Neurological Disorders

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

Crosstalk: Inflammation in Parkinson’s disease (PD) in a humanized in vitro model

Funding Type: 
Early Translational II
Grant Number: 
TR2-01778
ICOC Funds Committed: 
$2 472 839
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Collaborative Funder: 
Germany
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
Parkinson’s Disease (PD) is the most common neurodegenerative movement disorder. It is characterized by motor impairment such as slowness of movements, shaking and gait disturbances. Age is the most consistent risk factor for PD, and as we have an aging population, it is of upmost importance that we find therapies to limit the social, economic and emotional burden of this disease. Most of the studies to find better drugs for PD have been done in rodents. However, many of these drugs failed when tested in PD patients. One problem is that we can only investigate the diseased neurons of the brain after the PD patients have died. We propose to use skin cells from PD patients and reprogram these into neurons and other surrounding cells in the brain called glia. This is a model to study the disease while the patient is still alive. We will investigate how the glial surrounding cells affect the survival of neurons. We will also test drugs that are protective for glial cells and neurons. Overall, this approach is advantageous because it allows for the study of pathological development of PD in a human system. The goal of this project is to identify key molecular events involved at early stages in PD and exploit these as potential points of therapeutic intervention.
Statement of Benefit to California: 
The goal of this proposal is to create human cell-based models for neurodegenerative disease using transgenic human embryonic stem cells and induced pluripotent stem cells reprogrammed from skin samples of highly clinically characterized Parkinson’s Disease (PD) patients and age-matched controls. Given that age is the most consistent risk factor for PD, and we have an aging population, it is of utmost importance that we unravel the cellular, molecular, and genetic causes of the highly specific cell death characteristic of PD. New drugs can be developed out of these studies that will also benefit the citizens of the State of California. In addition, if our strategy can go into preclinical development, this approach would most likely be performed in a pharmaceutical company based in California.
Progress Report: 
  • In the first year of our CIRM Early Translational II Award we have largely accomplished the first two aims put forth in our proposal “Crosstalk: Inflammation in Parkinson’s disease (PD) in a humanized in vitro model.” Dr. Juergen Winkler, in Erlangen, Germany, has enrolled 10 patients and 6 controls in this project, most of which have had a biopsy of their skin cells sent to The Salk Institute in La Jolla. In Dr. Gage’s lab at The Salk Institute these patient fibroblasts are being reprogrammed into induced pluripotent stem cells (iPSCs), and initial attempts at differentiation into dopaminergic neurons are underway. Additionally, patient blood cells have been sent from Dr. Winkler’s clinic to the lab of Dr. Glass at UC San Diego, where their gene expression profile is being determined. In this initial reporting period we are successfully building the cellular tools necessary to investigate the role of nuclear receptors and inflammation in Parkinson’s Disease.
  • In the second year of our CIRM Early Translational II Award we are making substantial progress towards completing all three aims put forth in our proposal. Dr. Juergen Winkler, our German collaborator, has completed the patient recruitment phase of this project, and skin cells from all 16 subjects (10 with PD and six controls) have been reprogrammed into induced pluripotent stem cells (iPSCs) at the Salk Institute in La Jolla. The patient-specific iPSCs have been differentiated into well-characterized neural stem cells, which the Gage lab is further differentiating into both dopaminergic neurons and astrocytes. In addition to collecting patient skin cells, Dr. Winkler’s group has collected blood cells which are currently being analyzed for gene expression differences by Dr. Glass’ lab at UCSD using state-of-the-art RNA sequencing technology. We have identified a compound that is anti-inflammatory in human cells that we will test on the patient-specific cells once we finish building the cellular tools required to investigate the role of nuclear receptors and inflammation in Parkinson’s Disease.

Neural restricted, FAC-sorted, human neural stem cells to treat traumatic brain injury

