Therapeutic potentials of human embryonic stem cells in Parkinson's disease.

The loss of dopaminergic neurons of the substantia nigra is the pathological hallmark characteristic of Parkinson's disease (PD). The strategy of replacing these degenerating neurons with other cells that produce dopamine has been the main approach in the cell transplantation field for PD research. The isolation, differentiation, and long-term cultivation of human embryonic stem cells and the therapeutic research discovery made in relation to the beneficial properties of neurotrophic and neural growth factors has advanced the transplantation field beyond dopamine-producing cells. The present review addresses recent advances in human embryonic stem cell experimentation in relation to treating PD, as well as cell transplantation techniques in conjunction with alternative therapeutics.

TNF-alpha knockout and minocycline treatment attenuates blood-brain barrier leakage in MPTP-treated mice.

Following intraparenchymal injection of the dopamine (DA) neurotoxin 6-hydroxydopamine, we previously demonstrated passage of fluoresceinisothiocyanate-labeled albumin (FITC-LA) from blood into the substantia nigra (SN) and striatum suggesting damage to the blood-brain barrier (BBB). The factors contributing to the BBB leakage could have included neuroinflammation, loss of DA neuron control of barrier function, or a combination of both. In order to determine which factor(s) was responsible, we assessed BBB integrity using the FITC-LA technique in wild-type (WT), tumor necrosis factor alpha (TNF-alpha) knockout (KO), and minocycline (an inhibitor of microglia activation) treated mice 72 h following treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Compared with WT mice, TNF-alpha KO mice treated with MPTP showed reduced FITC-LA leakage, decreased numbers of activated microglia, and reduced proinflammatory cytokines (TNF-alpha and interleukin 1beta) associated with significant MPTP-induced DA neuron loss. In contrast, minocycline treated animals did not exhibit significant MPTP-induced DA neuron loss although their FITC-LA leakage, numbers of activated microglia, and MPTP-induced cytokines were markedly attenuated. Since both TNF-alpha KO and minocycline treatment attenuated MPTP-induced BBB dysfunction, microglial activation, and cytokine increases, but had differential effects on DA neuron loss, it appears that neuroinflammation and not DA neuron loss was responsible for disrupting the blood-brain barrier integrity.

Progressive dopamine neuron loss in Parkinson's disease: the multiple hit hypothesis.

Animal models have been an essential tool for researchers and clinicians in their efforts to study and treat Parkinson's disease (PD). Thus, the various ways 6-hydroxydopamine is employed, the use of MPTP in rodents and nonhuman primates, the prenatal exposure to bacterial endotoxin, the postnatal exposure to environmental toxins such as paraquat and rotenone, the assessment of dopamine (DA) neurons in genetic knockout mouse, and even the behavioral analysis of fruit flies and worms have added significantly to our knowledge base of PD--or have they? Are these animal models manifesting a true model of PD? Have the 7786 published studies (to date) on PD with animal models led to a clearer understanding of its etiology, treatment, or progression? In this review we critically assess this question. We begin with a succinct history of the major contributions, which have led to the current animal models of PD. We then evaluate the primary issue of the progressive loss of DA neurons, which, except for a few studies, has not been addressed in animal models of PD, even though this is the major pathological characteristic of the disease. Lastly, we discuss the possibility that more than one risk factor for PD may be necessary to develop an animal model that shows synergy--the progressive loss of DA neurons. Thus, the multiple hit hypothesis of PD-that is, the effect of more then one risk factor-may be the start of new era in animal models of PD that is one step closer to mimicking the pathology of PD in humans.

Cytokines produced by cultured human umbilical cord blood (HUCB) cells: implications for brain repair.

The potential therapeutic benefits from human umbilical cord blood (HUCB) cells for the treatment of injuries, diseases, and neurodegeneration are becoming increasingly recognized. The transplantation or infusion of cord blood cells in various animal models, such as ischemia/stroke, traumatic brain injury, myocardial infarction, Parkinson's disease, and amyotropic lateral sclerosis, has resulted in amelioration of behavioral deficits, and with some diseases, a prolonged lifespan decreased neuropathology. Previously, we reported the migration of HUCB cells to ischemic brain supernatant (tissue extracts) is time-dependent, and the expression of specific chemokines responds to this migration pattern. The mechanism(s) responsible for these effects are unknown. The expression of cytokines and chemokines produced by HUCB cells (under various culturing conditions) was investigated in this study. IL-8, MCP-1, and IL-1alpha were consistently expressed by the HUCB mononuclear cells regardless of the culture condition. These results provide insights to factors that may be partially responsible for the functional improvements seen in the animal models of injury investigating the therapeutic use of HUCB cells.

