ognizant Communication Corporation

The Regenerative Medicine Journal

VOLUME 14, NUMBER 4, 2005

Cell Transplantation, Vol. 14, pp. 173-182, 2005
0963-6897/05 $20.00 + 00
Copyright © 2005 Cognizant Comm. Corp.
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Magnetic Resonance Tracking of Human CD34+ Progenitor Cells Separated by Means of Immunomagnetic Selection and Transplanted Into Injured Rat Brain

Pavla Jendelová,1,2,4 Vít Herynek,2,3 Lucia Urdziková,1 Katerina Glogarová,1,2 Sárka Rahmatová,7 Ivan Fales,7 Benita Andersson,1 Pavel Procházka,5 Josef Zamecník,6 Tomás Eckschlager,5 Petr Kobylka,2,7 Milan Hájek,2,3 and Eva Syková1,2,4

1Institute of Experimental Medicine ASCR, Prague, Czech Republic
2Center for Cell Therapy and Tissue Repair, Charles University, Prague, Czech Republic
3MR-Unit, Department of Diagnostic and Interventional Radiology, Institute for Clinical and Experimental Medicine, Prague, Czech Republic
4Department of Neuroscience, Charles University, Second Medical Faculty, Prague, Czech Republic
5Department of Paediatric Haematology and Oncology and 6Department of Pathology and Molecular Medicine, Charles University, Second Medical Faculty, Prague, Czech Republic
7Institute of Hematology and Blood Transfusion, Prague, Czech Republic

Magnetic resonance imaging (MRI) provides a noninvasive method for studying the fate of transplanted cells in vivo. We studied whether superparamagnetic nanoparticles (CD34 microbeads), used clinically for specific magnetic sorting, can be used as a magnetic cell label for in vivo cell visualization. Human cells from peripheral blood were selected by CliniMACS® CD34 Selection Technology (Miltenyi). Purified CD34+ cells were implanted into rats with a cortical photochemical lesion, contralaterally to the lesion. Twenty-four hours after grafting, the implanted cells were detected in the contralateral hemisphere as a hypointense spot on T2 weighted images; the hypointensity of the implant decreased during the first week. At the lesion site we observed a hypointensive signal 10 days after grafting that persisted for the next 3 weeks, until the end of the experiment. Prussian blue and anti-human nuclei staining confirmed the presence of magnetically labeled human cells in the corpus callosum and in the lesion 4 weeks after grafting. CD34+ cells were also found in the subventricular zone (SVZ). Human DNA (a human-specific 850 base pair fragment of a-satellite DNA from human chromosome 17) was detected in brain tissue sections from the lesion using PCR, confirming the presence of human cells. Our results show that CD34 microbeads superparamagnetic nanoparticles can be used as a magnetic cell label for in vivo cell visualization. The fact that microbeads coated with different commercially available antibodies can bind to specific cell types opens extensive possibilities for cell tracking in vivo.

Key words: Cell transplantation; Magnetic resonance; Contrast agents; Hematopoietic stem cells; Photochemical lesion; Migration

Address correspondence to Pavla Jendelová, Ph.D., Institute of Experimental Medicine ASCR, Víde?ská 1083, 142 20 Prague 4, Czech Republic. Tel: +420-224436781; Fax: + 420-224436799; E-mail: pavla.jendelova@lfmotol.cuni.cz

Cell Transplantation, Vol. 14, pp. 183-192, 2005
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Neural Stem Cells Implanted Into MPTP-Treated Monkeys Increase the Size of Endogenous Tyrosine Hydroxylase-Positive Cells Found in the Striatum: A Return to Control Measures

Kimberly B. Bjugstad,1 D. Eugene Redmond, Jr.,2,3 Yang D. Teng,4,5 J. D. Elsworth,3 R. H. Roth,3 B. C. Blanchard,1 Evan Y. Snyder,4,6 and John R. Sladek, Jr.1

1Department of Psychiatry, University of Colorado Health Sciences Center, Denver, CO
Departments of 2Neurosurgery and 3Psychiatry, Yale Medical School, New Haven, CT
Departments of 4Neurology and 5Neurosurgery, Harvard Medical School, Boston, MA
6Burnham Institute, La Jolla, CA

