ognizant Communication Corporation


VOLUME 12, NUMBER 3, 2003

Cell Transplantation, Vol. 12, pp. 201-213, 2003
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Neural Differentiation and Incorporation of Bone Marrow-Derived Multipotent Adult Progenitor Cells After Single Cell Transplantation Into Blastocyst Stage Mouse Embryos

C. Dirk Keene,1,2 Xilma R. Ortiz-Gonzalez,1,2 Yuehua Jiang,3 David A. Largaespada,4 Catherine M. Verfaillie,3 and Walter C. Low1,2

1Department of Neurosurgery, 2Graduate Program in Neuroscience, 3Stem Cell Institute, Division of Hematology, Oncology and Transplantation, Department of Medicine, and 4Department of Genetics, Cell Biology and Development, University of Minnesota Medical School, Minneapolis, MN 55455

Previously we reported the characterization of multipotent adult progenitor cells (MAPCs) isolated from the bone marrow of rodents. In that study, single murine MAPCs derived from ROSA-26, b-galactosidase (b-Gal)-positive transgenic mice were injected into E3.5 C57/Bl6 mouse blastocysts. The resultant chimeric blastocysts were then implanted into pseudopregnant females and were allowed to develop naturally through birth and into adulthood. Chimeric mice were sacrificed 6 to 20 weeks after birth, and were processed for histological analysis. b-Galactosidase activity was identified in all organs and tissues examined, and tissue-specific differentiation and engraftment was confirmed by colabeling with antibodies that recognize b-Gal and tissue-specific markers. In the present study we have examined neural engraftment derived from the clonal expansion of a single MAPC during rodent development, and characterized the neural phenotype of MAPCs in the resultant chimeric animals. Donor cell-derived b-Gal activity was evident throughout the brain. Double and triple immunofluorescent labeling studies revealed MAPC-derived neurons (NeuN/b-Gal) and astrocytes (GFAP/b-Gal) in the cortex, striatum, medial septal nucleus, hippocampus, cerebellum, substantia nigra, and thalamus. More specifically, donor-derived neurons contributed to each of the cellular layers of the cortex; the pyramidal and granule cell layers, as well as the hilus, of the hippocampus; Purkinje and granule cell layers in the cerebellum; and GABAergic cells in the caudate and putamen. This study characterizes the potential for MAPCs to differentiate into specific neuronal and glial phenotypes, and to integrate normally during development, after implantation into blastocysts, and provides additional evidence that MAPCs exhibit properties similar to embryonic stem cells.

Key words: Stem cell; Blastocyst transplantation; Plasticity; Neural differentiation; b-Galactosidase; ROSA-26

Address correspondence to Walter C. Low, Ph.D., Department of Neurosurgery, University of Minnesota Medical School, 2001 6th Street SE, Minneapolis, MN 55455. Tel: (612) 626-9200; Fax: (612) 626-9201; E-mail: lowwalt@umn.edu

Cell Transplantation, Vol. 12, pp. 215-223, 2003
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The Effects of Various Concentrations of FGF-2 on the Proliferation and Neuronal Yield of Murine Embryonic Neural Precursor Cells In Vitro

Claire M. Kelly, Rike Zietlow, Steven B. Dunnett, and Anne E. Rosser

Brain Repair Group, School of Biosciences, Cardiff University, Museum Avenue, Cardiff CF10 3US, UK

Embryonic neural precursors (ENPs), also termed neural stem cells or "neurospheres," are an attractive potential source of tissue for neural transplantation, because of their capacity to expand in number in vitro while retaining the ability to develop into the major phenotypes of the CNS. ENPs are isolated from the developing brain and proliferate in the presence of mitogens such as FGF-2 and EGF. Subsequent withdrawal of these mitogens and exposure to a suitable substrate results in differentiation into the major cell types of the CNS. As well as its role in precursor cell expansion, FGF-2 also plays a key role in the division of astrocytes, and in neuronal differentiation. Thus, it is important to establish the optimal concentrations of this factor for expansion and differentiation of neuronal phenotypes. Here we explore the effect of FGF-2 concentrations ranging from 1 to 20 ng/ml on the expansion and differentiation capacity of ENPs isolated from the cortex and striatum of E14 mice. ENP expansion was seen under all conditions, but was greatest at 10 and 20 ng/ml and least at 1 ng/ml. The numbers of neurons (as a proportion of total cell number) differentiating from these ENP populations appeared to be greatest at 1 ng/ml. However, once adjustments were made for the amount of expansion at each dose, final neuronal yield was maximum at the highest concentration of FGF-2 used (20 ng/ml).

