Neurological Disorders

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

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.

Alteration of pre-mRNA splicing during stem cell differentiation

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: 
Mutations in one of the duplicated Survival of Motor Neuron (SMN) genes lead to the progressive loss of motor neurons and subsequent development of Spinal Muscular Atrophy (SMA), a common, usually fatal, pediatric disease. Homozygous absence of the telomeric copy (SMN1) correlates with development of SMA because differential splicing of the centromeric copy (SMN2) predominantly produces a biologically inactive protein isoform. The low amounts of SMN produced from SMN2 are adequate for a fetus to develop, but are insufficient to maintain healthy motor neurons throughout life. One of the biological functions of SMN is to catalyze the biosynthesis of components of the spliceosome, an enzymatic machinery that removes introns from pre-mRNAs. Indeed using animal models it was demonstrated that purified building blocks of the spliceosome are sufficient to rescue developmental defect caused by reduced levels of SMN. These experiments demonstrated that elevated levels of the splicing machinery are essential for proper differentiation of stem cells into motor neurons. Based on these observations we hypothesize that alternative pre-mRNA splicing of several genes set in stone a developmental pathway that imprints unique gene expression features throughout motor neuron differentiation and maintenance. In this application we propose to test this hypothesis by analyzing genome-wide alternative pre-mRNA splicing during the differentiation of human embryonic stem cells into motor neurons. Verification of the hypothesis will provide new insights into the biological processes that specify longevity of motor neurons and direct future research to identify alternative targets for SMA and other neurological disease therapy.
Statement of Benefit to California: 
Recent experiments demonstrated that pre-mRNA splicing is required to establish gene expression profiles that dictate the longevity of developing motor neurons. The ability to differentiate human embryonic stem cells into motor neurons in cell culture permits an evaluation of gene expression and alternative pre-mRNA splicing throughout the differentiation process. This unique opportunity is available in part because the state of California actively supports research using human embryonic stem cells. The results obtained from this proposal will establish gene expression and alternative splicing maps that link defined developmental events with gene expression and motor neuron differentiation. The resulting signature profiles will enable future studies investigating motor neuron longevity and they will likely identity new molecular targets to battle various motor neuron diseases that have in common premature motor neuron death. As such, the information gained from the proposed experiments would maintain California’s leading status in stem cell research and may provide the intellectual framework for the development of new therapeutic developments in the private sector. An additional benefit to California citizens could be the availability of cutting edge technologies in clinical trials carried out at California's research centers.
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.

Manipulation of Hedgehog signaling in early human embryos

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: 
We propose deriving a new generation of embryonic stem cell lines with greater potential than current conventional lines. Until recently, all human embryonic stem cell (hESC) lines have been derived from one of two clearly discernible cell types, the inner cell mass (ICM), of the blastocyst stage embryo. ICM-derived hESC are able to differentiate to a number of tissue types. Nevertheless, such conventional lines range widely in how easily they give rise to adult cell types. Investigations with ‘twin’ hESC lines, i.e. lines made from splitting a single ICM, show that cells at this stage of development give rise to lines with distinct differentiation preferences. Thus a given protocol to generate a therapeutically desired cell type has a variable chance of success when applied to any particular ICM-derived hESC line. Recently, a novel technical advance has allowed hESC to be derived from cells of significantly younger embryos (i.e. from blastomeres of 8-10 cell cleavage stage embryos). These cleavage stage hESCs are superior to conventional lines in the ease/efficiency with which they could be induced to differentiate a variety of cell types. As yet, however, only 2 such lines have been established. Through our work in mice, we have developed a protocol to keep cleavage stage embryos from advancing through the usual program leading to blastocyst formation. We hypothesize that culturing human blastomeres under such conditions will allow more efficient derivation of cleavage stage hESC. Compared to the hESC lines in circulation today, blastomere-derived hESC lines are potentially far superior in their differentiative capacities. Such properties could greatly unify and advance the field in efforts to generate cells types of therapeutic interest.
Statement of Benefit to California: 
The central hope of Proposition 71 is that human embryonic stem cell (hESC) research will alleviate devastating medical conditions such as diabetes, Parkinson's, Alzheimer's and cancer. Realization of this goal requires solving the problem of being able to reliably generate, in culture, a given cell type from any hESC line. Currently it is still not possible to differentiate (or generate) any given cell type from any given hESC line. Our proposal to create a new generation of hESC line from cells of younger embryos may move the field closer to this goal. Recent technical advances made by the private sector on the East Coast have allowed derivation of the first two hESC lines from blastomeres (cells) of 8-10 cell embryos, i.e. cells that are only about 3 days post fertilization as opposed to 6 days in conventional hESC derivations. These two novel lines have shown a greater efficiency and capacity for differentiation than conventional lines in a number of assays. We have developed a protocol in mice that suggests a modification of this new method may improve on such blastomere-derived hESC derivations. We are therefore poised to take a lead in research creating a new generation of hESC lines that may supercede those currently in circulation, including a major source of lines made by private foundations on the East Coast. Given such a leading edge, and with one of the highest concentrations of college graduates in the nation, the state of California could transform the fields of embryonic stem cell research and medical discovery.
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.

