Neurological Disorders

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

Reconstruction of Pathways involved in cardiomyocyte differentiation from embryonic stem cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00333
ICOC Funds Committed: 
$0
Disease Focus: 
Amyotrophic Lateral Sclerosis
Neurological Disorders
Spinal Cord Injury
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Heart disease is the leading cause of mortality and decline in the quality of life in the world. Current therapies are unable to restore function to damaged heart tissue. Recent scientific developments demonstrate the ability of human embryonic stem cells to form cardiomyocytes and this gives rise to the hope that we will be able to use these cells to differentiate and replace damaged myocardium. Despite this preliminary evidence and promise, we are far from being able to successfully accomplish regeneration of mature myocardium. This is largely due to our lack of understanding of all the factors that are involved in the differentiation process and our ability to manipulate the regeneration process. Our current understanding of the differentiation process indicates that there are at least four distinct stages of differentiation leading to cardiomyocytes, namely: (1) establishment of organizing centers; (2) mesendoderm induction; (3) establishment of cardiac precursors; and (4) terminal differentiation of beating cardiomyocytes. Our first quest is to identify what are the factors associated with each stage of differentiation leading to cardiomyocytes. We will carry out measurements of several biological macromolecules that are anticipated to play a role in sending signals for differentiation. This will be followed by systematic reconstruction of biochemical pathways so that we get a systems-level perspective on the differentiation process. In addition, we plan to embark on quantitative modeling of cardiomyogenesis to provide a firm basis for stem cell-based therapy. Quantitative modeling of cardiomyogenesis is certain to provide insights into the etiology of complex congenital heart defects (CHDs). A comprehensive quantitative approach will provide explanations for the malformations beyond that which can be obtained by single gene manipulations in typical experimental systems because of the ability to take into account the interconnected networks of signal transduction pathways. As important as this research is for pediatric cardiology, the significance for adult cardiology is perhaps more profound because it offers the promise of improving prospects for therapeutic intervention. In support of this view, adult cardiac disease, such as ventricular hypertrophy, reiterates molecular genetic pathways that are characteristic of embryonic development, and thus the predictions based on quantitative modeling and in vitro testing might define targets for pharmaceutical intervention.
Statement of Benefit to California: 
In general, cardiovascular disease typically refers to a wide variety of heart and blood vessel diseases, including coronary heart disease, hypertension, stroke, and rheumatic heart disease. In 2000 nearly 200,000 Californians were hospitalized on account of heart disease. The statewide hospitalization rate due to heart disease in California is 6.4 per thousand in 2000. In addition to heart related illnesses, Stroke, hypercholesterolemia, high blood pressure and stroke accounted for another 30 percent of residents in California. Nearly 100,000 deaths resulted from these pathologies. In almost every case damage to the heart preceded death. Several risk factors besides genetics and heredity play a role in heart diseases. While prevention by healthy life styles and avoidance of risk factors is the first choice, this may not be feasible for all the residents of California. Ability of embryonic stem cells to differentiate into cardiac cells provides the hope that therapy by regeneration of damaged heart is a definitely possibility for the future. However, our understanding of the processes that lead a stem cell to differentiate into a cardiac lineage, is very primitive and any efforts at stem cell therapy mandate that we have a molecular and systemic understanding of cardiomyogenesis. This is the major objective of our proposal. Based on obtaining a detailed molecular picture of pathways that lead to cardiac differentiation from stem cells, we will be able to design small molecules/drugs that would trigger such differentiation in pathology. Further, such understanding will also aid preventive measures that will lead to reduction of the number of heart-related fatalities in California.
Progress Report: 
  • A main goal of research in our laboratory is to identify strategies to promote neural repair in spinal cord injury and related neurological conditions. On the one hand, we have been using mouse models of spinal cord injury to study a long-standing puzzle in the field, namely, why axons, the fibers that connect nerve cells, do not regenerate after injury to the brain and the spinal cord. On the other hand, relevant to this CIRM SEED grant, we have started to explore the developmental and therapeutic potential of human embryonic stem cells (hESCs) for neural repair. We do this by first developing a method to genetically manipulate a HUES line of hESCs. The advent of hESCs has offered enormous potential for regenerative medicine and for basic understanding of human biology. To attain the full potential of hESCs as a tool both for therapeutic development and for basic research, we need to greatly enhance and expand our ability to genetically manipulate hESCs. A major goal for our SEED grant-sponsored research is to establish methods to genetically manipulate the HUES series of hESC lines, which are gaining wide utility in the research community due to the advantages on their growth characteristics over previously developed hESC lines. The first gene that we targeted in HUES cells, Fezf2, is critical for the development of the corticospinal tract, which plays important roles in fine motor control in humans and hence represents an important target for recovery and repair after spinal cord injury. By introducing a fluorescent reporter to the Fezf2 locus, we are now able to monitor the differentiation of hESCs into Fezf2-expressing neuronal lineages. This work has been published. A second goal is to start to explore the developmental and therapeutic potential of these cells and cells that derived from these cells in the brain and spinal cord. We are currently utilizing the cell line genetically engineered above to develop an efficient method to differentiate HUES cells into subcerebral neurons. Results so far have been encouraging. Efforts are also underway to overexpress Fezf2 as a complementary approach to drive the differentiation of HUES cells into specific neuronal types. Together, these studies will lay down the foundation for therapeutic development with HUES cells and their more differentiated derivatives for neurological disorders including spinal cord injury where neural regeneration can be beneficial. The CIRM SEED grant has allowed us to pursue a new, exciting path of research that we would have not pursued had we not been awarded the grant. Furthermore, the CIRM funded research has opened a new window of opportunity for us to explore genetic engineering of hESCs to model human neurological conditions in future.