Key words: Neural precursors; Neural stem cells; Rodent; FGF-2

Address correspondence to Anne E. Rosser, Brain Repair Group, School of Biosciences, Cardiff University, Museum Avenue, Cardiff CF10 3US, UK. Tel: +44 29 20876654; E-mail: RosserAE@cf.ac.uk

Cell Transplantation, Vol. 12, pp. 225-234, 2003
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Melatonin-Secreting Pineal Gland: A Novel Tissue Source for Neural Transplantation Therapy in Stroke

C. V. Borlongan,1,2 I. Sumaya,3 D. Moss,3 M. Kumazaki,4 T. Sakurai,4 H. Hida,4 and H. Nishino4

1Neurology/Institute of Molecular Medicine & Genetics/School of Graduate Studies, Medical College of Georgia, Augusta, GA
2Research and Affiliations Service Line, Augusta VAMC, Augusta, GA
3Psychology, University of Texas, El Paso, TX
4Physiology, Nagoya City University Medical School, Nagoya, Japan

Chronic systemic melatonin treatment attenuates abnormalities produced by occlusion of middle cerebral artery (MCA) in adult rats. Because the pineal gland secretes high levels of melatonin, we examined in the present study whether transplantation of pineal gland exerted similar protective effects in MCA-occluded adult rats. Animals underwent same-day MCA occlusion and either intrastriatal transplantation of pineal gland (harvested from 2-month-old rats) or vehicle infusion. Behavioral tests (from day of surgery to 3 days posttransplantation) revealed that transplanted stroke rats displayed significantly less motor asymmetrical behaviors than vehicle-infused stroke rats. Histological analysis at 3 days posttransplantation revealed that transplanted stroke rats had significantly smaller cerebral infarction than vehicle-infused rats. Additional experiments showed that pinealectomy affected transplantation outcome, in that transplantation of pineal gland only protected against stroke-induced deficits in stroke animals with intact pineal gland, but not in pinealectomized stroke rats. Interestingly, nonpinealectomized vehicle-infused stroke rats, as well as pinealectomized transplanted stroke rats, had significantly lower melatonin levels in the cerebrospinal fluid than nonpinealectomized transplanted stroke rats. We conclude that intracerebral transplantation of pineal gland, in the presence of host intact pineal gland, protected against stroke, possibly through secretion of melatonin.

Key words: Free radical scavenger; Neuroprotection; Cerebral ischemia; Oxidative stress; Cerebrospinal fluid; Cognitive performance; Locomotor behavior

Address correspondence to C. V. Borlongan, Department of Neurology, BI-3080, Medical College of Georgia, 1120 15th Street, Augusta, GA 30912-3200. Tel: (706) 733-0188, Ext. 2485; Fax: (706) 721-7619; E-mail: cborlong@neuro.mcg.edu

Cell Transplantation, Vol. 12, pp. 235-241, 2003
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Ultrastructural Characterization of Dissociated Embryonic Ventral Mesencephalic Tissue Treated With Neuroprotectants

Young Hwan Ahn,* Mia Emgård,** and Patrik Brundin

Section for Neuronal Survival, Wallenberg Neuroscience Center, BMC A10, SE-221 84 Lund, Sweden

Poor survival and differentiation of grafted dopamine neurons limits the application of clinical transplantation in Parkinson's disease. The survival of grafted dopamine neurons is only improved by a factor of 2-3 by adding neuroprotectants during tissue preparation. We used dye exclusion cell viability and electron microscopy to investigate the effects of the caspase inhibitor ac-YVAD-cmk and the lazaroid tirilazad mesylate on ultrastructural changes in dissociated embryonic mesencephalic cells. In addition, we examined whether the neuroprotectants selectively counteracted specific signs of neurodegeneration. Cell viability decreased significantly over time in both control and treated cell suspensions, but the number of viable cells remaining was significantly higher in tirilazad mesylate-treated cell suspensions. In control samples, the proportion of cells with an ultrastructure consistent with healthy cells decreased from 70%, immediately after dissociation, to 30% after 8 h of incubation. Similar changes were also observed in cell suspensions treated with neuroprotectants. Thus, the neuroprotectants examined did not block the development of specific morphological signs of neurodegeneration. However, when also taking into account that dead cells lysed and disappeared from each cell suspension with time, we found that the total number of remaining viable cells with healthy nuclear chromatin or intact membrane integrity was significantly higher in the tirilazad mesylate-treated group. The results indicate that tirilazad mesylate protects only a small subpopulation of embryonic mesencephalic cells from degeneration induced by mechanical trauma during tissue dissection and dissociation.

