Neurological Disorders

Coding Dimension ID: 
303
Coding Dimension path name: 
Neurological Disorders
Funding Type: 
Basic Biology I
Grant Number: 
RB1-01367
Investigator: 
ICOC Funds Committed: 
$1 363 262
Disease Focus: 
Amyotrophic Lateral Sclerosis
Neurological Disorders
Spinal Muscular Atrophy
Spinal Cord Injury
Genetic Disorder
Pediatrics
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
oldStatus: 
Active
Public Abstract: 

One of the main objectives of stem cell biology is to create physiologically relevant cell types that can be used to either facilitate the study of or directly treat human disease. Tremendous progress towards these goals has been made in the area of motor neuron disease and spinal cord injury through the findings that motor neurons can be generated from human embryonic stem cells and induced pluripotent stem cells. These advances have made possible the creation of motor neurons from patients afflicted with neurodegenerative diseases such as amyotrophic lateral sclerosis and spinal muscular atrophy that can be studied in the laboratory to determine the root causes of these diseases. In addition, stem cell-derived motor neurons could potentially serve as replacement cells that could be introduced into the spinal cord to recover motor functions in these patients, as well as those suffering from spinal cord injuries. A major assumption, however, is that human embryonic and induced pluripotent cell-derived motor neurons are identical to their normal counterparts. Despite its relevance, few studies of human motor neuron development have been carried out, and little information on the genetic and functional similarities between stem cell- and embryo-derived motor neurons has been obtained. The proposed research will provide important new insights into the profile of human motor neurons that must be recapitulated by stem cell studies. This approach is critical given that most of our knowledge on human motor neuron development is based on animal models. In addition, work with mouse embryonic stem cell-derived motor neurons has revealed limitations in the motor neuron subtypes that can be generated in culture, something others and we have also observed in human embryonic and induced pluripotent stem cell-derived motor neurons. The differences between embryo and stem cell-derived motor neurons are currently unknown, though our preliminary studies suggest that this deficiency may result from the inability of stem cell-derived motor neurons to express key regulators of motor neuron development. We will directly test this hypothesis by examining whether artificially expressing some of these important motor neuron fate determinants can alter the classes of motor neurons formed in culture and thereby broaden their innervation potential. Since most motor neuron diseases tend to affect certain motor neuron populations more than others, and that the pattern of motor innervation is highly specific to the type of cells formed, these studies will significantly advance our understanding of how the full repertoire of motor neuron subtypes may be created from stem cells to build disease models and generate therapeutically beneficial cells.

Statement of Benefit to California: 

Neurological diseases are among the most debilitating medical conditions that affect millions of Californians each year, and many more worldwide. Few effective treatments for these diseases currently exist, in part because we know very little about the mechanisms underlying these conditions. Through the use of human embryonic stem cell and induced pluripotent stem cell technologies, it is now possible to create neurons from patients suffering from a variety of neurological disorders that can serve as the basis for cell culture-based models to study disease pathologies in an experimentally accessible setting. Our proposed research seeks to develop the means to form different classes of neurons, confirm their physiological identities, and establish a system for studying their neurological activity in a cell culture setting. The generation of these models will constitute an important step towards understanding the basis of neurological illnesses and developing a platform for the discovery of drugs that can alter disease progression and improve the productivity and quality of life for many Californians. Moreover, progress in this field will help solidify the leadership role of California in bringing stem cell research to the clinic, and stimulate the future growth of the biotechnology and pharmaceutical industries within the state.