Stroke-induced migration of human umbilical cord blood cells: time course and cytokines.

The therapeutic window for treatment of individuals after stroke is narrow, regardless of the treatment regime; extension of this window would provide a major therapeutic advance. In prior reports, we demonstrated significant improvements in the behavioral defects of rats that received human umbilical cord blood (HUCB) cells 24 h after a middle cerebral arterial occlusion. These effects paralleled the recruitment of these cells to the site of tissue damage. While the administration of HUCB cells 24 h after stroke was effective, the optimal time to administer these cells after stroke has not been established. Here, we investigated the migration of HUCB cells to ischemic tissue extracts. After ischemic assault, brain tissue was homogenized, and the supernatants were assayed for their ability to attract HUCB mononuclear cells as well as for levels of several cytokines. We demonstrate increased migratory activity of HUCB cells toward the extracts harvested at 24-72 h after stroke. The extracts possessed increased levels of certain cytokines and chemokines, suggesting their participation in HUCB cell migration. The results from this study are promising in that the current 3-h therapeutic window for the treatment of stroke victims, using approved anticoagulant treatment, may be extended with the use of HUCB cell therapy 24-72 h post stroke. Last, the chemokines present in the supernatant provide a sound starting point to start examining the mechanisms responsible for the in vivo migration of HUCB cells after the induction of stroke.

Tumorigenicity issues of embryonic carcinoma-derived stem cells: relevance to surgical trials using NT2 and hNT neural cells.

Cell therapy is a rapidly moving field with new cells, cell lines, and tissue-engineered constructs being developed globally. As these novel cells are further developed for transplantation studies, it is important to understand their safety profiles both prior to and posttransplantation in animals and humans. Embryonic carcinoma-derived cells are considered an important alternative to stem cells. The NTera2/D1 teratocarcinoma cell-line (or NT2-N cells) gives rise to neuron-like cells called hNT neurons after exposure to retinoic acid. NT2 cells form tumors upon transplantation into the rodent. However, when the NT2 cells are treated with retinoic acid to produce hNT cells, they terminally differentiate into post-mitotic neurons with no sign of tumorigenicity. Preliminary human transplantation studies in the brain of stroke patients also demonstrated a lack of tumorigenicity of these cells. This review focuses on the use of hNT neurons in cell transplantation for the treatment in central nervous system (CNS) diseases, disorders, or injuries and on the mechanism involved in retinoic acid exposure, final differentiation state, and subsequent tumorigenicity issues that must be considered prior to widespread clinical use.

Transplantation of human umbilical cord blood cells in the repair of CNS diseases.

Cell transplantation therapies have been used to treat certain neurodegenerative diseases such as Parkinson's and Huntington's disease. However, ethical concerns over the use of fetal tissues, and the inherent complexities of standardising the procurement, processing and transplantation methods of this tissue, have prompted the search for a source of cells that have less ethical stigmatisations, are readily available and can be easily standardised. Several sources of human cells that meet these principles have been under investigation. Cells from human umbilical cord blood (HUCB) are one source that is consistent with these principles; therefore, they have become of great interest in the field of cellular repair/replacement for the treatment of CNS diseases and injury. This review will focus on the advantages of HUCB cells as a source for cellular transplantation therapies, recent studies that have examined the potential of these cells in vitro to be directed towards neural phenotypes, and in vivo studies that have investigated the functional recovery of animals in a number of models of CNS injury and disease following administration of HUCB cells.

Use of human umbilical cord blood (HUCB) cells to repair the damaged brain.

Neurodegenerative diseases as well as acute center nervous system (CNS) injuries remains a problematic and frustrating area of medicine in terms of treatments and cures, which is mostly due to the complex circuitry of the CNS along with our limited knowledge. Therapeutically, the last two and a half decades have offered new hope for those suffering from neurodegenerative diseases or injuries with advent of new drug discoveries and cellular therapies. Cell transplantation is a compelling and potential treatment for certain neurological and neurodegenerative diseases as well as for acute injuries to the spinal cord and brain. The hematopoietic system offers an alternative source of cells that is easily obtainable, abundant, and reliable when compared to cells obtained from fetal or embryonic origins. Human umbilical cord blood (HUCB) cells have been used clinically for over ten years to treat both malignant and non-malignant diseases. With in the last five years these cells have been used pre-clinically in animal models of brain and spinal cord injuries, in which functional recovery have been shown. This paper reviews the advantages, utilization, and progress of HUCB cells in the field of cellular transplantation and repair.