Human Embryonic Stem Cells and Neural Crest Plasticity

Funding Type: 
SEED Grant
Grant Number: 
RS1-00333
ICOC Funds Committed: 
$0
Disease Focus: 
Amyotrophic Lateral Sclerosis
Neurological Disorders
Spinal Cord Injury
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Craniofacial anomalies and bone defects pose a difficult and challenging problem both for the doctor and for patients along with their families. A great demand exists for the repair of craniofacial bone defects. Orofacial defects repaired with grafts obtained from an orofacial donor site are usually more successful than those from non-orofacial sites. However, the efficacy is limited by high cost, donor morbidity, and scarcity of orofacial tissue sources. Some skeletal diseases such as cherubism, hyperparathyroid jaw tumor syndrome and craniofacial fibrous dysplasia affect only orofacial bones. Studies of the craniofacial skeleton also indicate that molecular mechanisms controlling skeletogenesis in the head are unique and distinctive from those occurring in other body sites. Neural crest cells are multipotent stem cells that contribute to a diverse array of tissues throughout the embryo. During craniofacial development, cranial neural crest contributes extensively to the formation of mesenchymal structures in the head and neck, such as orofacial bone, cartilage, tooth and cranial nerve ganglia. The majority of orofacial skeleton is neural crest derived. In this application, we propose to derive cranial neural crest-like progenitor cells from human embryonic stem cells and subsequently induce bone formation by these cranial neural crest-like cells. Bone tissues generated from cranial neural crest-like cells share similar developmental origin with craniofacial bones, thereby representing a superior therapeutic tissue source for craniofacial bone repair. The results from this proposal will be used to optimize strategies for maintenance of stem cell populations while improving our ability to stimulate the development of cell specific lineages needed for the repair and regeneration of defects in craniofacial tissues.
Statement of Benefit to California: 
Craniofacial anomalies and bone defects pose a difficult and challenging problem both for the doctor and for patients along with their families. A great demand exists for the repair of craniofacial bone defects. Orofacial defects repaired with grafts obtained from an orofacial donor site are usually more successful than those from non-orofacial sites. However, the efficacy is limited by high cost, donor morbidity, and scarcity of orofacial tissue sources. This proposal will benefit the people and the state of California by deriving cranial neural crest-like progenitor cells from human embryonic stem cells. Bone tissues generated from cranial neural crest-like cells share similar developmental origin with craniofacial bones, thereby representing a superior therapeutic tissue source for craniofacial bone repair.
Progress Report: 
  • A main goal of research in our laboratory is to identify strategies to promote neural repair in spinal cord injury and related neurological conditions. On the one hand, we have been using mouse models of spinal cord injury to study a long-standing puzzle in the field, namely, why axons, the fibers that connect nerve cells, do not regenerate after injury to the brain and the spinal cord. On the other hand, relevant to this CIRM SEED grant, we have started to explore the developmental and therapeutic potential of human embryonic stem cells (hESCs) for neural repair. We do this by first developing a method to genetically manipulate a HUES line of hESCs. The advent of hESCs has offered enormous potential for regenerative medicine and for basic understanding of human biology. To attain the full potential of hESCs as a tool both for therapeutic development and for basic research, we need to greatly enhance and expand our ability to genetically manipulate hESCs. A major goal for our SEED grant-sponsored research is to establish methods to genetically manipulate the HUES series of hESC lines, which are gaining wide utility in the research community due to the advantages on their growth characteristics over previously developed hESC lines. The first gene that we targeted in HUES cells, Fezf2, is critical for the development of the corticospinal tract, which plays important roles in fine motor control in humans and hence represents an important target for recovery and repair after spinal cord injury. By introducing a fluorescent reporter to the Fezf2 locus, we are now able to monitor the differentiation of hESCs into Fezf2-expressing neuronal lineages. This work has been published. A second goal is to start to explore the developmental and therapeutic potential of these cells and cells that derived from these cells in the brain and spinal cord. We are currently utilizing the cell line genetically engineered above to develop an efficient method to differentiate HUES cells into subcerebral neurons. Results so far have been encouraging. Efforts are also underway to overexpress Fezf2 as a complementary approach to drive the differentiation of HUES cells into specific neuronal types. Together, these studies will lay down the foundation for therapeutic development with HUES cells and their more differentiated derivatives for neurological disorders including spinal cord injury where neural regeneration can be beneficial. The CIRM SEED grant has allowed us to pursue a new, exciting path of research that we would have not pursued had we not been awarded the grant. Furthermore, the CIRM funded research has opened a new window of opportunity for us to explore genetic engineering of hESCs to model human neurological conditions in future.