Instructive Biomaterials for Stem Cell Differentiation

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: 
Embryonic stem cells (hESCs) have the potential to differentiate into all adult cell types and will have profound applications in tissue engineering and regenerative medicine. However, a thorough understanding of how to control stem cells so that a wide range of different cells, bone, muscle, nerve, etc. can be obtained is not known. It is proposed to simulate conditions in the human body by combining for the first time both mechanical and biological control in the differentiation of stem cell. Using state of the art polymer chemistry, cell scaffolds which combine all of these features will be prepared and the right conditions for each type of stem cells determined. Due to the huge range of possible variables we will also take advantage of highly parallel arrays of scaffolds which will allow thousands of different conditions to be evaluated at the same time, greatly increasing the speed of discovery. This interdisciplinary represents a new paradigm that will be used to screen for conditions that reproducibly and specifically generate all cell types from a single parent source of stem cells.
Statement of Benefit to California: 
The ability to produce desired cells lines for the treatment and study of disease is of paramount importance for California from both a social and economic viewpoint. In this proposal we will initially design arrays of synthetic polymer scaffolds that will be used to control the differentiation of retinal cells that are useful in treating Age-related macular Degeneration as well as Parkinson’s disease. Both of these diseases are particularly relevant for California’s population due to climate and population demographics. The techniques that will be developed to optimize these polymer scaffolds will allow thousands of different conditions to be examined at the same time which represents a new approach in this field that will allow the differentiation of stem cells into virtually any other cell to be accurately controlled and predicted. This represents a significant business opportunity for California and the implications for tissue engineering to repair traumatic injury and treat disease is profound.
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.

Metabolomic Signatures of Pluripotent Cell Lines

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: 
Stem cells are the hope for many people suffering from some form of organ or tissue dysfunction. The renowned capacity of stem cells and their ability to give rise to multiple types of adult cells (pluripotent) makes them so appealing for cell therapies. Stem cells originally derived from early embryos, often referred to us, as embryonic stem (ES) cells. They have extensive growth potential, self-replication capacity, can mature into adult cells, and hence can reconstitute damaged tissue when are injected to the organism. When isolated from early embryos, ES cells can be maintained in a dish under specific conditions without loosing their ability to expand indefinitely. A critical requirement for maintenance of ES cells in cultures is a layer of murine cells that supply unidentified signals vital to the ES cells. This requirement, obviously involves a risk for clinical therapies. Therefore, there is considerable effort ongoing by many investigators to define these factors. When needed, ES cells can be removed from the murine cell layer and spontaneously and without the ability to control it, can form all adult tissues. This event occurs in a very disorganized way resulting in “monster” tumors. Intensive investigation controlling the fate of the cells still is undergoing. In this project, we are suggesting to identify and characterize the factors that are differentiating ES cells from their murine layer cells, and from their matured cells. The main goal is to control the factors that are responsible to their pluripotentacy, and enriched the factors that are participating in the maturation of the cells. In order to get matured functional cells, particularly liver cells. Controlling maturation of ES cells into active matured liver cells is proposed as ideal technique for end-stage liver diseases, because their ability to expand extensively, differentiate into all mature liver cells, and reconstitute liver tissue when transplanted. In this project, we are proposing to analyze and identify factors that are differentiating cells in their embryonic stem state from cells that were already committed to liver cells. In a three well designed specific aims, we are suggesting to examine changes in factors within ES cells before and after they have committed to differentiate into liver cells as a function of time and culture conditions. With state of the art technologies and an interdisciplinary research teams this project goals are to be compliance with the highest medical and ethical standards.
Statement of Benefit to California: 
This proposed project is directly relevant to the mission of proposition 71 and specifically will benefit the State of California and its citizens in the following ways: A. The knowledge and data from this project will directly improve the California health care system and reduce the long-term health care cost burden on California through the development of therapies that treat disease and injuries with the ultimate goal to cure them. B. The knowledge and data from the proposed project will specifically provide opportunity for the state of California to benefit from royalties, patents and licensing fees that result from this project. C. This project will benefit the state of California economy by creating jobs and projects to its citizens, will generate millions of dollars in new tax revenues to the state. D. The knowledge and data from this project potentially will advance biotech industry in the state of California E. This project will benefit the scientists, researchers and medical doctors working on this specific project by bringing them to a world-class recognition and leadership in the stem cell research.
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.

