Stroke

Coding Dimension ID: 
317
Coding Dimension path name: 
Neurological Disorders / Stroke

Enhancing healing via Wnt-protein mediated activation of endogenous stem cells

Funding Type: 
Early Translational I
Grant Number: 
TR1-01249
ICOC Funds Committed: 
$6 762 954
Disease Focus: 
Bone or Cartilage Disease
Stroke
Neurological Disorders
Heart Disease
Neurological Disorders
Skin Disease
Stem Cell Use: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 
All adult tissues contain stem cells. Some tissues, like bone marrow and skin, harbor more adult stem cells; other tissues, like muscle, have fewer. When a tissue or organ is injured these stem cells possess a remarkable ability to divide and multiply. In the end, the ability of a tissue to repair itself seems to depend on how many stem cells reside in a particular tissue, and the state of those stem cells. For example, stress, disease, and aging all diminish the capacity of adult stem cells to self-renew and to proliferate, which in turn hinders tissue regeneration. Our strategy is to commandeer the molecular machinery responsible for adult stem cell self-renewal and proliferation and by doing so, stimulate the endogenous program of tissue regeneration. This approach takes advantage of the solution that Nature itself developed for repairing damaged or diseased tissues, and controls adult stem cell proliferation in a localized, highly controlled fashion. This strategy circumvents the immunological, medical, and ethical hurdles that exist when exogenous stem cells are introduced into a human. When utilizing this strategy the goal of reaching clinical trials in human patients within 5 years becomes realistic. Specifically, we will target the growing problem of neurologic, musculoskeletal, cardiovascular, and wound healing diseases by local delivery of a protein that promotes the body’s inherent ability to repair and regenerate tissues. We have evidence that this class of proteins, when delivered locally to an injury site, is able to stimulate adult tissue stem cells to grow and repair/replace the deficient tissue following injury. We have developed technologies to package the protein in a specialized manner that preserves its biological activity but simultaneously restricts its diffusion to unintended regions of the body. For example, when we treat a skeletal injury with this packaged protein we augment the natural ability to heal bone by 350%; and when this protein is delivered to the heart immediately after an infarction cardiac output is improved and complications related to scarring are reduced. This remarkable capacity to augment tissue healing is not limited to bones and the heart: the same powerful effect can be elicited in the brain, and skin injuries. The disease targets of stroke, bone fractures, heart attacks, and skin wounds and ulcers represent an enormous health care burden now, but this burden is expected to skyrocket because our population is quickly aging. Thus, our proposal addresses a present and ongoing challenge to healthcare for the majority of Californians, with a novel therapeutic strategy that mimics the body’s inherent repair mechanisms.
Statement of Benefit to California: 
Californians represent 1 in 7 Americans, and make up the single largest healthcare market in the United States. The diseases and injuries that affect Californians affect the rest of the US, and the world. For example, stroke is the third leading cause of death, with more than 700,000 people affected every year. It is a leading cause of serious long-term disability, with an estimated 5.4 million stroke survivors currently alive today. Symptoms of musculoskeletal disease are the number two most cited reasons for visit to a physician. Musculoskeletal disease is the leading cause of work-related and physical disability in the United States, with arthritis being the leading chronic condition reported by the elderly. In adults over the age of 70, 40% suffer from osteoarthritis of the knee and of these nearly 80% have limitation of movement. By 2030, nearly 67 million US adults will be diagnosed with arthritis. Cardiovascular disease is the leading cause of death, and is a major cause of disability worldwide. The annual socioeconomic burden posed by cardiovascular disease is estimated to exceed $400 billion annually and remains a major cause of health disparities and rising health care costs. Skin wounds from burns, trauma, or surgery, and chronic wounds associated with diabetes or pressure ulcer, exact a staggering toll on our healthcare system: Burns alone affect 1.25M Americans each year, and the economic global burden of these injuries approaches $50B/yr. In California alone, the annual healthcare expenditures for stroke, skeletal repair, heart attacks, and skin wound healing are staggering and exceed 700,000 cases, 3.5M hospital days, and $34B. We have developed a novel, protein-based therapeutic platform to accelerate and enhance tissue regeneration through activation of adult stem cells. This technology takes advantage of a powerful stem cell factor that is essential for the development and repair of most of the body’s tissues. We have generated the first stable, biologically active recombinant Wnt pathway agonist, and showed that this protein has the ability to activate adult stem cells after tissue injury. Thus, our developmental candidate leverages the body’s natural response to injury. We have generated exciting preclinical results in a variety of animals models including stroke, skeletal repair, heart attack, and skin wounding. If successful, this early translational award would have enormous benefits for the citizens of California and beyond.
Progress Report: 
  • In the first year of CIRM funding our objectives were to optimize the activity of the Wnt protein for use in the body and then to test, in a variety of injury models, the effects of this lipid-packaged form of Wnt. We have made considerable progress on both of these fronts. For example, in Roel Nusse and Jill Helms’ groups, we have been able to generate large amounts of the mouse form of Wnt3a protein and package it into liposomal vesicles, which can then be used by all investigators in their studies of injury and repair. Also, Roel Nusse succeeded in generating human Wnt3a protein. This is a major accomplishment since our ultimate goal is to develop this regenerative medicine tool for use in humans. In Jill Helms’ lab we made steady progress in standardizing the activity of the liposomal Wnt3a formulation, and this is critically important for all subsequent studies that will compare the efficacy of this treatment across multiple injury repair scenarios.
  • Each group began testing the effects of liposomal Wnt3a treatment for their particular application. For example, in Theo Palmer’s group, the investigators tested how liposomal Wnt3a affected cells in the brain following a stroke. We previously found that Wnt3A promotes the growth of neural stem cells in a petri dish and we are now trying to determine if delivery of Wnt3A can enhance the activity of endogenous stem cells in the brain and improve the level of recovery following stroke. Research in the first year examined toxicity of a liposome formulation used to deliver Wnt3a and we found it to be well tolerated after injection into the brains of mice. We also find that liposomal Wnt3a can promote the production of new neurons following stroke. The ongoing research involves experiments to determine if these changes in stem cell activity are accompanied by improved neurological function. In Jill Helms’ group, the investigators tested how liposomal Wnt3a affected cells in a bone injury site. We made a significant discovery this year, by demonstrating that liposomal Wnt3a stimulates the proliferation of skeletal progenitor cells and accelerates their differentiation into osteoblasts (published in Science Translational Medicine 2010). We also started testing liposomal Wnt3a for safety and toxicity issues, both of which are important prerequisites for use of liposomal Wnt3a in humans. Following a heart attack (i.e., myocardial infarction) we found that endogenous Wnt signaling peaks between post-infarct day 5-7. We also found that small aggregates of cardiac cells called cardiospheres respond to Wnt in a dose-responsive manner. In skin wounds, we tested the effect of boosting Wnt signaling during skin wound healing. We found that the injection of Wnt liposomes into wounds enhanced the regeneration of hair follicles, which would otherwise not regenerate and make a scar instead. The speed and strength of wound closure are now being measured.
  • In aggregate, our work on this project continues to move forward with a number of great successes, and encouraging data to support our hypothesis that augmenting Wnt signaling following tissue injury will provide beneficial effects.
  • In the second year of CIRM funding our objectives were to optimize packaging of the developmental candidate, Wnt3a protein, and then to continue to test its efficacy to enhance tissue healing. We continue to make considerable progress on the stated objectives. In Roel Nusse’s laboratory, human Wnt3a protein is now being produced using an FDA-approved cell line, and Jill Helms’ lab the protein is effectively packaged into lipid particles that delay degradation of the protein when it is introduced into the body.
  • Each group has continued to test the effects of liposomal Wnt3a treatment for their particular application. In Theo Palmer’s group we have studied how liposomal Wnt3a affects neurogenesis following stroke. We now know that liposomal Wnt3a transiently stimulates neural progenitor cell proliferation. We don’t see any functional improvement after stroke, though, which is our primary objective.
  • In Jill Helms’ group we’ve now shown that liposomal Wnt3a enhances fracture healing and osseointegration of dental and orthopedic implants and now we demonstrate that liposomal Wnt3a also can improve the bone-forming capacity of bone marrow grafts, especially when they are taken from aged animals.
  • We’ve also tested the ability of liposomal Wnt3a to improve heart function after a heart attack (i.e., myocardial infarction). Small aggregates of cardiac progenitor cells called cardiospheres proliferate to Wnt3a in a dose-responsive manner, and we see an initial improvement in cardiac function after treatment of cells with liposomal Wnt3a. the long-term improvements, however, are not significant and this remains our ultimate goal. In skin wounds, we tested the effect of boosting Wnt signaling during wound healing. We found that the injection of liposomal Wnt3a into wounds enhanced the regeneration of hair follicles, which would otherwise not regenerate and make a scar instead. The speed of wound closure is also enhanced in regions of the skin where there are hair follicles.
  • In aggregate, our work continues to move forward with a number of critical successes, and encouraging data to support our hypothesis that augmenting Wnt signaling following tissue injury will provide beneficial effects.
  • Every adult tissue harbors stem cells. Some tissues, like bone marrow and skin, have more adult stem cells and other tissues, like muscle or brain, have fewer. When a tissue is injured, these stem cells divide and multiply but only to a limited extent. In the end, the ability of a tissue to repair itself seems to depend on how many stem cells reside in a particular tissue, and the state of those stem cells. For example, stress, disease, and aging all diminish the capacity of adult stem cells to respond to injury, which in turn hinders tissue healing. One of the great unmet challenges for regenerative medicine is to devise ways to increase the numbers of these “endogenous” stem cells, and revive their ability to self-renew and proliferate.
  • The scientific basis for our work rests upon our demonstration that a naturally occurring stem cell growth factor, Wnt3a, can be packaged and delivered in such a way that it is robustly stimulates stem cells within an injured tissue to divide and self-renew. This, in turn, leads to unprecedented tissue healing in a wide array of bone injuries especially in aged animals. As California’s population ages, the cost to treat such skeletal injuries in the elderly will skyrocket. Thus, our work addresses a present and ongoing challenge to healthcare for the majority of Californians and the world, and we do it by mimicking the body’s natural response to injury and repair.
  • To our knowledge, there is no existing technology that displays such effectiveness, or that holds such potential for the stem cell-based treatment of skeletal injuries, as does a L-Wnt3a strategy. Because this approach directly activates the body’s own stem cells, it avoids many of the pitfalls associated with the introduction of foreign stem cells or virally reprogrammed autologous stem cells into the human body. In summary, our data show that L-Wnt3a constitutes a viable therapeutic approach for the treatment of skeletal injuries, especially those in individuals with diminished healing potential.
  • This progress report covers the period between Sep 01 2012through Aug 31 2013, and summarizes the work accomplished under ET funding TR1-01249. Under this award we developed a Wnt protein-based platform for activating a patient’s own stem cells for the purpose of tissue regeneration.
  • At the beginning of our grant period we generated research grade human WNT3A protein in quantities sufficient for all our discovery experiments. We then tested the ability of this WNT protein therapeutic to improve the healing response in animal models of stroke, heart attack, skin wounding, and bone fracture. These experimental models recapitulated some of the most prevalent and debilitating human diseases that collectively, affect millions of Californians.
  • At the end of year 2, we assembled an external review panel to select the promising clinical indication. The scientific advisory board unanimously selected skeletal repair as the leading indication. The WNT protein is notoriously difficult to purify; consequently in year 3 we developed new methods to streamline the purification of WNT proteins, and the packaging of the WNT protein into liposomal vesicles that stabilized the protein for in vivo use.
  • In years 3 and 4 we continued to accrue strong scientific evidence in both large and small animal models that a WNT protein therapeutic accelerates bone regeneration in critical size bony non-unions, in fractures, and in cases of implant osseointegration. In this last year of funding, we clarified and characterized the mechanism of action of the WNT protein, by showing that it activates endogenous stem cells, which in turn leads to faster healing of a range of different skeletal defects.
  • In this last year we also identified a therapeutic dose range for the WNT protein, and developed a route and method of delivery that was simultaneously effective and yet limited the body’s exposure to this potent stem cell factor. We initiated preliminary safety studies to identify potential risks, and compared the effects of WNT treatment with other commercially available bone growth factors. In sum, we succeeded in moving our early translational candidate from exploratory studies to validation, and are now ready to enter into the IND-enabling phase of therapeutic candidate development.
  • This progress report covers the period between Sep 01 2013 through April 30 2014, and summarizes the work accomplished under ET funding TR101249. Under this award we developed a Wnt protein-based platform for activating a patient’s own stem cells for purposes of tissue regeneration.
  • At the beginning of our grant period we generated research grade human WNT3A protein in quantities sufficient for all our discovery experiments. We then tested the ability of this WNT protein therapeutic to improve the healing response in animal models of stroke, heart attack, skin wounding, and bone fracture. These experimental models recapitulated some of the most prevalent and debilitating human diseases that collectively, affect millions of Californians. At the conclusion of Year 2 an external review panel was assembled and charged with the selection of a single lead indication for further development. The scientific advisory board unanimously selected skeletal repair as the lead indication.
  • In year 3 we accrued addition scientific evidence, using both large and small animal models, demonstrating that a WNT protein therapeutic accelerated bone healing. Also, we developed new methods to streamline the purification of WNT proteins, and improved our method of packaging of the WNT protein into liposomal vesicles (e.g., L-WNT3A) for in vivo use.
  • In year 4 we clarified the mechanism of action of L-WNT3A, by demonstrating that it activates endogenous stem cells and therefore leads to accelerated bone healing. We also continued our development studies, by identifying a therapeutic dose range for L-WNT3A, as well as a route and method of delivery that is both effective and safe. We initiated preliminary safety studies to identify potential risks, and compared the effects of L-WNT3A with other, commercially available bone growth factors.
  • In year 5 we initiated two new preclinical studies aimed at demonstrating the disease-modifying activity of L-WNT3A in spinal fusion and osteonecrosis. These two new indications were chosen by a CIRM review panel because they represent an unmet need in California and the nation. We also initiated development of a scalable manufacturing and formulation process for both the WNT3A protein and L-WNT3A formulation. These two milestones were emphasized by the CIRM review panel to represent major challenges to commercialization of L-WNT3A; consequently, accomplishment of these milestones is a critical yardstick by which progress towards an IND filing can be assessed.

