Neurological Disorders

Coding Dimension ID: 
303
Coding Dimension path name: 
Neurological Disorders

Optimization of guidance response in human embryonic stem cell derived midbrain dopaminergic neurons in development and disease

Funding Type: 
SEED Grant
Grant Number: 
RS1-00271
ICOC Funds Committed: 
$633 170
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
A promising approach to alleviating the symptoms of Parkinson’s disease is to transplant healthy dopaminergic neurons into the brains of these patients. Due to the large number of transplant neurons required for each patient and the difficulty in obtaining these neurons from human tissue, the most viable transplantation strategy will utilize not fetal dopaminergic neurons but dopaminergic neurons derived from human stem cell lines. While transplantation has been promising, it has had limited success, in part due to the ability of the new neurons to find their correct targets in the brain. This incorrect targeting may be due to the lack of appropriate growth and guidance cues as well as to inflammation in the brain that occurs in response to transplantation, or to a combination of the two. Cytokines released upon inflammation can affect the ability of the new neurons to connect, and thus ultimately will affect their biological function. In out laboratory we have had ongoing efforts to determine the which guidance molecules are required for proper targeting of dopaminergic neurons during normal development and we have identified necessary cues. We now plan to extend these studies to determine how these critical guidance cues affect human stem cell derived dopaminergic neurons, the cells that will be used in transplantation. In addition, we will examine how these guidance cues affect both normal and stem cell derived dopaminergic neurons under conditions that are similar to the diseased and transplanted brain, specifically when the brain is inflamed. Ultimately, an understanding of how the environment of the transplanted brain influences the ability of the healthy new neurons to connect to their correct targets will lead to genetic, and/or drug-based strategies for optimizing transplantation therapy.
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 onParkinson’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 (Van Den Eeden et al., 2003), 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 ES 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. In addition to the direct benefit in improving PD therapies, discoveries from this work are also likely to generate substantial intellectual property and further boost clinical and biotechnical development efforts in California.
Progress Report: 
  • A promising approach to alleviating the symptoms of Parkinson's disease is to transplant healthy dopaminergic neurons into the brains of these patients. Due to the large number of transplant neurons required for each patient and the difficulty in obtaining these neurons from human tissue, the most viable transplantation strategy will utilize not fetal dopaminergic neurons but dopaminergic neurons derived from human stem cell lines. While transplantation has been promising, it has had limited success, in part due to the ability of the new neurons to find their correct targets in the brain. This incorrect targeting may be due to the lack of appropriate growth and guidance cues as well as to inflammation in the brain that occurs in response to transplantation, or to a combination of the two. Cytokines released upon inflammation can affect the ability of the new neurons to connect, and thus ultimately will affect their biological function. In out laboratory we have been examining which guidance molecules are required for proper targeting of dopaminergic neurons during normal development and have identified necessary cues. We have now extended these studies to determine that two of the molecules have dramitc effects on dopaminergic neurons made from human embryonic stem cellls and that at least in vitro, cytokines do not mask these effects. Ultimately, an understanding of how the environment of the transplanted brain influences the ability of the healthy new neurons to connect to their correct targets will lead to genetic, and/or drug-based strategies for optimizing transplantation therapy.

Development of human ES cell lines as a model system for Alzheimer disease drug discovery

