NEURAL STEM CELLS
(Lay Summary)

Neural stem cells (NSCs) have the ability to self-renew and differentiate into the primary phenotypes in the central nervous system. NSCs have shown great promise for the treatment of many neurological diseases, and the ability of neural stem cells to home to diseased areas of the brain and their capacity to differentiate into all neural phenotypes provide potentially powerful tools for the treatment of both neurologic disorders (Shah 2006).  However a full understanding of the molecular mechanisms regulating cell migration and the pathways of NSC differentiation is essential if these cells are to be used for therapeutic applications.

NSCs were first isolated and proliferated via epidermal growth factor (EGF) in 1992 (Reynolds 1992).  NSCs are isolated from brain tissue and are most commonly found in the hippocampus and the subventricular zone.  NSC have the potential to regenerate and, if provided with the proper microenvironment, can differentiate into neural precursor cells (NPC) and to a variety of different cell types including the neural cells (neurons, astrocytes, and oligodendrocytes), as well as erythrocytes, leukocytes and skeletal muscle (Sugaya 2005).

NSCs cultured in serum-free medium form aggregates, or neurospheres. Many studies have shown that these neurospheres formed in vitro include a mixture of NSC stem cells, neural progenitor cells and more highly differentiated cells such as neuronal and glial cells.  Undifferentiated neural stem cells express high levels of the intermediate filament proteins vimentin and nestin and are regulated multiple developmental signaling pathways such as Wnt, Sonic Hedgehog, PTEN (Sanai 2005).  Additionally, proliferating neural stem cells usually show little or no expression of mature cell markers (Yang 2006).  Wang et al. claim the neurosphere system is dynamic, and their results will serve as a stepping-stone to more in-depth studies of the neurosphere microenvironment (Wang 2006).

Kim (2006) compared bone marrow mesenchymal stem cells (BMSCs) and peripheral blood derived mesenchymal stem cells (PMSCs) to evaluate their potential to differentiate to neural stem cells.  Their study demonstrated that PMSCs in a rat model proliferated to putative neural stem cells (nestin positive neurospheres) and differentiated into neuronal or glial cells in vitro.  Thus, autologous PBSCs could serve as a readily isolated and easily proliferated source for cell therapies, and one that poses little risk of rejection upon autotransplantation.

Current treatments of neurodegenerative anomalies may alleviate symptoms, but they are not curative and provide only temporary relief of symptoms. The discovery of neurogenesis in the adult brain and the regenerative potential of neural stem cells holds the promise for restoration of neural populations.  Several applications of NSCs are being pursued, including use of cells activated within endogenous sources or from transplantation of donor cells.  FGF-2, IGF-1 and VEGF, in the microenvironment of the subgranular zone (SGZ) are prime contributors to the reduced neurogenic potential (Brinton 2006).

The less invasive application is expansion of endogenous NSCs.  NSCs respond to environmental signals, such as dietary regulation, exercise, and hormone therapy, all of which can stimulate neurogenesis.  NSCs are induced to divide then migrate to the area of damaged neurons where they develop the normal characteristics of damaged cells and replace them.  The second method is transplantation of NSCs or progenitors from a donor into the damaged neural area, with the intent of NSCs differentiating to appropriate cell types of the microenvironment rendered dysfunctional disease or trauma (Haughey 2002).  Several techniques exist for isolating and identifying NSC based on the tissue or cell types.  To isolate NSCs that form specialized neural cells requires observation of markers on the differentiated cells (Wolswijk 2002).  One study claims that the neurotrophins derived from the transplanted NSC modulates the expression of the major histocompatibility complex in the injured microenvironment to facilitate neurological recovery (Sun 2006).  Some of the many neurodegenerative conditions that NSC may have potential use for are discussed below.

Alzheimer’s Disease (AD) is a form of dementia affecting as many as five million Americans, yet there are currently no diagnostic tools to determine if a living person has AD. Alzheimer’s disease (AD) is characterized by plaque formation, neuronal loss, and cognitive decline. The two types of AD are sporadic, with an age of onset in the 60s or later, and familial, a genetic linked predisposition, the latter of which affects less than 10% of AD patients, but has an earlier age of onset.  The symptoms begin with forgetfulness to loss of memories not associated with general aging and intellectual skill deterioration, erratic behavior, and loss of bodily functions (Oliveira 2005).

