3 Mary B. Newman,1,2 Samuel Saporta,1,4 Juan Sanchez-Ramos,1,2,5 and Paul R. Sanberg1,2,3

1Center for Aging and Brain Repair, Departments of 2Neurosurgery, 3Neurology, and 4Anatomy, University of South Florida College of Medicine, Tampa, FL
5James Haley VA Hospital, Tampa, FL

Recently, our laboratory began to characterize the mononuclear cells from human umbilical cord blood (HUCB) both in vitro and in vivo. These cryopreserved human cells are available in unlimited quantities and it is believed that they may represent a source of cells with possible therapeutic and practical value. Our previous molecular and immunocytochemical studies on cultured HUCB cells revealed their ability to respond to nerve growth factor (NGF) by increased expression of neural markers typical for nervous system-derived stem cells. In addition, the DNA microarray detected downregulation of several genes associated with development of blood cell lines. To further explore the survival and phenotypic properties of HUCB cells we transplanted them into the developing rat brain, which is known to provide a conducive environment for development of neural phenotypes. Prior to transplantation, HUCB cells were either cultured with DMEM and fetal bovine serum or were exposed to retinoic acid (RA) and nerve growth factor (NGF). Neonatal pups (1 day old) received unilateral injection of cell suspension into the anterior part of subventricular zone. One month after transplantation animals were perfused, their brains cryosectioned, and immunocytochemistry was performed for identification of neural phenotypes. Our results clearly demonstrated that approximately 20% of transplanted HUCB survived (without immunosuppression) within the neonatal brain. Additionally, double-labeling with cell-type-specific markers revealed that some HUCB-derived cells (recognized by anti-human nuclei labeling) were immunopositive for glial fibrillary acidic protein (GFAP) and few donor cells expressed the neuronal marker TuJ1 (class III b-tubulin). These findings suggest that at least some of the transplanted HUCB cells differentiated into cells with distinct glial or neuronal phenotypes after being exposed to instructive signals from the developing brain.

Key words: Human umbilical cord blood; Transplantation; Neonatal rat; Subventricular zone; Neural markers

Address correspondence to Tanja Zigova, Ph.D., Department of Neurosurgery, College of Medicine, University of South Florida, 12901 B.B. Downs Blvd., Tampa, FL 33612. Tel: (813) 974-5724; Fax: (813) 974-3078; E-mail: tzigova@hsc.usf.edu

Cell Transplantation, Vol. 11, pp. 275-281, 2002
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Intravenous Administration of Human Umbilical Cord Blood Reduces Neurological Deficit in the Rat After Traumatic Brain Injury

Dunyue Lu,1 Paul R. Sanberg,4 Asim Mahmood,1 Yi Li,2 Lei Wang,2 Juan Sanchez-Ramos,5 and Michael Chopp2,3

Departments of 1Neurosurgery and 2Neurology, Henry Ford Health Sciences Center, Detroit, MI
3Department of Physics, Oakland University, Rochester, MI
Center for Aging and Repair, Departments of 4Neurosurgery and 5Neurology, University of South Florida, Tampa, FL

We measured the effect of treatment of traumatic brain injury (TBI) in the rat with human umbilical cord blood (HUCB) administered IV. HUCB cells were injected into the tail vein 24 h after TBI and the rats were sacrificed at day 28 after the treatment. The Rotarod test and the neurological severity score (NSS) were used to evaluate neurological function. The distribution of the donor cells in the brain, heart, lung, kidney, liver, spleen, bone marrow, and muscle were analyzed in recipient rats using immunohistochemical staining and laser confocal microscopy. HUCB cells injected IV significantly reduced motor and neurological deficits compared with control groups by day 28 after the treatment. The cells preferentially entered the brain and migrated into the parenchyma of the injured brain and expressed the neuronal markers, NeuN and MAP-2, and the astrocytic marker, GFAP. Some HUCB cells integrated into the vascular walls within the boundary zone of the injured area. Our data suggest that IV administration of HUCB may be useful in the treatment of TBI.

Key words: Traumatic brain injury (TBI); Rat; Human umbilical cord blood (HUCB); Intravenous administration

Address correspondence to Michael Chopp, Ph.D., Henry Ford Hospital, Neurology Department, 2799 West Grand Boulevard, Detroit, MI 48202. Tel: (313) 916-3936; Fax: (313) 916-1318; E-mail: chopp@neuro.hfh.edu

Cell Transplantation, Vol. 11, pp. 283-295, 2002
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Fibronectin Promotes Survival and Migration of Primary Neural Stem Cells Transplanted Into the Traumatically Injured Mouse Brain

Matthew C. Tate,1 Deborah A. Shear,3 Stuart W. Hoffman,4 Donald G. Stein,2,4 David R. Archer,5 and Michelle C. LaPlaca1

1Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA 30332
Departments of 2Neurology, 3Psychology, 4Emergency Medicine, and 5Pediatrics, Emory University, Atlanta, GA 30322

Multipotential stem cells are an attractive choice for cell therapy after traumatic brain injury (TBI), as replacement of multiple cell types may be required for functional recovery. In the present study, neural stem cells (NSCs) derived from the germinal zone of E14.5 GFP-expressing mouse brains were cultured as neurospheres in FGF2-enhanced medium. When FGF2 was removed in vitro, NSCs expressed phenotypic markers for neurons, astrocytes, and oligodendrocytes and exhibited migratory behavior in the presence of adsorbed fibronectin (FN). NSCs (105 cells) were transplanted into mouse brains 1 week after a unilateral, controlled, cortical contusion (depth = 1 mm, velocity = 6 m/s, duration = 150 ms) (n = 19). NSCs were injected either directly into the injury cavity with or without an injectable FN-based scaffold [collagen I (CnI)/FN gel; n = 14] or into the striatum below the injury cavity (n = 5). At all time points examined (1 week to 3 months posttransplant), GFP+ cells were confined to the ipsilateral host brain tissue. At 1 week, cells injected into the injury cavity lined the injury penumbra while cells inserted directly into the striatum remained in or around the needle track. Striatal transplants had a lower number of surviving GFP+ cells relative to cavity injections at the 1 week time point (p < 0.01). At the longer survival times (3 weeks-3 months), 63-76% of transplanted cells migrated into the fimbria hippocampus regardless of injection site, perhaps due to cues from the degenerating hippocampus. Furthermore, cells injected into the cavity within a FN-containing matrix showed increased survival and migration at 3 weeks (p < 0.05 for both) relative to injections of cells alone. These results suggest that FGF2-responsive NSCs present a promising approach for cellular therapy following trauma and that the transplant location and environment may play an important role in graft survival and integration.

Key words: Traumatic brain injury; Tissue engineering; Fibronectin; Extracellular matrix; Neural progenitor cells; Stem cells

Address correspondence to Michelle C. LaPlaca, Ph.D., Coulter Department of Biomedical Engineering, 315 Ferst Dr., Atlanta, GA 30332-0535. Tel: (404) 385-0629; Fax: (404) 894-2295; E-mail: michelle.laplaca@bme.gatech.edu

Cell Transplantation, Vol. 11, pp. 297-307, 2002
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TrkC Overexpression Enhances Survival and Migration of Neural Stem Cell Transplants in the Rat Spinal Cord

Daniel A. Castellanos, Pantelis Tsoulfas, Beata R. Frydel, Shyam Gajavelli, Jean-Claude Bes, and Jacqueline Sagen

The Miami Project to Cure Paralysis, University of Miami School of Medicine, Miami, FL 33136

Although CNS axons have the capacity to regenerate after spinal cord injury when provided with a permissive substrate, the lack of appropriate synaptic target sites for regenerating fibers may limit restoration of spinal circuitry. Studies in our laboratory are focused on utilizing neural stem cells to provide new synaptic target sites for regenerating spinal axons following injury. As an initial step, rat neural precursor cells genetically engineered to overexpress the tyrosine kinase C (trkC) neurotrophin receptor were transplanted into the intact rat spinal cord to evaluate their survival and differentiation. Cells were either pretreated in vitro prior to transplantation with trkC ligand neurotrophin-3 (NT-3) to initiate differentiation or exposed to NT-3 in vivo following transplantation via gelfoam or Oxycel©. Both treatments enhanced survival of trkC-overexpressing stem cells to nearly 100%, in comparison with approximately 30-50% when either NT-3 or trkC was omitted. In addition, increased migration of trkC-overexpressing cells throughout the spinal gray matter was noted, particularly following in vivo NT-3 exposure. The combined trkC expression and NT-3 treatment appeared to reduce astrocytic differentiation of transplanted neural precursors. Decreased cavitation and increased b-tubulin fibers were noted in the vicinity of transplanted cells, although the majority of transplanted cells appeared to remain in an undifferentiated state. These findings suggest that genetically engineered neural stem cells in combination with neurotrophin treatment may be a useful addition to strategies for repair of spinal neurocircuitry following injury.

Key words: Neural transplantation; Neural precursor cells; Spinal cord; Stem cells; Neurotrophin-3

Address correspondence to Jacqueline Sagen, Ph.D., The Miami Project to Cure Paralysis, University of Miami School of Medicine, Lois Pope LIFE Center, 1095 N.W. 14th Terrace (R-48), Miami, FL 33136. Tel: (305) 243-5618; Fax: (305) 243-3923; E-mail: jsagen@miami.edu