Progress Report: 
  • The main goals of this project are to evaluate the similarities and differences between human stem cell-derived spinal motor neurons and their fetal counterparts, and to refine the techniques used to make these cells to facilitate motor neuron disease research and create therapeutically beneficial cells. In the first year of this project, we have confirmed that motor neuron generated from stem cells exhibit many molecular and physiological changes over time that closely mirror the formation of motor neurons during normal human development. There are some subtle differences, however, and our ongoing work will explore whether these discrepancies have any functional relevance. In carrying out these experiments, we also discovered new techniques by which we can create more diverse populations of motor neurons that better match the complexity seen in the spinal cord. Lastly, we have made significant progress in developing experimental assays to study the connections formed between stem cell-derived motor neurons and their muscle targets. We anticipate that these assays will serve as a valuable platform for modeling the pathology of human motor neuron diseases.
  • The main goals of this project are: 1) to evaluate the similarities and differences between human stem cell-derived spinal motor neurons and their fetal counterparts, and 2) to refine the techniques used to make these cells to facilitate motor neuron disease research and create therapeutically beneficial cells. In the second year of this project, we have documented that the initial stages of motor neuron development in stem cell cultures are very similar to the process of motor neuron formation during fetal development. However, stem cell-derived motor neurons appear to be more homogeneous than their fetal counterparts and lack several defining characteristics of mature cells. We are currently investigating the basis of these differences and whether there are any consequences on the function of the stem cell-derived neurons. We have also developed methods for evaluating the communication of stem cell-derived motor neurons with muscle cells. We anticipate that this assay platform will be valuable for modeling the pathology of neurodegenerative diseases that affect motor function. Lastly, we have obtained evidence that the forced expression of genes associated with specific motor neuron groups can strongly influence their trajectory and rate of motor axon growth, and improve innervation of limb muscles.
  • The main goals of our project are: 1) to evaluate the similarities and differences between human stem cell-derived spinal motor neurons and their fetal counterparts, and 2) to refine the techniques used to make these cells to facilitate motor neuron disease research and create therapeutically beneficial cells. In the third year of this project, we have assembled a nearly complete documentation of the developmental progression of human stem cell-derived motor neurons in cell culture compared to that seen in normal fetal development. From this analysis we conclude that the process of forming motor neurons in the culture setting faithfully replicates many aspects of their formation in the intact spinal cord. However, the types of motor neurons that are formed in stem cell cultures are more limited in their subtype diversity, which has implications for the utility of these cells as therapeutic agents and models to investigate disease mechanism. We have nevertheless found that we can extend the diversity of stem cell derived motor neurons by programming the cells to express specific proteins that promote the formation of different motor neuron subtypes. These findings suggest a general strategy for creating different functional classes of motor neurons for therapeutic uses and research applications. Lastly, we have developed two simple cell culture systems to measure the communication between motor neurons and muscle cells. Breakdown in this communication is thought to underlie many motor neuron diseases, and we anticipate that this platform will provide a means for studying the underlying pathology of these diseases, and facilitate the discovery of novel therapeutic agents.
  • The main goals of our project are: 1) to evaluate the similarities and differences between human stem cell-derived spinal motor neurons and their fetal counterparts, and 2) to refine the techniques used to make these cells to facilitate motor neuron disease research and create therapeutically beneficial cells. In the final period of this project, we have completed our documentation of the developmental progression of human stem cell-derived motor neurons in cell culture compared to that seen in normal fetal development. From this analysis we conclude that the process of forming motor neurons in the culture setting faithfully replicates many aspects of their formation in the intact spinal cord. However, the types of motor neurons that are formed in stem cell cultures are more limited in their subtype diversity, which has implications for the utility of these cells as therapeutic agents and models to investigate disease mechanism. We have nevertheless found that we can extend the diversity of stem cell derived motor neurons by programming the cells to express specific proteins that promote the formation of different motor neuron subtypes. These findings suggest a general strategy for creating different functional classes of motor neurons for therapeutic uses and research applications. Lastly, we have developed a novel cell culture system to measure the communication between motor neurons and muscle cells. Breakdown in this communication is thought to underlie many motor neuron diseases, and we anticipate that this platform will provide a means for studying the underlying pathology of these diseases, and facilitate the discovery of novel therapeutic agents.
Funding Type: 
Basic Biology I
Grant Number: 
RB1-01358
Investigator: 
Institution: 
Type: 
PI
ICOC Funds Committed: 
$1 407 076
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 

Parkinson’s disease (PD) is a neurodegenerative movement disorder that affects 1 in 100 people over the age of 60, one million people in the US and six million worldwide. Patients show a resting tremor, slowness of movement (bradykinesia), postural instability and rigidity. Parkinson's disease results primarily from the loss of neurons deep in the middle part of the brain (the midbrain), in particular neurons that produce dopamine (referred to as “dopaminergic”). There are actually two groups of midbrain dopaminergic (DA) neurons, and only one, those in the substantia nigra (SN) are highly susceptible to degeneration in Parkinson’s patients. There is a relative sparing of the second group and these are called ventral tegmental area (VTA) dopaminergic neurons. These two groups of neurons reside in different regions of the adult ventral midbrain and importantly, they deliver dopamine to their downstream neuronal targets in different ways. SN neurons deliver dopamine in small rapid squirts, like a sprinkler, whereas VTA neurons have a tap that provides a continuous stream of dopamine.