Exogenous and endogenous testing of NSC has found varying results.  Exogenous testing includes infusion of neural progenitor cells (NPC) has been shown to reverse the effects of age related memory loss.  Flax et al has shown that implantation of NSC into the brains of mice appear phenotypically and morphological normal (Flax 1998).  A study with rats had shown that infusion with nerve growth factor (NGF), a cytokine, prevented the degeneration of cholinergic neurons and may reverse age-related memory deficits (Oliveira AA, Hodges HM 2005).  However, in a human clinical trial, nerve growth factor was discontinued because of the adverse effects related to interaction with non-targeted structures (Blesch 2004).

In the aged and AD brain, both the pool of neural stem cells and their proliferative potential are markedly diminished. In parallel, the level of potential regenerative factors is diminished in the brains of Alzheimer's patients compared to age-matched controls (Brinton2006).   Seladin-1 (Selective Alzheimer’s Disease Indicator-1) is an anti-apoptotic gene, which is down regulated in brain regions affected by Alzheimer’s disease (AD).  Susanna Benvenuti and her group claimed that hippocampus and subventricular cell compartments are the predominant source of seladin-1 in normal brain and they express less in AD because of an altered pool of multipotent cells (Benvenuti 2006).   Studies on human postmortem material and transgenic mice expressing amyloid precursor protein found in familial Alzheimer’s (AD) suggest that AD is associated with enhanced neurogenesis (Greenberg 2006).

Multiple sclerosis (MS) is a chronic de-myelinating disease involving inflammation and scarring in the central nervous system.  The etiology of MS unknown, but current theory is that a virus activates an autoimmune response to the myelin found in the CNS.  Current treatment involves prednisone in tapering treatments, β-interferon, and glatiramer aacetate all of which only slow the progression of the disease, but do not heal the affected areas.

Lamoury et al showed that undifferentiated MSC express typical neural cell markers nestin, MAP2, A2B5, GFAP, MBP, CNPase, GalC, O1, in culture and Oct-4 and Rex-1 are expressed by untreated MSC in a mouse model induced with MS disease (Lamoury 2006).  The role of microglial cells in demyelinating diseases such as MS is unknown.  One study has showed that high levels of IFN-gamma impeded oligodendrogenesis from adult neural stem/progenitor cells (Butovsky 2006) and another has identified myelin debris as the inhibitor of remyelination (Kotter 2006).

Spinal cord samples from patients with long-term MS were obtained and found to have many lesions.  It was also seen that patients with primary progressive and secondary progressive MS had similar numbers of the needed neural stem cells, and that immature neural cells were only found in lesions that contained higher numbers of the initial neural stem cells.  The belief is that there is a decrease of NSCs in older lesions and new generations of these neural cells are damaged, while the failure of nearby neural stem cells to migrate may contribute to a lack of re-myelination (Wolswijk F 2002).  Dubois-Dalcq (2005) reviewed recent NSC studies and established that cell therapies alone seem unable to remyelinate neuronal cell on a scale large enough for thorough MS therapy. 

Parkinson’s disease (PD) is a neurodegenerative, age-related disorder, with initial onset occurring at about 60 years of age.  Approximately one million people in the United States are affected.  PD is characterized by a significant decrease of dopamine in the corpus striatum, which is caused by degeneration of dopaminergic neurons in the substantia nigra.  Clinical manifestations of PD are tremor, rigidity, disrupted gait and unstable posture.  The primary treatment of PD is Levodopa, an amino acid metabolic precursor of dopamine.  Benefits are limited and decline after chronic administration.  However, there is optimism that neural transplant therapies may one day enable complete circuit reconstruction and thus functional benefit for patients with PD (Olanow 2004).

Zeng et al. established that there is no remarkable immunological rejection when neural stem cells are transplanted into the brain of a PD diseased rat (Zeng 2006).  Wei has shown that NSCs transplanted to the striatum engraft with good success, and give rise to neurons, astrocytes and oligodendrocytes (Liu 2006).  One study using human embryonic NSCs cloned by gene transfer in a PD diseased rat model showed that NCSs exert neurogeneic and neuroprotective effects on dopaminergic neurons (Yasuhara 2006).  Sigrid Schwarz’s group recently showed that immunosuppression with cyclosporine did not improve function and survival of neuroprogenitor cells transplanted into the striatum of lesioned rats (Schwarz 2006).

Damage to the spinal cord, either from trauma (the major cause of SCI) or disease, can lead to varying degrees of loss of function (sensory or motor).  In the United States, there are roughly half a million individuals, mostly younger men, who are directly affected by SCI.  Damage from SCI is mostly permanent, with a very small percentage regaining full recovery. SCI often result in swelling of the spinal cord due to a variety of factors, including inflammatory cytokines that decreases over several days or weeks sometimes resulting in some functional recovery (Okano 2005).  Treatment for acute SCI involves inhibiting further damage to the spinal cord, often by realignment of the vertebral column. Treatment for chronic SCI is mostly aimed at treating secondary complications and at improving quality of life. Several trials are underway to find new treatments for SCI, including new drugs and embryonic stem cells.