High-throughput Optimization of Stem Cell Microenvironment in 3D

Funding Type: 
Basic Biology II
Grant Number: 
RB2-01637
ICOC Funds Committed: 
$0
Disease Focus: 
Alzheimer's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
Public Abstract: 
Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and recently developed human induced pluripotent stem cells (iPSCs), hold great promise as attractive cell sources for tissue regeneration. Unlike other types of cells, hPSCs can self-renew indefinitely and possess the potential to differentiate into any type of cells in our body. Before hPSCs can be used for therapeutic purposes, methods must be developed to control their differentiation into functional mature cell types. Stem cells reside in a highly complex niche in vivo where they constantly respond to microenvironmental cues including soluble factors, extracellular matrix, adjacent cells and mechanical signals. To fully realize the therapeutic potential of hPSCs, it is critical to understand the mechanisms by which they receive information from microenvironment and how such interactions alter hPSC functions. While the effect of individual type of microenvironmental cues on stem cell behavior has been studied in great depth, little is known about how the complex interplay of multiple types of microenvironmental cues would influence stem cell behavior. In addition, conventional iterative approach typically requires large amounts of cells and materials, and is slow an inefficient in discovery. To address these limitations, high-throughput screening has emerged as a novel approach to achieve rapid discovery with reduced materials and costs. However, most high-throughput studies on cell-material interactions to date have been performed on two-dimensional environments, while the architecture of the stem cell niche itself is three-dimensional. Recent research have clearly emphasized dimensionality as a critical determinant for regulating cell behavior, and systematic evaluation of stem cell responses to complex 3D signaling environment remains challenging. Through working at the interface of biology, material science, and engineering, here we propose to develop novel 3D combinatorial systems to understand how microenvironmental signals influences stem cells fate decision in 3D, and to rapidly optimize stem cell niche using high-throughput strategies. Such studies can greatly accelerate the clinical applications of hPSCs by elucidating the mechanisms underlying the control of hPSC differentiation. The outcome of proposed work can also aid in the synthesis of culture microenvironments that emulate stem cell niche in vivo, and would have broad applications in areas such as tissue regeneration and drug delivery.
Statement of Benefit to California: 
California is the most populated State in the US and many Californians suffer from diseases and injuries that lead to tissue loss and organ failure. With the rise of average life expectancy in our population, the number of Californians that suffer from devastating diseases will continue to increase. Human pluripotent stem cells (hPSCs) represent a promising candidate as cell sources for tissue repair and regenerative medicine. However, before they can be broadly used for therapeutic purposes, methods must be developed to control their differentiation into functional mature cell types. This proposal aims to elucidate the fundamental mechanisms by which hPSCs respond to the complex microenvironmental cues, and the outcomes of the proposed work will greatly accelerate the clinical translation of hPSCs for treating many Californian patients. Furthermore, the discovery from the proposed work will strengthen the leadership role of California in stem cell research. Our findings could also provide outstanding opportunities to stimulate the growth of biotechnology and pharmaceutical industries within the State as well as creating new job opportunities.
Progress Report: 
  • We are interested in identifying soluble protein factors in blood which can either promote or inhibit stem cell activity in the brain. Through a previous aging study and the transfer of blood from young to old mice and vice versa we had identified several proteins which correlated with reduced stem cell function and neurogenesis in young mice exposed to old blood. Over the past year we studied two factors, CCL11/eotaxin and beta2-microglobulin in more detail in tissue culture and in mice. We could demonstrate that both factors administered into the systemic environment of mice reduce neurogenesis in a brain region involved in learning and memory. We have also begun to test the effect of these factors on human neural stem cells and we started experiments to try to identify protein factors which can enhance neurogenesis.
  • While age-related cognitive dysfunction and dementia in humans are clearly distinct entities and affect different brain regions, the aging brain shows the telltale molecular and cellular changes that characterize most neurodegenerative diseases. Remarkably, the aging brain remains plastic and exercise or dietary changes can increase cognitive function in humans and animals, with animal brains showing a reversal of some of the aforementioned biological changes associated with aging. We showed recently that blood-borne factors coming outside the brain can inhibit or promote adult neurogenesis in an age-dependent fashion in mice. Accordingly, exposing an old mouse to a young systemic environment or to plasma from young mice increased neurogenesis, synaptic plasticity, and improved contextual fear conditioning and spatial learning and memory. Preliminary proteomic studies show several proteins with stem cell activity increase in old “rejuvenated” mice supporting the notion that young blood may contain increased levels of beneficial factors with regenerative capacity. We believe we have identified some of these factors now and tested them on cultured mouse and human neural stem cell derived cells. Preliminary data suggest that these factors have beneficial effects and we will test whether these effects hold true in living mice.
  • Cognitive function in humans declines in essentially all domains starting around age 50-60 and neurodegeneration and Alzheimer’s disease seems to be inevitable in all but a few who survive to very old age. Mice with a fraction of the human lifespan show similar cognitive deterioration indicating that specific biological processes rather than time alone are responsible for brain aging. While age-related cognitive dysfunction and dementia in humans are clearly distinct entities the aging brain shows the telltale molecular and cellular changes that characterize most neurodegenerative diseases including synaptic loss, dysfunctional autophagy, increased inflammation, and protein aggregation. Remarkably, the aging brain remains plastic and exercise or dietary changes can increase cognitive function in humans and animals. Using heterochronic parabiosis or systemic application of plasma we showed recently that blood-borne factors present in the systemic milieu can rejuvenate brains of old mice. Accordingly, exposing an old mouse to a young systemic environment or to plasma from young mice increased neurogenesis, synaptic plasticity, and improved contextual fear conditioning and spatial learning and memory. Unbiased genome-wide transcriptome studies from our lab show that hippocampi from old “rejuvenated” mice display increased expression of a synaptic plasticity network which includes increases in c-fos, egr-1, and several ion channels. In our most recent studies, plasma from young but not old humans reduced neuroinflammation in brains of immunodeficient mice (these mice allow us to avoid an immune response against human plasma). Together, these studies lend strong support to the existence of factors with beneficial, “rejuvenating” activity in young plasma and they offer the opportunity to try to identify such factors.
  • Cognitive function in humans declines in essentially all domains starting around age 50-60 and neurodegeneration and dementia seem to be inevitable in all but a few who survive to very old age. Mice with a fraction of the human lifespan show similar cognitive deterioration indicating that specific biological processes rather than time alone are responsible for brain aging. While age-related cognitive dysfunction and dementia in humans are clearly distinct entities and affect different brain regions the aging brain shows the telltale molecular and cellular changes that characterize most neurodegenerative diseases including synaptic loss, dysfunctional autophagy, increased inflammation, and protein aggregation. Remarkably, the aging brain remains plastic and exercise or dietary changes can increase cognitive function in humans and animals, with animal brains showing a reversal of some of the aforementioned biological changes associated with aging. Using heterochronic parabiosis we showed recently that blood-borne factors present in the systemic milieu can inhibit or promote adult neurogenesis in an age-dependent fashion in mice. Accordingly, exposing an old mouse to a young systemic environment or to plasma from young mice increased neurogenesis, synaptic plasticity, and improved contextual fear conditioning and spatial learning and memory. Over the past three years we discovered that factors in blood can actively change the number of new neurons that are being generated in the brain and that local cells in areas were neurons are generated respond to cues from the blood. We have started to identify some of these factors and hope they will allow us to regulate the activity of neural stem cells in the brain and hopefully improve cognition in diseases such as Alzheimer's.