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

Funding Type: 
SEED Grant
Grant Number: 
RS1-00331
ICOC Funds Committed: 
$0
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Cell Line Generation: 
Embryonic Stem Cell
Public Abstract: 
Effective treatments for motor neuron diseases, such as Amyotrophic Lateral Sclerosis (ALS), Spinal Muscular Atrophy (SMA), Kennedy's Disease and Hereditary Spastic Paraplegias (HSP), have yet to be developed. Currently the only FDA-approved drug for treating motor neuron diseases is riluzole. However, riluzole has only moderate success in prolonging patient survival. One possible reason for the lack of effective treatments for motor neuron diseases is that drugs for such diseases were previously developed for humans based on animal models which may not accurately reflect motor neuron diseases in humans. Therefore, establishing a human system to study motor neuron diseases is a crucial step in developing successful therapeutic treatments. Human Embryonic Stem (hES) cells constantly self-renew and have the potential to grow into any type of cell in the human body. Since little hES research has been conducted, it is not clear whether these cells can be used in scientific models to understand cell death in motor neuron diseases. Therefore, in this study we propose to steer hES cells into becoming motor neurons so that we may gain a better understanding of the aforementioned motor neuron diseases. Because of their self-renewing properties and their ability to be fostered in a scientific environment, our studies of hES cells could provide a useful source of knowledge to develop therapies for patients with motor neuron diseases or spinal cord injuries. Aim 1: We will take motor neurons from hES cell lines and study their molecular and cellular properties. These studies will allow us to identify how motor neurons express specific genes and proteins. In addition, we will be able to evaulate how effective hES cell-derived motor neurons are in developing treatments for motor neuron diseases. We will also improve the mothod used by the scientific community to induce motor neurons from hES cells. The goal is to develop a reliable method to use hES cells for deriving motor neurons useful for basic research and clinical applications. Aim 2: We will evaluate toxicity in hES cell-derived motor neurons to test whether human cell cultures can be used as experimental models to investigate motor neuron diseases. We will first analyze how well functional ionotropic glutamate receptors (the basic building blocks of motor neurons in the brain) in these hES-derived motor neurons are expressed using both a pharmacological approach and by recording the electrical activities of motor neurons (electrophysiology). We will then investigate cell death in motor neurons using specific toxins that will target glutamate receptors and transporters. We will also determine the effect of drugs in protecting these cells from toxin-induced death.
Statement of Benefit to California: 
The goal of our proposed research is to use hES cells to develop a reliable method for deriving motor neurons. Since hES cells can self-renew and be maintained in vitro the motor neurons derived from hES cells could provide useful knowledge that could be applied to treatments for patients with motor neuron diseases or spinal cord injuries. This proposed research will benefit the State of California and its citizens in several ways. The most obvious way in which this research will benefit Californians is through the reduction of pain and suffering undergone by Californian patients suffering from motor neuron diseases or spinal cord injuries. Caretakers and relatives of those with motor neuron-related medical problems will also experience the benefits. As the state with the largest population in the nation, California spends a enormous amount of money on healthcare for its residents. In the future, finding a cure for neurodegenerative diseases like ALS will significantly reduce state healthcare costs by lessoning the amount of time patients are treated for such diseases. In addition, conducting research in the stem cell field will create job opportunities and attract highly skilled personnel to California. Other states will not be able to attract these workers because of federal restrictions on stem cell research funding. The technologies, drugs, and patents which will result from our studies will allow Californians to be the first people in the country to benefit from stem cell research with respect to its ability to positively affect healthcare, the economy and technological advances.
Progress Report: 
  • Parkinson’s disease (PD) is the most frequent neurodegenerative movement disorder caused by damage of dopamine-producing nerve cells (DA neuron) in patient brain. The main symptoms of PD are age-dependent tremors (shakiness). There is no cure for PD despite administration of levodopa can help to control symptoms.

  • Most of PD cases are sporadic in the general population. However, about 10-15% of PD cases show familial history. Genetic studies of familial cases resulted in identification of PD-linked gene changes, namely mutations, in six different genes, including α-synuclein, LRRK2, uchL1, parkin, PINK1, and DJ-1. Nevertheless, it is not known how abnormality in these genes cause PD. Our long-term research goal is to understand PD pathogenesis at cellular and molecular levels via studying functions of these PD-linked genes and dysfunction of their disease-associated genetic variants.

  • A proper experimental model plays critical roles in defining pathogenic mechanisms of diseases and for developing therapy. A number of cellular and animal models have been developed for PD research. Nevertheless, a model closely resembling generation processes of human DA nerve cells is not available because human neurons are unable to continuously propagate in culture. Nevertheless, human embryonic stem cells (hESCs) provide an opportunity to fulfill the task. hESCs can grow and be programmed to generate DA nerve cells. In this study, we propose to create a PD model using hESCs.

  • During the funding period, we have generated a number of human ES cell lines overexpressing α-synuclein and two disease-associated α-synuclein mutants. These cells are being used to determine the cellular and molecular effects of the disease genes on human ES cells and the PD affected dopaminergic neurons made from these cells. We have found that normal and disease α-synucleins have little effect on hESC growth and differentiation. We will continue to investigate roles of this protein in modulating PD affected dopaminergic neurons. Completion of this study will allow us to study the pathological mechanism of PD and to design strategies to treat the disease.