With regards to NSC, researchers have been focusing on cell transplantation therapies rather than activation of endogenous NSC.  Activation of endogenous NSC may prove to be fruitful once these stem cells can be coerced to differentiate into the desired progenitors.  Varying results have been observed so much more research must be done before anything can be confirmed (Enzmann 2005, Setoguchi 2004).
During the initial trauma of SCI, various inflammatory cytokines that exhibit a neurotoxic effect, which would not allow new neurons to form, are unregulated. Implanting NSC/NPC several days after injury showed favorable results in mice and also in primates, where improvements of motor function were seen and differentiation of NSC/NPC into the three neural cell types (Iwanami 2005). 

Studies have shown transplantation of NSCs into the injured spinal cord, with or without GDNF led to differentiated astrocytic population (Macias 2006).  Neuronal-restricted precursors (NRPs) and glial-restricted precursors (GRPs) can be labeled in vitro with the super paramagnetic iron oxide contrast agent Feridex (Lepore 2006).  In other studies, researchers have proposed paraplegic adult rat with a T8 spinal cord transection to be standard experimental model for spinal cord reconstruction (Fernandez 2006). A mixture of more restricted neural precursors might be better suited than highly immature NSCs for neural replacement strategies after central nervous system (CNS) injuries (Alexanian 2006).

Stroke is the third most common death cause and one of the prime causes of permanent disability and healthcare consumption.  The National Institute of Neurological Disorders reports that there are an estimated 700,000 cases of stroke each year, 75% of which occur in individuals who are 65 or older.  A stroke results from either a blood clot that blocks blood vessels supplying blood to the brain (ischemic stroke), or by the bursting of blood vessels within the brain (hemorrhagic stroke).  Both types of strokes cause a sudden obstruction of blood flow to the brain, which leads to cellular death of brain tissue.  The most common form of stroke is the result of occlusion of the middle cerebral artery (MCA), which directs the majority of blood supplied by the internal carotid artery to the forebrain. Since a stroke can cause cell death in many different areas of the brain, it leads to a variety of disabilities ranging from the loss of memory, problems with thought processing, the loss of balance and coordination, and even paralysis.  The risk of having a stroke increases in those who have high blood pressure, smoke cigarettes, have diabetes, or are obese.

Although presently there is no cure for a stroke, neural stem cells are being used to produce new brain cells to replace those that die during a stroke.  While transplantation of neuron-generating cells into injured areas is one feasible way to replace dead cells, it is not risk free and can lead to seizures and further obstruction of blood vessels (Savitz 2004).  A safer method to produce new brain cells is to use the Stem Cell Factor (SCF) or SDF-1a to attract NSC to areas of brain injury where they can begin to divide and produce new brain cells (Imitola 2004).  Kenneth Pollock’s group has derived human neural stem cell line, CTX0E03, from human somatic stem cells following genetic modification with a conditional immortalizing gene, c-myc ERTAM.  This study has shown that delayed grafting of this human stem cell line can promote functional recovery in adult rats after stroke (Pollock 2006).

Traumatic brain injury (TBI) is a major health problem that can severely affect the quality of life at any age but is especially prevalent among male adolescents and young adults. Unlike neurodegenerative diseases CNS related injuries such as brain and spinal cord injuries require immediate intervention.  Timing and methods of intervention, whether traditional or experimental, are critical to stabilize the patient and prevent any further damage.  Symptoms of a traumatic brain injury can be mild, moderate, or severe, depending on the extent of damage to the brain.  40% of surviving TBI patients suffers long-term disabilities of cognition, sensation, movement and emotion resulting from loss of brain cells.  The extensive loss of neural cells produced by TBI contributes substantially to functional impairments.

Transplantation of neural progenitor cells has been shown to improve neuromotor function and restore neurons in the hippocampus following fluid-percussion brain injury in rats (Philips 2001).  Additionally, human fetal neural stem cells have been shown to improve cognitive function of TBI-induced rats when grafted into the hippocampus after a moderate brain injury (Gao 2006).

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Summarized by:  Joyce Kim, Jason Moore and Ajitha Patlolla, Graduate Course in Stem Cell Biology, Fall 2006

Teaching Assistant: Steve Greco