Funding Type: 
Early Translational II
Grant Number: 
TR2-01767
ICOC Funds Committed: 
$1 708 549
Disease Focus: 
Neurological Disorders
Trauma
Collaborative Funder: 
Maryland
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
Traumatic brain injury (TBI) affects 1.4 million Americans a year; 175,000 in California. When the brain is injured, nerve cells near the site of injury die due to the initial trauma and interruption of blood flow. Secondary damage occurs as neighboring tissue is injured by the inflammatory response to the initial injury, leading to a larger area of damage. This damage happens to both neurons, the electrically active cells, and oligodendrocytes, the cell which makes the myelin insulation. A TBI patient typically loses cognitive function in one or more domains associated with the damage (e.g. attention deficits with frontal damage, or learning and memory deficits associated with temporal lobe/hippocampal damage); post-traumatic seizures are also common. Currently, no treatments have been shown to be beneficial in alleviating the cognitive problems following even a mild TBI. Neural stem cells (NSCs) provide a cell population that is promising as a therapeutic for neurotrauma. One idea is that transplanting NSCs into an injury would provide “cell replacement”; the stem cells would differentiate into new neurons and new oligodendrocytes and fill in for lost host cells. We have successfully used “sorted“ human NSCs in rodent models of spinal cord injury, showing that hNSCs migrate, proliferate, differentiate into oligodendrocytes and neurons, integrate with the host, and restore locomotor function. Killing the NSCs abolishes functional improvements, showing that integration of hNSCs mediates recovery. Two Phase I FDA trials support the potential of using sorted hNSC for brain therapy and were partially supported by studies in my lab. NSCs may also improve outcome by helping the host tissue repair itself, or by providing trophic support for newly born neurons following injury. Recently, transplantation of rodent-derived NSCs into a model of TBI showed limited, but significant improvements in some outcome measures. These results argue for the need to develop human-derived NSCs that can be used for TBI. We will establish and characterize multiple “sorted” and “non-sorted“ human NSC lines starting from 3 human ES lines. We will determine their neural potential in cell culture, and use the best 2 lines in an animal model of TBI, measuring learning, memory and seizure activity following TBI; then correlating these outcomes to tissue modifying effects. Ultimately, the proposed work may generate one or more human NSC lines suitable to use for TBI and/or other CNS injuries or disorders. A small reduction in the size of the injury or restoration of just some nerve fibers to their targets beyond the injury could have significant implications for a patient’s quality of life and considerable economic impact to the people of California. If successful over the 3-year grant, additional funding of this approach may enable a clinical trial within the next five years given success in the Phase I FDA approved trials of sorted hNSCs for other nervous system disorders.
Statement of Benefit to California: 
The Centers for Disease Control and Prevention estimate that traumatic brain injury (TBI) affects 1.4 million Americans every year. This equates to ~175,000 Californian’s suffering a TBI each year. Additionally, at least 5.3 million Americans currently have a long-term or a lifelong need for help to perform activities of daily living as a result of suffering a TBI previously. Forty percent of patients who are hospitalized with a TBI had at least one unmet need for services one year after their injury. One example is a need to improve their memory and problem solving skills. TBI can also cause epilepsy and increases the risk for conditions such as Alzheimer's disease, Parkinson's disease, and other brain disorders that become more prevalent with age. The combined direct medical costs and indirect costs such as lost productivity due to TBI totaled an estimated $60 billion in the United States in 2000 (when the most recent data was available). This translates to ~$7.5 billion in costs each year just to Californians. The proposed research seeks to generate several human neural-restricted stem cell lines from ES cells. These “sorted” neural-restricted stem cell lines should have greatly reduced or no tumor forming capability, making them ideally suited for clinical use. After verifying that these lines are multipotent (e.g. they can make neurons, astrocytes and oligodendrocytes), we will test their efficacy to improve outcomes in TBI on a number of measures, including learning and memory, seizure activity, tissue sparing, preservation of host neurons, and improvements in white matter pathology. Of benefit to California is that these same outcome measures in a rodent model of TBI can also be assessed in humans with TBI, potentially speeding the translational from laboratory to clinical application. A small reduction in the size of the injury, or restoration of just some nerve fibers to their targets beyond the injury, or moderate improvement in learning and memory post-TBI, or a reduction in the number or severity of seizures could have significant implications for a patient’s quality of life and considerable economic impact to the people of California. Additionally, the cell lines we have chosen to work with are unencumbered with IP issues that would prevent us, or others, from using these cell lines to test in other central nervous system disorders. Two of the cell lines have already been manufactured to “GMP” standards, which would speed up the translation of this work from the laboratory to the clinic. Finally, if successful, these lines would be potentially useful for treating a variety of central nervous system disorders in addition to TBI, including Alzheimer’s disease, Parkinson’s disease, stroke, autism, spinal cord injury, and/or multiple sclerosis.