Chromosome instability due to telomere loss in human embryonic stem cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00271
ICOC Funds Committed: 
$0
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Human embryonic stem cells (hESCs) have important potential in the treatment of human disease. Because they can change into a large number of different cell types, they may be useful in restoring a variety of damaged tissues. One potentially harmful side effect of hESC therapy is cancer due to unregulationed growth of the hESCs introduced in the body. hESCs have the potential to grow almost indefinitely. Therefore if they should become "transformed" into cancer cells while being cultured in the laboratory, they may cause cancer in the individuals into which they are injected. Transformation of normal cells into cancer cells can occur through changes in their DNA, which contains the information telling cells to grow or not to grow. Because multiple changes must occur for cells to begin the unchecked growth of cancer cells, the likelihood of cancer is low. However, some cellular changes can increase the rate at which subsequent changes occur, which greatly increases the probability that a cell will acquire all of the changes necessary to become a cancer cell. This increased rate of changes in DNA is called genomic instability, which is proposed to be an early step in many cancers. One mechanism by which genomic instabiiity can occur is through the loss of the caps that protect the ends of chromosomes that contain the DNA. Loss of these caps, called telomeres, can make the DNA highly unstable. This proposal will study whether the loss of telomeres is a cause of instability in hESCs during their growth in the laboratory. Information on this process will allow steps to be taken to avoid this potential harmful effect during hESC therapy.
Statement of Benefit to California: 
Human embryonic stem cells (hESCs) have important potential in the treatment of human disease. Because they can change into a large number of different cell types, they may be useful in restoring a variety of damaged tissues. This study will investigate a potentially harmful side-effect involving genetic changes that may occur during growth of hESCs in the laboratory that could lead to cancer when they are injected into people. Understanding the process involved in generating these genetic changes will allow scientists to avoid them and limit the likelihood of these complications in the clinic.
Progress Report: 
  • A promising approach to alleviating the symptoms of Parkinson's disease is to transplant healthy dopaminergic neurons into the brains of these patients. Due to the large number of transplant neurons required for each patient and the difficulty in obtaining these neurons from human tissue, the most viable transplantation strategy will utilize not fetal dopaminergic neurons but dopaminergic neurons derived from human stem cell lines. While transplantation has been promising, it has had limited success, in part due to the ability of the new neurons to find their correct targets in the brain. This incorrect targeting may be due to the lack of appropriate growth and guidance cues as well as to inflammation in the brain that occurs in response to transplantation, or to a combination of the two. Cytokines released upon inflammation can affect the ability of the new neurons to connect, and thus ultimately will affect their biological function. In out laboratory we have been examining which guidance molecules are required for proper targeting of dopaminergic neurons during normal development and have identified necessary cues. We have now extended these studies to determine that two of the molecules have dramitc effects on dopaminergic neurons made from human embryonic stem cellls and that at least in vitro, cytokines do not mask these effects. Ultimately, an understanding of how the environment of the transplanted brain influences the ability of the healthy new neurons to connect to their correct targets will lead to genetic, and/or drug-based strategies for optimizing transplantation therapy.

Comprehensive study of the osteogenic potential of human embryonic stem cells - are they equivalent to liposuctioned fat and bone marrow derived stem cells?

Funding Type: 
SEED Grant
Grant Number: 
RS1-00271
ICOC Funds Committed: 
$0
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Bony defects of the face, skull, or long bone may result from trauma, destruction by tumors, or congenital causes like cleft palate. These defects are encountered regularly by plastic surgeons and current methods of reconstruction primarily involve bone grafts harvested from another part of the body (autologous bone) and re-planted into the defect. However, the amount of donor bone is limited and the grafts are difficult to shape. In addition, autologous bone adds an additional operative procedure and can result in pain, hemorrhage, fracture, and nerve injury. To solve these problems, our laboratory has been developing a bone graft substitute since 1990. In other words, we are trying to grow new bone in a pre-determined three-dimensional shape. Growing new bone instead of transferring it from one part of the body to another would dramatically reduce operative time, hospitalization, and morbidity. There are three ingredients necessary to grow new bone 1) a 3-D delivery scaffold 2) stem cells 3) factors that turn the stem cells into bone. A scaffold must be biocompatible and biodegradable and allow for cells to attach, multiply, and turn into bone. Our lab has been using a material called poly-lactide-co-glycolide (PLGA) to create scaffolds that degrade in the body into lactic and glycolic acid which are naturally found. PLGA is FDA approved and is currently used in dissolvable suture material. Osteoblasts, the cells that make bone, adhere to, multiply, and form bone on 3-D PLGA scaffolds. Another critical component of synthesizing bone is the stem cell source. Ideally, one could harvest stem cells from an individual, seed them onto a biodegradable scaffold of the necessary shape, turn the stem cells into bone, and implant the bone back into the same individual. We have used bone marrow stem cells (BMSC) grown on a 3-D PLGA culture to grow new bone in vitro (in cell culture) and in vivo (in a living organism) to heal cranial defects in rabbits. Liposuctioned fat cells have also been shown to form new bone in 3-D culture and in animal defects. There have been only a few studies investigating hESC’s ability to form bone on a 2-D plate or in a collagen gel, but no studies have looked at their ability to form bone on a 3-D scaffold. Our first goal is to seed hESCs on our 3-D PLGA scaffold and form bone. We will perform this experiment side by side with human liposuctioned fat and bone marrow derived stem cells for comparison. Our second goal is to supplement osteogenic media with different concentrations of Vitamin D and/or retinoic acid to determine an ideal growth media for maximal bone formation. Our third goal is to look at the specific mechanisms through which hESCs form bone and a blood supply. We will analyze the differences in gene expression of hESCs grown on 3-D culture and compare them with human fat and bone marrow derived stem cells. Understanding these mechanisms is critical to developing a bone graft.
Statement of Benefit to California: 
In 2001, there were 24,298 operations for craniofacial trauma in the United States, 227,500 births with craniofacial defects resulting in 37,732 operations to repair congenital craniofacial defects. It has been calculated that annually, approximately 3.6 million craniofacial cases were treated in the medical system [Snowden et al., 2003]. The rate per 100,000 of congenitial anomalies is 36.1, craniofacial trauma 119.5, and neoplasms 94 for an annual cost of approximately 23,206 million dollars[Snowden et al., 2003]. California is no exception to the types of trauma, congenital defects, or head and neck cancers that result in craniofacial skeleton defects. With the passing of the California helmet law, there is also decreased mortality of motorcyclists and bicyclists resulting in greater number survivors who might require repair of cranial and facial trauma defects. A bone graft substitute would have widespread use for the citizens of California. The development of a bone graft substitute would eliminate the pain and complications of graft harvesting, such as excessive blood loss, infection, and fracture. A bone graft substitute would significantly reduce operating time and hospital stay due to prolonged donor site pain and morbidity. Our purpose is to use an FDA approved biodegradeable and biocompatible 3-D scaffold seeded with stem cells exposed to osteo-inductive agents to grow new bone. Our study will significantly improve our knowledge about new bone formation, the osteogenic potential of different stem cell types, and likely result in a clinically viable and cost-effective bone graft substitute for use in the population of California
Progress Report: 
  • A promising approach to alleviating the symptoms of Parkinson's disease is to transplant healthy dopaminergic neurons into the brains of these patients. Due to the large number of transplant neurons required for each patient and the difficulty in obtaining these neurons from human tissue, the most viable transplantation strategy will utilize not fetal dopaminergic neurons but dopaminergic neurons derived from human stem cell lines. While transplantation has been promising, it has had limited success, in part due to the ability of the new neurons to find their correct targets in the brain. This incorrect targeting may be due to the lack of appropriate growth and guidance cues as well as to inflammation in the brain that occurs in response to transplantation, or to a combination of the two. Cytokines released upon inflammation can affect the ability of the new neurons to connect, and thus ultimately will affect their biological function. In out laboratory we have been examining which guidance molecules are required for proper targeting of dopaminergic neurons during normal development and have identified necessary cues. We have now extended these studies to determine that two of the molecules have dramitc effects on dopaminergic neurons made from human embryonic stem cellls and that at least in vitro, cytokines do not mask these effects. Ultimately, an understanding of how the environment of the transplanted brain influences the ability of the healthy new neurons to connect to their correct targets will lead to genetic, and/or drug-based strategies for optimizing transplantation therapy.