Human stem cell derived oligodendrocytes for treatment of stroke and MS

Funding Type: 
Comprehensive Grant
Grant Number: 
RC1-00135
ICOC Funds Committed: 
$2 566 701
Disease Focus: 
Multiple Sclerosis
Neurological Disorders
Stroke
Immune Disease
Stem Cell Use: 
Adult Stem Cell
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
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.
Statement of Benefit to California: 
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.
Progress Report: 
  • 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.

MEF2C-Directed Neurogenesis From Human Embryonic Stem Cells

Funding Type: 
Comprehensive Grant
Grant Number: 
RC1-00125
ICOC Funds Committed: 
$3 035 996
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Stroke
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Cell Line Generation: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Understanding differentiation of human embryonic stem cells (hESCs) provides insight into early human development and will help directing hESC differentiation for future cell-based therapies of Parkinson’s disease, stroke and other neurodegenerative conditions. The PI’s laboratory was the first to clone and characterize the transcription factor MEF2C, a protein that can direct the orchestra of genes to produce a particular type of cell, in this case a nerve cell (or neuron). We have demonstrated that MEF2C directs the differentiation of mouse ES cells into neurons and suppresses glial fate. MEF2C also helps keep new nerve cells alive, which is very helpful for their successful transplantation. However, little is known about the role of MEF2C in human neurogenesis, that is, its ability to direct hESC differentiation into neuronal lineages such as dopaminergic neurons to treat Parkinson’s disease and its therapeutic potential to promote the generation of nerve cells in stem cell transplantation experiments. The goal of this application is to fill these gaps. The co-PI’s laboratory has recently developed a unique procedure for the efficient differentiation of hESCs into a uniform population of neural precursor cells (NPCs), which are progenitor cells that develop from embryonic stem cells and can form different kinds of mature cells in the nervous system. Here, we will investigate if MEF2C can instruct hESC-derived NPCs to differentiate into nerve cells, including dopaminergic nerve cells for Parkinson’s disease or other types of neurons that are lost after a stroke. Moreover, we will transplant hESC-NPCs engineered with MEF2C to try to treat animal models of stroke and Parkinson’s disease. We will characterize known and novel MEF2C target genes to identify critical components in the MEF2C transcriptional network in the clinically relevant cell population of hESC-derived neural precursor cells (hESC-NPCs). Specifically we will: 1) determine the function of MEF2C during in vitro neurogenesis (generation of new nerve cells) from hESC-NPCs; 2) investigate the therapeutic potential of MEF2C engineered hESC-NPCs in Parkinson’s and stroke models; 3) determine the MEF2C DNA (gene) binding sites and perform a “network” analysis of MEF2C target genes in order to understand how MEF2C works in driving the formation of new nerve cells from hESCs.
Statement of Benefit to California: 
Efficient and controlled neuronal differentiation from human embryonic stem cells (hESCs) is mandatory for developing future clinical cell-based therapies. Strategies to direct differentiation towards neuronal vs. glial fate are critical for the development of a uniform population of desired neuronal specificities (e.g., dopaminergic neurons for Parkinson’s disease (PD)). Our laboratory was the first to clone and characterize the transcription factor MEF2C, the major isoform of MEF2 found in the developing brain. Based on our encouraging preliminary results that were obtained with mouse (m)ESC-derived and human fetal brain-derived neural precursors, we propose to investigate if MEF2C enhances neurogenesis from hESCs. In addition to neurogenic activity, we have shown that MEF2C exhibits an anti-apoptotic (that is, anti-death) effect and therefore increases cell survival. This dual function of MEF2C is extremely valuable for the purpose of transplantation of MEF2C-engineererd neural precursors. Additionally, we found MEF2 binding sites in the Nurr1 promoter region, which in the proper cell context, should enhance dopaminergic (DA) neuronal differentiation. We hypothesize that hESC-derived neural precursors engineered with MEF2C will selectively differentiate into neurons, which will be resistant to apoptotic death and not form tumors such as teratomas. We believe that our proposed research will lead us to a better understanding of the role of MEF2C in hESC differentiation to neurons. These results will lead to novel and effective means to direct hESCs to become neurons and to resist cell death. This information will ultimately lead to novel, stem cell-based therapies to treat stroke and neurodegenerative diseases such as Parkinson’s. We also believe that an effective, straightforward, and broadly understandable way to describe the benefits to the citizens of the State of California that will flow from the stem cell research we propose to conduct is to couch the work in the familiar, everyday business concept of “Return on Investment.” The novel therapies and reconstructions that will be developed and accomplished as a result of our research program and the many related programs that will follow will provide direct benefits to the health of California citizens. In addition, this program and its many complementary programs will generate potentially very large, tangible monetary benefits to the citizens of California. These financial benefits will derive directly from two sources. The first source will be the sale and licensing of the intellectual property rights that will accrue to the state and its citizens from this and the many other stem cell research programs that will be financed by CIRM. The second source will be the many different kinds of tax revenues that will be generated from the increased bio-science and bio-manufacturing businesses that will be attracted to California by the success of CIRM.
Progress Report: 
  • In Year 02 of this grant, we have continued to refine the techniques developed for producing nerve cells from human embryonic stem cells (hESC). Central to our grant proposal is the expression of an active form of a protein called MEF2C, which we insert into the stem cells at a young age. MEF2C is a transcription factor, which is a molecule that regulates how RNA is converted to a protein. MEF2C regulates the production of proteins that are specifically found in neurons, and it plays an important role in making a stem cell into a nerve cell. Specific improvements this year in culture conditions have resulted in our being able to direct a much higher percentage of hESCs into precursors of nerve cells, and it is at this stage that the cells are most appropriate for insertion of MEF2C. Following this, we can transplant the stem cells, destined to become nerve cells, in to the brain in rodent models of stroke and Parkinson’s disease. We have also made very good progress in producing dopaminergic nerve cells, the specific type of cell that dies in Parkinson’s disease. In addition, our improved methods are completely free of any animal products, so they represent a step forward in developing cells as a treatment for human diseases.
  • Building upon these advances in our techniques, we have transplanted cells into a rat model of Parkinson’s disease and shown that a large percentage of the cells become dopaminergic nerve cells in the brain. Additionally, rats receiving these cell transplants show greater improvements in motor skills compared to rats receiving similar cells without the inserted MEF2C factor. These findings complement our results presented in the first year’s progress report showing that transplantation of these MEF2C-expressing cells into a mouse model of stroke resulted in less damage to the brain. Together these results indicate the utility and versatility of these cells “programmed” by expression of the inserted MEF2C gene.
  • Finally, in Year 02 we report on our efforts to discover the mechanism by which the MEF2C gene prevents cell death and drives stem cells to become nerve cells. We have performed microarray analyses, which measure the expression levels of various genes, e.g., how much of each protein is produced from a gene. This approach includes 24,000 of the possible ~30,000 gene sequences expressed in human cells and tissues. These experiments were performed on stem cells with the inserted MEF2C gene just as the cells were making the decision to become a nerve cell. We observed a decrease in the activity of several genes that are known to make stem cells proliferate (divide and multiply), rather than becoming a differentiated nerve cell. This finding is consistent with the known role of MEF2C, which causes cells to stop proliferating and start differentiating into nerve cells. Without insertion of MEF2C into the stem cells, they mostly continue proliferating. We also saw that many genes, which are not expressed in mature nerve cells, were coordinately down regulated. These results may suggest a new role of MEF2C as a factor for shutting down gene expression, thereby helping to promote the formation of new nerve cells. We are continuing our investigations into the mechanism of MEF2C actions in neuronal differentiation and function as well as our transplantation experiments in stroke and Parkinson’s disease models in the coming year.
  • We initially discovered that mouse embryonic stem cell (ESC)-derived neural progenitor cells forced to express the transcription factor MEF2C were protected from dying and were also given signals to differentiate almost exclusively into neurons (J Neurosci 2008; 28:6557-68). Under the CIRM grant, we have investigated the role of MEF2C and consequences of its forced expression in neural differentiation of human ES cells, including identification of specific genes under MEF2C regulation. We have also used rodent models of Parkinson’s disease and stroke to evaluate the therapeutic potential of human ESC-derived neural progenitors forced to express active MEF2C (MEF2CA).