Role of p120-catenin in restricting reprogramming of human keratinocytes

Funding Type: 
Basic Biology II
Grant Number: 
RB2-01637
ICOC Funds Committed: 
$0
Disease Focus: 
Alzheimer's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
Public Abstract: 
Adult human cells such as skin cells can be turn into “reprogrammed” stem cells that acquire properties of pluripotency - the ability to become multiple types of specialized cells of the body. These reprogrammed stem cells are called induced pluripotent stem cells (iPSCs). Generation of iPSCs may enable the development of new therapies for many human diseases such as Alzheimer's diseases, spinal cord injury, stroke, burns, heart disease, type I diabetes, osteoarthritis, and rheumatoid arthritis. For example, type I diabetes occurs when the pancreatic beta cells that produce insulin are damaged or die. The only current cure is a pancreatic transplant from a recently deceased donor, but the demand for transplants far outweighs the supply. If reprogramming becomes possible, skin cells can be easily taken from a patient with type I diabetes and converted to iPSCs which can then be grown into the insulin-producing beta cells. These beta cells genetically match the patient’s cells and can be transplanted back into the patient’s body without any risk of rejection. This would potentially cure the disease. Therefore, the reprogramming technology has enormous therapeutic potential. The most widely used method for reprogramming is delivering certain stem cell-associated genes or proteins called reprogramming factors (RFs) into adult human cells. The major drawback of this type of approach is the low efficiency of reprogramming, which compromises the clinical use of this powerful approach. Recently, it has been found that stimulation of cells with a protein called Wnt3a increases efficiency of the conversion of adult cells to iPSCs, but the efficiency is still too low for clinical use. However, adult cells containing less amount of a protein called p53 has higher efficiency of reprogramming. These observations suggest existence of barriers in the cell to restrict reprogramming. Our proposed study is to determine whether p120-catenin serves as a barrier to the reprogramming. To achieve this goal, we will use skin cells (keratinocytes) in which p120-catenin expression is deleted or reduced. We will then reprogram these cells by introducing RFs and/or Wnt, and examine the reprogramming efficiency. If the cells lacking p120-catenin or with low level of p120-catenin are more easily reprogrammed than are cells with normal level of p120-catenin, is would suggest that p120-catenin serves as barrier to the reprogramming. We will further determine how p120-catenin functions as a barrier by examining the interaction between p120-catenin and Wnt regulated events. Skin cells which are relatively easy to obtain and reprogram would be an ideal source of iPSCs. Successful completion of this study will provide information that helps develop new strategies such as inhibition of p120-catenin to improve the reprogramming efficiency. This would overcome existing hurdles that hamper the application of the technology useful in transplantation therapy of many human diseases.
Statement of Benefit to California: 
Chronic degenerative diseases, such as Alzheimer's diseases, type I diabetes, heart disease, osteoarthritis, rheumatoid arthritis, etc., afflict a significant number of California’s population. Today, donated organs or tissues which match host’s immune system are often used to replace ailing or destroyed tissue of the patient, but the need for transplantable tissues and organs far outweighs the available supply. Right now, over 21,000 patients in Californian are waiting for life-saving transplants. That's 21 percent of the more than 100,000 people waiting across our country. One third of them will die before receiving a transplant. Stem cells can give rise to a variety of cell types, which offer the possibility of a renewable source of replacement cells and tissues to treat degenerative diseases. Adult somatic cells can be converted to induced pluripotent stem cells (iPSCs). This process is called reprogramming. The reprogrammed iPSCs have the potential to become any type of cells in the body. The iPSCs are remarkably similar to embryonic stem cells and would offer all the benefits of embryonic stem cells without the controversial use of embryos. For example, it may become possible to generate healthy heart muscle cells from patient’s own tissue in the laboratory and then transplant those cells into patients with chronic heart disease without risk of immune rejection. However, the low efficiency of the reprogramming limited clinical application of this powerful method. Our study is aimed at determining whether p120-catenin serves as barrier to prevent somatic cells from dedifferentiation (reprogramming). This study will uncover the role for p120-catenin in regulating somatic reprogramming and provide information for developing strategy about how to make reprogramming more efficient so that iPSCs can be generated on a large scale for transplantation, which is beneficial to the patients with degenerative diseases in California. The improved reprogramming technology can be commercialized by the biotech industry in California to generate revenue and create new job opportunities.
Progress Report: 
