Multiple sclerosis (MS) is an autoimmune disease in which the myelin sheath that insulates neurons is destroyed, resulting in loss of proper neuronal function. Existing treatments for MS are based on strategies that suppress the immune response. While these drugs do provide benefit by reducing relapses and delaying progression (but have significant side effects), the disease invariably progresses. We are pursuing an alternative therapy aimed at regeneration of the myelin sheath through drugs that act on an endogenous stem cell population in the central nervous system termed oligodendrocyte precursor cells (OPCs). Remission in MS is largely dependent upon OPCs migrating to sites of injury and subsequently differentiating into oligodendrocytes – the cells that synthesize myelin and are capable of neuronal repair. Previous studies indicate that in progressive MS, OPCs are abundantly present at sites of damage but fail to differentiate to oligodendrocytes. As such, drug-like molecules capable of inducing OPC differentiation should have significant potential, used alone or in combination with existing immunomodulatory agents, for the treatment of MS. The objective of this project is to identify a development candidate (DC) for the treatment of multiple sclerosis (MS) that functions by directly stimulating the differentiation of the adult stem cells required for remyelination.
Multiple Sclerosis (MS) is a painful, neurodegenerative disease that leads to an impairment of physical and cognitive abilities. Patients with MS are often forced to stop working because their condition becomes so limiting. MS can interfere with a patient's ability to even perform simple routine daily activities, resulting in a decreased quality of life. Existing treatments for MS delay disease progression and minimize symptoms, however, the disease invariably progresses to a state of chronic demyelination. The goal of this project is to identify novel promyelinating drugs, based on differentiation of an endogenous stem cell population. Such drugs would be used in combination with existing immunosuppressive drugs to prevent disease progression and restore proper neuronal activity. More effective MS treatment strategies represent a major unmet medical need that could impact the roughly 50,000 Californians suffering from this disease. Clearly the development of a promyelinating therapeutic would have a significant impact on the well-being of Californians and reduce the negative economic impact on the state resulting from this degenerative disease.
- Multiple sclerosis (MS) is an autoimmune disease characterized by the destruction of the myelin sheath that insulates neurons, resulting in loss of proper neuronal function. Existing treatments for MS are based exclusively on strategies that suppress the immune response. We are pursuing an alternative stem cell-based therapeutic approach aimed at enhancing regeneration of the myelin sheath. Specifically, we are focused on the identification of drug-like molecules capable of inducing oligodendrocyte precursor cell (OPC) differentiation. To date, we have identified a series approved drugs that effectively induce OPC differentiation under tissue culture conditions. Additionally, we have demonstrated that several of these drug candidates reduce MS-like symptoms in relevant rodent models of the disease. We are currently conducting detailed pharmacology experiments to determine which of the identified molecules will serve as the best candidate for future clinical development.
- The aim of this project is to identify and characterize molecules that induce the repair of lesions in multiple sclerosis. Molecules that induce the selective differentiation of oligodendrocyte precursor cells to oligodendrocytes and thereby lead to remyelination of axons are being characterized with respect to their in vitro activity and in vivo efficacy in relevant animal models, alone and in combination with immunosuppressive drugs. This work may lead to a new regenerative therapy for multiple sclerosis that is complementary to the current immune-focused therapies.
Multiple Sclerosis (MS) is a disease of the central nervous system (CNS) caused by inflammation and loss of cells that produce myelin, which normally insulates and protects nerve cells. MS is a leading cause of neurological disability among young adults in North America. Current treatments for MS include drugs such as interferons and corticosteroids that modulate the ability of immune system cells to invade the CNS. These therapies often have unsatisfactory outcomes, with continued progression of neurologic disability over time. This is most likely due to irreversible tissue injury resulting from permanent loss of myelin and nerve destruction. The limited ability of the body to repair damaged nerve tissue highlights a critically important and unmet need for MS patients. The long-term goal of our research is to develop a stem cell-based therapy that will not only halt ongoing loss of myelin but also lead to remyelination and repair of damaged nerve tissue. Our preliminary data in animal models of human MS are very promising and suggest that this goal is possible. Research efforts will concentrate on refining techniques for production and rigorous quality control of clinically-compatible transplantable cells generated from high-quality human pluripotent stem cell lines, and to verify the therapeutic activity of these cells. We will emphasize safety and development of the most therapeutically beneficial cell type for eventual use in patients with MS.