  • In the third year of the CIRM grant, we continued to refine our procedures for differentiating MEF2CA-expressing human ES cells growing in culture into neural progenitor cells (NPC) and fully developed neurons. We also investigated their electrophysiological characteristics and potential to develop into specific types of neurons. We found that not only do the MEF2CA-expressing NPCs become almost exclusively neurons, as we previously showed, but they also had a strong bias to develop into dopaminergic neurons, the type of neuron that dies in Parkinson’s disease. We also found that MEF2CA-expressing NPCs differentiated to maturity in culture dishes showed a wide variety of electrophysiological responses of normal mature neurons. We were able to record sodium currents and action potentials indicating that the neurons were capable of transmitting chemo-electrical signals. They also responded to GABA and NMDA (a glutamate mimic), which shows that the neurons can respond to the major signal-transmitting molecules in the brain.
  • Previously we showed that transplantation of the MEF2CA-expressing human ESC-derived NPCs into the brains of a rat model of Parkinson’s disease resulted in a much higher number of dopaminergic (DA) neurons and positive behavioral recovery compared to controls. We now report that evaluation of the MEF2CA-expressing cells showed a much higher expression level of a variety of proteins known to be important in DA neuron differentiation and that none of these cells become tumors or hyper proliferative. We have also transplanted NPCs into the brains of a rat stroke model. Our preliminary data analysis shows an improvement in the ability to walk a tapered beam in the rats transplanted with MEF2CA-expressing cells compared to controls. These results are evidence there may be a great advantage in the use of NPC expressing MEF2C for transplantation into various brain diseases and injuries.
  • We have also continued our investigations into the mechanisms of MEF2C activities in the hope of finding new drug targets to mimic it effects. We have identified interactive pathways in which MEF2C plays a role and found correlations between MEF2C expression levels and a variety of diseases. These will hopefully lead us to a better understanding of how to leverage our results to produce effective therapies for a broad spectrum of neurological diseases and traumas.
  • Our goals for this grant were to determine the role of the transcription factor MEF2C in neurogenesis, including all of the targets of this factor in the genome, use this knowledge to direct differentiation of human embryonic stem cells (hESC) into specific types of neurons, and investigate the transplantation of these cells into rodent models of Parkinson’s disease (PD) and stroke. During the tenure of this grant, we accomplished these goals to a very significant degree. Our investigations into the role of MEF2C in neurogenesis produced a large body of knowledge pertinent to its essential role in this process. This knowledge base was achieved through both monitoring expression levels of MEF2C during the entire process of neurogenesis and by knocking down its expression by use of siRNA. We now have a very detailed view of the temporal contribution of MEF2C as stem cells differentiate into neurons. Using this knowledge, we optimized a differentiation protocol for directing hESC into neuronal precursor cells and then initiated expression of a constitutively active MEF2 transcription factor (MEF2CA) via lentiviral technology. We discovered that the forced expression of MEF2CA provided a strong bias to neurons to differentiate along a dopaminergic (DA) lineage. Our network analysis for MEF2C confirmed that many of the known effector proteins for DA neurons are indeed targets for this transcription factor. Histological and electrophysiological investigations into the nature of these cells grown in vitro showed that they are indeed functional neurons displaying the anticipated qualities during the various stages of differentiation.
  • Our in vivo transplantation studies have been equally productive. Owing to the strong tendency of the MEF2CA-expressing cells to differentiate into DA neurons, we first investigated their effects on a rat PD model where the dopaminergic cells of the substantia nigra are ablated on one side of the brain by injection of 6-hydroxydopamine. In response to an injection of the dopamine analog apomorphine, these rats will turn in a circle and the readout is the number of turns in a 30 minute period measured on a rotometer. Fewer turns indicate that the rat has less pathology, i.e., is getting better. We transplanted hESC-derived neural progenitor cells (hESC-NPC) either expressing MEF2CA or not and monitored recovery of the rats. While rats receiving both preparations of stem cells showed considerable improvement, the ones receiving MEF2C-expressing cells did significantly better on the rotometer. Also, histologically the MEF2CA-expressing cells could all be seen to differentiate, whereas those that did not express MEF2CA were often found in an undifferentiated state, which potentially posses a problem of continuing proliferation in the brain and tumor formation. Thus, the forced expression of MEF2CA forced the cells to differentiate and prevented uncontrolled cell division. An additional advantage was that the remaining endogenous DA neurons showed much greater density of fibers in the vicinity of the transplanted cells, suggesting that there was an additional benefit of factor secretion. Thus, the MEF2CA genetically modified cells appear to have significant advantages for transplantation for PD.
  • We are also investigating the use of the MEF2CA-expressing hESC-NPC in rat and mouse models of stroke. Preliminary data shows that in both systems we see behavioral improvements following the transplantations with these cells. In the period of the no cost extension, we will complete these studies and characterize the types of neurons these transplanted cells become and their role in reversing the pathology caused by the brain ischemia from stroke. Our hypothesis is that there is a strong bias toward the DA neuron phenotype produced by the expression of MEF2CA, but that this is overridden by the context within the brain. Therefore, in a stroke model, the context of damage to the cortex provides signals to the newly transplanted cells that they should migrate to the damaged area and become cells appropriate to that region, not DA neurons. We will test this hypothesis in the remaining months of the grant.
  • Our goals for this grant were to determine the role of the transcription factor MEF2C in neurogenesis, including all of the targets of this factor in the genome, use this knowledge to direct differentiation of human embryonic stem cells (hESC) into specific types of neurons, and investigate the transplantation of these cells into rodent models of Parkinson’s disease (PD) and stroke. During the tenure of this grant, we accomplished these goals to a very significant degree. Our investigations into the role of MEF2C in neurogenesis produced a large body of knowledge pertinent to its essential role in this process. This knowledge base was achieved through both monitoring expression levels of MEF2C during the entire process of neurogenesis and by knocking down its expression by use of siRNA. We now have a very detailed view of the temporal contribution of MEF2C as stem cells differentiate into neurons. Using this knowledge, we optimized a differentiation protocol for directing hESC into neuronal precursor cells and then initiated expression of a constitutively active MEF2 transcription factor (MEF2CA) via lentiviral technology. We discovered that the forced expression of MEF2CA provided a strong bias to neurons to differentiate along a dopaminergic (DA) lineage. Our network analysis for MEF2C confirmed that many of the known effector proteins for DA neurons are indeed targets for this transcription factor. Histological and electrophysiological investigations into the nature of these cells grown in vitro showed that they are indeed functional neurons displaying the anticipated qualities during the various stages of differentiation.
  • Our in vivo transplantation studies have been equally productive. Owing to the strong tendency of the MEF2CA-expressing cells to differentiate into DA neurons, we first investigated their effects on a rat PD model where the dopaminergic cells of the substantia nigra are ablated on one side of the brain by injection of 6-hydroxydopamine. In response to an injection of the dopamine analog apomorphine, these rats will turn in a circle and the readout is the number of turns in a 30 minute period measured on a rotometer. Fewer turns indicate that the rat has less pathology, i.e., is getting better. We transplanted hESC-derived neural progenitor cells (hESC-NPC) either expressing MEF2CA or not and monitored recovery of the rats. While rats receiving both preparations of stem cells showed considerable improvement, the ones receiving MEF2C-expressing cells did significantly better on the rotometer. Also, histologically the MEF2CA-expressing cells could all be seen to differentiate, whereas those that did not express MEF2CA were often found in an undifferentiated state, which potentially posses a problem of continuing proliferation in the brain and tumor formation. Thus, the forced expression of MEF2CA forced the cells to differentiate and prevented uncontrolled cell division. An additional advantage was that the remaining endogenous DA neurons showed much greater density of fibers in the vicinity of the transplanted cells, suggesting that there was an additional benefit of factor secretion. Thus, the MEF2CA genetically modified cells appear to have significant advantages for transplantation for PD.
  • We are also investigating the use of the MEF2CA-expressing hESC-NPC in rat and mouse models of stroke. Preliminary data shows that in both systems we see behavioral improvements following the transplantations with these cells. In the period of the no cost extension, we will complete these studies and characterize the types of neurons these transplanted cells become and their role in reversing the pathology caused by the brain ischemia from stroke. Our hypothesis is that there is a strong bias toward the DA neuron phenotype produced by the expression of MEF2CA, but that this is overridden by the context within the brain. Therefore, in a stroke model, the context of damage to the cortex provides signals to the newly transplanted cells that they should migrate to the damaged area and become cells appropriate to that region, not DA neurons. We will test this hypothesis in the remaining months of the grant.