Funding Type: 
SEED Grant
Grant Number: 
RS1-00247
ICOC Funds Committed: 
$492 750
Disease Focus: 
Alzheimer's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Cell Line Generation: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Alzheimer disease (AD) is a progressive neurodegenerative disorder that currently affects over 4.5 million Americans. By the middle of the century, the prevalence of AD in the USA is projected to almost quadruple. As current therapies do not abate the underlying disease process, it is very likely that AD will continue to be a clinical, social, and economic burden. Progress has been made in our understanding of AD pathogenesis by studying transgenic mouse models of the disease and by utilizing primary neuronal cell cultures derived from rodents. However, key proteins that are critical to the pathogenesis of this disease exhibit many species-specific differences at both a biophysical and functional level. Additional species differences in other as yet unidentified AD-related proteins are likely to also exist. Thus, there is an urgent need to develop novel models of AD that recapitulate the complex array of human proteins involved in this disease. Cell culture-based models that allow for rapid high-throughput screening and the identification of novel compounds and drug targets are also critically needed. To that end we propose to model both sporadic and familial forms of AD by generating two novel human embryonic stem cell lines (hES cells). Differentiation of these lines along a neuronal lineage will provide researchers with an easily accessible and reproducible neuronal cell culture model of AD. These cells will also allow high-throughput screening and experimentation in neuronal cells with a species-relevant complement of human proteins. In Aim 1 we will develop and characterize hES cell lines designed to model both sporadic and familial forms of AD. To model sporadic AD we will stably transfect HUES7 hES cells (developed by Douglas Melton) with lentiviral constructs coding for human wild type amyloid precursor protein (APP-695) under control of the human APP promoter. APP is well expressed within hES cells and upregulated upon neuronal differentiation. To model familial AD and generate cells that exhibit a more aggressive formation of oligomeric A species we will also develop a second hES cell line stably transfected with human APP that includes the Arctic (E693G) mutation.In Aim 2 we will utilize our wild-type APP hES cells to perform a high-throughput siRNA screen. We will utilize AMAXA reverse-nucleofection in conjunction with a human druggable genome siRNA array (Dharmacon) that targets 7309 genes considered to be potential therapeutic targets. Following transfection conditioned media will be examined by a sensitive ELISA to identify novel targets that modulate A levels. In addition a Thioflavin S assay will determine any effects on A aggregation. Follow-up experiments will confirm promising candidates identified in the high-throughput screen. Taken together these studies aim to establish novel AD-specific hES cell lines and identify promising new therapeutic targets for this devastating disease.
Statement of Benefit to California: 
Alzheimer disease (AD) is a progressive neurodegenerative disorder that currently affects over 500 thousand Californians. As the baby-boomer generation ages the prevalence of AD in California is projected to almost quadruple such that 1 in every 45 individuals will be afflicted. As current therapies do not abate the underlying disease process, it is very likely that AD will continue to be a major clinical, social, and economic burden. Some estimates have even suggested that AD alone may bankrupt the current Californian health care system. Progress has been made in our understanding of AD by studying rodent-based models of the disease. However, key proteins that are critical to the disease exhibit many species-specific differences at both a biophysical and functional level. Thus, there is an urgent need to develop novel models of AD that exhibit the complex array of human proteins involved in this disease. Cell culture-based models that also allow for rapid high-throughput screening and the identification of novel compounds and drug targets are also in critical need. The proposed studies aim to utilize human embryonic stem (hES) cells to establish a novel cell culture based model of Alzheimer’s disease. Once developed these cells will provide Californian researchers with a unique tool to investigate genes and proteins that influence the progress of AD. In this proposal we will also utilize these hES cells to perform a high-throughput screen of over 7300 genes to identify multiple novel drug targets that may critically regulate the development of this disease. Taken together these studies aim to establish novel AD-specific hES cell lines that can be utilized by multiple Californian researchers to identify promising new therapeutic targets for this devastating disease.
Progress Report: 
  • Alzheimer’s disease (AD) is the most common age-related neurodegenerative disorder. It is characterized by an irreversible loss of neurons accompanied by the accumulation of extracellular amyloid plaques and intraneuronal neurofibrillary tangles. Currently, 5.3 million Americans are afflicted with this insidious disorder, including over 588,000 in the State of California alone. Mouse models of AD have contributed significantly to our understanding of the proteins and factors involved in the pathology of AD. However, there are critical differences between mouse and human cell physiology that likely dramatically influence the development of AD-related pathologies. Hence, there is an urgent need to develop novel human neuronal cell-based models of AD.
  • To achieve this goal, we have generated stable human embryonic stem cell (hES) lines over-expressing the gene for human amyloid precursor protein (APP). We succeeded in creating several lines of hES cells that stably express either wild-type (unaltered) APP or APP that includes rare familial mutations known to cause early-onset cases of AD. In each line, transgene expression is driven under control of the human APP proximal promoter. Mutant versions of APP utilized include the “Swedish” mutation which increases production of Aß and the “Arctic” mutation which increases the assembly and accumulation of synaptotoxic Aß oligomers and protofibrils. The generation of lines that harbor familial mutations in APP both provides an aggressive model of AD, to facilitate the identification of targets that modulate not only Aß production but also the assembly of toxic oligomeric species.
  • In addition to generating stable HUES7 and H9 cell lines over-expressing mutant and wild type forms of APP, we also succeeded in establishing a neuronal differentiation protocol which results in 80% of cells adopting a mature neuronal fate. Importantly, we have also verified by biochemical measures that APP-overexpressing cells produce significantly elevated levels of Aß. As a result we are now preparing to utilize these novel cell lines to identify and examine genes that regulate Aß production and hence the development of AD.
  • Alzheimer’s disease (AD) is the most common age-related neurodegenerative disorder. Currently, 5.3 million individuals are afflicted with this insidious disorder, including over 588,000 in the State of California alone. Unfortunately, existing therapies provide only palliative relief. Although transgenic mouse models and cell culture experiments have contributed significantly to our understanding of the proteins and factors involved in the pathology of AD, these approaches are beset by certain critical limitations. Most notably, mouse models by definition are not based on human cells and cell culture models have been limited to non-human or non-neuronal cells. Hence, there is an urgent need to develop a human neuronal cell-based model of AD. To address this need, we have engineered human embryonic stem cell lines to overexpress mutant human genes that cause early-onset familial AD. These novel stem cell lines will provide a valuable system to test therapies and enhance our understanding of the mechanisms that mediate this devastating disease. Interestingly, we have found that overexpression of these AD-related genes can trigger the rapid differentiation of human embryonic stem cells into neuronal cells. We have examined the mechanisms involved and anticipate that our findings may provide a novel and rapid method to generate neurons from embryonic stem cells.

New Chemokine-Derived Therapeutics Targeting Stem Cell Migration

Funding Type: 
SEED Grant
Grant Number: 
RS1-00225
ICOC Funds Committed: 
$759 000
Disease Focus: 
Spinal Cord Injury
Neurological Disorders
Stroke
Trauma
Stem Cell Use: 
Adult Stem Cell
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
This proposal describes a sharply-focused, timely, and rigorous effort to develop new therapies for the treatment of injuries of the Central Nervous System (CNS). The underlying hypothesis for this proposal is that chemokines and their receptors (particularly those involved in inflammatory cascades) actually play important roles in mediating the directed migration of human neural stem cells (hNSCs) to, as well as engagement and interaction with, sites of CNS injury, and that understanding and manipulating the molecular mechanism of chemokine-mediated stem cell homing and engagement will lead to new, better targeted, more specific, and more efficacious chemokine-mediated stem cell-based repair strategies for CNS injury. In recent preliminary studies, we have discovered and demonstrated the important role of chemokine SDF-1-alpha and its receptor CXCR4 in mediating the directed migration of hNSCs to sites of CNS injury. To manipulate this SDF-1-alpha/CXCR4 pathway in stem cell migration, we have developed Synthetically and Modularly Modified Chemokines (SMM-chemokines) as highly potent and specific therapeutic leads. Here in this renewal application we propose to extend our research into a new area of stem cell biology and medicine involving chemokine receptors such as CXCR4 and its ligand SDF-1. Specifically, we will design more potent and specific analogs of SDF-1-alpha to direct the migration of beneficial stem cells toward the injury sites for the repair process.
Statement of Benefit to California: 
This proposal describes a sharply-focused, timely, and rigorous effort to develop new therapies for the treatment of injuries of the Central Nervous System (CNS). CNS injuries and related disorders such as stroke, traumatic brain injury and spinal cord injury are significant health issues in the nation including the state of California. The new stem cell-based therapies to be developed from this application will have important clinical application in patients with these diseases in California.
Progress Report: 
  • Human neural stem cells (hNSCs) expressing CXCR4 have been found to migrate in vivo toward an infarcted area that are representative of central nervous system (CNS) injuries, where local reactive astrocytes and vascular endothelium up-regulate the SDF-1α secretion level and generate a concentration gradient. Exposure of hNSCs to SDF-1α and the consequent induction of CXCR4-mediated signaling triggers a series of intracellular processes associated with fundamental aspects of survival, proliferation and more importantly, proper lamination and migration during the early stages of brain development [1]. To date, there is no crystal structure available for chemokine receptors [2, 3]. Structural and modeling studies of SDF-1α and D-(1~10)-L-(11~69)-vMIP-II in complexes with CXCR4 TM helical regions led us to a plausible “two-pocket” model for CXCR4 interaction with agonists or antagonists. [4-6] In this study, we extended the employment of this model into the novel design strategy for highly potent and selective CXCR4 agonist molecules, with potentials in activating CXCR4-mediated hNSC migration by mimicking a benign version of the proinflamatory signal triggered by SDF-1α. Successful verification of directed, extensive migration of hNSCs, both in vitro and in transplanted uninjured adult mouse brains, with the latter manifesting significant advantages over the natural CXCR4 agonist SDF-1α in terms of both distribution and stability in mouse brains, strongly supports the effectiveness and high potentials of these de novo designed CXCR4 agonist molecules in optimizing directed migration of transplanted human stem cells during the reparative therapeutics for a broad range of neurodegenerative diseases in a more foreseeable future.
  • Our final progress report is divided into 3 subsections, each addressing progress in the 3 fundamental areas of investigation for the successful completion of this project:
  • (1) De-novo design and synthesis of CXCR4-specific SDF-1α analogs.
  • (2) In vitro studies on validating biological potencies of molecules in (1) in activating CXCR4 down-stream signaling.
  • (3) In vivo studies on migration of transplanted neural precursor cells (NPCs) in co-administration of molecules with validated biological activities in (2).