  • We are interested in identifying soluble protein factors in blood which can either promote or inhibit stem cell activity in the brain. Through a previous aging study and the transfer of blood from young to old mice and vice versa we had identified several proteins which correlated with reduced stem cell function and neurogenesis in young mice exposed to old blood. Over the past year we studied two factors, CCL11/eotaxin and beta2-microglobulin in more detail in tissue culture and in mice. We could demonstrate that both factors administered into the systemic environment of mice reduce neurogenesis in a brain region involved in learning and memory. We have also begun to test the effect of these factors on human neural stem cells and we started experiments to try to identify protein factors which can enhance neurogenesis.
  • While age-related cognitive dysfunction and dementia in humans are clearly distinct entities and affect different brain regions, the aging brain shows the telltale molecular and cellular changes that characterize most neurodegenerative diseases. Remarkably, the aging brain remains plastic and exercise or dietary changes can increase cognitive function in humans and animals, with animal brains showing a reversal of some of the aforementioned biological changes associated with aging. We showed recently that blood-borne factors coming outside the brain can inhibit or promote adult neurogenesis in an age-dependent fashion in mice. Accordingly, exposing an old mouse to a young systemic environment or to plasma from young mice increased neurogenesis, synaptic plasticity, and improved contextual fear conditioning and spatial learning and memory. Preliminary proteomic studies show several proteins with stem cell activity increase in old “rejuvenated” mice supporting the notion that young blood may contain increased levels of beneficial factors with regenerative capacity. We believe we have identified some of these factors now and tested them on cultured mouse and human neural stem cell derived cells. Preliminary data suggest that these factors have beneficial effects and we will test whether these effects hold true in living mice.
  • Cognitive function in humans declines in essentially all domains starting around age 50-60 and neurodegeneration and Alzheimer’s disease seems to be inevitable in all but a few who survive to very old age. Mice with a fraction of the human lifespan show similar cognitive deterioration indicating that specific biological processes rather than time alone are responsible for brain aging. While age-related cognitive dysfunction and dementia in humans are clearly distinct entities the aging brain shows the telltale molecular and cellular changes that characterize most neurodegenerative diseases including synaptic loss, dysfunctional autophagy, increased inflammation, and protein aggregation. Remarkably, the aging brain remains plastic and exercise or dietary changes can increase cognitive function in humans and animals. Using heterochronic parabiosis or systemic application of plasma we showed recently that blood-borne factors present in the systemic milieu can rejuvenate brains of old mice. Accordingly, exposing an old mouse to a young systemic environment or to plasma from young mice increased neurogenesis, synaptic plasticity, and improved contextual fear conditioning and spatial learning and memory. Unbiased genome-wide transcriptome studies from our lab show that hippocampi from old “rejuvenated” mice display increased expression of a synaptic plasticity network which includes increases in c-fos, egr-1, and several ion channels. In our most recent studies, plasma from young but not old humans reduced neuroinflammation in brains of immunodeficient mice (these mice allow us to avoid an immune response against human plasma). Together, these studies lend strong support to the existence of factors with beneficial, “rejuvenating” activity in young plasma and they offer the opportunity to try to identify such factors.
  • Cognitive function in humans declines in essentially all domains starting around age 50-60 and neurodegeneration and dementia seem to be inevitable in all but a few who survive to very old age. Mice with a fraction of the human lifespan show similar cognitive deterioration indicating that specific biological processes rather than time alone are responsible for brain aging. While age-related cognitive dysfunction and dementia in humans are clearly distinct entities and affect different brain regions the aging brain shows the telltale molecular and cellular changes that characterize most neurodegenerative diseases including synaptic loss, dysfunctional autophagy, increased inflammation, and protein aggregation. Remarkably, the aging brain remains plastic and exercise or dietary changes can increase cognitive function in humans and animals, with animal brains showing a reversal of some of the aforementioned biological changes associated with aging. Using heterochronic parabiosis we showed recently that blood-borne factors present in the systemic milieu can inhibit or promote adult neurogenesis in an age-dependent fashion in mice. Accordingly, exposing an old mouse to a young systemic environment or to plasma from young mice increased neurogenesis, synaptic plasticity, and improved contextual fear conditioning and spatial learning and memory. Over the past three years we discovered that factors in blood can actively change the number of new neurons that are being generated in the brain and that local cells in areas were neurons are generated respond to cues from the blood. We have started to identify some of these factors and hope they will allow us to regulate the activity of neural stem cells in the brain and hopefully improve cognition in diseases such as Alzheimer's.