Epigenetic gene regulation during the differentiation of human embryonic stem cells: Impact on neural repair

Funding Type: 
Comprehensive Grant
Grant Number: 
RC1-00111
ICOC Funds Committed: 
$2 516 613
Disease Focus: 
Stroke
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Closed
Public Abstract: 
Human embryonic stem cells (hESCs) have the potential to become all sorts of cells in human body including nerve cells. Moreover, hESCs can be expanded in culture plates into a large quantity, thus serving as an ideal source for cell transplantation in clinical use. However, the existing hESC lines are not fully characterized in terms of their potential to become specific cell types such as nerve cells. It is also unclear if the nerve cells that are derived from hESCs are totally normal when tested in cell transplantation experiments. One of the goals for our proposal is to compare the quality and the potential of eight lines of hESCs in their capacity to become nerve cells. To measure if the nerve cells that are derived from hESCs are normal when compared to the nerve cells in normal human beings, we will examine the levels of gene expression and the mechanisms that control gene expression in hESC-derived nerve cells. Specifically, we will examine the pattern of DNA modification, namely DNA methylation, in the DNA of nerve cells. This DNA modification is involved in the inhibition of gene expression. It is known that if DNA methylation pattern is abnormal, it can lead to human diseases including cancer and mental retardation disorders. We will use a DNA microarray technology to identify DNA methylation pattern in the critical regions where gene expression is controlled. Our recent results suggest that increased DNA methylation is observed in hESC-derived nerve cells. In this proposal, we will also test if we can balance the level of DNA methylation through pharmacological treatment of enzymes that are responsible for DNA methylation. Finally, we will test if hESC-derived nerve cells can repair the brain after injury . A mouse stroke model will be used for testing the mechanisms stem cell-mediated repair and recovery in the injured brain and for selecting the best nerve cells for cell transplantation. Our study will pave the way for the future use of hESC-derived nerve cells in clinical treatment of nerve injury and neurodegenerative diseases such as stroke and Parkinson’s disease.
Statement of Benefit to California: 
Neurodegenerative diseases such as stroke are the leading cause of adult disability. Stroke produces an area of damage in the brain which frequently causes the loss of crucial brain functions such as sensory and movement control, language skills, and cognition capability. Stem cell transplantation has emerged as a method that may improve recovery in these brain areas. Studies of stem cell transplantation after stroke have been limited because many of the transplanted cells do not survive, the appropriate regions for transplantation have not been identified, and the mechanisms by which transplanted stem cells improve recovery have not been determined. Also, there have been no studies of human embryonic stem cell transplantation after stroke. For the use of stem cell therapy in stroke patients, human embryonic stem cell lines have to be grown and tested for their efficacy in repairing the brain after stroke. We have recently found that the process of growing human embryonic stem cells in culture introduces genetic modifications in some of these cell lines that may decrease survival of the cells in the brain and impair their ability to repair the injured brain. The experiments in this grant will determine which human embryonic stem cell lines do not undergo this negative genetic modification. The optimum human embryonic stem cell lines will then be systematically tested for the location in the stroke brain that produces survival and integration, and the mechanisms of repair that these cells mediate in the brain after stroke. These studies will specifically test the role of human embryonic stem cells in improving sensory and movement functions after stroke. In summary, these studies will establish protocols for the proper growth of human embryonic stem cell lines, the lines that are most effective for repairing the brain after stroke, and the principles behind how human embryonic stem cells repair the brain. These results are applicable to other kinds of neurodegenerative conditions, such as Parkinsons, Alzheimer’s and Huntington’s diseases, and to the growth and culture of human embryonic stem cells in general for repair of disease of other human tissues.
Progress Report: 
  • Summary of Research Progress:
  • Our research aims to identify the optimal culture conditions and the best hESC lines for the derivation of nerve lineage cells in therapeutic cell transplantation. Toward this goal, we propose to compare the behavior of nerve cell differentiation in multiple lines of hESCs in one laboratory setting. We will further characterize molecular changes during directed cell differentiation and identify the cells that exhibit a pattern of DNA modification, namely DNA methylation, similar to primary neural cells in human brain. In the case of DNA hypermethylation, pharmacological treatment and genetic manipulation will be applied to correct the methylation defects by blocking enzymes involved in DNA methylation. Finally, cell transplantation in a mouse stroke model will be used to study the mechanisms and efficacy of different types of hESC-derived neural cells in neural repair.
  • In the past year, we have made progress in guiding several lines of human stem cells into nerve cells. We are now ready to compare the property of different lines of nerve cells such as the efficiency of nerve cell differentiation and the preferential production of specific nerve cells in culture. We also begin to produce and characterize a new type of human stem cells, namely induced pluripotent cells that are obtained by converting somatic cells into stem cell through reprogramming. We also test the pattern of DNA methylation in different lines of human stem cells. By engineering stem cells carrying different levels of methylation, we aim to find the optimal levels of DNA methylation for efficient nerve cell differentiation. Finally, we also made excellent progress on the procedure of cell transplantation. We have found a suitable substrate that can be used to enhance neuronal survival after cell transplantation and we expect to publish a research paper in this new method of cell transplantation.
  • Summary of Research Progress:
  • Our research aims to identify the optimal culture conditions and the best hESC lines for the derivation of nerve lineage cells in therapeutic cell transplantation. Toward this goal, we propose to compare the behavior of nerve cell differentiation in multiple lines of hESCs in one laboratory setting. We will further characterize molecular changes during directed cell differentiation and identify the cells that exhibit a pattern of DNA modification, namely DNA methylation, similar to primary neural cells in human brain. In the case of DNA hypermethylation, pharmacological treatment and genetic manipulation will be applied to correct the methylation defects by blocking enzymes involved in DNA methylation. Finally, cell transplantation in a mouse stroke model will be used to study the mechanisms and efficacy of different types of hESC-derived neural cells in neural repair.
  • In the past year, we have made great progress in converting several lines of human stem cells into nerve cells. We have compared the property of different lines of nerve cells such as the efficiency of nerve cell differentiation and the preferential production of specific nerve cells in culture. We also begin to produce and characterize a new type of human stem cells, namely induced pluripotent cells that are obtained by converting somatic cells into stem cell through reprogramming. We also test the pattern of DNA methylation in different lines of human stem cells. By engineering stem cells carrying different levels of methylation, we aim to find the optimal levels of DNA methylation for efficient nerve cell differentiation. Finally, we also made excellent progress on the procedure of cell transplantation. We have found a suitable substrate that can be used to enhance neuronal survival after cell transplantation and we expect to publish a research paper in this new method of cell transplantation.
  • Our research aims to identify the optimal culture conditions and the best hESC lines for the derivation of nerve lineage cells in therapeutic cell transplantation. Toward this goal, we propose to compare the behavior of nerve cell differentiation in multiple lines of hESCs in one laboratory setting. We will further characterize molecular changes during directed cell differentiation and identify the cells that exhibit a pattern of DNA modification, namely DNA methylation, similar to primary neural cells in human brain. In the case of DNA hypermethylation, pharmacological treatment and genetic manipulation will be applied to correct the methylation defects by blocking enzymes involved in DNA methylation. Finally, cell transplantation in a mouse stroke model will be used to study the mechanisms and efficacy of different types of hESC-derived neural cells in neural repair.
  • In the past year, we have made great progress in converting several lines of human stem cells into nerve cells. We have compared the property of different lines of nerve cells such as the efficiency of nerve cell differentiation and the preferential production of specific nerve cells in culture. We also succeeded in making a new type of human stem cells, namely induced pluripotent cells that are obtained by converting somatic cells into stem cell through reprogramming. We have tested the pattern of DNA methylation in different lines of human stem cells, including mutant cell lines from patients who exhibit defects in DNA methylaiton. Finally, we also made excellent progress on the procedure of cell transplantation and we characterized gene expression and epigenetic changes in transplanted nerve cells from human embryonic stem cells. Our studies allow us to optimize methods of neural cell differentiation and transplantation. We plan to publish additional two research papers in the near future.

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).