Identifying small molecules that stimulate the differentiation of hESCs into dopamine-producing neurons

Funding Type: 
SEED Grant
Grant Number: 
RS1-00215
ICOC Funds Committed: 
$564 309
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
In this application, we propose to identify small molecule compounds that can stimulate human embryonic stem cells to become dopamine-producing neurons. These neurons degenerate in Parkinson’s disease, and currently have very limited availability, thus hindering the cell replacement therapy for treating Parkinson’s disease. Our proposed research, if successful, will lead to the identification of small molecule compounds that can not only stimulate cultured human embryonic stem cells to become DA neurons, but may also stimulate endogenous brain stem cells to regenerate, since the small molecule compounds can be made readily available to the brain due to their ability to cross the blood-brain barrier. In addition, these small molecule compounds may serve as important research tools, which can tell us the fundamental biology of the human embryonic stem cells.
Statement of Benefit to California: 
The proposed research will potentially lead to a cure for the devastating neurodegenerative, movement disorder, Parkinson’s disease. The proposed research will potentially provide important research tools to better understand hESCs. Such improved understanding of hESCs may lead to better treatments for a variety of diseases, in which a stem-cell based therapy could make a difference.
Progress Report: 
  • Parkinson’s disease is the most common movement disorder due to the degeneration of brain dopaminergic neurons. One strategy to combat the disease is to replenish these neurons in the patients, either through transplantation of stem cell-derived dopaminergic neurons, or through promoting endogenous dopaminergic neuronal production or survival. We have carried out a small molecule based screen to identify compounds that can affect the development and survival of dopaminergic neurons from pluripotent stem cells. The small molecules that we have identified will not only serve as important research tools for understanding dopaminergic neuron development and survival, but potentially could also lead to therapeutics in the induction of dopaminergic neurons for treating Parkinson’s disease.

Generation of forebrain neurons from human embryonic stem cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00205
ICOC Funds Committed: 
$612 075
Disease Focus: 
Aging
Alzheimer's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
The goal of this proposal is to generate forebrain neurons from human embryonic stem cells. Our general strategy is to sequentially expose ES cells to signals that lead to differentiation along a neuronal lineage, and to select for cells that display characteristics of forebrain neurons. These cells would then be used in transplantation experiments to determine if they are able to make synaptic connections with host neurons. If successful these experiments would provide a therapeutic strategy for the treatment of Alzheimer’s disease and other disorders that are characterized by loss of forebrain neurons. Currently there is no effective treatments for Alzheimer’s disease, and with an aging baby-boomer population, the incidence of this disease is likely to increase sharply. One of the few promising avenues to treat Alzheimer’s is the possibility of cell replacement therapy in which the neurons lost could be replaced by transplanted neurons. Embryonic stem cells, which have the ability to differentiate into various cells of the body, could be a key component of such a therapy if we can successfully differentiate them into forebrain neurons.
Statement of Benefit to California: 
Alzheimer’s disease is a devastating sporadic neurological disorder that places all of us at risk. As the California population ages, there will be a significant increase in the incidence of Alzheimer’s disease, and the medical and financial cost on the state will be severe. There are currently no effective treatments for this disorder, and one of the few promises is the possibility of transplantation therapy to replace the neurons that are lost in the disease. Being able to generate forebrain neurons from human embryonic stem cells would provide a key tool in the fight against this disease. Needless to say, the development of an effective cell replacement therapy would not only be of immense medical significance as we care for our senior population, it will also greatly relieve the financial burden associated with the care of Alzheimer’s patients, which is often borne by the state.
Progress Report: 
  • The goal of this proposal was to generate forebrain neurons from human embryonic stem cells. Our general strategy was to sequentially expose ES cells to signals that would lead the cells to acquire characteristics typical of differentiated brain cells that are lost in disorders such as Alzheimer's Disease. The most important advance of the research was our ability to achieve this goal. We now have a well-developed protocol that can be used to generate forebrain cells in culture. We have found that these cells not only express genes typical of these cells, they extend axons and dendrites and can make synaptic connections. These cells could be very useful for transplantation studies, as well as for developing cell culture models of Alzheimer's disease. Finally, we have discovered that the same protocol is effective in generating forebrain neurons from iPS cells, attesting to the general usefulness of this strategy.