One in seven Americans lives in California, and these people make up the single largest health care market in the United States. The diseases and injuries that affect Californians affect the rest of the US and the world. Many of these diseases involve degeneration of healthy cells and tissues, including neuronal tissue in diseases such as Multiple Sclerosis (MS). The best estimates indicate that there are 400,000 people diagnosed with MS in the USA and 2.2 million worldwide. In California, there are approximately 160,000 people with MS – roughly half of MS patients in the US live in California. MS is a life-long, chronic disease diagnosed primarily in young adults who have a virtually normal life expectancy but suffer from progressive loss of motor and cognitive function. Consequently, the economic, social and medical costs associated with the disease are significant. Estimates place the annual cost of MS in the United States in the billions of dollars. The development of a stem cell therapy for treatment of MS patients will not only alleviate ongoing suffering but also allow people afflicted with this disease to return to work and contribute to the economic stabilization of California. Moreover, a stem cell-based therapy that will provide sustained recovery will reduce recurrence and the ever-growing cost burden to the California medical community.
- The team has been highly productive during the first year of work on this award. A major goal of the project is to evaluate the efficacy of neural progenitor cell transplantation to promote remyelination following virus induced central nervous system damage. With intracranial infection by the virus mouse hepatitis virus (MHV), mice develop paralysis due to immune mediated destruction of cells that generate myelin. Using protocols developed in the Loring laboratory, neural precursor cells (NPC) were derived from the human embryonic stem cell line H9. Mice developing paralysis due to intracranial infection with MHV were subject to intraspinal transplantation of these NPC, resulting in significant clinical recovery beginning at 2-3 weeks following transplant. This clinical effect of NPC transplantation remained out to six months, suggesting that these NPC are effective for long-term repair following demyelination. Despite this striking recovery, these human ES cell derived NPC were rapidly rejected. Several protocols for the generation of NPC for transplantation have been characterized, with the greatest clinical impact observed for NPC cultures bearing a high level of expression of TGF beta I and TGF beta II. These findings support the hypothesis that transplanted NPC reprogram the immune system within the central nervous system (CNS), leading to the activation of endogenous NPC and other repair mechanisms. Thus, it may not be necessary to induce complete immune suppression in order to promote remyelination and CNS repair following NPC transplantation for demyelinating diseases such as multiple sclerosis.
- The team has been highly productive during the first two years of work on this award. A major goal of the project is to evaluate the efficacy of neural progenitor cell transplantation to promote remyelination following virus induced central nervous system damage. With intracranial infection by the virus mouse hepatitis virus (MHV), mice develop paralysis due to immune mediated destruction of cells that generate myelin. Using protocols developed in the Loring laboratory, neural precursor cells (NPC) were derived from the human embryonic stem cell line H9. Mice developing paralysis due to intracranial infection with MHV were subject to intraspinal transplantation of these NPC, resulting in significant clinical recovery beginning at 2-3 weeks following transplant. This clinical effect of NPC transplantation remained out to six months, suggesting that these NPC are effective for long-term repair following demyelination. Despite this striking recovery, these human ES cell derived NPC were rapidly rejected. Several protocols for the generation of NPC for transplantation have been characterized, with the greatest clinical impact observed for NPC cultures bearing a high level of expression of TGF beta I and TGF beta II. These findings support the hypothesis that transplanted NPC reprogram the immune system within the central nervous system (CNS), leading to the activation of endogenous NPC and other repair mechanisms. Thus, it may not be necessary to induce complete immune suppression in order to promote remyelination and CNS repair following NPC transplantation for demyelinating diseases such as multiple sclerosis. In addition, the group is currently assessing the impact of NPCs in experimental autoimmune encephalomyelitis (EAE), an autoimmune model of MS. Initial results suggest that NPCs also reduce the severity of disease in this model, and studies are underway to determine the mechanism(s) by which NPCs promote clinical recovery during EAE.