Human umbilical cord blood (HUCB) cells for central nervous system repair.

Cellular therapy is a compelling and potential treatment for certain neurological and neurodegenerative diseases as well as a viable treatment for acute injury to the spinal cord and brain. The hematopoietic system offers alternative sources for stem cells compared to those of fetal or embryonic origin. Bone marrow stromal and umbilical cord cells have been used in pre-clinical models of brain injury, directed to differentiate into neural phenotypes, and have been related to functional recovery after engraftment in central nervous system (CNS) injury models. This paper reviews the advantages, utilization and progress of human umbilical cord blood (HUCB) cells in the neural cell transplantation and repair field.

Nicotinic acetylcholine receptors on NT2 precursor cells and hNT (NT2-N) neurons.

This is the first report, to our knowledge, of prominent, natural expression of nAChR alpha4, alpha6 and alpha9 subunits in a human, neuronally-committed cell line. We performed studies with specific reference to the expression of nicotinic acetylcholine receptors (nAChR) to further characterize a human, postmitotic, transplantable, with a neuronal phenotype, cell line called hNT (also called NT2-N). hNT cells acquire a distinctive neuronal phenotype upon differentiation from their NT2 precursors. Immunocytochemical studies showed that NT2 cells were strongly immunopositive for alpha4 or alpha7 subunits, moderately immunopositive for alpha3/alpha5 subunits, and weakly immunopositive for beta2 or beta4 subunits, whereas hNT neurons showed positive, strong-to-moderate immunostaining for all of these nAChR subunits. Reverse transcription-polymerase chain reaction (RT-PCR) mRNA analyses indicated that levels of alpha7 subunit messages were similar in both NT2 and hNT cells, whereas alpha2, alpha10, and beta3 subunit transcripts were not detected. Levels of alpha3, alpha5, and beta4 subunit messages were lower in hNT neurons than in NT2 precursors. However, alpha4 and beta2 subunit messages were present in NT2 precursors but were greatly induced in hNT neurons. Levels of alpha6 and alpha9 subunit messages, not detectable in NT2 precursors, rose to high levels in hNT neurons. hNT cell nAChR subunit message levels were comparable to (alpha4, alpha5, beta4) or higher than (alpha6, alpha9, beta2) levels in adult human brain. NT2 and hNT cells may provide an excellent model for studies of neurogenesis, roles played by nAChR in differentiation and neurodegeneration, and effects of neuronal differentiation on nAChR expression.

Nicotine's oxidative and antioxidant properties in CNS.

Nicotine has been reported to be therapeutic in some patients with certain neurodegenerative diseases and to have neuroprotective effects in the central nervous system. However, nicotine administration may result in oxidative stress by inducing the generation of reactive oxygen species in the periphery and central nervous system. There is also evidence suggesting that nicotine may have antioxidant properties in the central nervous system. The antioxidant properties of nicotine may be intracellular through the activation of the nicotinic receptors or extracellular by acting as a radical scavenger in that it binds to iron. The possibility that nicotine might be used to treat some symptoms of certain neurodegenerative diseases underlies the necessity to determine whether nicotine has pro-oxidant, antioxidant or properties of both. This review discusses the studies that have addressed this issue, the behavioral effects of nicotine, and the possible mechanisms of action that result from nicotine administration or nicotinic receptor activation.

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 beta-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.

Anxiolytic effects of mecamylamine in two animal models of anxiety.

Clinical and preclinical evidence suggests that mecamylamine, a nicotinic receptor antagonist, may have anxiolytic properties. The purpose of this study was to further investigate the anxiolytic properties of mecamylamine in rats as measured by the Elevated Plus Maze and the Social Interaction models of anxiety and to determine if manipulation of the testing environment (either brightly lit or dimly lit conditions) influenced the results. Results indicated that mecamylamine had significant anxiolytic effects in both the Elevated Plus Maze and Social Interaction Tests and that these effects were dependent on dose administered and the level of anxiety produced under different testing conditions. If confirmed by further clinical research, nicotinic receptor antagonists like mecamylamine may represent a novel class of anxiolytics.