Key words: Lazaroid; Caspase inhibitor; Ultrastructure; Embryonic mesencephalic; Viability; Survival

Address correspondence to Patrik Brundin, Section for Neuronal Survival, Wallenberg Neuroscience Center, BMC A10, Lund University, SE-221 84 Lund, Sweden. Tel: +46 46 222 0563; Fax: +46 46 222 0531; E-mail: patrik.brundin@neurol.lu.se

*Present address: Department of Neurosurgery, Ajou University School of Medicine, Suwon 442-721, Korea.

**Present address: Department of Veterinary Morphophysiology and Animal Production (DIMORFIPA), University of Bologna, Via Tolara di Sopra 50, 40064 Ozzano Emilia, Bologna, Italy.

Cell Transplantation, Vol. 12, pp. 243-255, 2003
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Fetal Lateral Ganglionic Eminence Attracts One of Two Morphologically Different Types of Tyrosine Hydroxylase-Positive Nerve Fibers Formed by Cultured Ventral Mesencephalon

Saga Johansson1 and Ingrid Strömberg2

1Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden
2Deparment of Integrative Medical Biology, Umeå University, Umeå, Sweden

The purpose of this study was to investigate the influence of fetal lateral ganglionic eminence (LGE) on nerve fiber outgrowth formed by fetal ventral mesencephalon (VM). Organotypic tissue cultures of fetal VM and LGE plated as single or cocultures were employed. Survival time was 3-21 days in vitro. Nerve fiber outgrowth and migration of astrocytes were analyzed using immunohistochemistry for tyrosine hydroxylase (TH) and S100. In addition, cultures were labeled with the TUNEL technique and with antibodies directed against neurofilament (NF) in order to study apoptosis and retraction of nerve fibers, respectively. The results revealed two morphologically different types of TH-positive outgrowth growing into the substrate. The initially formed TH-positive outgrowth radiated continuously without changing direction, while a second wave of TH-positive outgrowth became obvious when the initial growth already had reached a distance of approximately 1000 mm. The second wave of TH-positive outgrowth radiated from the tissue, but at a certain distance changed direction and formed a network surrounding the culture. The initially formed TH-positive growth was not associated with the presence of S100-positive astrocytes and avoided to grow into the LGE. At longer time points the first wave of TH-positive nerve fibers appeared dotted, with disrupted NF-immunoreactive fibers and in most cultures these long distance growing fibers had disappeared at 21 days in vitro. The second wave of TH-positive nerve fibers was growing onto a layer of glia and never reached the distance of the first wave. LGE became innervated by TH-positive fibers at the time point for when the second wave of TH-positive growth had been initiated, and the innervation appeared in TH-dense patches that also showed a high density of S100-positive astrocytes. Significantly increased TUNEL activity within LGE portion of cocultures was observed when TH-positive fibers entered the LGE and formed patches. In conclusion, two morphologically different types of TH-positive outgrowth were found and the initially formed fibers neither targeted the LGE nor were they guided by glial cells, but their potential to grow for long distances was high.

