Tanja Zigova

Intraventricular Administration of BDNF Increases the Number of Newly Generated Neurons in the Adult Olfactory Bulb

We have previously demonstrated that the most rostral part of the subventricular zone (SVZ) is a source of neuronal progenitor cells whose progeny are destined to become interneurons of the olfactory bulb. To determine whether the number of newly generated neurons in the adult olfactory bulb could be increased by the administration of an exogenous factor, brain-derived neurotrophic factor (BDNF) was infused for 12 days into the right lateral ventricle of adult rat brains. The production of new cells was monitored by either the intraventricular infusion or intraperitoneal injection of the cell proliferation marker BrdU. In both experimental paradigms we observed significantly more BrdU-labeled cells in the olfactory bulbs on the BDNF-infused side than in the olfactory bulb of PBS-infused animals. Analysis of the BDNF-infused brains of animals injected intraperitoneally with BrdU demonstrated a 100% increase in the number of BrdU-labeled cells in the bulb, the preponderance ( approximately 90%) of which were double-labeled with a neuron-specific antibody. These results demonstrate that the generation and/or survival of new neurons in the adult brain can be increased substantially by an exogenous factor. Furthermore, the SVZ, and in particular the rostral part, may constitute a reserve pool of progenitor cells available for neuronal replacement in the diseased or damaged brain.

Infusion of Human Umbilical Cord Blood Cells in a Rat Model of Stroke Dose-Dependently Rescues Behavioral Deficits and Reduces Infarct Volume

Background and Purpose— Intravenously delivered human umbilical cord blood cells (HUCBC) have been previously shown to improve functional recovery of stroked rats. To extend these findings, we examined the behavioral recovery and stroke infarct volume in the presence of increasing doses of HUCBC after permanent middle cerebral artery occlusion (MCAO). Methods— Rats were subjected to MCAO and allowed to recover for 24 hours before intravenous infusion of 104 up to 3 to 5×107 HUCBC. Behavioral tests (spontaneous activity, step test, elevated body swing test) were performed 1 week before MCAO and at 2 and 4 weeks after HUCBC infusion. On completion of behavioral testing, animals were euthanized and brain infarct volumes quantified. HUCBC were identified by immunofluorescence for human nuclei and by polymerase chain reaction (PCR) using primers specific for human glycerol 3-phosphate dehydrogenase. Results— At 4 weeks after infusion, there was a significant recovery in behavioral performance when 106 or more HUCBC were delivered (p=0.001 to p=0.05). Infarct volume measurements revealed an inverse relationship between HUCBC dose and damage volume, which reached significance at the higher HUCBC doses (107 cells, p<0.01; 3 to 5×107 cells, p<0.05). Moreover, HUCBC were localized by immunohistochemistry and PCR analysis only in the injured brain hemisphere and spleen. Conclusions— These results extend previous observations of HUCBC infusion in the MCAO rat stroke model by demonstrating a dose relationship between HUCBC, behavioral improvement, and neuronal sparing.

Intravenous Administration of Human Umbilical Cord Blood Cells in a Mouse Model of Amyotrophic Lateral Sclerosis: Distribution, Migration, and Differentiation

Amyotrophic lateral sclerosis (ALS), a multifactorial disease characterized by diffuse motor neuron degeneration, has proven to be a difficult target for stem cell therapy. The primary aim of this study was to determine the long-term effects of intravenous mononuclear human umbilical cord blood cells on disease progression in a well-defined mouse model of ALS. In addition, we rigorously examined the distribution of transplanted cells inside and outside the central nervous system (CNS), migration of transplanted cells to degenerating areas in the brain and spinal cord, and their immunophenotype. Human umbilical cord blood (hUCB) cells (10(6)) were delivered intravenously into presymptomatic G93A mice. The major findings in our study were that cord blood transfusion into the systemic circulation of G93A mice delayed disease progression at least 2-3 weeks and increased lifespan of diseased mice. In addition, transplanted cells survived 10-12 weeks after infusion while they entered regions of motor neuron degeneration in the brain and spinal cord. There, the cells migrated into the parenchyma of the brain and spinal cord and expressed neural markers [Nestin, III Beta-Tubulin (TuJ1), and glial fibrillary acidic protein (GFAP)]. Infused cord blood cells were also widely distributed in peripheral organs, mainly the spleen. Transplanted cells also were recovered in the peripheral circulation, possibly providing an additional cell supply. Our results indicate that cord blood may have therapeutic potential in this noninvasive cell-based treatment of ALS by providing cell replacement and protection of motor neurons. Replacement of damaged neurons by progeny of cord blood stem cells is probably not the only mechanism by which hUCB exert their effect, since low numbers of cells expressed neural antigens. Most likely, cord blood efficacy is partially due to neuroprotection by modulation of the autoimmune process.

