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Human stem cell-derived motor neurons as an experimental model for ALS

Funding Type: 
SEED Grant
Grant Number: 
Funds requested: 
$752 999
Funding Recommendations: 
Not recommended
Grant approved: 
Public Abstract: 
Effective treatments for motor neuron diseases, such as Amyotrophic Lateral Sclerosis (ALS), Spinal Muscular Atrophy (SMA), Kennedy's Disease and Hereditary Spastic Paraplegias (HSP), have yet to be developed. Currently the only FDA-approved drug for treating motor neuron diseases is riluzole. However, riluzole has only moderate success in prolonging patient survival. One possible reason for the lack of effective treatments for motor neuron diseases is that drugs for such diseases were previously developed for humans based on animal models which may not accurately reflect motor neuron diseases in humans. Therefore, establishing a human system to study motor neuron diseases is a crucial step in developing successful therapeutic treatments. Human Embryonic Stem (hES) cells constantly self-renew and have the potential to grow into any type of cell in the human body. Since little hES research has been conducted, it is not clear whether these cells can be used in scientific models to understand cell death in motor neuron diseases. Therefore, in this study we propose to steer hES cells into becoming motor neurons so that we may gain a better understanding of the aforementioned motor neuron diseases. Because of their self-renewing properties and their ability to be fostered in a scientific environment, our studies of hES cells could provide a useful source of knowledge to develop therapies for patients with motor neuron diseases or spinal cord injuries. Aim 1: We will take motor neurons from hES cell lines and study their molecular and cellular properties. These studies will allow us to identify how motor neurons express specific genes and proteins. In addition, we will be able to evaulate how effective hES cell-derived motor neurons are in developing treatments for motor neuron diseases. We will also improve the mothod used by the scientific community to induce motor neurons from hES cells. The goal is to develop a reliable method to use hES cells for deriving motor neurons useful for basic research and clinical applications. Aim 2: We will evaluate toxicity in hES cell-derived motor neurons to test whether human cell cultures can be used as experimental models to investigate motor neuron diseases. We will first analyze how well functional ionotropic glutamate receptors (the basic building blocks of motor neurons in the brain) in these hES-derived motor neurons are expressed using both a pharmacological approach and by recording the electrical activities of motor neurons (electrophysiology). We will then investigate cell death in motor neurons using specific toxins that will target glutamate receptors and transporters. We will also determine the effect of drugs in protecting these cells from toxin-induced death.
Statement of Benefit to California: 
The goal of our proposed research is to use hES cells to develop a reliable method for deriving motor neurons. Since hES cells can self-renew and be maintained in vitro the motor neurons derived from hES cells could provide useful knowledge that could be applied to treatments for patients with motor neuron diseases or spinal cord injuries. This proposed research will benefit the State of California and its citizens in several ways. The most obvious way in which this research will benefit Californians is through the reduction of pain and suffering undergone by Californian patients suffering from motor neuron diseases or spinal cord injuries. Caretakers and relatives of those with motor neuron-related medical problems will also experience the benefits. As the state with the largest population in the nation, California spends a enormous amount of money on healthcare for its residents. In the future, finding a cure for neurodegenerative diseases like ALS will significantly reduce state healthcare costs by lessoning the amount of time patients are treated for such diseases. In addition, conducting research in the stem cell field will create job opportunities and attract highly skilled personnel to California. Other states will not be able to attract these workers because of federal restrictions on stem cell research funding. The technologies, drugs, and patents which will result from our studies will allow Californians to be the first people in the country to benefit from stem cell research with respect to its ability to positively affect healthcare, the economy and technological advances.
Review Summary: 
SYNOPSIS: The goal of this study is to characterize gene expression and glutamate neurotransmission in hESC-derived motor neurons(MNs). The applicant provides preliminary data that include transcription profiles using laser capture microscopy of mouse embryonic spinal MNs. First, the applicant will characterize two non-NIH approved lines (in order to avoid influences from mouse feeder cells) at 5, 15, and 25 days after hES cell derived neuroectodermal cells are treated with RA for further differentiation with respect to transcription profiling and immunocytochemistry. The applicant will then test the functionality of mature hESC-derived MNs and also provide an in vitro model of glutamate toxicity. Finally, the applicant will investigate subtype-specific expression of functional ionotropic glutamate receptors using electrophysiological methods following incubation with agonists and antagonists and specific toxins that target different glutamate receptors and transporters. SIGNIFICANCE AND INNOVATION: The applicant plans to study motor neurons isolated from hESC’s by gene profiling and immunocytochemistry and then explore the toxic effects of glutamate on these cells and neuroprotective strategies. The proposed approaches are quite standard in the field and not especially innovative. Nonetheless the topic has importance to ALS and potentially to neuroprotective mechanisms of human motor neurons. STRENGTHS: • The preliminary microarray data showing significant differences between expressed genes in neonatal MNs vs. adult MNs vs. pooled spinal cord cells are of interest • The use of the Illumina Sentrix Human Ref-8 Expression Bead Chips will provide a broad unbiased screen of genes that are expressed in hESCs • The applicant has experience culturing murine motor neurons and studying their pharmacology in vitro WEAKNESSES: • The preliminary data related to the microarray studies are of interest, but there are a number of questions that are not addressed. What specific genes were found to be overexpressed in neonatal spinal cord MNs? How many of the genes that were found to be overexpressed had confirmation by real time-RT-PCR or immunohistochemistry? How will the applicant confirm that the overexpression of particular genes in hESCs is real? • Although the Illumina Sentrix Human Ref-8 Expression Bead Chips will provide a broad unbiased screen of genes that are expressed in hESCs, this aim is clearly a descriptive one • It was not clear which gene products were planned to be investigated by immmunocytochemistry since the list provided is a somewhat small one. • The rationale for the role of glutamate neurotransmission is not clearly provided. If the PI wants to examine functionality of the cells, are there other ways besides glutamate neurotransmission that should be pursued? Are there other toxicities that should be examined besides glutamate neurotoxicity? • The application does not detail anticipated pitfalls and plans to overcome or circumvent these difficulties. • The applicant has modest productivity.

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