Progress Report: 
  • In the first year of this Early Translation Award for traumatic brain injury (TBI), our goal was to develop the stem cells lines necessary to begin testing of stem cells in an animal model of TBI in year 2. If we are fortunate to demonstrate that the stem cell products are effective in animal models of TBI, these cells will need to be grown in a way that is acceptable to the FDA for future use in man. Xenofree means that the cells are not exposed to possible animal product contaminants (e.g. serum or blood products) and that every component that the cells were exposed to is chemically defined and can be traced to the original source.
  • First, we obtained three separate embryonic stem (ES) cell lines from Sheffield, UK and imported them to the United States. These lines where then thawed and grown in “xenofree” cell culture conditions. Many labs have had difficulty transitioning human ES cells to xenofree conditions without introducing genetic defects in the cell lines or killing the cells. We were able to work out the correct conditions for all three ES cell lines to be grown xenofree. We were also successful in converting two of the three ES lines into neural stem cells (the subtype of stem cell needed for transplanting into brain tissue). These neural stem cells (NSCs) were further purified by labeling them for a stem cell surface marker present on NSCs (called CD133) and then magnetically sorting out just the CD133 positive cells and continuing to grow them. This approach is thought to enrich the stem cell population for NSCs and eliminate any remaining non-differentiated ES cells (which have an added risk of forming tumors if injected into animals or man). We successfully “sorted” both Shef cell lines and we now have four candidate populations of sorted and unsorted Shef4 and Shef6 cells. We grew these cells in culture and tested whether they differentiated into neuronal precursor or glial precursor cells. Quantification of the type of cells they turn into after 2 weeks showed that the four cell populations were different. These differences were even more apparent when looking at the cells in a microscope. At the end of year one, we have four different populations of neural stem cells which are growing in defined xenofree conditions, are frozen down in master cell banks, and which are genetically normal. There are sufficient quantities of these human neural stem cells (hNSC) to complete the remaining aims of the ETA grant over the remaining two years.
  • In the first year we also trained staff in the surgical procedures required to produce controlled cortical impact injuries in Athymic nude rats (ATNs), a type of rat that has no immune system. These procedures were necessary because no one has ever used ATN rats to model TBI. Our goal in year two is to transplant hNSCs into rats with TBI. If the rats had a normal immune system, their bodies would detect the foreign human cells and reject them. Also, because no one has ever tested TBI in ATN rats, we needed to find out if ATN rats respond like regular rats to the injury and if they have similar, predictable deficits on the cognitive tasks we plan to use in year 2 to measure whether hNCSs improve the animal’s recovery or not. This training and these pilot tests in ATN rats were completed successfully. Finally, the hypothesis is that by “sorting” the hNSCs to be CD133 positive, we are making the stem cell population safer for transplantation. This will be tested in year 2 using a tumorigenicity assay. We worked out how to conduct these assays in year 1 using a population of ES cells known to cause tumors so that we will have a positive control to compare the hNSCs to in year 2.
  • In summary, we met all of our goals and milestones for year 1 and are poised to make good progress in year 2.
  • The goal of this project is to take three human embryonic stem cell lines (Shef3, Shef4, and Shef6), transition them to multipotent neural stem cell (hNSC) populations, sort/enrich these hNSC stem/progenitor populations, and then test these cell lines for efficacy in a rat model of controlled cortical impact (CCI) model of traumatic brain injury (TBI). Our strategy is to develop xenofree culture methods for the transition of hESC to NSCs, use magnetic activated cell sorting (MAC) for the cell surface markers CD133+/CD34- to enrich the hNSC populations for stem/progenitor cells, test these sorted vs unsorted cell lines in tumorigenicity assays, and use the best two non-tumorigenic lines in a CCI model of TBI. Efficacy will be assessed on a battery of cognitive tests, via a reduction in spontaneous seizure, and in histological outcomes.
  • At the Two Year time-point in the grant, we have (A) generated 6 hNSC populations, (B) completed short-term teratoma assays which demonstrate that none of our hNSC populations form teratomas in either of two transplantation sites (sub cutaneous into the leg or intracranially into the brain, (C) established parameters for graded contusion traumatic brain injuries in ATN rats that (D) yield long-term (≥8 weeks) deficits in both learning and memory on the Morris Water Maze. (E) We have also determined that TBI yields an altered response on a conditioned taste aversion task (neophobia) and on the elevated plus maze compared to sham controls. (F) Determined that unsorted hNSCs (both Shef4 and Shef6) do not survive long-term in uninjured brain and (G) transplanted two large cohorts of TBI injured animals with Shef6 sorted NSCs of high passage, Shef6 sorted hNSCs of low passage, sham animals, and animals with a vehicle control. These two cohorts are too large to run simultaneously, so they are being run in parallel. Animals from both cohorts will complete functional all assessments by the end of June 2013.