Novel role for Akt1 activation/translocation in mitochondria as regulator of hES cells

Funding Type: 
Basic Biology II
Grant Number: 
RB2-01602
ICOC Funds Committed: 
$0
Disease Focus: 
Epilepsy
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
The main objective of the CIRM Basic Biology Awards II is to provide funding for cutting-edge stem cell research and to tackle significant unresolved issues pertinent to understanding the biology of human embryonic stem cells and the control of stem cell fate. Our laboratory recently discovered that we can modulate the expression of genes that regulates cell fate by activating a key factor (Akt) in the power plant (mitochondria) of human embryonic stem cells. This is a novel mechanism that has not been described in the human embryonic stem cells. Potential ability to adjust cell fate through modulation of energy production will add a novel method to interrogate and manipulate the regulatory network that defines the identity of human embryonic stem cells. The proposed studies will investigate how Akt interacts with other molecules in the power plant in human embryonic stem cells, how this new paradigm regulates energy production, and how such mechanism regulates stem cell fate and survival. Future therapeutic application of stem cells will rely heavily on delicate control of cell fate. We will test our hypothesis that human embryonic stem cell gene expression and cell fate can be altered by adjusting the power plant of stem cells. The results of this project will provide opportunities to identify new targets that can be used to manipulate embryonic stem cell fate in the future, and will advance our understanding of the relationship between regulation of energy production and stem cell fate.
Statement of Benefit to California: 
A primary goal of CIRM is to translate basic stem cell research to clinical applications. The disability and loss of earning power and personal freedom resulting from a disease or disorder are devastating and create a financial burden for California in addition to the suffering caused to patients and their families. Therapies using human embryonic stem cells (hES cells) have the potential to change millions of lives. For the potential of hES cells to be realized, we have to decode basic mechanisms that direct stem cell development into specialized cells. Future application of stem cells will rely heavily on delicate control of cell fate. Potential ability to adjust cell fate through modulation of cellular signaling will add new method to manipulate the regulatory network that defines the identity of human embryonic stem cells. Potential benefits of this project to the Citizens of California include: 1. Development of new methods based on modulation of mitochondria function to direct the stem cell fate and stem cell viability, which may eventually be used to develop new strategies to design cell replacement therapies for human diseases. 2. Systemically screening and identifying new protein targets that may be used to develop new drugs and agents to promote stem cell function, thereby developing new treatment methods. 3. Transfer of new technologies and intellectual property to the public realm with resulting IP revenues coming into the state. 4. Creation of new biotechnology spin-off companies based on generated intellectual property. 5. Creating interdisciplinary research teams that will have a competitive edge for obtaining funding from out of state. 6. Creation of new jobs in the biotechnology sector.
Progress Report: 
  • We have been developing new methods to identify the products of stems cells that are differentiated in tissue culture dished. We are focusing on generating a specific type of neuron - cortical interneuron. To this end, we have identified specific sequences in the human genome that drive gene expression in the immature cortical interneurons. Results from the first year of our work provide evidence that our method to use these gene expression elements is working to help us identify cortical interneurons.
  • We have identified 5 gene regulatory elements (enhancers) that can promote gene expression in a specific type of neuronal precursor and neuron. We found that these enhancers can be used to aid in the identification and isolation of these types of cells from embryonic stem cells. In other studies, our group is testing the feasibility of using these types of cells to ameliorate neurological disorders, such as epilepsy.
  • We have identified 5 gene regulatory elements (enhancers) that can promote gene expression in a specific type of neuronal precursor and neuron. We found that these enhancers can be used to aid in the identification and isolation of these types of cells from embryonic stem cells. In other studies, our group is testing the feasibility of using these types of cells to ameliorate neurological disorders.