Strokes that affect the nerves cells, i.e., “gray matter”, consistently receive the most attention. However, the kind of strokes that affecting the “wiring” of the brain, i.e., “white matter”, cause nearly as much disability. The most severe disability is caused when the stroke is in the wiring (axons) that connect the brain and spinal cord; as many as 150,000 patients are disabled per year in the US from this type of stroke. Although oligodendrocytes (“oligos”) are the white matter cells that produce the lipid rich axonal insulator called myelin) are preferentially damaged during these events, stem cell-derived oligos have not been tested for their efficacy in preclinical (animal) trials. These same white matter tracts (located underneath the gray matter, called subcortical) are also the primary sites of injury in MS, where multifocal inflammatory attack is responsible for stripping the insulating myelin sheaths from axons resulting in axonal dysfunction and degeneration. Attempts to treat MS-like lesions in animals using undifferentiated stem cell transplants are promising, but most evidence suggests that these approaches work by changing the inflammation response (immunomodulation) rather than myelin regeneration. While immunomodulation is unlikely to be sufficient to treat the disease completely, MS may not be amenable to localized oligo transplantation since it is such a multifocal process. This has led to new emphasis on approaches designed to maximize the response of endogenous oligo precursors that may be able to regenerate myelin if stimulated. We hypothesize that by exploiting novel features of oligo differentiation in vitro (that we have discovered and that are described in our preliminary data) that we will be able to improve our ability to generate oligo lineage cells from human embryonic stem cells and neural stem cells for transplantation, and also to develop approaches to maximize oligo development from endogenous precursors at the site of injury in the brain. This proposal will build on our recent successes in driving oligo precursor production from multipotential mouse neural stem cells by expressing regulatory transcription factors, and apply this approach to human embryonic and neural stem cells to produce cells that will be tested for their ability to ameliorate brain damage in rodent models of human stroke. Furthermore, we hope to develop approaches that may facilitate endogenous recruitment of oligo precursors to produce mature oligos, which may prove a viable regenerative approach to treat a variety of white matter diseases including MS and stroke.
Diseases associated with disruption of oligodendrocyte function and integrity (such as subcortical ischemic stroke and multiple sclerosis) are major causes of morbidity and mortality. Stroke is the third leading cause of death and the leading cause of permanent disability in the United States, costing over $50 billion dollars annually, as approximately 150,000 chronic stroke patients survive the acute event and are left with permanent, severe motor and/or sensory deficits. While much less common, multiple sclerosis (MS) is the primary non-traumatic cause of neurologic disability in young adults. Most patients are diagnosed in their 20s-40s and live for many decades after diagnosis with increasing needs for expensive services, medications and ultimately long-term care. Existing strategies for stem cell based therapies include both strategies to replace lost cells and to augment regeneration after injury, but most of these efforts have emphasized the role of undifferentiated stem cells in treatment despite the realization that the main nexus of injury in both diseases is frequently a differentiated cell type – the oligodendrocyte. This project will use new insights into the development of oligodendrocytes from the laboratories of the investigators to find ways to improve production of oligodendrocytes from human ES cells and human neural stem cells, test whether these cells can improve the clinical outcome in rodent models of stroke and MS after transplantation and search for new molecular treatments that would augment the regeneration of oligodendrocytes from resident brain stem cells after injury. This is the first step to translating the basic fundamental understanding of oligodendrocyte development into viable therapies for important human diseases that are major burdens on the citizens of California.
- Over the last year we have succeeded in generating nearly pure cultures of human ES cell derived oligodendrocyte precursors from two different human ES cell lines. We are now also testing whether manipulation of transcription factors or morphogenic signaling pathways regulates the ability of these cells to differentiate into oligodendrocytes that produce myelin. We are testing these cells in a rodent stroke model to determine if they survive in the region of the stroke. If they survive, we will test whether they help to treat the strokes. We are also testing cells in transplantation into a developmental ischemia model and a model for genetic failure to produce myelin.