hESC-Derived Motor Neurons For the Treatment of Cervical Spinal Cord Injury

Funding Type: 
Comprehensive Grant
Grant Number: 
RC1-00345
ICOC Funds Committed: 
$2 396 932
Disease Focus: 
Amyotrophic Lateral Sclerosis
Neurological Disorders
Spinal Muscular Atrophy
Spinal Cord Injury
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Cervical spinal cord injuries result in a loss of upper limb function because the cells within the spinal cord that control upper limb muscles are destroyed. The goal of this research program is to create a renewable human source of these cells, to restore upper limb function in both acute and chronic spinal cord injuries. There are two primary challenges to the realization of this goal: 1) a source of these human cells in high purity, and 2) functional integration of these cells in the body after transplantation. Human embryonic stem cells (hESCs) can form any cell in the body, and can reproduce themselves almost indefinitely to generate large quantities of human tissue. One of the greatest challenges of hESC research is to find ways to restrict hESCs such that they generate large amounts on only one cell type in high purity such that they could be used to replace lost cells in disease or trauma. Our laboratory was the first laboratory in the world to develop a method to restrict hESCs such that they generate large amounts of only one cell type in high purity. That cell type is called an oligodendrocyte, which insulates connections in the spinal cord to allow them to conduct electricity. Transplantation of these cells was useful for treating spinal cord injuries in rats if the treatment was given one week after the injury. That treatment is being developed for use in humans. Recent studies in our laboratory indicate that we have succeeded in restricting hESCs to generate large quantities of a different cell type in the spinal cord, that which controls upper limb muscles. We have generated large quantities of these human cells, grown them with human muscle, and demonstrated that they connect and control the human muscle. The cells also express markers that are appropriate for this cell type. Here we propose to generate these cells in high purity from hESCs and genetically modify them so that they can be induced to grow over inhibitory environments that exist in the injured spinal cord. We will then determine whether these human cells have the ability to regenerate the injured tissue in the spinal cord, and restore lost function. All of our studies will be conducted in an FDA-compliant manner, which will speed the translation of our results to humans if we are successful. The studies outlined in this proposal represent a novel approach to treating spinal cord injury, which might work for both acute and chronic injuries.
Statement of Benefit to California: 
This research plan will position California for international competitiveness in this emerging area of biotechnology, as our research strategy addresses critical scientific problems limiting the development of this sector in California and abroad. High purity cultures of hESC-derivatives enable transplantation approaches to disease, drug discovery, and predictive toxicology. This research plan will lead to the development and thorough characterization of a renewable source of human motor neurons that enables these 3 strategies as they pertain to acute spinal cord injury, chronic spinal cord injury, amyotrophic lateral sclerosis, polio, and spinal muscular atrophy. Clinically relevant scientific advance leads to the development of biotechnology companies, creating jobs and taxation. The treatment and care of individuals with disease or trauma-induced disorders of the central nervous system represents a significant economic burden to the State of California. If successful, our research plan will form the basis of a clinical strategy to improve the function and quality of life of spinal cord injured individuals, which may lessen the cost that the State bears in terms of patient care.
Progress Report: 
  • We have completed the first two AIMs of our proposal on time, and on budget, and we reported on these AIMs in our previous progress report. During this reporting period we have made progress on AIMs 3, 4 and 5. In AIM 3, we transplanted hESC-derived motor neuron progenitor cells into sites of motor neuron death in adult rats. We experienced minor technical difficulties that have set us back by a few months, due to sub-optimal expression of a growth factor in muscles, which is necessary to draw motor neuron axons out to muscles. We have fixed the problem and have confirmed long term growth factor expression in muscles. We have also confirmed that our toxin model induces motor neuron death using several methods, that transplanted motor neurons survive and connect with the spinal cord, and standardized all testing protocols to determine whether transplants along with growth factor addition to muscles will benefit the behavior of the treated animals. Our final experiment is in progress. This delay will not alter the project costs.
  • With regards to AIM 4, we are well ahead of schedule. This AIM was to begin in Year 3, but we began the experiments in Year 2. In this AIM, we transplanted hESC-derived motor neuron progenitor cells into sites of spinal cord injury in adult rats. We have confirmed that transplanted motor neurons survive and connect with the spinal cord, that transplantation enhances the survival of the host spinal cord that otherwise would have been lost, that transplantation enhances axon branching of the host spinal cord, and that these ‘nursing’ effects cause behavioral improvement of locomotion. Our increased productivity has not affected the budget.
  • With regards to AIM 5, are on track and on budget. We have generated FDA-compliant documents for all of the studies listed above.
  • We are on schedule with our research plan, having made progress on the last two AIMs of the proposal according to schedule. The goal of the 4th AIM was to transplant cells to the spinal cord of rats and see if they connect to muscle in the limbs that had been engineered to express an attractant for the processes of the cells in the spinal cord. We confirmed that we can induce the muscle in the limbs to express the attractant, and have the cells in the spinal cord survive, differentiate appropriately, become connected in the spinal cord to other circuits, and extend processes. In addition, we have evidence that these treatments benefit the locomotor ability of the rats. We wrote a scientific article concerning some of this work, and it was accepted for publication in an excellent journal. The goal of the 5th AIM was to document regulatory oversight for the project, to ensure compliance with FDA policies. We have generated FDA-compliant paperwork for all of our studies to date. Thus, our progress is in line with the original proposal.
  • This study tested the hypothesis that high purity motor neurons (MNs) derived from human embryonic stem cells could benefit spinal cord injury. In the first AIM, we proved that MNs could extend processes to muscle and cause it’s contraction, in a dish. In the second AIM, we proved that we could enhance process extension to muscle, in a dish. In the third and fourth AIMs, we proved that MNs transplanted into the diseased or injured spinal cord could integrate and benefit the function and spinal cord tissue structure of animals. In neither case did we see projection of MN processes to muscles, despite the provision of a MN process attractant in the muscles. Nonetheless, MN transplantation reduced tissue loss that normally results from injury or disease, and enhanced regeneration of the spinal cord and functional recovery of the animals.