Neural stem cells (NSC) have been shown to migrate towards damaged areas, produce trophic factors, and replace lost cells in ways that might be therapeutic for Parkinson's disease (PD). However, there is very little information on the effects of NSC on endogenous cell populations. In the current study, effects of implanted human NSC (hNSC) on endogenous tyrosine hydroxylase-positive cells (TH+ cells) after treatment with 1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine (MPTP) were explored in nonhuman primates. After MPTP damage and in PD, the primate brain is characterized by decreased numbers of dopamine neurons in the substantia nigra (SN) and an increase in neurons expressing TH in the caudate nucleus. To determine how implanted NSC might affect these cell populations, 11 St. Kitts African green monkeys were treated with the selective dopaminergic neurotoxin, MPTP. Human NSC were implanted into the left and right caudate nucleus and the right SN of eight of the MPTP-treated monkeys. At either 4 or 7 months after NSC implants, the brains were removed and the size and number of TH+ cells in the target areas were assessed. The results were compared to data obtained from normal untreated control monkeys and to the three unimplanted MPTP-treated monkeys. The majority of hNSC were found bilaterally along the nigrostriatal pathway and in the substantia nigra, while relatively few were found in the caudate. In the presence of NSC, the number and size of caudate TH+ cells returned to non-MPTP-treated control levels. MPTP-induced and hNSC-induced changes in the putamen were less apparent. We conclude that after MPTP treatment in the primate, hNSC prevent the MPTP-induced upregulation of TH+ cells in the caudate and putamen, indicating that hNSC may be beneficial to maintaining a normal striatal environment.

Key words: Neural stem cells; Parkinson's disease; MPTP; Dopamine; Substantia nigra; Caudate

Address correspondence to Kimberly B. Bjugstad, Department of Psychiatry, Mail Stop F8342, UCHSC at Fitzsimmons, P.O. Box 6511, Aurora, CO 80045. Tel: (303) 724-3041; Fax: (303) 724-3049; E-mail: kimberly.bjugstad@uchsc.edu

Cell Transplantation, Vol. 14, pp. 193-202, 2005
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Brain Transplantation of Neural Stem Cells Cotransduced With Tyrosine Hydroxylase and GTP Cyclohydrolase 1 in Parkinsonian Rats

M. Y. Ryu,1,2 M. A. Lee,2 Y. H. Ahn,1 K. S. Kim,2 S. H. Yoon,1 E. Y. Snyder,3 K. G. Cho,1,2 and S. U. Kim2,4

1Department of Neurosurgery and 2Brain Disease Research Center, Ajou University School of Medicine, Suwon, Korea
3Burnham Institute, La Jolla, CA
4Division of Neurology, Department of Medicine, University of British Columbia, Vancouver, BC, Canada

Neural stem cells (NSCs) of the central nervous system (CNS) recently have attracted a great deal of interest not only because of their importance in basic research on neural development, but also in terms of their therapeutic potential in neurological diseases, such as Parkinson's disease (PD). To examine if genetically modified NSCs are a suitable source for the cell and gene therapy of PD, an immortalized mouse NSC line, C17.2, was transduced with tyrosine hydroxylase (TH) gene and with GTP cyclohydrolase 1 (GTPCH1) gene, which are important enzymes in dopamine biosynthesis. The expression of TH in transduced C17.2-THGC cells was confirmed by RT-PCR, Western blot analysis, and immunocytochemistry, and expression of GTPCH1 by RT-PCR. The level of L-DOPA released by C17.2-THGC cells, as determined by HPLC assay, was 3793 pmol/106 cells, which is 760-fold higher than that produced by C17.2-TH cells, indicating that GTPCH1 expression is important for L-DOPA production by transduced C17.2 cells. Following the implantation of C17.2-THGcC NSCs into the striata of parkinsonian rats, a marked improvement in amphetamine-induced turning behavior was observed in parkinsonian rats grafted with C17.2-THGC cells but not in the control rats grafted with C17.2 cells. These results indicate that genetically modified NSCs grafted into the brain of the parkinsonian rats are capable of survival, migration, and neuronal differentiation. Collectively, these results suggest that NSCs have great potential as a source of cells for cell therapy and an effective vehicle for therapeutic gene transfer in Parkinson's disease.