Embryonic-Derived Neural Stem Cells for Treatment of Motor Sequelae following Sub-cortical Stroke

Funding Type: 
Disease Team Research I
Grant Number: 
DR1-01480
ICOC Funds Committed: 
$20 000 000
Disease Focus: 
Stroke
Neurological Disorders
Collaborative Funder: 
Germany
Stem Cell Use: 
Embryonic Stem Cell
Cell Line Generation: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
A stroke kills brain cells by interrupting blood flow. The most common “ischemic stroke” is due to blockage in blood flow from a clot or narrowing in an artery. Brain cells deprived of oxygen can die within minutes. The loss of physical and mental functions after stroke is often permanent and includes loss of movement, or motor, control. Stroke is the number one cause of disability, the second leading cause of dementia, and the third leading cause of death in adults. Lack of movement or motor control leads to job loss and withdrawal from pre-stroke community interactions in most patients and institutionalization in up to one-third of stroke victims. The most effective treatment for stroke, thrombolytics or “clot-busters”, can be administered only within 4.5 hours of the onset of stroke. This narrow time window severely limits the number of stroke victims that may benefit from this treatment. This proposal develops a new therapy for stroke based on embryonic stem cells. Because our (and others’) laboratory research has shown that stem cells can augment the brain’s natural repair processes after stroke, these cells widen the stroke treatment opportunity by targeting the restorative or recovery phase (weeks or months after stroke instead of several hours). Embryonic stem cells can grow in a culture dish, but have the ability to produce any tissue in the body. We have developed a technique that allows us to restrict the potential of embryonic stem cells to producing cell types that are found in the brain, making them “neural stem cells”. These are more appropriate for treating stroke and may have lower potential for forming tumors. When these neural stem cells are transplanted into the brains of mice or rats one week after a stroke, the animals are able to regain strength in their limbs. Based on these findings, we propose in this grant to further develop these neural stem cells into a clinical development program for stroke in humans at the end of this grant period. This proposal develops a multidisciplinary team that will rigorously test the effectiveness of stem cell delivery in several models of stroke, while simultaneously developing processes for the precise manufacture, testing and regulatory approval of a stem cell therapy intended for human use. Each step in this process consists of definite milestones that must be achieved, and provides measurable assessment of progress toward therapy development. To accomplish this task, the team consists of stroke physician/scientists, pharmacologists, toxicologists, experts in FDA regulatory approval and key collaborations with biotechnology firms active in this area. This California-based team has a track record of close interactions and brings prior stroke clinical trial and basic science experience to the proposed translation of a stem cell therapy for stroke.
Statement of Benefit to California: 
The State of California has made a historic investment in harnessing the potential of stem cells for regenerative therapy. While initially focused on developing new stem cell technologies, CIRM has recognized that translational progress from laboratory to clinic must also be fostered, for this is ultimately how Californians will benefit from their investment. Our focus on developing a neuro-restorative therapy for treatment of motor sequelae following sub-cortical stroke contains several benefits to California. The foremost benefit will be the development of a novel form of therapy for a major medical burden: The estimated economic burden for stroke exceeds $56.8 billion per year in the US, with 55% of this amount supporting chronic care of stroke survivors (1). While the stroke incidence markedly increases in the next half-century, death rates from stroke have declined. These statistics translate into an expected large increase in disabled stroke survivors (1) that will have a significant impact on many aspects of life for the average Californian. Stroke is the third greatest cause of death, and a leading cause of disability, among Californians. Compared to the nation, California has slightly above average rates for stroke (2). Treatments that improve repair and recovery in stroke will reduce this clinical burden. The team that has been recruited for this grant is made of uniquely qualified members, some of whom were involved in the development, manufacturing and regulatory aspects of the first clinical trial for safety of neural stem cells for stroke. Thus not only is the proposed work addressing a need that affects most Californians, it will result in the ability to initiate clinical studies of stem cells for stroke recovery from a consortium of academic and biotechnology groups in California. 1. Carmichael, ST. (2008) Themes and strategies for studying the biology of stroke recovery in the poststroke epoch. Stroke 39(4):1380-8. 2. Reynen DJ, Kamigaki AS, Pheatt N, Chaput LA. The Burden of Cardiovascular Disease in California: A Report of the California Heart Disease and Stroke Prevention Program. Sacramento, CA: California Department of Public Health, 2007.
Progress Report: 
  • A stroke kills brain cells by interrupting blood flow. The most common “ischemic stroke” is due to blockage in blood flow from a clot or narrowing in an artery. Brain cells deprived of oxygen can die within minutes. The loss of physical and mental functions after stroke is often permanent and includes loss of movement, or motor, control. Stroke is the number one cause of disability, the second leading cause of dementia, and the third leading cause of death in adults. Lack of movement or motor control leads to job loss and withdrawal from pre-stroke community interactions in most patients and institutionalization in up to one-third of stroke victims. The most effective treatment for stroke, thrombolytics or “clot-busters”, can be administered only within 4.5 hours of the onset of stroke. This narrow time window severely limits the number of stroke victims that may benefit from this treatment. This proposal develops a new therapy for stroke based on embryonic stem cells. Because our (and others’) laboratory research has shown that stem cells can augment the brain’s natural repair processes after stroke, these cells widen the stroke treatment opportunity by targeting the restorative or recovery phase (weeks or months after stroke instead of several hours).
  • Embryonic stem cells can grow in a culture dish, but have the ability to produce any tissue in the body. We have developed a technique that allows us to restrict the potential of embryonic stem cells to producing cell types that are found in the brain, making them “neural stem cells”. These are more appropriate for treating stroke and may have lower potential for forming tumors. When these neural stem cells are transplanted into the brains of mice or rats one week after a stroke, the animals are able to regain strength in their limbs. Based on these findings, we propose in this grant to further develop these neural stem cells into a clinical development program for stroke in humans at the end of this grant period.
  • A multidisciplinary team is working rigorously to test the effectiveness of stem cell delivery in several models of stroke, while simultaneously developing processes for the precise manufacture, testing and regulatory approval of a stem cell therapy intended for human use. Each step in this process consists of definite milestones that are being achieved, providing measurable assessment of progress toward therapy development. To accomplish this task, the team consists of stroke physician/scientists, pharmacologists, toxicologists, experts in FDA regulatory approval and key collaborations with a biotechnology manufacturer active in this area. This California-based team has a track record of close interactions and brings prior stroke clinical trial and basic science experience to the proposed translation of a stem cell therapy for stroke.
  • In the first year of this program, the cells have been translated from an encouraging research level to a product which can be manufactured under conditions suitable for human administration. This has included optimization of the production process, development of reliable tests to confirm cell identity and function, and characterization of the cells utilizing these tests. In animal models in two additional laboratories , improvement in motor function following stroke has been confirmed. The method of administration has also been carefully studied. It has been determined that the cells will be administered around the area of stroke injury rather than directly into the middle of the stroke area. These results encourage the translation of this product from research into clinical trials for the treatment of motor deficit following stroke.
  • A stroke kills brain cells by interrupting blood flow. The most common “ischemic stroke” is due to blockage in blood flow from a clot or narrowing in an artery. Brain cells deprived of oxygen can die within minutes. The loss of physical and mental functions after stroke is often permanent and includes loss of movement, or motor, control. Stroke is the number one cause of disability, the second leading cause of dementia, and the third leading cause of death in adults. Lack of movement or motor control leads to job loss and withdrawal from pre-stroke community interactions in most patients and institutionalization in up to one-third of stroke victims. The most effective treatment for stroke, thrombolytics or “clot-busters”, can be administered only within 4.5 hours of the onset of stroke. This narrow time window severely limits the number of stroke victims that may benefit from this treatment. This proposal develops a new therapy for stroke based on embryonic stem cells. Because our (and others’) laboratory research has shown that stem cells can augment the brain’s natural repair processes after stroke, these cells widen the stroke treatment opportunity by targeting the restorative or recovery phase (weeks or months after stroke instead of several hours).
  • Embryonic stem cells can grow in a culture dish, but have the ability to produce any tissue in the body. We have developed a technique that allows us to restrict the potential of embryonic stem cells to producing cell types that are found in the brain, making them “neural stem cells”. These are more appropriate for treating stroke and may have lower potential for forming tumors. When these neural stem cells are transplanted into the brains of mice or rats one week after a stroke, the animals are able to regain strength in their limbs. Based on these findings, we propose in this grant to further develop these neural stem cells into a clinical development program for stroke in humans at the end
  • of this grant period.
  • A multidisciplinary team is working rigorously to test the effectiveness of stem cell delivery in several models of stroke, while simultaneously developing processes for the precise manufacture, testing and regulatory approval of a stem cell therapy intended for human use. Each step in this process consists
  • of definite milestones that are being achieved, providing measurable assessment of progress toward therapy development. To accomplish this task, the team consists of stroke physician/scientists, pharmacologists, toxicologists, experts in FDA regulatory approval and key collaborations with a biotechnology manufacturer active in this area. This California-based team has a track record of close interactions and brings prior stroke clinical trial and basic science experience to the proposed translation of a stem cell therapy for stroke.
  • A stroke kills brain cells by interrupting blood flow. The most common “ischemic stroke” is due to blockage in blood flow from a clot or narrowing in an artery. Brain cells deprived of oxygen can die within minutes. The loss of physical and mental functions after stroke is often permanent and includes loss of movement, or motor control. Stroke is the number one cause of disability, the second leading cause of dementia, and the third leading cause of death in adults. Lack of movement or motor control leads to job loss and withdrawal from pre-stroke community interactions in most patients and institutionalization in up to one-third of stroke victims. The most effective treatment for stroke, thrombolytics or “clot-busters”, can be administered only within 4.5 hours of the onset of stroke. This narrow time window severely limits the number of stroke victims that may benefit from this treatment. This proposal develops a new therapy for stroke based on embryonic stem cells. Because our (and others’) laboratory research has shown that stem cells can augment the brain’s natural repair processes after stroke, these cells widen the stroke treatment opportunity by targeting the restorative or recovery phase (weeks or months after stroke instead of several hours).
  • Embryonic stem cells can grow in a culture dish, but have the ability to produce any tissue in the body. We have developed a technique that allows us to restrict the potential of embryonic stem cells to producing cell types that are found in the brain, making them “neural stem cells”. These are more appropriate for treating stroke and may have lower potential for forming tumors. When these neural stem cells are transplanted into the brains of mice or rats one week after a stroke, the animals are able to regain strength in their limbs. Based on these findings this grant is supporting conduct of IND-enabling work to initiate a clinical development program for stroke in humans by the end of this grant period.
  • A multidisciplinary team is working rigorously to test the effectiveness of stem cell delivery in several models of stroke, while enabling precise manufacture, testing and regulatory clearance of a first in human clinical trial. Defined milestones are being achieved, providing measurable assessment of progress toward therapy development. Definitive manufacturing and pharmacology studies are underway and regulatory filings are in progress. The team consists of stroke physician/scientists, pharmacologists, toxicologists, experts in FDA regulatory and key collaborations with a biotechnology manufacturer active in this area. This California-based team has a track record of close interactions and brings prior stroke clinical trial and basic science experience to the proposed translation of a stem cell therapy for stroke.
  • A stroke kills brain cells by interrupting blood flow. The most common 'ischemic stroke' is due to blockage in blood flow from a clot or narrowing in an artery. Brain cells deprived of oxygen can die within minutes. The loss of physical and mental functions after stroke is often permanent and includes loss of movement, or motor, control. Stroke is the number one cause of disability, the second leading cause of dementia, and the third leading cause of death in adults. Lack of movement or motor control leads to job loss and withdrawal from pre-stroke community interactions in most patients and institutionalization in up to one-third of stroke victims. The most effective treatment for stroke, thrombolytics or 'clot-busters', can be administered only within 4.5 hours of the onset of stroke. This narrow time window severely limits the number of stroke victims that may benefit from this treatment. This proposal develops a new therapy for stroke based on embryonic stem cells. Because our (and others') laboratory research has shown that stem cells can augment the brain's natural repair processes after stroke, these cells widen the stroke treatment opportunity by targeting the restorative or recovery phase (weeks or months after stroke instead of several hours).
  • Embryonic stem cells can grow in a culture dish, but have the ability to produce any tissue in the body. We have developed a technique that allows us to restrict the potential of embryonic stem cells to producing cell types that are found in the brain, making them 'neural stem cells'. These are more appropriate for treating stroke and may have lower potential for forming tumors. When these neural stem cells are transplanted into the brains of mice or rats one week after a stroke, the animals are able to regain strength in their limbs. Based on these findings this grant is supporting conduct of IND-enabling work to initiate a clinical development program for stroke in humans by the end of this grant period.
  • A multidisciplinary team is working to test the effectiveness of stem cell delivery in several models of stroke, while enabling precise manufacture, testing and regulatory clearance of a first in human clinical trial. Defined milestones are being achieved, providing measurable assessment of progress toward therapy development. Definitive manufacturing and pharmacology studies are underway and regulatory filings are in progress. The team consists of stroke physicians/scientists, pharmacologists, toxicologists, experts in FDA regulatory and key collaborations with a biotechnology manufacturer active in this area. This California-based team has a track record of close interactions and brings prior stroke clinical trial and basic science experience to the proposed translation of a stem cell therapy for stroke.