Derivation and characterization of dopamine neurons from human embryonic stem cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00271
ICOC Funds Committed: 
$0
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Parkinson's disease is a debilitating chronic and progressive disease characterized by muscle rigidity, tremor, slowing of movement progressing to inability to move, and various difficulties with speech, posture, and cognition. It is generally agreed that the loss of a particular type of brain cell called the dopamine neuron is responsible. Drug therapy is useful in early stages but becomes less effective as the disease progresses. Dopamine cell transplantation has been successful to some degree, but has been limited by the number of cells available for transplant as well as the long term functional viability of such cells. Recently it has been shown that the exposure of human embryonic stem cells to a cocktail of growth factors leads to the production of substantial numbers of dopamine cells. Thus human embryonic stem cells may yield an inexhaustible supply of such neurons. However, the efficacy of such cells in transplant therapy depends on such cells exhibiting the necessary characteristics of dopamine cells, most importantly the ability to produce and release dopamine when transplanted into the brain. The work proposed here will allow a complete characterization of the properties of such cells in terms of their neurochemistry, degree of activity and ability to make and release dopamine. Using a variety of markers we have developed and are developing in live cells, we will be able to isolate a homogeneous population of dopamine cells produced from human embryonic stem cells and optimized for their ability to replace the function of the lost dopamine neurons in Parkinson's disease. We see this as a necessary "quality control" step to assure the maximum therapeutic value of such cells. Eventually we will transplant such functionally verified dopamine cells into animal models of Parkinson's disease as the next step to their eventual therapeutic use in human Parkinsonian patients.
Statement of Benefit to California: 
Parkinson's disease is a debilitating chronic and progressive disease characterized by muscle rigidity, tremor, slowing of movement progressing to inability to move, and various difficulties with speech, posture, and cognition. The incidence of newly diagnosed Parkinson's disease in California is estimated at about 13/100,000 citizens per year. Assuming a population of 36 million in California, that yields 468,000 newly diagnosed cases of Parkinson's disease every year in California. Clearly this is one of the most prevalent neurological disorders in California. This translates into a tremendous decrease in productivity of the work force, let alone the immense cost in medical care, and the personal cost in human suffering. The ability to transform human embryonic stem cells into dopamine neurons holds probably the greatest potential for cure of this terrible disease. However, stem cells do not uniformly turn into dopamine neurons when exposed to appropriate growth factors. Thus this proposal is aimed at optimizing the transformation by finding the best conditions for transformation, followed by functional characterization of those cells by a wide variety of neurochemical and physiological assays. The result will be a homogeneous population of functionally optimized dopamine neurons that will be maximally useful for transplantation first into animal models of Parkinson's and ultimately into human patients.
Progress Report: 
  • A promising approach to alleviating the symptoms of Parkinson's disease is to transplant healthy dopaminergic neurons into the brains of these patients. Due to the large number of transplant neurons required for each patient and the difficulty in obtaining these neurons from human tissue, the most viable transplantation strategy will utilize not fetal dopaminergic neurons but dopaminergic neurons derived from human stem cell lines. While transplantation has been promising, it has had limited success, in part due to the ability of the new neurons to find their correct targets in the brain. This incorrect targeting may be due to the lack of appropriate growth and guidance cues as well as to inflammation in the brain that occurs in response to transplantation, or to a combination of the two. Cytokines released upon inflammation can affect the ability of the new neurons to connect, and thus ultimately will affect their biological function. In out laboratory we have been examining which guidance molecules are required for proper targeting of dopaminergic neurons during normal development and have identified necessary cues. We have now extended these studies to determine that two of the molecules have dramitc effects on dopaminergic neurons made from human embryonic stem cellls and that at least in vitro, cytokines do not mask these effects. Ultimately, an understanding of how the environment of the transplanted brain influences the ability of the healthy new neurons to connect to their correct targets will lead to genetic, and/or drug-based strategies for optimizing transplantation therapy.