Key words: Neural stem cell; C17.2 cell line; Parkinson's disease; Tyrosine hydroxylase; GTP cyclohydrolase 1; L-DOPA; Brain transplantation

Address correspondence to Seung U. Kim, M.D., Ph.D., Division of Neurology, Department of Medicine, University of British Columbia, Vancouver, BC V6T 2B5, Canada. Tel: 604-822-7145; Fax: 604-822-7897; E-mail: sukim@interchange.ubc.ca or to Kyung G. Cho, M.D., Ph.D., Department of Neurosurgery, Ajou University School of Medicine, Suwon 442-721, Korea. Tel: 82-31-219-5661; Fax: 82-31-216-6658; E-mail: sandori@ajou.ac.kr

Cell Transplantation, Vol. 14, pp. 203-211, 2005
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NMDA Antagonist Peptide Supplementation Enhances Pain Alleviation by Adrenal Medullary Transplants

Farinaz NasiriNezhad* and Jacqueline Sagen

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

Spinal transplantation of adrenal medullary chromaffin cells has been shown to decrease pain responses in several animal models. Improved potency may be possible by engineering cells to produce greater levels of naturally derived analgesics. As an initial screen for potential candidates, adrenal medullary transplants were evaluated in combination with exogenously administered neuropeptides in rodent pain models. Histogranin is a 15-amino acid peptide that exhibits NMDA receptor antagonist activity. The stable derivative [Ser1]histogranin (SHG) can attenuate pain symptoms in some animal models. The formalin model for neurogenic inflammatory pain and the chronic constriction injury (CCI) model for neuropathic pain were used to evaluate the combined effects of chromaffin cell transplantation and intrathecal (IT) SHG injections. Animals were implanted with either adrenal medullary or control striated muscle tissue in the spinal subarachnoid space. For evaluation of formalin responses, animals were pretreated with SHG (0.5, 1.0, 3.0 mg) followed by an intraplantar injection of formalin, and flinching responses were quantified. Pretreatment with SHG had no significant effect on flinching behavior in control animals at lower doses, with incomplete attenuation only at the highest dose. In contrast, 0.5 mg SHG significantly reduced flinching responses in animals with adrenal medullary transplants, and 1.0 mg nearly completely eliminated flinching in these animals in the tonic phase. For evaluation of effects on neuropathic pain, animals received transplants 1 week following CCI, and were tested for thermal and mechanical hyperalgesia and cold allodynia before and following SHG treatment. The addition of low doses of SHG nearly completely eliminated neuropathic pain symptoms in adrenal medullary transplanted animals, while in control transplanted animals only thermal hyperalgesia was attenuated, at the highest dose of SHG. These results suggest that SHG can augment adrenal medullary transplants, and the combination may result in improved effectiveness and range in the treatment of chronic pain syndromes.

Key words: Histogranin; Spinal cord; Neuropathic pain; Inflammatory pain; Formalin test

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

*Current address: Department of Physiology. Basic Science Center, Medical School, Iran University of Medical Sciences, Tehran, Iran

Cell Transplantation, Vol. 14, pp. 213-223, 2005
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Blueberry Extract Enhances Survival of Intraocular Hippocampal Transplants

Lauren Willis,1 Paula Bickford,2 Vandana Zaman,1 Alfred Moore,1 and Ann-Charlotte Granholm1

1Department of Neurosciences and the Center on Aging, Medical University of South Carolina, Charleston, SC
2Department of Neurosurgery, University of South Florida, Tampa, FL