Development of Single Cell MRI Technology using Genetically-Encoded Iron-Based Reporters

Funding Type: 
Tools and Technologies II
Grant Number: 
RT2-02018
ICOC Funds Committed: 
$1 930 608
Disease Focus: 
Stroke
Neurological Disorders
Stem Cell Use: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 
Clinical application of cell transplantation therapy requires a means of non-invasively monitoring these cells in the patient. Several imaging modalities, including MRI, bioluminescence imaging, and positron emission tomography have been used to track stem cells in vivo. For MR imaging, cells are pre-loaded with molecules or particles that substantially alter the image brightness; the most common such labelling strategy employs iron oxide particles. Several studies have shown the ability of MRI to longitudinally track transplanted iron-labeled cells in different animal models, including stroke and cancer. But there are drawbacks to this kind of labeling. Division of cells will result in the dilution of particles and loss of signal. False signal can be detected from dying cells or if the cells of interest are ingested by other cells. To overcome these roadblocks in the drive toward clinical implementation of stem cell tracking, it is now believed that a genetic labeling approach will be necessary, whereby specific protein expression causes the formation of suitable contrast agents. Such endogenous and persistent generation of cellular contrast would be particularly valuable to the field of stem cell therapy, where the homing ability of transplanted stem cells, long-term viability, and capacity for differentiation are all known to strongly influence therapeutic outcomes. However, genetic labeling or "gene reporter" strategies that permit sensitive detection of rare cells, non-invasively and deep in tissue, have not yet been developed. This is therefore the translational bottleneck that we propose to address in this grant, through the development and validation of a novel high-sensitivity MRI gene reporter technology. There have been recent reports of gene-mediated cellular production of magnetic iron-oxide nanoparticles of the same composition as the synthetic iron oxide particles used widely in exogenous labeling studies. It is an extension of this strategy, combined with our own strengths in developing high-sensitivity MRI technology, that we propose to apply to the task of single cell tracking of metastatic cancer cells and neural stem cells. If we are successful with the proposed studies, we will have substantially advanced the field of in vivo cellular imaging, by providing a stable cell tracking technology that could be used to study events occurring at arbitrary depth in tissue (unlike optical methods) and over unlimited time duration and arbitrary number of cell divisions (unlike conventional cellular MRI). With the ability to track not only the fate (migration, homing and proliferation) but also the viability and function of very small numbers of stem cells will come new knowledge of the behavior of these cells in a far more relevant micro-environment compared with current in vitro models, and yet with far better visualization and cell detection sensitivity compared with other in vivo imaging methods.
Statement of Benefit to California: 
Stem cell therapy has enormous promise to become a viable therapy for a range of illnesses, including stroke, other cardiovascular diseases, and neurological diseases. Progress in the development of these therapies depends on the ability to monitor cell delivery, migration and therapeutic action at the disease site, using imaging and other non-invasive technologies. If breakthroughs could be made along these lines, it would not only be of enormous benefit to the citizens of the state of California, but would also greatly reduce healthcare costs. From a broader research perspective, the state of California is the front-runner in stem cell research, having gathered not only private investments, as demonstrated by the numerous biotechnology companies that are developing innovative tools, but also extensive public funds that allows the state, through CIRM, to sponsor stem cell research in public and private institutions. In order to preserve the leadership position and encourage research on stem cells, CIRM is calling for research proposals to develop innovative tools and technologies that will overcome current roadblocks in translational stem cell research. This proposal will benefit the state by providing important new technology that will be valuable for both basic and translational stem cell research. A key bottleneck to the further development and translation of new stem cell therapies is the inability to track stem cells through a human body. It is possible to image stem cells using embedded optical fluorescence labels, but optical imaging does not permit tracking of cells deep in tissue. Other imaging modalities and their associated cellular labels (for example positron emission tomography) have also been used to track cells but do not have the sensitivity to detect rare or single cells. Finally, MRI has been used to track cells deep in tissue, down to the single cell level, but only by pre-loading cells with a non-renewable supply of iron oxide nanoparticles, which prevents long-term tracking and assessment of cell viability and function. We propose here to develop MRI technology and a new form of genetically-encoded, long-term cell labeling technology, to a much more advanced state than available at present. This will make it possible to use MRI to detect and follow cancer and stem cells as they migrate to and proliferate at the site of interest, even starting from the single cell stage. This will provide a technology that will help stem cell researchers, first and foremost in California, to understand stem cell behavior in a realistic in vivo environment. This technology will be translatable to future human stem cell research studies.
Progress Report: 
  • We have made good progress in the first year. This project involves four separate scientific teams, brought together for the first time, representing diverse backgrounds ranging from magnetic resonance imaging (MRI) physics and cell tracking (Dr. Rutt), microbiology (Dr. Matin), nano and magnetic characterization (Dr. Moler) and stem cell imaging in stroke models (Dr. Guzman). Substantial progress has been made by all four teams, and we are starting to see important interactions between the teams. An overall summary of progress is that we have evaluated three different bacterial genes (magA, mms6, mamB) in one mammalian cell line (MDA-MB-231BR) and have shown significant iron accumulation in vitro with two of these genes, which is a very positive result implying that these genes may have the required characteristics to act as "reporter genes" for MRI-based tracking of cells labeled with these genes. MR imaging of mouse brain specimens has yielded promising results and in vivo imaging experiments are underway at medium MRI field strength (3 Tesla). At the same time, we are ramping up our higher field, higher sensitivity MR imaging methods and will be ready to evaluate the different variations of our MR reporter gene at 7 Tesla (the highest magnetic field widely available for human MRI) in the near future. Finally, methods to perform quantitative characterization of our reporter cells are being developed, with the goal of being able to characterize magnetic properties down to the single cell level, and also to be able to assess iron loading levels down to the single level in brain tissue slices.
  • We have made good progress in the second year. This project involves four separate scientific teams, brought together for the first time for this project, representing diverse backgrounds ranging from magnetic resonance imaging (MRI) physics and cell tracking (Dr. Rutt), microbiology (Dr. Matin), nano and magnetic characterization (Dr. Moler) and imaging reporter development and testing in small animal models of disease (Dr. Contag). Substantial progress has been made by all four teams, and we are starting to see important interactions between the teams.
  • An overall summary of progress is that we have been evaluating three different bacterial genes (magA, mms6, mamB) in two mammalian cell lines (MDA-MB-231BR and DAOY). In year I we had shown significant iron accumulation in vitro with two of these genes, which was a very positive result implying that these genes may have the required characteristics to act as "reporter genes" for MRI-based tracking of cells labeled with these genes. In year 2, we diversified and intensified the efforts to achieve expression of one or more of the bacterial genes in different cell lines, using different genetic constructs. We began a concerted effort to achieve optical labeling such that we could visualize the gene expression and to identify sub-cellular localization of the report gene products.
  • We obtained promising results from MR imaging of mouse brain. In vivo imaging experiments were accomplished at medium MRI field strength (3 Tesla). At the same time, we ramped up our higher field, higher sensitivity MR imaging methods and began to evaluate the sensitivity gains enabled at the higher magnetic field strength of 7 Tesla (the highest magnetic field widely available for human MRI
  • Finally, methods to perform quantitative characterization of our reporter cells were developed, with the goal of being able to characterize magnetic properties down to the single cell level, and also to be able to assess iron loading levels down to the single level in brain tissue slices.
  • We have made good progress in the third year. This project involves four separate scientific teams, brought together for the first time for this project, representing diverse backgrounds ranging from magnetic resonance imaging (MRI) physics and cell tracking (Dr. Rutt), microbiology (Dr. Matin), nano and magnetic characterization (Dr. Moler) and imaging reporter development and testing in small animal models of disease (Dr. Contag). Substantial progress has been made by all four teams, and we have benefited from important interactions between all teams in this third year.
  • An overall summary of progress is that we evaluated several iron-binding bacterial genes (magA, mamB, mms6, mms13), both singly and doubly, in two mammalian cell lines (MDA-MB-231BR and DAOY). In year 2, we diversified and intensified the efforts to achieve expression of one or more of the bacterial genes in different cell lines, using different genetic constructs. We completed an effort to achieve optical labeling such that we could visualize the gene expression and to identify sub-cellular localization of the report gene products. In year 3, while continuing to face challenges with single gene constructs, we succeeded in finding substantial iron uptake in cells containing unique double gene expression, notably magA and mms13.
  • We completed much of the development of our higher field, higher sensitivity MR imaging methods and evaluated the sensitivity gains enabled at the higher magnetic field strength of 7 Tesla (the highest magnetic field widely available for human MRI).
  • Finally, we demonstrated novel nanomagnetic methods to characterize our reporter cells, able to characterize magnetic properties down to the single cell level.