- Our proposal centers on developing novel effective methods to generate oligodendrocytes from human ES cells. We focus on identifying signaling pathways (using studies in rodent neural stem cells) that can be adapted to human ES cells and used to regulate the efficiency of oligodendrocyte specification and differentiation from human ES cells. We then hope to use these human ES cell derived oligodendrocytes to determine whether transplantation of these cells is feasible in well characterized animal models associated with damage to oligodendrocytes. Over the last year we have made major progress toward these goals.
- First, we have completed and submitted for publication two studies identifying the roles of Wnts and Sox10 in regulating the development of oligodendrocytes both during brain development and during stem cell differentiation in vitro. One of these papers is in the final stages of consideration after revision and the other is submitted awaiting reviews.
- Second, we have developed a novel method for culturing human ES cell derived oligodendrocyte precursors. This is based on modifications of published methods but leads to greatly enhanced purity of final oligodendrocytes in our cultures (about 80% oligodendrocytes and 20% astrocytes). We have used this culture approach to address the role of sonic hedgehog in the differentiation of oligodendrocytes from human oligodendrocyte progenitors and have identified sonic hedgehog as a major regulator of oligodendrocyte differentiation and myelin production. This is quite distinct from rodent neural cells where sonic hedgehog doesn't appear to have this function. This will provide a novel therapeutic target to affect oligodendrocyte maturation and regeneration in disease models and will be of great utility for studying the function of mature human oligodendrocytes. This work is in preparation for submission.
- Third, we have made some significant progress in our transplantation studies. We completed studies transplanting human ES derived oligodendrocyte progenitors into a rodent model of focal stroke and found that at 1 week post stroke and 2 weeks post stroke the survival of oligodendrocytes from these transplants is very minimal. Thus, we have discontinued this work because of this feasibility issue. We have moved on to examine studies of transplantation into newborn rodents with hypoxic injury and with dysmyelination becahse of the shiverer mutation. The progress here is good. The hypoxia model we are using is a chronic (up to 1 week) exposure to low oxygen tension of P2 mice, which is known to cause oligodendrocyte injury. We are initially characterizing the injury to oligodendrocytes at various durations of hypoxic exposure so that we can identify the best time point to transplant our cells into the brains. We are using immunodeficient mice to decrease the chances of rejection of the transplanted cells. In addition, we are generating a mouse colony with the shiverer allele combined with an immunodeficiency allele in order to be able to transplant cells in this model. In the meantime, we are determining the survival of transplanted cells into newborn mice to identify technical factors that will need to be overcome to allow efficient transplantation and to determine if our human cells participate in differentiation in these mice. Preliminarily we have found good survival of oligodendrocyte lineage cells after transplantation into P2 mice and the expression of myelin antigens after an appropriate period of development in vivo. This is very encouraging.
- In the last year we have continued our efforts to transplant oligodendrocyte progenitors obtained by differentiation of human ES cells. Our progress in this area has been mixed because of substantial technical hurdles in consistent production of the oligodendrocyte progenitors from frozen stocks of cells. This will necessitate a no-cost extension for a small portion of the work to allow completion of the analysis of already transplanted animals.
- We have made substantial progress as well in showing that these cells are capable of myelinating axons effectively in vitro. In addition, we've found that the human ES derived oligodendrocytes are capable of myelinating artificial nanofibers in vitro as well. This may serve as a useful platform in the future for drug discovery or other high throughput studies.
- We have also identified an important novel molecular regulator of oligodendrocyte number and development and this work will continue into the future.
- In this NCE period we were completing studies with animals that had received neonatal ischemic injury and were implanted with human ES cell derived cells of the oligodendrocyte lineage. These experiments showed that the cells survive and have oligodendrocyte lineage markers for three weeks post injection. Longer survival experiments are still ongoing.
Multiple sclerosis (MS) is the most common neurologic disease affecting young adults under the age of 40 with the majority of MS patients diagnosed in the second or third decade of life. MS is characterized by the gradual loss of the myelin sheath that surrounds and insulates axons that allow for the conduction of nerve impulses – a process known as demyelination. For unknown reasons, the ability to remyelinate axons is impaired in MS patients making recovery of motor skills difficult. Therefore, developing novel and effective approaches to remyelinate axons in MS patients would dramatically improve the quality of life of many MS patients. The experiments described in this research proposal utilize a well-accepted model of MS to further characterize the potential clinical applicability of human embryonic stem cells (hESCs) to remyelinate axons. Such knowledge is crucial in order to increase our understanding of stem cells with regards to treatment of numerous human diseases including MS.