Neurogenesis in Alzheimer's Disease: A-beta, Friend or Foe?

Funding Type: 
SEED Grant
Grant Number: 
RS1-00271
ICOC Funds Committed: 
$0
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Alzheimer's disease (AD), a late-onset disease manifests impairment and loss of neurons over years before the onset of clinical symptoms such as, learning and memory loss. Studies suggest that the brain attempts to replace these nerve cells throughout the course of the disease with new cells, but results are insufficient. It may be that not enough new cells are generated, or that they die due to the disease process itself, and prior to restoration of function. In AD, a major factor of the disease is production of a toxic molecule, the A-beta peptide. We plan to test if this molecule stimulates new nerve cell production, but also eventually is toxic to those cells as they develop more neuron-like functions. As these cells move into the diseased area of the brain, they must assume contact with other existing nerve cells that are essential for memory and learning functions. This step may also not occur in a normal way in AD. Because we cannot test for these activities in the AD-affected patients, and because animal or tissue culture models are inadequate, we propose to use human embryonic stem cells (hESCs) that have been treated to become neural stem cells (NSCs) and expose them to the toxic A-beta peptide and determine if they both increase in numbers and then become more fully like neurons in the brain. This latter function include migration of the new neurons moving towards normal nerve cells that have been previously damaged, for example, exposure to the toxic A-beta peptide. These studies will provide some important information on the ultimate possibility of stem cell replacement of neurons loss during the disease course.
Statement of Benefit to California: 
Alzheimer's disease, a progressive neurological process leading to dementia is devastating to the patient and family. The average duration of disease is 8 years, and is both costly for families and the community, and labor intensive for the caregivers. Our preliminary studies indicate that process of new nerve cell generation is attempted by the brain itself, but insufficient. Possibility of embryonic stem cell biology offers the ultimate possibility of replacement of lost or impaired nerve cells. This proposal attempts to understanding how this replacement can occur and allow the patient to maintain normal function, especially of memory and cognition sufficient for activities of daily living. Defining critical, several steps in neurogenesis can be best accomplished with hESCs. Exposing these cells to the toxic molecule A-beta provides a means to define if the cells become neurons and are maintained or lost due to the A-beta effect itself. If the latter is true, than drugs to block this later step could be tested on these cells. If these cells fail to replace the lost nerve cells, and connect up with remaining cells, then research targeted to promote their integration should be pursued. All of these studies lead to the ultimate goal of repairing the brain and halting the neuron loss or decreasing the rate of loss and the functional decline of the patient.
Progress Report: 
  • A promising approach to alleviating the symptoms of Parkinson's disease is to transplant healthy dopaminergic neurons into the brains of these patients. Due to the large number of transplant neurons required for each patient and the difficulty in obtaining these neurons from human tissue, the most viable transplantation strategy will utilize not fetal dopaminergic neurons but dopaminergic neurons derived from human stem cell lines. While transplantation has been promising, it has had limited success, in part due to the ability of the new neurons to find their correct targets in the brain. This incorrect targeting may be due to the lack of appropriate growth and guidance cues as well as to inflammation in the brain that occurs in response to transplantation, or to a combination of the two. Cytokines released upon inflammation can affect the ability of the new neurons to connect, and thus ultimately will affect their biological function. In out laboratory we have been examining which guidance molecules are required for proper targeting of dopaminergic neurons during normal development and have identified necessary cues. We have now extended these studies to determine that two of the molecules have dramitc effects on dopaminergic neurons made from human embryonic stem cellls and that at least in vitro, cytokines do not mask these effects. Ultimately, an understanding of how the environment of the transplanted brain influences the ability of the healthy new neurons to connect to their correct targets will lead to genetic, and/or drug-based strategies for optimizing transplantation therapy.