In vitro differentiation of hESCs into corticospinal motor neurons

Funding Type: 
SEED Grant
Grant Number: 
RS1-00170
ICOC Funds Committed: 
$500 000
Disease Focus: 
Amyotrophic Lateral Sclerosis
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Amyotrophic lateral sclerosis (ALS) is a rapidly progressive, fatal neurological disease that leads to the degeneration of motor neurons in the brain and in the spinal cord. There are currently 20,000 ALS patients in the United States, and 5,000 new patients are diagnosed every year. Unfortunately no cure has been found for ALS. The only medication approved by the FDA to treat ALS can only slow the disease’s progression and prolong life by a few months in some patients. Thus it is critical to explore other therapeutic strategies for the treatment of ALS such as cell replacement strategy. Because of the ability to generate many different cell types, human embryonic stem cells (hESCs) may potentially serve as a renewable source of cells for replacing the damaged cells in diseases. However, transplanting ESCs directly may cause tumor growth in patients. To support cell transplants, it is important to develop methods to differentiate hESCs into the specific cell types affected by the disease. In this application, we propose to develop an effective method to differentiate hESCs into corticospinal motor neurons (CSMNs), the neurons in the cerebral cortex that degenerate in ALS. We will test whether these CSMNs generated from hESCs in culture conditions can form proper connections to the spinal cord when transplanted into mouse brains. To direct hESCs to become the CSMNs, it is critical to establish a reliable method to identify human CSMNs. Recent progress in developmental neuroscience have identified genes that are specifically expressed in the CSMNs in mice. However no information is available for identifying human CSMNs. We hypothesize that CSMN genes in mice will be reliable markers for human CSMNs. To test this hypothesis we will investigate whether mouse CSMN markers are specifically expressed in the human CSMNs. The therapeutic application of hESCs to replace damaged CSMNs in ALS depends on the ability to direct hESCs to develop into CSMNs. Currently a reliable condition to direct hESCs to differentiate into CSMNs has not been established. We will attempt to differentiate hESCs into CSMNs based on the knowledge gained from studying the development of nervous system. We will achieve this goal in two steps: first we will culture hESCs in a condition to make them become progenitors cells of the most anterior region of the brain; then we will culture these progenitors to become neurons of the cerebral cortex, particularly the CSMNs. We will study the identities of these neurons using the CSMN markers that we have proposed to identify. To apply the cell replacement strategy to treat ALS, it will be critical to test if human CSMNs generated from cultured hESCs can form proper connections in an animal model. We will transplant the CSMNs developed from hESCs into the brains of mice and test whether they can form connections to the spinal cord. When carried out, the proposed research will directly benefit cell replacement therapy for ALS.
Statement of Benefit to California: 
Amyotrophic lateral sclerosis (ALS) is a rapidly progressive, fatal neurological disease that leads to the degeneration of motor neurons in the brain and in the spinal cord. There are currently 20,000 ALS patients in the United States, and 5,000 new patients are diagnosed every year. Unfortunately no cure has been found for ALS. The only medication approved by the FDA to treat ALS can only slow the disease’s progression and prolong life by a few months in some patients. Thus it is critical to explore other therapeutic strategies for the treatment of ALS such as cell replacement strategy. Because of the ability to generate many different types of cells, human embryonic stem cells (hESCs) may potentially serve as a renewable source of cells for replacing the damaged cells in diseases. However, transplanting ESCs directly may cause tumor growth in patients. To support cell transplants, it is important to develop methods to differentiate hESCs into the specific cell types affected by the disease. In this application, we propose to develop an effective method to differentiate hESCs into corticospinal motor neurons (CSMNs), the neurons in the cerebral cortex that degenerate in ALS. We will test whether these CSMNs generated from hESCs in culture conditions can form proper connections to the spinal cord when transplanted into mouse brains. Everyday, 15 people die from ALS. For patients diagnozied with ALS, time is running out very fast. It is critical to explore novel therapeutic strategies for this rapidly progressive and fatal disease. The research proposed in this application may provide the basis for a novel cell replacement therapy for ALS, thus it will greatly benefit the State of California and everyone in the State.
Progress Report: 
  • Corticospinal motor neurons are affected in motor neuron diseases and damaged in spinal cord injuries. In this grant application, we proposed to induce human embryonic stem cells to generate corticospinal motor neurons. In this past grant period, we have generated neurons that express the corticospinal motor neuron genes. We are currently characterizing the cell types of theses neurons in detail. In the near future we will transplant them into the brains in mice to test whether they can form functional neural circuits.
  • In the past grant period, we have been continuing to generate brain neurons from cultured human embryonic stems. We have been determining what types of neurons are generated using our protocol. We are testing the functions of these neurons.