Key words: Substantia nigra; Lateral ganglionic eminence (LGE); Organotypic culture; Astrocytes

Address correspondence to Dr. Ingrid Strömberg, Department of Integrative Medical Biology, Umeå University, S-901 87 Umeå, Sweden. Tel: +46-90-786 6585; Fax: +46-90-786 6608; E-mail: ingrid.stromberg@histocel.umu.se

Cell Transplantation, Vol. 12, pp. 257-264, 2003
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The Role of Pretraining on Skilled Forelimb Use in an Animal Model of Huntington's Disease

R. A. Fricker-Gates, R. Smith, J. Muhith, and S. B. Dunnett

Brain Repair Group, School of Biosciences, Cardiff University, UK

After a unilateral striatal lesion, animals have generally been seen to have a bilateral impairment in paw reaching, with the contralateral paw being more affected. However, most studies to date have not used a pretraining paradigm to assess maximal capacity for paw reaching, to compare with any lesion-induced loss. This study compared animals that were pretrained with naive animals in their ability to paw reach after a striatal lesion, to address the role of the striatum in either acquisition or execution of this motor task. All lesioned animals showed a significant decrease in reaching ability with their contralateral paw compared with the ipsilateral paw. Pretrained lesioned animals showed a clear lesion deficit with the contralateral paw immediately after lesion, and no impairment whatsoever with the ipsilateral paw. Naive lesioned animals showed delayed acquisition of the task with both paws, possibly due to postural deficits, and a lasting deficit on the contralateral side. The variability of performance between animals was higher in the naive lesioned group. These results suggest that animals should be pretrained on the staircase task prior to lesion to enable maximum sensitivity in detecting both loss and recovery of function of skilled forelimb use.

Key words: Striatal lesion; Skilled forelimb use; Quinolinic acid; Motor functions

Address correspondence to Rosemary A. Gates, School of Biosciences, Cardiff University, Museum Avenue, P.O. Box 911, Cardiff CF10 3US, Wales, UK. Tel: 029 2087 5188; Fax: 029 2087 6749; E-mail: gatesra@cf.ac.uk

Cell Transplantation, Vol. 12, pp. 265-277, 2003
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Axonal Regeneration Across Long Gaps in Silicone Chambers Filled With Schwann Cells Overexpressing High Molecular Weight FGF-2

M. Timmer,1,2 S. Robben,1 F. Müller-Ostermeyer,1 G. Nikkhah,2 and C. Grothe1

1Department of Neuroanatomy, Center of Anatomy, OE 4140, Hannover Medical School, Carl-Neuberg-Strasse 1, D-30623 Hannover, Germany
2Department of Neurosurgery, University Hospital, Breisacher Strasse 64, 79106 Freiburg, Germany

Basic fibroblast growth factor (FGF-2) has been shown to enhance the survival and neurite extension of various types of neurons including spinal ganglion neurons. In addition, endogenous FGF-2 and FGF receptors are upregulated following peripheral nerve lesion in ganglia and at the lesion site. FGF-2 protein is expressed in different isoforms (18 kDa, 21 kDa, 23 kDa) and differentially regulated after nerve injury. In the rat we analyzed the regenerative capacity of the high molecular weight (HMW) FGF-2 isoforms (21/23 kDa) to support the regeneration of the axotomized adult sciatic nerve across long gaps. The nerve stumps were inserted into the opposite ends of a silicone chamber resulting in an interstump gap of 15 mm. Silicone tubes were filled with Matrigel or a mixture of Schwann cells (SC) and Matrigel. SC were prepared from newborn rats and transfected to overexpress HMW FGF-2. Four weeks after the operation procedure, channels were analyzed with regard to tissue cables bridging both nerve stumps and myelinated axons distal to the original proximal nerve stump. Peripheral nerves interposed with HMW Schwann cells displayed significantly enhanced nerve regeneration, with the greatest number of tissue cables containing myelinated axons and the highest number of myelinated axons. These results suggest that a cellular substrate together with a source of a trophic factor could be a promising tool to promote nerve regeneration and, therefore, become useful also for a clinical approach to repair long gaps.

Key words: Fibroblast growth factor-2; Isoforms; Genetically modified Schwann cells; Peripheral nerve regeneration; Rat; Sciatic nerve; Tubulization

Address correspondence to Prof. Dr. C. Grothe, Department of Neuroanatomy, Center of Anatomy, OE 4140, Hannover Medical School, Carl-Neuberg-Strasse 1, D-30623 Hannover, Germany. Tel.: +49-511-532-2896; Fax: +49-511-532-2880; E-mail: Grothe.Claudia@MH-Hannover.de

Cell Transplantation, Vol. 12, pp. 279-290, 2003
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Transection of the Adult Rat Spinal Cord Upregulates EphB3 Receptor and Ligand Expression