Pluripotent state and its relationship to cancer stem cells in human gliomas

Funding Type: 
Basic Biology II
Grant Number: 
RB2-01602
ICOC Funds Committed: 
$0
Disease Focus: 
Epilepsy
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Glioblastomas are the most common and lethal form of intracranial tumors. They account for approximately 70% of the 22,500 new cases of malignant primary brain tumors that are diagnosed in adults in the United States each year. These cancers exhibit a relentless malignant progression characterized by widespread invasion throughout the brain, resistance to traditional and newer targeted therapeutic approaches, destruction of normal brain tissue, and certain death. The median age of patients at the time of diagnosis is 64 years. Despite optimal treatment, and improving standard of care the median survival is only 12 to 15 months for patients with glioblastomas. Glioblastoma multiforme (GBM), the most malignant of all brain tumors resuls from the sequential accumulation of genetic aberrations. To understand the role of some or all of the altered genes found in the human gliomas, we have recently used a novel method of using viral vectors to introduce the mutated genes directly into the specific part or cells in the brain. Remarkably we are able to completely recapture the disease of GBM, found in humans. Thus we have an excellent model system to understand and mechanistically decipher the formation of cancer stem cells. In our mouse model systems and xenotransplantation with human GBM cells, as few as 10 cells can lead to the formation of gliomas. Thus on principle every GBM cell is potentially a cancer initiating stem cell. To understand the role of cancer stem cells in glioblastomas, in the next three years we would like to first recapitulate the genetic traits of human GBM in mouse models. We would then like to pursue the time course in which brain tumors develop to get an idea of how soon the tumors grow and more importantly if the different regions of the brain form the same or different tumors. Another important property of the GBMs is that they have very extensive blood supply; therefore it is a good candidate for treatment with drugs that inhibit blood vessel formation. Interestingly, the tumor cells have learned to transdifferentiate into cells that line the walls of blood vessels, thereby rendering the drugs effecting formation of blood vessels ineffective. Finally, since as few as 10-100 GBM tumor cells are capable of initiating new tumors, they offer an excellent opportunity to study the pluripotency of GBMs and their relationship to cancer stem cells We believe that the proposed experiments provide an excellent opportunity to help us understand the molecular mechanisms of formation of GBM and its relationship to formation of pluripotent cancer stem cells.
Statement of Benefit to California: 
There is a growing body of scientific literature which suggests that many cancers have stem cells which, like pluripotent stem cells, have the ability to replicate and differentiate into specific cancer phenotypes. The clinical implications are that unless such cancer stem cells are not eliminated, tumors will grow back again leading to a relapse. The actual percentage of the stem cells in different types of tumors varies quite a bit. Our results, albeit preliminary, indicate that in glioblastoma multiforme (GBM)—one of the most devastating brain tumors with a life expectancy of just over 12-15 months—every tumor cell has the potential to induce gliomas. Moreover these gliomas undergo transdifferentiation to endothelial cells, lining the blood vessels, thus frustrating the use of anti-angiogenic drugs. The work proposed here in our CIRM Basic Biology Awards II application has the potential to not only identify the mechanism of gliomogenesis, but offers an excellent opportunity to generate therapeutic entities. The State of California has excellent academic and biotechnology institutions that will be very glad to further develop some of the genes that we identify in our pursuit to understand the mechanism of GBM formation. Cancer is a very big health burden on the state budget and takes a major toll on the families of cancer patients, especially something as malignant as GBM. Ever more, we need to spend state resources wisely, and finding ways to reduce the continually increasing cost of long-term medical care is critical. The work proposed here seeks to do just that by creating outright cures for diseases that if left untreated require substantial and prolonged medical expenditures and incredible suffering for the patients and their families. In other regards keeping the State of California at the forefront of medical breakthroughs and strengthening our biomedical and biotechnology industries. We are a leading force in these fields, not only across the nation but also worldwide.
Progress Report: 
  • We have been developing new methods to identify the products of stems cells that are differentiated in tissue culture dished. We are focusing on generating a specific type of neuron - cortical interneuron. To this end, we have identified specific sequences in the human genome that drive gene expression in the immature cortical interneurons. Results from the first year of our work provide evidence that our method to use these gene expression elements is working to help us identify cortical interneurons.
  • We have identified 5 gene regulatory elements (enhancers) that can promote gene expression in a specific type of neuronal precursor and neuron. We found that these enhancers can be used to aid in the identification and isolation of these types of cells from embryonic stem cells. In other studies, our group is testing the feasibility of using these types of cells to ameliorate neurological disorders, such as epilepsy.
  • We have identified 5 gene regulatory elements (enhancers) that can promote gene expression in a specific type of neuronal precursor and neuron. We found that these enhancers can be used to aid in the identification and isolation of these types of cells from embryonic stem cells. In other studies, our group is testing the feasibility of using these types of cells to ameliorate neurological disorders.