MGE Enhancers to Select for Interneuron Precursors Produced from Human ES Cells

Funding Type: 
Basic Biology II
Grant Number: 
RB2-01602
ICOC Funds Committed: 
$1 387 800
Disease Focus: 
Epilepsy
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
There are now viable experimental approaches to elucidate the genetic and molecular mechanisms that underlie severe brain disorders through the generation of stem cells, called iPS cells, from the skin of patients. Scientists are now challenged to develop methods to program iPS cells to become the specific types of brain cells that are most relevant to each specific brain disease. For instance, there is evidence that defects in cortical interneurons contribute to epilepsy, autism and schizophrenia. The experiments proposed in this grant application aim to understand basic mechanisms that underlie the development of cortical interneurons. We are discovering regulatory elements (called enhancers) in the human genome that control gene expression in developing interneurons. We have three experimental Aims. In Aim 1, we will study when and where these enhancers are expressed during mouse brain development. We will concentrate on identifying enhancers that control gene expression during development of specific types of cortical interneurons, although we hope to use this approach for additional cell types. Once we identify and characterize where and when these enhancers are active, in Aim 2 we will use the enhancers as tools in human stem cells to produce specific types of cortical interneurons in the test tube. The enhancers will be used to express proteins in the stem cells that will enable us purify only those cells that have specific properties (e.g. properties of cortical interneurons). In Aim 3 we will explore whether the human brain produces cortical interneurons in the same way as the mouse brain; this information is essential to identify molecular markers on the developing interneurons that could be used for further characterization and purification of the interneurons that we care generating in Aim 2. We want to emphasize that while the experiments focus on cortical interneuron subtypes, our work has general implications for the other types of brain cells our labs study, such as cortical and striatal neurons. In sum, the basic science mechanisms that we will discover will provide novel insights into how to generate specific types of neurons that can be used to study and treat brain diseases.
Statement of Benefit to California: 
Large numbers of California residents are stricken with severe medical disorders affecting the function of their brain. These include epilepsy, Parkinson’s Disease, Alzheimer’s Disease, Huntington’s Disease, Autism and Schizophrenia. For instance, a recent report from the Center for Disease Control and Prevention [www.cdc.gov/epilepsy/] estimates that 1 out of 100 adults have epilepsy. In California, epilepsy is one of the most common disabling neurological conditions, with approximately 140,000 affected individuals. The annual cost estimates to treat epilepsy range from $12 to $16 billion in the U.S. Currenlty up to one-third of these patients are not receiving adequate treatment, and may benefit from a cell-based transplantation therapy that we are currently exploring with our work in mice. There are now viable experimental approaches to elucidate the genetic and molecular mechanisms that underlie these neuropsychiatric disorders through the generation a stem cells, called iPS cells, from the skin of patients. Scientists are now challenged to develop methods to program iPS cells to become the specific types of brain cells that are most relevant to each specific brain disease. For instance, there is evidence that defects in cortical interneurons contribute to epilepsy, autism and schizophrenia. The experiments proposed in this grant application aim to understand basic mechanisms that underlie the development of cortical interneurons. We are discovering regulatory elements (called enhancers) in the human genome that control gene expression in developing interneurons. Our experiments will help us understand fundamental mechanisms that govern development of these cells. Furthermore, we have designed experiments that harness these enhancers to drive the production of specific subtypes of these cells from human stem cells. This will open the door to making these types of neurons from iPS cells to study human disease, and potentially to the production of these neurons for transplantation into patients whose interneurons are deficient in regulating their brain function. Furthermore, the approach we describe is general and readily applicable to the generation of other brain cells. Thus, the results from these studies will provide essential and novel basic information for understanding and potentially treating severe brain disorders.
Progress Report: 
  • We have been developing new methods to identify the products of stems cells that are differentiated in tissue culture dished. We are focusing on generating a specific type of neuron - cortical interneuron. To this end, we have identified specific sequences in the human genome that drive gene expression in the immature cortical interneurons. Results from the first year of our work provide evidence that our method to use these gene expression elements is working to help us identify cortical interneurons.
  • We have identified 5 gene regulatory elements (enhancers) that can promote gene expression in a specific type of neuronal precursor and neuron. We found that these enhancers can be used to aid in the identification and isolation of these types of cells from embryonic stem cells. In other studies, our group is testing the feasibility of using these types of cells to ameliorate neurological disorders, such as epilepsy.
  • We have identified 5 gene regulatory elements (enhancers) that can promote gene expression in a specific type of neuronal precursor and neuron. We found that these enhancers can be used to aid in the identification and isolation of these types of cells from embryonic stem cells. In other studies, our group is testing the feasibility of using these types of cells to ameliorate neurological disorders.