Christopher A. Willson,1,2 Jorge D. Miranda,4 Roy D. Foster,1,2 Stephen M. Onifer,1,2,3 and Scott R. Whittemore1,2,3

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
4Department of Physiology, University of Puerto Rico School of Medicine, San Juan, PR 00936

Eph receptors and ligands represent two families of proteins that control axonal guidance during development. Recent work has shown that several Eph receptors are expressed postnatally. Because the Eph molecules represent a class of axon guidance molecules that are mainly inhibitory to axonal growth, we investigated whether EphB3 expression was upregulated in both spinal cord and four supraspinal nuclei (locus coeruleus, vestibular, raphe pallidus, and red) 1 week after a complete spinal cord thoracic transection. Injured rats had a significant increase in EphB3 mRNA and protein expression in the spinal cord. The increased EphB3 expression was colocalized with GFAP staining and indicated that astrocytes play a role in EphB3 expression after spinal cord injury. No change in EphB3 expression was seen in supraspinal brain nuclei, which further demonstrated that changes in expression were due to changes in the local microenvironment at the injury site. The expression of EphB3 was colocalized to regions of the CNS that had a high level of EphB3 binding ligands. These data indicate upregulation of EphB3 expression after injury may also contribute to an environment in the spinal cord that is inhibitory to axonal regeneration.

Key words: Axon guidance; Immunohistochemistry; In situ hybridization; Receptor protein tyrosine kinases

Address correspondence to Scott R. Whittemore, Ph.D., Kentucky Spinal Cord Injury Research Center, Neurological Surgery, MDR Room 616, 511 S. Floyd Street, University of Louisville, Louisville, KY 40202. Tel: (502) 852-0711; Fax: (502) 852-5148; E-mail: swhittemore@louisville.edu

Cell Transplantation, Vol. 12, pp. 291-303, 2003
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The Noradrenergic System of Aged GDNF Heterozygous Mice

V. Zaman,1 Z. Li,4 L. Middaugh,3 S. Ramamoorthy,1 B. Rohrer,1,2 M. E. Nelson,1 A. C. Tomac,5 B. J. Hoffer,5 G. A. Gerhardt,6 and A. Ch. Granholm1

1Department of Physiology and Neuroscience, 2Department of Ophthalmology, 3Department of Psychiatry, and 4Department of Neurology, Medical University of South Carolina, Charleston, SC 29425
5National Institute on Drug Abuse, IRP, Baltimore, MD 21224
6Chandler Medical Center, Department of Anatomy & Neurobiology, University of Kentucky, Lexington, KY 40536-0098

Glial cell line-derived neurotrophic factor (GDNF) is a trophic factor for noradrenergic (NE) neurons of the pontine nucleus locus coeruleus (LC). Decreased function of the LC-NE neurons has been found during normal aging and in neurodegenerative disorders. We have previously shown that GDNF participates in the differentiation of LC-NE neurons during development. However, the continued role of GDNF for LC-NE neurons during maturation and aging has not been addressed. We examined alterations in aged mice that were heterozygous for the GDNF gene (Gdnf+/-). Wild-type (Gdnf+/+) and Gdnf+/- mice (18 months old) were tested for locomotor activity and brain tissues were collected for measuring norepinephrine levels and uptake, as well as for morphological analysis. Spontaneous locomotion was reduced in Gdnf+/- mice in comparison with Gdnf+/+ mice. The reduced locomotor activity of Gdnf+/- mice was accompanied by reductions in NE transporter activity in the cerebellum and brain stem as well as decreased norepinephrine tissue levels in the LC. Tyrosine hydroxylase (TH) immunostaining demonstrated morphological alterations of LC-NE cell bodies and abnormal TH-positive fibers in the hippocampus, cerebellum, and frontal cortex of Gdnf+/- mice. These findings suggest that the LC-NE system of Gdnf+/- mice is impaired and suggest that GDNF plays an important role in continued maintenance of this neuronal system throughout life.

Key words: Locus coeruleus; Noradrenergic system; Glial cell line-derived neurotrophic factor (GDNF); Aging; Neurotrophic factors

Address correspondence to Dr. A. Ch. Granholm, Director, Center on Aging, Department of Physiology and Neuroscience, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC 29425. Tel: (843) 792-4527; Fax: (843) 297-0652; E-mail:Granholm@musc.edu