A novel approach for pancreatic beta-cell differentiation in vitro and in vivo

Funding Type: 
SEED Grant
Grant Number: 
RS1-00271
ICOC Funds Committed: 
$0
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Our bodies are made of about 2 billions of different cell types that have their own functions. Diseases like diabetes are largely caused by a breakdown in cell function or by cell death. The major issue of diabetes is an inability to control the level of glucose (sugar) in the blood. Blood glucose levels are normally controlled by insulin produced by islet cells of pancreas. In people with Type I diabetes islet cells are being destroyed by autoimmune system, which is mainly occurred in children. A lifetime of diabetes results in severe and debilitating consequences including kidney failure, adult blindness, nerve damage and cardiovascular disease (leading to limb amputations, heart attack and stroke). The majority of current constructive techniques rely on supply of donor tissues for replacement; however, the major hurdle is finding enough number of human islets to be transplanted into all the people who is waiting on the list for this treatment. The supply of islets donated from cadaver will never be balanced with demand. There is currently no available cure for diabetes that would prevent the occurrence of these consequences. This reality has driven researchers and clinicians to find alternate strategies to cure diabetes. An ideal pancreas, which reproduces the physiological response of the normal pancreas to the glucose changing, would drastically reduces the occurrence of secondary illness and improve the quality of live of diabetic people. Embryonic stem cells (ESC) can make all cell types existing in human body, however at the moment they can not be used for clinical application because the risk of tumor formation and the lack of knowledge for efficient differentiation in insulin-producing cells. We propose to create a novel human cellular system that will address these unmet need, especially for cell therapy. Our goal is to prune all bad characteristics of ESC by selecting a new population of cells from ESC, which can not only maintain to grow indefinitely but also produce enough number of clinical grade cells to make up the destroyed insulin-secreting cells. Our new cells will be called pruned-ESC. In order to demonstrate that insulin producing cells from our pruned-ESC be safely used, we will combine gene and cell therapies for curing Type I diabetes. We will insert an active master gene into pruned-ESC, which is crucial for normal development of pancreas, particularly for insulin-producing cells. Pruned-ESC with a master gene will be given to Type I diabetic mice to evaluate the feasibility of our system for clinical application. We believe that our combined gene and cell technology along with pruned-ESC will allow us to investigate that reasons of Type I diabetes and offer an evidence that is Type I diabetes can be curable in the near future.
Statement of Benefit to California: 
Diabetes mellitus (DM) is estimated to affect approximately 18.2 million people in the US alone, and more than 150 million people worldwide. California’s ethnically diverse population is disproportionately affected by diabetes. The overall prevalence of diabetes among California adults is increasing. A study by UCLA Center for Health Policy Research, comparing the years 2001 and 2003, showed that in 2003 nearly 1.7 million California adults age 18 and over (6.6%) has been diagnostic with diabetes in 2003, up from 1.5 million (6.2%) in 2001. By the year 2020, the prevalence of diabetes in California is expected to exceed four million people. Without normal glucose homeostasis over the long term, complications involving the eyes, kidneys, nerves and cardiovascular system are common, which reduce quality of life and significantly increase morbidity, mortality, and cost. For this reason new therapeutic solutions for diabetes should be a priority for California. Stem cell research is among the hottest fields in today’s medical research because these cells have the potential to replace cells that are dysfunctional or lost. The use of stem cells for curing disease and ending disabilities may change the medical treatment in this century. However, there are some major roadblocks that need to be resolved before these cells are implemented in ordinary medical care. Reliable methods for isolation and expansion of stem cells need to be established, efficient differentiation protocols need to be developed, and stem cell plasticity causing tumorigenicity needs to be controlled. This proposal aims to address all of these roadblocks. We will use a novel approach to select a new stem cell population from human embryonic stem cells, which retain differentiation capability but lose propensity for teratoma formation. Our overall goal is to evaluate the potential use of this novel stem cell system for the treatment of a devastating, yet incurable, disease, diabetes mellitus. If we succeed this project our developed system may become the basic of new treatment solutions for diabetes, giving the California a clear competitive advantage over other states in area of stem cell research.
Progress Report: 
  • A promising approach to alleviating the symptoms of Parkinson's disease is to transplant healthy dopaminergic neurons into the brains of these patients. Due to the large number of transplant neurons required for each patient and the difficulty in obtaining these neurons from human tissue, the most viable transplantation strategy will utilize not fetal dopaminergic neurons but dopaminergic neurons derived from human stem cell lines. While transplantation has been promising, it has had limited success, in part due to the ability of the new neurons to find their correct targets in the brain. This incorrect targeting may be due to the lack of appropriate growth and guidance cues as well as to inflammation in the brain that occurs in response to transplantation, or to a combination of the two. Cytokines released upon inflammation can affect the ability of the new neurons to connect, and thus ultimately will affect their biological function. In out laboratory we have been examining which guidance molecules are required for proper targeting of dopaminergic neurons during normal development and have identified necessary cues. We have now extended these studies to determine that two of the molecules have dramitc effects on dopaminergic neurons made from human embryonic stem cellls and that at least in vitro, cytokines do not mask these effects. Ultimately, an understanding of how the environment of the transplanted brain influences the ability of the healthy new neurons to connect to their correct targets will lead to genetic, and/or drug-based strategies for optimizing transplantation therapy.

Formation of Personalized Embryonic Stem-Like Cells by In Vitro Epigenetic Cell Reprogramming

Funding Type: 
SEED Grant
Grant Number: 
RS1-00225
ICOC Funds Committed: 
$0
Disease Focus: 
Spinal Cord Injury
Neurological Disorders
Stroke
Trauma
Stem Cell Use: 
Adult Stem Cell
Embryonic Stem Cell
Public Abstract: 
Embryonic stem (ES) cells have great promise for treating many human diseases and it is believed that this cell therapy someday will revolutionize medicine. However, the rejection of the introduced ES cells by the patient’s immune system is a big challenge lying ahead when applying the currently available human ES cell lines to clinical trials. The recent breakthrough of creating human embryonic stem cells (ESCs) from human embryos by therapeutic cloning has offered a resolution and highlights the possibility of making so called “autologous” cell lines specific to individual patients. However, this technique is not without problems because the successful rate of cloning is extremely low. Furthermore, the technical difficulties make it hard to be broadly employed in common research and clinical facilities. We thus propose to develop a simple and efficient method to replace therapeutic cloning in creating personalized stem cells. We will achieve this goal by using a two-step “cell reprogramming” procedure. Specifically, cells are collected from the skin of the patient and are converted into embryonic stem-like (ESL) cells using defined ES factors, which will induce a complete cell reprogramming in skin cells by activating a panel of ES-specific genes and silence genes specifically expressed in skin cells. These ESL cells have the same features as ES cells and are capable of self-renewing and differentiating into all the adult cell types for clinical cell therapy. Most importantly, they are stem cells derived from the patient and will not cause any side-effects related to immune rejection by the patient’s defense system. As a result, these patient-derived stem cells may function better when implanted in diseased organs in cell therapy than currently available ES cell lines that are cloned from early embryos. Thus, there is every reason to hope that this revolutionary new approach will result in radically improved ways to create human stem cells for treatment of disease.
Statement of Benefit to California: 
This project proposes to develop an efficient cell rejuvenating method to create patient-specific pluripotent cells used in cell regeneration therapy. We believe this project will make the following contributions to the stem cell research for the state of California. 1. The project will provide a simple and useful complement, rather than human therapeutic cloning, to reprogram somatic nuclei in creating customized stem cells. This approach is cost-effective and time-saving, and it may eventually lead to an alternative approach for creating genetically tailored human embryonic stem (ES) cell lines for use in stem cell research and treatment of human diseases. 2. These pluripotent cell lines are patient-specific and are unlikely to be rejected by the patient’s immune system when transplanted into the body. Thus, they may be much safer than ES cells that are derived the embryo when applied to clinical cell therapy. 3. Identification and characterization of reprogramming factors in ES cell extracts will benefit biomedical and genetic studies aimed at understanding how to reprogram differentiated cells to an embryonic state and thereby increase their developmental potential. 4. With this powerful in vitro cell reprogramming technique, it is highly possible to create a stem cell bank in California that holds thousands of ES cell Lines with varied HLA types. These stem cell lines will be immediately available for basic research and clinical studies.
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).