Role of the microenvironment in human iPS and NSC fate and tumorigenesis

Funding Type: 
Basic Biology II
Grant Number: 
RB2-01496
ICOC Funds Committed: 
$1 284 921
Disease Focus: 
Spinal Cord Injury
Neurological Disorders
Collaborative Funder: 
Japan
Stem Cell Use: 
Adult Stem Cell
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
Multipotent Neural Stem Cells (NSC) can be derived from adult central nervous system (CNS) tissue, embryonic stem cells (ESC), or iPSC and provide a partially committed cell population that has not exhibited evidence of tumorigenesis after long term CNS transplantation. Transplantation of NSC from these different sources has been shown by multiple investigators in different CNS injury and disease paradigms to promote recovery or ameliorate disease. Additionally, both {REDACTED} groups have shown that human NSCs transplanted in the subacute period after spinal cord injury promote functional recovery. While the role of the host immune response has been considered in the context of immune-rejection, predominantly regarding the T-cell response, the consequence of an ongoing inflammatory response within the context of the tissue microenvironment for cell fate, migration, and integration/efficacy has been largely overlooked. Critically, the tumorigeneis, fate, migration, and integration/repair potential of a stem cell is driven by: 1) the intrinsic properties of cell programming, e.g., the type and source of cell / means used to derive the cell, and maintenance/differentiation of the cell in vitro; and 2) the extrinsic factors the cell encounters. Variations in the intrinsic properties of the cell may affect the potential of that cell for uncontrolled proliferation or the response of the cell to extrinsic factors that it later encounters, defining its fate, migration, and integration/repair potential. The {REDACTED} group has demonstrated that iPS-derived neurospheres (iPS-NS) exhibit a surprisingly large degree of variation in tumorigenesis potential after CNS transplantation, which is correlated with tissue source as well as differentiation and NS forming capacity. Moreover, the intrinsic properties of hNSC populations derived from different cell sources have not been broadly characterized; in fact, {REDACTED} has published the first data in the field demonstrating the differences in fate and integration/repair potential between primary and secondary neurospheres generated via in vitro differentiation of mouse or human ESC and iPSC. In parallel, {REDACTED} has shown profound differences in the response of NSC derived from human tissue versus hESC to extrinsic signals. Together, these data suggest that both characterization of the intrinsic properties of NSCs derived from different sources is essential for our understanding of the basic biology of these cells. Investigation of molecules and signaling pathways directing hNSC fate choices in the injured CNS microenvironment will yield new insight into the mechanisms of fate and migration decisions in these cell populations.
Statement of Benefit to California: 
Multipotent Neural Stem Cells (NSC) can be derived from adult central nervous system (CNS) tissue, embryonic stem cells (ESC), or induced pluripotent cells (iPSC) and provide a partially committed cell population that has not exhibited evidence of tumorigenesis after long term CNS transplantation. Transplantation of NSC from these different sources has been shown by multiple investigators in different CNS injury and disease paradigms to promote recovery or ameliorate disease. Accordingly, stem cell based therapeutics such as these have the potential to treat a variety of traumatic, congenital, and acquired human conditions. However, while much progress has been made, translational research with human stem cell populations will remain limited by the progress of the fundamental understanding of the basic biology of these cells. The {REDACTED} group has pioneered understanding the critical role of timing in considering cell transplantation therapies. More recently, this group has focused on the neural induction of mouse- and human-derived iPSC and tested the potential of these cell populations for spinal cord injury treatment in animal models. {REDACTED} has established the NOD-scid mouse as a model for experimental neurotransplantation for xenograft studies, characterizing the relationship between transplant timing, engraftment outcome, cell fate, host remyelination, and functional recovery. Recently, this group has focused on how the innate inflammatory response influences cell fate and migration. In this collaborative proposal, researchers from California and Japan propose to combine their expertise to characterize and investigate some of the most fundamental aspects of the intrinsic properties of, and extrinsic factors influencing, human induced pluripotent (hiPSC) and human embryonic (hESC) stem cells, pooling knowledge and expertise in stem cell and animal model paradigms. The experiments proposed investigate the basic cellular and molecular mechanisms underlying the role of the host environment in stem cell fate regulation, and the relationship between reprogramming and tumorigenic/fate potential of hiPS-NSC in vitro and after transplantation, and key to this collaborative effort, the interface of these two aspects of basic stem cell biology. Critically, this international collaboration combines the expertise of two of the most advanced laboratories in translational stem cell biology to address several key unresolved questions in the control of cell fate, and will promote sharing of resources, data, and techniques between these labs to advance the field. Ultimately, the collaborative work proposed may permit the development of strategies to refine cellular reprogramming techniques, alter in vitro differentiation strategies, or manipulate the microenvironment to maximize the window for potential stem cell-based neurotherapeutics.
Progress Report: 