Expression of neural markers in human umbilical cord blood.

A population of cells derived from human and rodent bone marrow has been shown by several groups of investigators to give rise to glia and neuron-like cells. Here we show that human umbilical cord blood cells treated with retinoic acid (RA) and nerve growth factor (NGF) exhibited a change in phenotype and expressed molecular markers usually associated with neurons and glia. Musashi-1 and beta-tubulin III, proteins found in early neuronal development, were expressed in the induced cord blood cells. Other molecules associated with neurons in the literature, such as glypican 4 and pleiotrophin mRNA, were detected using DNA microarray analysis and confirmed independently with reverse transcriptase polymerase chain reaction (RT-PCR). Glial fibrillary acidic protein (GFAP) and its mRNA were also detected in both the induced and untreated cord blood cells. Umbilical cord blood appears to be more versatile than previously known and may have therapeutic potential for neuronal replacement or gene delivery in neurodegenerative diseases, trauma, and genetic disorders.

Neuronal Progenitor Cells Derived from the Anterior Subventricular Zone of the Neonatal Rat Forebrain Continue to Proliferatein Vitroand Express a Neuronal Phenotype

A discrete area of the anterior part of the subventricular zone, or SVZa, of the postnatal forebrain is composed of progenitor cells that are dissimilar to those elsewhere in the CNS. In vivo SVZa progenitor cells retain the ability for division, even though they are phenotypically neurons. To characterize further the properties of SVZa cells, we have analyzed their characteristics in vitro using cell-type specific antibodies and their proliferative capacity by the incorporation of bromodeoxyuridine. At 2 h in vitro, as well as after 1 day in vitro, virtually all SVZa cells isolated from the neonatal forebrain express TuJ1, an antibody that recognizes neuron-specific tubulin, and are GFAP-negative. Likewise, the preponderance of SVZa cells express the neuron-specific markers N-CAM and MAP-2 when examined after 1 day in culture. The majority of SVZa cells cultured for as long as 8 days also possessed a neuronal phenotype. In addition, process-bearing TuJ1-positive SVZa cells continued to proliferate throughout the entire culture period. Thus, the neuronal progenitor cells of the SVZa constitute a unique cell population with characteristics distinct from the cells of other germinal zones.

Human umbilical cord blood cells express neural antigens after transplantation into the developing rat brain.

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 β-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.

Dopaminergic and GABAergic interneurons of the olfactory bulb are derived from the neonatal subventricular zone.

Earlier studies in our laboratory have demonstrated that a discrete region of the anterior part of the neonatal subventricular zone (SVZa) contains exclusively neuronal progenitor cells. The descendants of the SVZa progenitor cells are destined for the granule cell and glomerular layers of the olfactory bulb, where they differentiate into granule and periglomerular cells, the interneurons of the olfactory bulb, respectively. In the present set of experiments we examined the neurotransmitter phenotype of the SVZa‐derived cells. In order to label SVZa‐derived cells, the cell proliferation marker bromodeoxyuridine (BrdU) was injected into the SVZa of postnatal day 2 (P2) rats. After 3 weeks, by which time most of the SVZ‐aderived cells have migrated to their final destination in the bulb, the animals were perfused and their brains processed for immunohistochemistry. To identify the neurotransmitter phenotype of the SVZa‐derived cells, sagittal sections of the forebrain, including the olfactory bulb, were double‐labeled with an antibody to BrdU in conjunction with an antibody to γ‐amino‐butyric acid (GABA) or tyrosine hydroxylase (TH), the rate limiting enzyme in the synthesis of dopamine. Using simultaneous indirect immunofluorescence to detect the presence of single‐ and double‐labeled cells, we found that 59% and 51% of the BrdU‐positive cells were immunoreactive for GABA in the granule cell and glomerular layers, respectively. In addition, 10% of the BrdU‐positive periglomerular cells were immunoreactive for TH. The presence of double‐labeled (BrdU‐positive/GABA‐positive and BrdU‐positive/TH‐positive) cells in the olfactory bulb, demonstrates that the SVZa is a source of the GABAergic and dopaminergic interneurons of the olfactory bulb during postnatal development.

Human umbilical cord blood progenitors: the potential of these hematopoietic cells to become neural.