Elucidating Chemotropic Responses of Human Embryonic Stem Cells to Guidance Cues

Funding Type: 
SEED Grant
Grant Number: 
RS1-00215
ICOC Funds Committed: 
$0
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Parkinson’s disease is caused by the death of neurons in the substantia nigra. These neurons extend projections, called axons, to another region of the brain called the corpus striatum. Here, they release dopamine, an essential chemical required for smooth and coordinated movement. Loss of 80% of the dopamine producing cells leads to the symptoms of Parkinson’s disease which include muscle rigidity, tremor, and uncoordinated movement. Current therapy involves supplementing the dwindling dopamine production with drugs, but this therapy loses effectiveness over time. Surgical options also exist, such as neurostimulation from battery-operated implants, but surgical complications occur in addition to problems associated with implanted hardware such as broken or dislocated wires. Successful long-term treatment has been attained for a subset of patients using fetal midbrain tissue grafts that reconstitute the axonal pathway, but general use of this therapy has been limited by requirements for fresh human fetal tissue. Consequently, there are clear advantages to using sources of dopaminergic neurons for transplantation, such as human embryonic stem cells (hESC), that can be perpetually grown in tissue culture Great strides toward treatment have recently been made in our ability to reliably differentiate hESCs into dopaminergic neurons, but ultimately, therapeutic success will require reconstituting the precise neuronal connections with engrafted hESC-derived dopaminergic neurons. In the nervous system, neurons and their axons make appropriate connections by following cues present in the extracellular environment. Studies on dopaminergic neurons have shown that they are guided by two families of environmental cues called Netrins and Slits. In this application, we propose to investigate the response of hESC-derived dopaminergic neurons to the Netrin and Slit cues. Neurons respond to cues based on the expression of receptors on their cell surface. Consequently, in Aim I, we propose to determine the profile of receptors expressed on dopaminergic neurons. Studies on mouse embryonic stem cells suggest that at least one receptor for each family of cue will be expressed by hESCs. In Aim II, we propose to evaluate how hESCs respond to Netrin and Slit cues, using an assay in which aggregates of hESC-derived dopaminergic neurons are placed in a 3-dimensional matrix near a point source of Netrin and Slit. We will determine how axons of these dopaminergic neurons respond to cues by recording the migration behavior of the axons, toward or away from, the point source. If the dopaminergic neurons do not respond, we have previously identified methods to stimulate the neurons to generate a response. Finally in Aim III, we propose to observe the response of hESC-derived dopaminergic neurons to Netrins and Slits that are present in adult brains by transplanting the neurons into mice and evaluating the response of their axons one and four weeks post-transplantation.
Statement of Benefit to California: 
The citizens of California voted to support research on potential stem cell therapies that can be used to treat serious medical conditions that cripple millions of Americans. Parkinson’s disease is one of these conditions. The experiments proposed in this application tackle key issues that must be resolved before neurons derived from human embryonic stem cells can be successfully used to treat this neural disorder. Recently, researchers have developed ways to reliably differentiate human embryonic stem cells into the type of neurons, dopaminergic, that degenerate in patients with Parkinson’s disease. This application addresses the next step required for the development of successful therapeutic strategies. These strategies will require that transplanted dopaminergic neurons respond robustly and appropriately to environmental cues in the patient’s brain so that they supply the crucial chemical, dopamine, to the correct target. Parkinson’s disease is one of the illnesses in which a stem cell-based therapy is within grasp. An early success in using human stem cells to treat a devastating illness will be inspirational to California citizens.
Progress Report: 
  • Parkinson’s disease is the most common movement disorder due to the degeneration of brain dopaminergic neurons. One strategy to combat the disease is to replenish these neurons in the patients, either through transplantation of stem cell-derived dopaminergic neurons, or through promoting endogenous dopaminergic neuronal production or survival. We have carried out a small molecule based screen to identify compounds that can affect the development and survival of dopaminergic neurons from pluripotent stem cells. The small molecules that we have identified will not only serve as important research tools for understanding dopaminergic neuron development and survival, but potentially could also lead to therapeutics in the induction of dopaminergic neurons for treating Parkinson’s disease.

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