Development of a Hydrogel Matrix for Stem Cell Growth and Neural Repair after Stroke

Funding Type: 
Tools and Technologies II
Grant Number: 
RT2-01881
ICOC Funds Committed: 
$1 825 613
Disease Focus: 
Stroke
Neurological Disorders
Stem Cell Use: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
Stroke is the leading cause of adult disability. Most patients survive their initial stroke, but do not recover fully. Because of incomplete recovery, up to 1/3 of stroke patients are taken from independence to a nursing home or assisted living environment, and most are left with some disability in strength or control of the arms or legs. There is no treatment that promotes brain repair and recovery in this disease. Recent studies have shown that stem cell transplantation into the brain can promote repair and recovery in animal models of stroke. However, a stem cell therapy for stroke has not reached the clinic. There are at least three limitations to the development of a human stroke stem cell therapy: most of the transplanted cells die, most of the cells that survive do not interact with the surrounding brain, and the process of injecting stem cells into the brain may damage the normal brain tissue that is near the stroke site. The studies in this grant develop a novel investigative team and research approach to achieve a solution to these limits. Using the combined expertise of engineering, stem cell biology and stroke scientists the studies in this grant will develop tissue bioengineering systems for a stem cell therapy in stroke. The studies will develop a biopolymer hydrogel that provides a pro-growth and pro-survival environment for stem cells when injected with them into the brain. This approach has three unique aspects. First, the hydrogel system utilizes biological components that mimic the normal brain environment and releases specific growth factors that enhance transplanted stem cell survival. Second, these growth factors will also likely stimulate the normal brain to undergo repair and recovery, providing a dual mechanism for neural repair after stroke. Third, this approach allows targeting of the stroke cavity for a stem cell transplant, and not normal brain. The stroke cavity is an ideal target for a stroke stem cell therapy, as it is a cavity and can receive a stem cell transplant without displacing normal brain, and it lies adjacent to the site in the brain of most recovery in this disease—placing the stem cell transplant near the target brain region for repair in stroke. The progress from stroke stem cell research has identified stem cell transplantation as a promising treatment for stroke. The research in this grant develops a next generation in stem cell therapies for the brain by combining new bioengineering techniques to develop an integrated hydrogel/stem cell system for transplantation, survival and neural repair in this disease.
Statement of Benefit to California: 
Advances in the early treatment of stroke have led to a decline in the death rate from this disease. At the same time, the overall incidence of stroke is projected to substantially increase because of the aging population. These two facts mean that stroke will not be lethal, but instead produce a greater number of disabled survivors. A 2006 estimate placed over half of the annual cost in stroke as committed to disabled stroke survivors, and exceeding $30 billion per year in the United States. The studies in this grant develop a novel stem cell therapy in stroke by focusing on one major bottleneck in this disease: the inability of most stem cell therapies to survive and repair the injured brain. With its large population California accounts for roughly 24% of all stroke hospital discharges in the Unites States. The development of a new stem cell therapy approach for this disease will lead to a direct benefit to the State of California.
Progress Report: 
  • This grant develops a tissue bioengineering approach to stem cell transplantation as a treatment for brain repair and recovery in stroke. Stem cell transplantation has shown promise as a therapy that promotes recovery in stroke. Stem cell transplantation in stroke has been limited by poor survival of the transplanted cells. The studies in this grant utilize a multidisciplinary team of bioengineers, neuroscientists/neurologists and stem cell biologists to develop an approach in which stem or progenitor cells can be transplanted into the site of the stroke within a biopolymer hydrogel that provides an environment which supports cell survival and treatment of the injured brain. These hydrogels need to contain naturally occurring brain molecules, so that they do not release foreign or toxic components when they degrade. Further, the hydrogels have to remain liquid so that the injection approach can be minimally invasive, and then gel within the brain. In the past year the fundamental properties of the hydrogels have been determined and the optimal physical characteristics, such as elasticity, identified. Hydrogels have been modified to contain molecules which stem or progenitor cells will recognize and support survival, and to contain growth factors that will both immediately release and, using a novel nanoparticle approach, more slowly release. These have been tested in culture systems and advanced to testing in rodent stroke models. This grant also tests the concept that the stem/progenitor cell that is more closely related to the area within the brain that receives the transplant will provide a greater degree of neural repair and recovery. Progress has been made in the past year in differentiating induced pluripotent stem cells along a lineage that more closely resembles the part of the brain injured in this stroke model, the cerebral cortex.
  • This grant determines the effect of a tissue bioengineering approach to stem cell survival and engraftment after stroke, as means of improving functional recovery in this disease. Stem cell transplantation in stroke has been limited by the poor survival of transplanted cells and their lack of differentiation in the brain. These studies use a biopolymer hydrogel, made of naturally occurring molecules, to provide a pro-survival matrix to the transplanted cells. The studies in the past year developed the chemical characteristics of the hydrogel that promote survival of the cells. These characteristics include the modification of the hydrogel so that it contains specific amounts of protein signals which resemble those seen in the normal stem cell environment. Systematic variation of the levels of these protein signals determined an optimal concentration to promote stem cell survival in vitro. Next, the studies identified the chemistry and release characteristics from the hydrogel of stem cell growth factors that normally promotes survival and differentiation of stem cells. Two growth factors have been tested, with the release characteristics more completely defined with one specific growth factor. The studies then progressed to determine which hydrogels supported stem cell survival in vivo in a mouse model of stroke. Tests of several hydrogels determined that some provide poor cell survival, but one that combines the protein signals, or “motifs”, that were studied in vitro provided improved survival in vivo. These hydrogels did not provoke any additional scarring or inflammation in surrounding tissue after stroke. Studies in the coming year will now determine if these stem cell/hydrogel matrices promote recovery of function after stroke, testing both the protein motif hydrogels and those that contain these motifs plus specific growth factors.
  • This grant determines the effect of a tissue bioengineering approach to stem cell survival and engraftment after stroke, as means of improving functional recovery in this disease. Stem cell transplantation in stroke has been limited by the poor survival of transplanted cells and their lack of differentiation in the brain. These studies use a biopolymer hydrogel, made of naturally occurring molecules, to provide a pro-survival matrix to the transplanted cells. The studies in past years developed the two chemical characteristics of hydrogels that contain recognition or signal elements for stem cells: “protein motifs” that resemble molecules in the normal stem cell environment and growth factors that normally communicate to stem cells in the brain. The hydrogels were engineered so that they contain these familiar stem cell protein motifs and growth factors and release the growth factors over a slow and sustained time course. In the past year on this grant, we tested the effects of hydrogels that had the combined characteristics of these protein motifs and growth factors, at varying concentrations, for their effect on induced pluripotent neural precursor cells (iPS-NPCs) in culture. We identified an optimum concentration for cell survival and for differentiation into immature neurons. We then initiated studies of the effects of this optimized hydrogel in vivo in a mouse model of stroke. These studies are ongoing. They will determine the cell biological effect of this hydrogel on adjacent tissue and on the transplanted cells—determining how the hydrogel enhances engraftment of the transplant. The behavioral studies, also under way, will determine if this optimized hydrogel/iPS-NPC transplant enhances recovery of movement, or motor, function after stroke.

Defining the Isoform-Specific Effects of Apolipoprotein E on the Development of iPS Cells into Functional Neurons in Vitro and in Vivo