The mononuclear fraction from human umbilical cord blood (HUCB) contains a significant number of stem/progenitor cells that in theory could be come any cell in the body, including neurons. Taking into consideration that transdifferentiation would be a very rare event and also knowing that overlapping genetic programs for hematopoiesis and neuropoiesis exist, we undertook a characterization of the HUCB mononuclear fraction, including analysis of cellular subpopulations and their morphology, cell viability, proliferation, and expression of neural and hematopoietic antigens. Two cell populations were apparent—adherent and floating fractions. The adherent fraction was mainly lymphocytes (∼53%) expressing hematopoietic antigens. Upon replate, the floating population had many cells that expressed stem cell antigens. More of the cells in this subfraction expressed neural proteins. Neurotrophin receptors trkB and trkC were present in both cell fractions, although expression was higher in the floating fraction. Our initial characterization suggests that a subpopulation of cells exists within the HUCB mononuclear fraction that seems to have the potential to become neural cells, which could then be used in the development of cell‐based therapies for brain injuries and diseases.

Neuronal progenitor cells of the neonatal subventricular zone differentiate and disperse following transplantation into the adult rat striatum

We have investigated the suitability of a recently identified and characterized population of neuronal progenitor cells for their potential use in the replacement of degenerating or damaged neurons in the mammalian brain. The unique population of neuronal progenitor cells is situated in a well-delineated region of the anterior part of the neonatal subventricular zone (referred to as SVZa). This region can be separated from the remaining proliferative, gliogenic, subventricular zone encircling the lateral ventricles of the forebrain. Because the neurons arising from the highly enriched neurogenic progenitor cell population of the SVZa ordinarily migrate considerable distances and ultimately express the neurotransmitters GABA and dopamine, we have examined whether they could serve as an alternative source of tissue for neural transplantation. SVZa cells from postnatal day 0-2 rats, prelabeled by intraperitoneal injections of the cell proliferation marker BrdU, were implanted into the striatum of adult rats approximately 1 mo after unilateral denervation by 6-OHDA. To examine the spatio-temporal distribution and phenotype of the transplanted SVZa cells, the experimental recipients were perfused at short (less than 1 wk), intermediate (2-3 wk) and long (5 mo) postimplantation times. The host brains were sectioned and stained with an antibody to BrdU and one of several cell-type specific markers to determine the phenotypic characteristics of the transplanted SVZa cells. To identify neurons we used the neuron-specific antibody TuJ1, or antimembrane-associated protein 2 (MAP-2), and anti-GFAP was used to identify astrocytic glia. At all studied intervals the majority of the surviving SVZa cells exhibited a neuronal phenotype. Moreover, morphologically they could be distinguished from the cells of the host striatum because they resembled the intrinsic granule cells of the olfactory bulb, their usual fate. At longer times, a greater number of the transplanted SVZa cells had migrated from their site of implantation, often towards an outlying blood vessel, and the density of cells within the core of the transplant was reduced. Furthermore, there were rarely signs of transplant rejection or a glial scar surrounding the transplant. In the core of the transplant there were low numbers of GFAP-positive cells, indicating that the transplanted SVZa cells, predominantly TuJ1-positive/MAP2-positive, express a neuronal phenotype. Collectively, the propensity of the SVZa cells to express a neuronal phenotype and to survive and integrate in the striatal environment suggest that they may be useful in the reconstruction of the brain following CNS injury or disease.

Lithium Chloride Induces the Expression of Tyrosine Hydroxylase in hNT Neurons

In the present study, several doses of lithium chloride were tested for their ability to induce the expression of tyrosine hydroxylase (TH) in neurons derived from a human teratocarcinoma cell line (hNT) after 5 and 10 days in vitro (DIV). Following immunocytochemical staining for tyrosine hydroxylase, the percentage of TH-positive neurons was determined and morphometric analysis, including mean soma profile area and neuritic length, was performed. hNT neurons responded to lithium treatment in a dose-dependent manner. In 5 DIV, the most effective dose of lithium chloride (1.0 mM) increased the number of TH-positive neurons approximately sixfold. In addition, both TH-positive hNT neuron mean soma profile area and neurite length were significantly larger than controls by 60 and 70%, respectively. Moreover, even after withdrawal of lithium chloride on day 5, the number of TH-positive neurons in 10 DIV cultures remained significantly increased. These data suggest that hNT cells are indeed responsive to lithium exposure and may serve as a continual source of TH-expressing neurons in new therapeutic approaches to degenerative brain disease.