California is the most populated state in the USA. As such, the costs of medical care for the treatment of patients with chronic diseases such as multiple sclerosis (MS) represents a significant and growing problem. MS is the most common neurologic disease affecting young adults under the age of 40 with the majority of MS patients diagnosed in the second or third decade of life. Given the population of California, there are many MS patients living in the state and the numbers will undoubtedly grow. It is unusual for MS patients to die from the disease and many will live normal life spans but will develop an increasing array of medical problems stemming from the progression of neurologic damage associated with MS. MS is characterized by the gradual loss of the myelin sheath that surrounds and insulates axons that allow for the conduction of nerve impulses – a process known as demyelination. For unknown reasons, the ability to remyelinate axons is impaired in MS patients making recovery of motor skills difficult. Therefore, developing novel and effective approaches to remyelinate axons in MS patients would dramatically alleviate some of the burden placed on the medical community by improving the quality of life of many MS patients. The experiments described in this research proposal utilize a well-accepted model of MS to further characterize the potential clinical applicability of human embryonic stem cells (hESCs) to remyelinate axons. Such knowledge is crucial in order to increase our understanding of stem cells with regards to treatment of human diseases with the ultimate goal of limiting patient suffering and reducing medical costs.
- Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) that results in demyelination and axonal loss, culminating in extensive disability through defects in neurologic function. The demyelination that defines MS pathology is progressive over time; however, studies indicate that myelin repair can occur during the course of disease in patients with MS and in animal models designed to mimic the immunopathogenesis of MS. While it is generally thought that endogenous oligodendrocyte precursor cells (OPCs) are largely responsible for spontaneous remyelination, it is unclear why these cells are only able to transiently induce myelin repair in the presence of ongoing disease. Along these lines, two therapies for demyelinating diseases look promising; implanting OPCs into sites of neuroinflammation that are directly capable of inducing remyelination of the damaged axons and/or modifying the local environment to stimulate and support remyelination by endogenous OPCs. Indeed, we have shown that human embryonic stem cell (hESC)-derived oligodendrocytes surgically implanted into the spinal cords of mice with virally induced demyelination promoted focal remyelination and axonal sparing. We are currently investigating how the implanted OPCs positionally migrate to areas of on-going demyelination and the role these cells play in repairing the damaged CNS. The purpose of this research is to identify the underlying mechanism(s) responsible for hESC-induced remyelination.
- Oligodendrocyte progenitor cells (OPCs) are important in mediating remyelination in response to demyelinating lesions. As such, OPCs represent an attractive cell population for use in cell replacement therapies to promote remyelination for treatment of human demyelinating diseases. High-purity OPCs have been generated from hESC and have been shown to initiate remyelination associated with improved motor skills in animal models of demyelination. We have previously determined that engraftment of hESC-derived OPCs into mice with established demyelination does not significantly improve clinical recovery nor reduce the severity of demyelination. Importantly, remyelination is limited following OPC transplantation. These findings highlight that the microenvironment is critical with regards to the remyelination potential of engrafted cells. In addition, we have determined that human OPCs are capable of migrating in response to proinflammatory molecules often associated with human neuroinflammatory diseases such as multiple sclerosis. This is an important observation in that it will likely be necessary for engrafted OPCs to be able to positionally navigate within tissue in order to move from the site of surgical transplantation to areas of damage to initiate repair and tissue remodeling. Finally, we have also made a novel discovery of a unique signaling pathway that protects OPCs from damage/death in response to treatment with proinflammatory cytokines. We believe this is an important and translationally relevant observation as OPCs are critical in contributing to remyelination and remyelination failure is an important clinical feature for many human demyelinating diseases inclusing spinal cord injury and MS. We have identified a putative protective ligand/receptor interaction affords protection from cytokine-induced apoptosis. These findings may reveal novel avenues for therapeutic intervention to prevent damage/death of OPCs and enhance remyelination.