  • Multipotent Neural Stem Cells (NSC) can be derived from adult and fetal central nervous system (CNS) tissue, embryonic stem cells (ESC), or iPSC and provide a partially committed cell population that has not exhibited evidence of tumorigenesis after long term CNS transplantation. Transplantation of NSC from these different sources has been shown by multiple investigators in different CNS injury and disease paradigms to promote recovery or ameliorate disease. Additionally, both Dr. Okano and Dr. Anderson’s groups have shown that human NSCs transplanted in the subacute period after spinal cord injury promote functional recovery. While the role of the host immune response has been considered in the context of immune-rejection, predominantly regarding the T-cell response, the consequence of an ongoing inflammatory response within the context of the tissue microenvironment for cell fate, migration, and integration/efficacy has been largely overlooked. Critically, the tumorigeneis, fate, migration, and integration/repair potential of a stem cell is driven by: 1) the intrinsic properties of cell programming, e.g., the type and source of cell / means used to derive the cell, and maintenance/differentiation of the cell in vitro; and 2) the extrinsic factors the cell encounters. Variations in the intrinsic properties of the cell may affect the potential of that cell for uncontrolled proliferation or the response of the cell to extrinsic factors that it later encounters, defining its fate, migration, and integration/repair potential. The Nakamura/Okano group has demonstrated that iPS-derived neurospheres (iPS-NS) exhibit a surprisingly large degree of variation in tumorigenesis potential after CNS transplantation, which is correlated with tissue source as well as differentiation and NS forming capacity. Moreover, the intrinsic properties of hNSC populations derived from different cell sources have not been broadly characterized; in fact, Dr. Okano’s group has published the first data in the field demonstrating the differences in fate and integration/repair potential between primary and secondary neurospheres generated via in vitro differentiation of mouse or human ESC and iPSC. In parallel, Dr. Anderson’s group has shown profound differences in the response of NSC derived from human fetal tissue versus hESC to extrinsic signals. Together, these data suggest that both characterization of the intrinsic properties of NSCs derived from different sources is essential for our understanding of the basic biology of these cells. Investigation of molecules and signaling pathways directing hNSC fate choices in the injured CNS microenvironment will yield new insight into the mechanisms of fate and migration decisions in these cell populations.
  • Progress has been excellent in the first year, as has communication between the groups.
  • The Nakamura/Okada/Okano laboratory has regularly shared and updated us on these important findings and the progress of Aim 1 at Keio University via emails, live phone conferences and face-to-face meetings. The latest meeting occurred at the International Stem Cell Meeting in Toronto (ISSCR, June 2011), where safety and efficacy data of the initial screenings of numerous hiPS cell lines are shared and discussed which will have a significant impact on which cell lines we will work with under Aims 2 and 3.
  • Additionally, the Anderson laboratory took the additional step of focusing on xeno-free cells for this grant, with the goal of advancing future knowledge of utility for clinical translation based on CIRM funding. Xeno-free cells are cells that are cultured under conditions in which they are not exposed to animal proteins. Towards this goal, we have successfully transitioned multiple ESC and iPSC lines to xeno-free conditions for both maintenance, and successfully differentiated these lines to a neural stem cell lineage under parallel conditions. Moreover, by taking this step we have significantly enhanced the comparability of different cell lines for intrinsic properties and extrinsic influences, enhancing the potential impact of this work in increasing our basic understanding of stem cell biology, and how to harness it. Finally, we have conducted the first of our experiments testing the role of cell intrinsic properties in defining responses to the in vitro and in vivo microenvironment. Our data suggest that there are clear differences in intrinsic properties between cell lines, consistent with our initial hypothesis.
  • Although the role of the host immune response has been considered in the context of immune-rejection, predominantly regarding the T-cell response, the consequence of an ongoing inflammatory response within the context of the tissue microenvironment for cell fate, migration, and integration/efficacy has been largely overlooked. While classical immunosuppressants alter the T-cell response, these drugs have minimal impact on other immune cells such as neutrophils (polymorphonuclear (PMN) leukocytes) and macrophages (MACs)/microglia, which makes up a significant part of the host environment after traumatic injuries to the CNS, such as spinal cord injury (SCI). Accordingly, there is little known about the basic biology of either the host microenvironment or inflammatory microenvironment in influencing and interacting with either endogenous or transplanted stem cell populations. Understanding the molecules and signaling pathways directing hNSC fate choices in the injured CNS microenvironment is critical. hNSC derived from hiPS-NSC and hESC will be tested. We have therefore established and characterized hiPS-NSC and hES-NSC derived from multiple origins and tested the specific role of innate inflammatory cells (i.e. PMNs and macrophages) and molecules in cell fate, migration and proliferation of these hiPS-NSC and hES-NSC lines in vitro. Thus far, these data have revealed clear cell line specific intrinsic differences in response to inflammatory factors, which we will further investigated in the coming funding period both in vitro and in vivo.
  • The fate, migration, and repair potential of a stem cell is driven by a combination of intrinsic properties, such as the type, source, and maintenance/differentiation of the cell in vitro, as well as extrinsic factors the cell encounters in the in vivo environment, such as proteins related to inflammation or the growth matrix. Variations in the intrinsic properties of the cell may affect the potential of that cell for uncontrolled proliferation or the response of the cell to extrinsic factors that it encounters in its environment. We have previously shown that neural stem cells derived from human fetal tissue are highly sensitive to extrinsic inflammatory signals in vitro and in vivo. In the current studies, we sought to determine whether neural stem cell populations derived from different sources respond to the same sorts of inflammatory signals, in other words, whether these extrinsic factors affect stem cells as a general principal. Accordingly, we sought to characterize the intrinsic properties of neural stem cells derived from different sources and exposed to extrinsic inflammatory signals, including human embryonic and induced pluripotent cell, as an essential component of understanding of basic stem cell biology. We found that, in fact, all neural stem cells derived from embryonic and induced pluripotent populations responded to inflammatory signals. However, we also found that cell line intrinsic properties exert a strong degree of control, in some cases resulting in opposing consequences for cell proliferation and fate. Critically, we found that in vitro characteristics of response to extrinsic inflammatory signals were predictive for the way different cell populations behaved in vivo after transplantation. These data may offer a new opportunity to screen stem cell populations in vitro for comparability and predicted in vivo translational properties, and reveal a new and critical set of interactions between intrinsic cell programming and response to the environment.