A major therapeutic strategy for Parkinsons’ patients is to produce DA neurons from human embryonic stem cells for use in transplantation therapy. However early human trials were disappointing, since a number of patients with grafts of human fetal neurons developed additional, highly undesirable motor dyskinesias. Why this occurred is not known, but one possibility is that the transplant mixture, which contained both SN and VTA DA neurons, provided too much or unregulated amounts of DA (from the VTA neurons), overloading or confusing the target region in the brain that usually receives dopamine from SN neurons in small, regular quantities. Future human trials will likely utilize DA neurons that have been made from human embryonic stem cells (hES). Since stem cells have the potential to develop into any type of cell in the body, these considerations suggest that we should devise a way to specifically produce SN neurons and not VTA neurons from stem cells for use in transplantation. However, although we can produce dopaminergic neurons from hES cells, to date the scientific community cannot distinguish SN from VTA neurons outside of their normal brain environment and therefore has no ability to produce one selectively and not the other. We do know, however, that these two populations of neurons normally form connections with different regions in the brain, and we propose to use this fact to identify molecular markers that distinguish SN from VTA neurons and to determine optimal conditions for the differentiation of hES to SN DA neurons, at the expense of VTA DA neurons. Our studies have the potential to significantly impact transplantation therapy by enabling the production of SN over VTA neurons from hES cells, and to generate hypotheses about molecules that might be useful for coaxing SN DA neurons to form appropriate connections within the transplanted brain.

Statement of Benefit to California: 

The goal of our work is to further optimize our ability to turn undifferentiated human stem cells into differentiated neurons that the brain can use as replacement for neurons damaged by disease. We focus on Parkinson’s disease, a neurodegenerative disease that afflicts 4-6 million people worldwide in all geographical locations, but which is more common in rural farm communities compared to urban areas, a criteria important for California's large farming population. In Parkinson’s patients, a small, well-defined subset of neurons, the midbrain dopaminergic neurons have died, and one therapeutic strategy is to transplant healthy replacement neurons to the patient. Our work will further our understanding of the biology of these neurons in normal animals. This will allow us to refine the process of turning human embryonic stem cells onto biologically active dopaminergic neurons that can be used in transplantation therapy. Our work will be of benefit to all Parkinson's patients including afflicted Californians. Further, this project will utilize California goods and services whenever possible.