Epigenetic switches: molecular mechanisms underlying transitions between epigenetic states

Funding Type: 
Basic Biology II
Grant Number: 
RB2-01602
ICOC Funds Committed: 
$0
Disease Focus: 
Epilepsy
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Life begins with single fertilized zygote that through the process of differentiation gives rise to every cell type in human body. Amazingly the embryonic stem cells and induced pluripotent cells retain that potential and thus provide exciting hope for therapeutic applications. In the process of differentiation cells “make a decision” and “learn” what their job in the body is and often have to remember that decision for a lifetime, while retaining the ability to interact with their environment. In our proposal aims at investigating the nature of this cellular memory and plasticity and at understanding the mechanism by which certain genes are turned on or turned off during differentiation. Scientific evidence suggests that an indexing system exists that is based on chemical modifications of proteins organizing DNA into chromatin to regulate gene expression. Some of these modifications act as signals marking particular genes to be active, while others mark inactive genes. This indexing system needs maintenance and there are specialized proteins, called chromatin modifying enzymes that maintain chromatin modifications, thus acting as a “memory”. However, chromatin modifying machines are also capable of switching one indexing state (e.g. active) to another (e.g. inactive) at a particular gene locus in response to a signaling event. We recently discovered a novel chromatin modifying complex, that may work in mediating such epigenetic switches during development and embryonic stem cell differentiation. We propose here to test the hypothesis that this complex enzyme is indeed a switch, to investigate the exact mechanism of the switch at the molecular level and determine which regions of genome are the targets for the switch complex at different times during differentiation. We anticipate that these studies will advance our understanding of basic biological processes underlying the developmental decisions of differentiating stem cells. Such advancements could contribute to improvements in stem cell derived therapies.
Statement of Benefit to California: 
Since the nature of basic science is to tackle the “unknown unknowns” we can’t make specific promises for short-term health benefits to the residents of California. Nevertheless, research proposed here addresses fundamental questions regarding mechanisms of gene regulation in stem cells and during human development and thus, in the long-term, it is likely to have a high impact on development of cell replacement therapies, for harnessing the potential of personalized medicine and for identification of novel drug targets to combat cancer and age-related diseases. Other tangible and immediate benefits for the community include: - creations of at least 2 new jobs in a high skill sector - contribution to the training of new workforce in a set of unique skills in human stem cell technology - creation of new intellectual property that would benefit local institution and by extension local community. - boosting local economy since we buy our supplies from local vendors whenever possible.
Progress Report: 
  • We have been developing new methods to identify the products of stems cells that are differentiated in tissue culture dished. We are focusing on generating a specific type of neuron - cortical interneuron. To this end, we have identified specific sequences in the human genome that drive gene expression in the immature cortical interneurons. Results from the first year of our work provide evidence that our method to use these gene expression elements is working to help us identify cortical interneurons.
  • We have identified 5 gene regulatory elements (enhancers) that can promote gene expression in a specific type of neuronal precursor and neuron. We found that these enhancers can be used to aid in the identification and isolation of these types of cells from embryonic stem cells. In other studies, our group is testing the feasibility of using these types of cells to ameliorate neurological disorders, such as epilepsy.
  • We have identified 5 gene regulatory elements (enhancers) that can promote gene expression in a specific type of neuronal precursor and neuron. We found that these enhancers can be used to aid in the identification and isolation of these types of cells from embryonic stem cells. In other studies, our group is testing the feasibility of using these types of cells to ameliorate neurological disorders.

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