Transplantation of neural tissue has been explored as a potential therapy to replace dead or dying cells in the brain, such as after brain injury or neurodegenerative disease. However, survival of transplanted tissue is poor, especially when the transplant recipient is of advanced age. Recent studies have demonstrated improvement of neuronal deficits in aged animals given a diet supplemented with blueberry extract. The present study focuses on the survival of fetal hippocampal transplants to young (4 months) or middle-aged (16 months) animals with or without dietary supplementation with blueberry extract. Results indicate that fetal hippocampus transplanted to middle-aged host animals exhibits poor survival characterized by reduced growth and compromised tissue organization. However, when middle-aged animals were maintained on a diet supplemented with 2% blueberry extract, hippocampal graft growth was significantly improved and cellular organization of grafts was comparable to that seen in tissue grafted to young host animals. Thus, the data suggest that factor(s) in blueberries may have significant effects on development and organization of this important brain region.

Key words: Hippocampal formation; Transplantation; Intraocular; Neuronal survival; Apoptosis; Organotypic development

Address correspondence to Lauren Willis, Department of Neurosciences, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC 29425. Tel: (843) 792-8872; E-mail: willisl@musc.edu

Cell Transplantation, Vol. 14, pp. 225-240, 2005
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Survival, Integration, and Axon Growth Support of Glia Transplanted Into the Chronically Contused Spinal Cord

D. J. Barakat,1 S. M. Gaglani,1 S. R. Neravetla,1 A. R. Sanchez,1 C. M. Andrade,1 Y. Pressman,1 R. Puzis,1 M. S. Garg,1 M. B. Bunge,1,2,3 and D. D. Pearse1,2

1The Miami Project to Cure Paralysis, Departments of 2Neurological Surgery, and 3Cell Biology and Anatomy, University of Miami Miller School of Medicine, Miami, FL 33136

Due to an ever-growing population of individuals with chronic spinal cord injury, there is a need for experimental models to translate efficacious regenerative and reparative acute therapies to chronic injury application. The present study assessed the ability of fluid grafts of either Schwann cells (SCs) or olfactory ensheathing glia (OEG) to facilitate the growth of supraspinal and afferent axons and promote restitution of hind limb function after transplantation into a 2-month-old, moderate, thoracic (T8) contusion in the rat. The use of cultured glial cells, transduced with lentiviral vectors encoding enhanced green fluorescent protein (EGFP), permitted long-term tracking of the cells following spinal cord transplantation to examine their survival, migration, and axonal association. At 3 months following grafting of 2 million SCs or OEG in 6 ml of DMEM/F12 medium into the injury site, stereological quantification of the three-dimensional reconstructed spinal cords revealed that an average of 17.1 ± 6.8% of the SCs and 2.3 ± 1.4% of the OEG survived from the number transplanted. In the OEG grafted spinal cord, a limited number of glia were unable to prevent central cavitation and were found in patches around the cavity rim. The transplanted SCs, however, formed a substantive graft within the injury site capable of supporting the ingrowth of numerous, densely packed neurofilament-positive axons. The SC grafts were able to support growth of both ascending calcitonin gene-related peptide (CGRP)-positive and supraspinal serotonergic axons and, although no biotinylated dextran amine (BDA)-traced corticospinal axons were present within the center of the grafts, the SC transplants significantly increased corticospinal axon numbers immediately rostral to the injury-graft site compared with injury-only controls. Moreover, SC grafted animals demonstrated modest, though significant, improvements in open field locomotion and exhibited less foot position errors (base of support and foot rotation). Whereas these results demonstrate that SC grafts survive, support axon growth, and can improve functional outcome after chronic contusive spinal cord injury, further development of OEG grafting procedures in this model and putative combination strategies with SC grafts need to be further explored to produce substantial improvements in axon growth and function.

Key words: Schwann cells; Olfactory ensheathing glia; Regeneration; Sensory; Spinal cord injury; Chronic; Contusion

Address correspondence to Dr. Damien Daniel Pearse, The Miami Project to Cure Paralysis, University of Miami School of Medicine, The Lois Pope Life Center, Locator code R-48, PO BOX 016960, Miami, FL 33101. Tel: (305) 243-7139; Fax: (305) 243-3923; E-mail: DPearse@miamiproject.med.miami.edu