Progress Report: 
  • Parkinson's disease results primarily from the loss of neurons deep in the middle part of the brain (the midbrain), in particular neurons that produce dopamine (referred to as “dopaminergic”). In this region of the midbrain there are actually two different groups of dopaminergic (DA) neurons, and only one of them, the neurons of the substantia nigra (SN) are highly susceptible to degeneration in patients with PD. There is a relative sparing of the second group of midbrain dopaminergic neurons, called the ventral tegmental area (VTA) dopaminergic neurons. These two groups of neurons reside close to each other in the brain and both make dopamine. They are virtually indistinguishable except for one major functional difference—they release dopamine, the transmitter that is lost in Parkinson’s patients, to their downstream neuronal targets in different ways. SN neurons deliver dopamine in small rapid squirts, like a sprinkler, whereas VTA neurons have a tap that provides a continuous stream of dopamine.
  • A major therapeutic strategy for patients with PD is to make new DA neurons from human embryonic stem cells (hES). As stem cells have the potential to develop into any type of cell in the body, these considerations suggest that we should devise a way to produce SN neurons in the absence of VTA neurons from stem cells for use in transplantation. At present although we can produce dopaminergic neurons from hES cells, the scientific community cannot distinguish SN from VTA neurons in vitro due to lack of molecular markers or a bioassay, and we are therefore unable to identify culture conditions that favor the production of one over the other,
  • In addition to releasing dopamine differently, SN and VTA neurons have axons that project to different regions of the striatum. It has been shown over the last decade that specific classes of guidance cues guide axons to their particular targets. One approach we have taken has been to investigate whether differences in axon guidance receptor expression and or responses to guidance cues in vitro might provide both markers and a bioassay that will distinguish SN from VTA neurons. Over the last year we have shown that VTA and SN neurons respond differentially to Netrin-1 and express different markers associated with the guidance cue family. We now have a bioassay and markers that distinguish these two populations of neurons in vitro and in the coming year we plan to utilize this information to identify cultures conditions that favor the production of SN over VTA neurons, from hES cells.
  • Parkinson’s disease results primarily from the loss of neurons deep in the middle part of the brain (the midbrain), in particular neurons that produce dopamine (referred to as “dopaminergic”). In this region of the midbrain there are actually two different groups of dopaminergic (DA) neurons, and only one of them, the neurons of the substantia nigra (SN) are highly susceptible to degeneration in patients with PD. There is a relative sparing of the second group of midbrain dopaminergic neurons, called the ventral tegmental area (VTA) dopaminergic neurons. These two groups of neurons reside close to each other in the brain and both make dopamine. They are virtually indistinguishable except for one major functional difference—they release dopamine, the transmitter that is lost in Parkinson’s patients, to their downstream neuronal targets in different ways. SN neurons deliver dopamine in small rapid squirts, like a sprinkler, whereas VTA neurons have a tap that provides a continuous stream of dopamine. 
A major therapeutic strategy for patients with PD is to make new DA neurons from human embryonic stem cells (hES). As stem cells have the potential to develop into any type of cell in the body, these considerations suggest that we should devise a way to produce SN neurons in the absence of VTA neurons from stem cells for use in transplantation. At present although we can produce dopaminergic neurons from hES cells, the scientific community cannot distinguish SN from VTA neurons in vitro due to lack of molecular markers or a bioassay, and we are therefore unable to identify culture conditions that favor the production of one over the other, 
In addition to releasing dopamine differently, SN and VTA neurons have axons that project to different regions of the striatum. It has been shown over the last decade that specific classes of guidance cues guide axons to their particular targets. One approach we have taken has been to investigate whether differences in axon guidance receptor expression and or responses to guidance cues in vitro might provide both markers and a bioassay that will distinguish SN from VTA neurons. We showed previously that VTA and SN neurons respond differentially to Netrin-1 and express different markers associated with the guidance cue family. Also, in this year using backlabeling, laser capture and microarray analysis of SN vs VTA neurons, we have identified a number of genes expressed in on or the other population. We now have a bioassay and markers that distinguish these two populations of neurons in vitro and in the coming year we plan to utilize this information to identify cultures conditions that favor the production of SN over VTA neurons, from hES cells.
  • Parkinson's disease (PD) is a neurodegenerative movement disorder that affects more than six million people worldwide. The main symptoms of the disease result from the loss of neurons from the midbrain that produce dopamine (referred to as "dopaminergic" or DA neurons).Human embryonic stem cells (hESC) offer an exciting opportunity to treat Parkinson’s disease by transplanting hESC-derived DA neurons to replace those that have died. There are actually two groups of midbrain DA neurons in the human brain. Those from the substantia nigra (SN) are highly susceptible to degeneration in Parkinson's patients while those from the ventral tegmental area (VTA) are not. These two types of neurons have similar features but have different functions and it is important to ensure that DA neurons from hESC are the correct SN type before they are used in therapy. The primary goal of this research was to study these two neuronal types in animals and determine if the distinguishing features discovered in mice or rats can be used to more easily recognize and purify SN-type DA neurons made from hESC.
  • One of the discoveries made in this research is that SN and VTA neurons show differences in how they make connections within the brain. We have been able to identify some of the molecules that guide each neuron to connect to it appropriate target and have found that SN and VTA neurons placed in the petri dish can be distinguished from each other by their response to guidance molecules. Work in the final period of this grant has focused on testing guidance response in hESC-derived DA neurons and we have found that many of the neurons produced from hESC do show SN-like responses to guidance molecules. This discovery is being further developed as a screening tool to help guide our ongoing efforts to make increasingly pure populations of DA neurons from hESC.
  • Future human trials will likely utilize such DA neurons but since embryonic stem cells have the potential to develop into any type of cell in the body, it is important to ensure that the production methods used to make a therapeutic product for Parkinson’s disease do indeed specifically produce SN neurons. Prior to the research supported under this CIRM grant, the scientific community was not able to distinguish SN from VTA neurons outside of their normal brain environment and therefore had no ability to confirm whether a method produced one type selectively and not the other. Further refinements of the assay tools developed in our research may provide a practical means of quantifying the purity of a DA neuron preparation. This would have a significant impact transplantation therapy as well as provide useful insights into the molecular mechanisms that underlie proper connectivity and function of SN and VTA DA neurons in humans.

Pages