Stem Cell-Derived Astrocyte Precursor Transplants in Amyotrophic Lateral Sclerosis

Funding Type: 
Disease Team Research I
Grant Number: 
DR1-01471
ICOC Funds Committed: 
$5 694 308
Disease Focus: 
Amyotrophic Lateral Sclerosis
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Cell Line Generation: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Amyotrophic lateral sclerosis (ALS), a lethal disease lacking effective treatments, is characterized by the loss of upper and lower motor neurons. 5-10% of ALS is familial, but the majority of ALS cases are sporadic with unknown causes. The lifetime risk is approximately 1 in 2000. This corresponds to ~30,000 affected individuals in the United States and ~5000 in the Collaborative Funding Partner country. There is currently only one FDA-approved compound, Rilutek, that extends lifespan by a maximum of three months. Although the causes of ALS are unknown and the presentation of the disease highly variable, common to all forms of ALS is the significant loss of motor neurons leading to muscle weakness, paralysis, respiratory failure and ultimately death. It is likely that many pathways are affected in the disease and focusing on a single pathway may have limited impact on survival. In addition, as ALS is diagnosed at a time that significant cell loss has occurred, an attempt to spare further cell loss would have significant impact on survival. Several findings support the approach of glial (cells surrounding the motor neurons) transplants. Despite the relative selectivity of motor neuron cell death in ALS, published studies demonstrate that glial transporters critical for the appropriate balance of glutamate surrounding the motor neurons are affected both in animal models and in tissue from sporadic and familial ALS. The significance of non-neuronal cells in the disease process has been well characterized using SOD1 mouse models representing many of the key aspects of the human disease. In addition, transplantation using glial-restricted precursors (GRPs) that differentiate into astrocytes in SOD1 mutant rats has been shown to increase survival. Motor neurons have a process, the axon, up to a meter in length which connects the cell body to its target, the muscle. The ability to appropriately rewire and ensure functional connections after motor neuron replacement remains a daunting task with no evidence to date that this will be possible in humans. Therefore, we will focus on the development of an ALS therapy based on hES-derived astrocyte precursor cell transplants to prevent the progression of ALS. Our proposed project will develop clinical grade stem-cell derived astrocyte precursor transplants for therapy in a prospective Phase I clinical trial. We will: 1) generate astrocyte precursors from three different sources of human embryonic stem cell (hESC) lines; 2) identify the hESC line and glial progenitor combination that has the best characteristics of minimal toxicity, best efficiency in generating astrocytes, and reducing disease phenotypes in vivo in a rat model of ALS; 3) manufacture the appropriate cells in a GMP facility required by the FDA; 4) work with our established clinical team to design a Phase I safety trial; and 5) submit an application for an invesitgational new drug (IND) within the next four years.
Statement of Benefit to California: 
Amyotrophic lateral sclerosis (ALS; also known as Lou Gehrig's Disease) is a common and devastating adult motor neuron disease that afflicts many Californians. In the absence of a cure, or an effective treatment, the cost of caring for patients with ALS is substantial, and the consequences on friends and family members similarly takes a devastating toll. Our goal is to develop a safe and effective cell transplant therapy for ALS by starting with human embryonic stem cells. If successful, this advance will hopefully diminish the cost of caring for the many Californians with ALS, extend their useful lives, and improve their quality of life. In addition, the development of this type of therapeutic approach in California will serve as an important proof of principle and stimulate the formation of businesses that seek to develop these types of therapies in California with consequent economic benefit.
Progress Report: 
  • Considerable progress was made on transitioning cells and cell production methods from research-scale to translational/clinical scale. Specifically, Year 1 activities were focused on transitioning from research to pilot-scale cell production methods, and characterization of the animal amyotrophic lateral sclerosis (ALS) disease model. These activities were essential because cellular therapy development is a multi-stage process with increasing stringency over time in terms of the increased focus on the details of the methods, stringent requirements for reagents/materials, greater scale, and more thorough product characterization during the transition from early research to an approved cellular therapy.
  • During Year 1, small-scale embryonic stem cell (ESC) growth and differentiation methods previously developed for research at Life Technologies were further developed at a larger pilot-scale, which provided enough cells to perform early animal pre-clinical studies and cell characterization. In addition to the increased scale of cell production, where possible, research grade reagents and materials were substituted with reagents and materials that would be required or preferred for producing a cell therapy for use in humans [produced under Good Manufacturing Practices (GMP), non-animal origin, well characterized]. These conditions are not ideal for many ESC lines, and only 1 of the 4 starting ESC lines was able to adapt successfully to these culture conditions. To increase the number of potential clinical ESC candidate cell lines, we acquired 2 additional ESC lines, UCFB6 and UCSFB7 from the University of California, San Francisco. Development is ongoing to ensure the cell processing methods are robust and scalable for the increased cell numbers required for the large-scale animal studies in Year 2. Cells from the pilot-scale production are being subjected to deep sequencing as part of the development of molecular characterization methods that may provide future quality control assays.
  • During Year 1, further studies of a rat ALS disease model were performed to: 1) optimize cell injection methods; 2) improve characterization of disease onset and progression in the rat model; 3) evaluate the utility of behavioral and electrophysiology tests for following the disease; and 4) evaluate histology methods for measuring neuron damage and detection of implanted cells, which will be used to optimize the large-scale efficacy studies planned for Year 2. We discovered that several time-consuming analysis approaches for efficacy evaluation could be replaced by simpler, more cost effective approaches. Additionally, the Year 1 studies tested and ensured that the team could handle an aggressive cell implant schedule, twice daily immunosuppression, demanding behavioral and electrophysiology assessments, and extensive histology evaluations.
  • Considerable progress was made on transitioning cells and cell production methods from research-scale to translational/clinical scale, including initial cell production in a GMP facility with GMP compatible production methods. Additionally, extensive characterization of the amyotrophic lateral sclerosis (ALS) disease animal model was completed and cells were evaluated for potential efficacy in this ALS disease animal model. These activities are key for continued progress in cellular therapy development, which is a multi-stage process that requires increasing focus on the details of the methods, stringent requirements for reagents/materials, greater scale, and more thorough product characterization during the transition to an approved cellular therapy.
  • Specifically, we made significant progress in three major areas:
  • First, we found evidence for efficacy using neural stem cells made at Life Technologies. In brief, during Year 1, the rat ALS disease model was shown to be a more aggressive disease model with an earlier disease onset and more rapid progression to end-stage and death than the model that had been used in previous studies. During Year 2, this more aggressive ALS disease model was further characterized with the identification of a reliable marker of disease onset, and demonstration that alpha motor neuron sparing by implanted cells could be detected and measured even, despite the aggressive nature of disease progression in this rat model.
  • We found that H9 NSCs produced by Life Technologies, when implanted into the rat ALS disease model, survived, migrated extensively into the area where alpha motor neurons are located, differentiated into cells that appear to be astrocytes, and provided a protective effect for the alpha motor neurons. This protective effect was determined by comparing the survival of alpha motor neurons on the side of the rat spinal cord where NSCs were implanted with the side of the spinal cord that did not have cells implanted. The side of the spinal cord where the NSCs were implanted showed approximately 10% more surviving alpha motor neurons than the matching side of the spinal cord that did not have cells implanted.
  • Second, cells from the various production methods were subjected to gene sequencing as part of the development of molecular characterization methods. This sequencing information was critical to identify whether cells produced by various methods were typical for the cell type, or exhibited qualities that indicated they were not optimal cell populations. These methods will be used to identify optimal markers for characterizing cell populations as part of current cell production development and for future quality control assays.
  • Third, during Year 2, Life Technologies further developed their pilot-scale embryonic stem cell (ESC) growth and differentiation methods to be more easily adaptable to cell production under Good Manufacturing Practices (GMP). This involved increasing the scale of cell production, and where possible, substituting reagent grade reagents and materials with reagents and materials that would be required or preferred for producing a cell therapy for use in humans (produced GMP, non-animal origin, well characterized). These conditions are not ideal for many ESC lines, and in Year 1, only one (H9) of the 4 starting ESC lines was successfully adapted to these culture conditions, however, 3 additional ESC lines were acquired to increase the number of potential clinical ESC candidate cell lines. One of these ESC lines (UCSFB7 from the University of California, San Francisco) was successfully adapted to the pilot ESC culture conditions, and resulted in the production of NSCs, and with AP production in progress. Because the research version of ESC line H9 has been used to successfully produce NSCs at Life Technologies, agreements are in progress for City of Hope for NSC cell production using the H9 ESCs, that have been banked under GMP conditions at City of Hope. In addition, pilot-scale cell production was initiated earlier than originally planned at the University of California, Davis GMP facility. The plan is to produce NSCs and APs under conditions that UC Davis has found to be successful in the past, and transition these methods to GMP compliance. To date, UC Davis has produced ESCs from 3 ESC lines [UCSF4, UCSF4.2 (a.k.a. UCSFB6) and UCSF4.3 (a.k.a. UCSFB7] and has produced NSCs from ESC line UCSF4. The UCSF4 NSCs are scheduled to be shipped to UCSD for testing in the ALS disease animal model in early June, 2012, and NSC production from ESC lines UCSF4.2 and UCSF4.3 is expected to begin in late June 2012.

Molecular Characterization of hESC and hIPSC-Derived Spinal Motor Neurons

Funding Type: 
Basic Biology I
Grant Number: 
RB1-01367
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.

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