Funding Type: 
New Faculty II
Grant Number: 
RN2-00952
ICOC Funds Committed: 
$2 847 600
Disease Focus: 
Stroke
Neurological Disorders
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
GOALS We propose to determine the effects of different forms of apoE on the development of induced pluripotent stem (iPS) cells into functional neurons. In Aim 1, iPS cells will be generated from skin cells of adult knock-in (KI) mice expressing different forms of human apoE and in humans with different apoE genotypes. In Aim 2, the development of the iPS cells into functional neurons in culture and in mouse brains will be compared. In Aim 3, the effects of different forms of apoE on the functional recovery of mice with acute brain injury treated with iPS cell–derived neural stem cells (NSCs) will be assessed. RATIONALE AND SIGNIFICANCE The central nervous system (CNS) has limited ability to regenerate and recover after injury. For this reason, recovery from acute and chronic neurological diseases, such as stroke and Alzheimer’s disease (AD), is often incomplete and disability results. Embryonic stem cells have great promise for treating or curing neurological diseases, but their therapeutic use is limited by ethical concerns and by rejection reactions after allogenic transplantation. The generation of iPS cells from somatic cells offers a way to potentially circumvent the ethical issues and to generate patient- and disease-specific stem cells for future therapy. In the CNS, apoE plays important roles in lipid homeostasis and in neuronal maintenance. However, apoE2, apoE3, and apoE4 differ in their ability to accomplish these tasks. ApoE4, the major genetic risk factor for AD, is associated with poor clinical outcome and more rapid progression or greater severity of head trauma, stroke, Parkinson’s disease, multiple sclerosis, and amyotrophic lateral sclerosis—all potential targets of stem cell therapy. This proposal builds on three novel findings in human apoE-KI mice. (1) NSCs express apoE. (2) ApoE plays a role in cell-fate determination (neuron vs astrocyte) of NSCs. (3) ApoE4 impairs the neuronal development of NSCs. Thus, we hypothesize that transplantation of iPS cells derived from apoE4 carriers (~20% of the general population and ~50% of AD patients) might not be beneficial or even detrimental for patients with neurological diseases. We propose in vitro and in vivo studies to assess the effects of different forms of apoE on the development of iPS cells into functional neurons and on the functional recovery of mice with acute brain injury treated with iPS cell-derived NSCs. These studies will shed light on the regulation of neuronal development of iPS cells and help to “optimize” future iPS cell therapy for neurological diseases. SPECIFIC AIMS Aim 1. To establish adult mouse and human iPS cell lines with different apoE genotypes. Aim 2. To determine the isoform-specific effects of apoE on the development of iPS cells into functional neurons in culture and in mouse brains. Aim 3. To assess the isoform-specific effects of apoE on the functional recovery of mice with acute (stroke) brain injury treated with iPS cell-derived NSCs.
Statement of Benefit to California: 
CONTRIBUTION TO THE CALFORNIA ECONOMY: A major goal of regenerative medicine is to repair damaged cells or tissue. My research focuses on (1) understanding the role of neuronal regeneration in central nervous system function and (2) developing stem cell therapy for acute and chronic neurological diseases, including stroke and Alzheimer's disease. Stroke and Alzheimer's disease are the leading causes of disability and dementia and are the fastest growing form of neurological diseases in California, in the USA, and worldwide. My research could benefit the California economy by creating jobs in the biomedical sector. Ultimately, this study could help reduce the adverse impact of neurological diseases. Thereby, I hope to increase the productivity and enhance the quality of life for Californians. The results of my studies will also help develop new technology that could contribute to the California biotechnology industry. The studies will characterize multiple lines of induced pluripotent stem (iPS) cells carrying apoE3, a protein protective to the brain, or apoE4, which is detrimental to the brain and is associated with increased risk of Alzheimer’s disease and other neurodegenerative disorders. These cell lines could be valuable for biotechnology companies and researchers who are screening for drug compounds targeting different neurological diseases. CONTRIBUTION TO THE HEALTH OF CALFORNIANS: The most important contribution of the studies will be to improve the health of Californians. Diseases that are the target of regenerative medicine, such as stroke and Alzheimer’s disease, are major causes of mortality and morbidity, resulting in billions of dollars in healthcare costs and lost productivity. As we continue our efforts in medical research, we hope to one day unlock the secrets of brain development and repair. This knowledge will help medical researchers develop beneficial therapies beyond what is currently available and potentially improve the quality of life and life expectancy of patients with neurological diseases, such as stroke and Alzheimer’s disease.
Progress Report: 
  • The goal of this proposal is to determine the isoform-specific effects of apolipoprotein (apo) E on the development of induced pluripotent stem (iPS) cells into functional neurons both in vitro and in mice. Toward this goal, we have made significant progress in Aims 1 and 2.
  • First, we further demonstrated that neural stem cells (NSCs) express apoE. ApoE-KO mice had significantly less hippocampal neurogenesis, but significantly more astrogenesis, than wildtype mice due to decreased Noggin expression in NSCs. In contrast, neuronal maturation in apoE4 knock-in (apoE4-KI) mice was impaired due to reduced survival and function of GABAergic interneurons in the hilus of the hippocampus, and a GABAA receptor potentiator rescued the apoE4-associated decrease in hippocampal neurogenesis. Thus, apoE plays an important role in hippocampal neurogenesis, and the apoE4 isoform impairs GABAergic input to newborn neurons, leading to decreased neurogenesis. A paper describing these data was published in Cell Stem Cell (Li G. et al. 2009, 5:634-645), which evidently is the 400th publication of CIRM-funded projects.
  • Second, we established mouse iPS cell lines from adult mouse fibroblasts of wildtype, apoE knockout (apoE-KO), human apoE2-KI, human apoE3-KI, and human apoE4-KI mice.
  • Finally, we developed NSC lines from mouse iPS cells with different apoE genotypes (wildtype mouse apoE, apoE-KO, apoE2, apoE3, and apoE4). These cell lines will be used to study the effects of apoE isoforms on neuronal development in vitro in culture and in vivo in mouse models.
  • The goal of this proposal is to determine the isoform-specific effects of apolipoprotein (apo) E on the development of induced pluripotent stem (iPS) cells into functional neurons both in vitro and in mice. Toward this goal, we have made significant progress in the past year, as summarized below.
  • First, We developed human iPS cells from skin fibroblasts of individuals with different apoE genotypes. We are fully characterizing these human iPS cell lines.
  • Second, We are establishing neural stem cell (NSC) lines from human iPS cells with different apoE genotypes. Some of the NSCs have been maintained in monolayer cultures for many generations. These NSCs will be used to study the effects of apoE isoforms on neuronal development in vitro in cultures and in vivo in mice.
  • Finally, we demonstrated that mouse apoE4-NSCs generated significantly fewer total neurons and fewer GABAergic interneurons than mouse apoE3-NSCs in culture. Thus, the detrimental effects of apoE4 on neurogenesis and GABAergic interneuron survival, as we observed in vivo in apoE4 knock-in mice (Li G. et al. Cell Stem Cell, 2009, 5:634-645), are recapitulated in cultures of mouse iPS cell–derived NSCs in vitro.
  • The goal of this proposal is to determine the isoform-specific effects of apolipoprotein (apo) E on the development of induced pluripotent stem (iPS) cells into functional neurons both in vitro and in mice. Toward this goal, we have made significant progress in all three aims in the past year, as summarized below.
  • 1) We have fully characterized two apoE3/3-hiPS cell lines and two apoE4/4-iPS cell lines.
  • 2) We have established NSC lines from human iPS cells with an apoE3/3 or apoE4/4 genotype. The hNSCs have been maintained in suspension or monolayer culture for multiple passages.
  • 3) We demonstrated that apoE4-hNSCs generated ~50% fewer GABAergic interneurons than apoE3-hNSCs in culture. Thus, the detrimental effects of apoE4 on GABAergic interneuron survival, as we observed in vivo in apoE4 knock-in mice (Li G. et al. Cell Stem Cell, 2009, 5:634-645), are recapitulated in cultures of human iPS cell-derived NSCs in vitro.
  • 4) We established protocols in our lab to differentiate human iPS cell-derived NSCs into different types of neurons in cultures.
  • The goal of this proposal is to determine the isoform-specific effects of apolipoprotein (apo) E on the development of induced pluripotent stem (iPS) cells into functional neurons both in vitro and in mice. Toward this goal, we have made significant progress in all three aims in the past year, as summarized below.
  • 1) We demonstrated that apoE4-miPSC-derived mNSCs had a greater “age-dependent (passage-dependent)” decrease in generation and/or survival of MAP2-positive neurons in cultures.
  • 2) We also demonstrated that apoE4-miPSC-derived mNSCs had an even greater “age-dependent (passage-dependent)” decrease in generation and/or survival of GAD67-positive GABAergic neurons, as seen in vivo in apoE4 knock-in mice (Li et al., Cell Stem Cell, 2009, 5:634–645).
  • 3) We expanded the pilot study reported last year and confirmed the detrimental effect of apoE4 on GABAergic interneuron development/survival of hiPS cell-derived hNSCs. ApoE4 also increased tau phosphorylation, one of the pathological hallmarks of Alzheimer’s disease, in neurons derived from apoE4-hiPS cells.
  • 4) We established a protocol to transplant apoE-miPS cell-derived mNSCs into mouse brains. The transplanted apoE-mNSCs developed into neurons and astrocytes and integrated into the neural circuitry.
  • The goal of this proposal is to determine the isoform-specific effects of apolipoprotein (apo) E on the development of pluripotent stem cells into functional neurons in vitro in culture and in vivo in mice for potential cell replacement therapy. Toward this goal, we have made significant progress in all three aims in the past year, as summarized below.
  • 1) We demonstrated that mouse GABAergic progenitors transplanted into the hilus of apoE3-KI and apoE4-KI mice developed into mature interneurons and functionally integrated into the hippocampal circuitry.
  • 2) We also demonstrated that transplantation of mouse GABAergic progenitors into the hilus of apoE4-KI mice rescued learning and memory deficits.
  • 3) Transplantation of mouse GABAergic progenitors into the hilus of hippocampus also rescued learning and memory deficits in apoE4-KI mice expressing Alzheimer’s disease-causing APP mutations.

Paracrine and synaptic mechanisms underlying neural stem cell-mediated stroke recovery

Funding Type: 
Basic Biology V
Grant Number: 
RB5-07363
ICOC Funds Committed: 
$1 178 370
Disease Focus: 
Stroke
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Stem cell therapy holds promise for the almost million Americans yearly who suffer a stroke. Preclinical data have shown that human neural stem cells (hNSCs) aid recovery after stroke, resulting in a major effort to advance stem cell therapy to the clinic, and we are currently transitioning our hNSC product to the clinic for stroke therapy. In this proposal we will explore how these cells improve lost function. We have already shown that injected hNSCs secrete factors that promote the gross rewiring of the brain, a major component of the spontaneous recovery observed after stroke. We now intend to focus on the connections between neurons, the synapses, which are a critical part of this rewiring process. We aim to quantify the effect of hNSCs on synapse density and function, and explore whether the stem cells secrete restorative synaptogenic factors or form functional synapses with pre-existing neurons. Our pursuit is made possible by our combination of state-of-the-art imaging techniques enabling us to visualize, characterize, and quantify these tiny synaptic structures and their interaction with the hNSCs. Furthermore, by engineering the hNSCs we can identify the factors they secrete in the brain and identify those which modulate synaptic connections. Our proposed studies will provide important insight into how transplanted stem cells induce recovery after stroke, with potential applicability to other brain diseases.
Statement of Benefit to California: 
Cerebrovascular stroke is the fourth leading cause of mortality in the United States and a significant source of long-term physical and cognitive disability that has devastating consequences to patients and their families. In California alone, over 9% of adults 65 years or older have had a stroke according to a 2005 study. In the next 20 years the societal toll is projected to amount to millions of patients and 18.8 billion dollars per year in direct medical costs. To date, there is no approved therapeutic agent for the recovery phase after stroke, making the long-term care of stroke patients a tremendous socioeconomic burden that will continue to rise as our aging population increases. Our laboratory and others have demonstrated the promise of stem cell transplantation to treat stroke. We are dedicated to developing human neural stem cells (hNSCs) as a novel neuro-restorative treatment for lost motor function after stroke. The goal of our proposed work is to further understand how transplanted hNSCs improve stroke recovery, as dissecting the mechanism of action of stem cells in the stroke brain will ultimately improve the chance of clinical success. This could potentially provide significant cost savings to California, but more importantly benefit the thousands of Californians and their families who struggle with the aftermath of stroke.

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