Human stem cell derived oligodendrocytes for treatment of stroke and MS

Funding Type: 
Comprehensive Grant
Grant Number: 
RC1-00135
ICOC Funds Committed: 
$2 566 701
Disease Focus: 
Multiple Sclerosis
Neurological Disorders
Stroke
Immune Disease
Stem Cell Use: 
Adult Stem Cell
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Strokes that affect the nerves cells, i.e., “gray matter”, consistently receive the most attention. However, the kind of strokes that affecting the “wiring” of the brain, i.e., “white matter”, cause nearly as much disability. The most severe disability is caused when the stroke is in the wiring (axons) that connect the brain and spinal cord; as many as 150,000 patients are disabled per year in the US from this type of stroke. Although oligodendrocytes (“oligos”) are the white matter cells that produce the lipid rich axonal insulator called myelin) are preferentially damaged during these events, stem cell-derived oligos have not been tested for their efficacy in preclinical (animal) trials. These same white matter tracts (located underneath the gray matter, called subcortical) are also the primary sites of injury in MS, where multifocal inflammatory attack is responsible for stripping the insulating myelin sheaths from axons resulting in axonal dysfunction and degeneration. Attempts to treat MS-like lesions in animals using undifferentiated stem cell transplants are promising, but most evidence suggests that these approaches work by changing the inflammation response (immunomodulation) rather than myelin regeneration. While immunomodulation is unlikely to be sufficient to treat the disease completely, MS may not be amenable to localized oligo transplantation since it is such a multifocal process. This has led to new emphasis on approaches designed to maximize the response of endogenous oligo precursors that may be able to regenerate myelin if stimulated. We hypothesize that by exploiting novel features of oligo differentiation in vitro (that we have discovered and that are described in our preliminary data) that we will be able to improve our ability to generate oligo lineage cells from human embryonic stem cells and neural stem cells for transplantation, and also to develop approaches to maximize oligo development from endogenous precursors at the site of injury in the brain. This proposal will build on our recent successes in driving oligo precursor production from multipotential mouse neural stem cells by expressing regulatory transcription factors, and apply this approach to human embryonic and neural stem cells to produce cells that will be tested for their ability to ameliorate brain damage in rodent models of human stroke. Furthermore, we hope to develop approaches that may facilitate endogenous recruitment of oligo precursors to produce mature oligos, which may prove a viable regenerative approach to treat a variety of white matter diseases including MS and stroke.
Statement of Benefit to California: 
Diseases associated with disruption of oligodendrocyte function and integrity (such as subcortical ischemic stroke and multiple sclerosis) are major causes of morbidity and mortality. Stroke is the third leading cause of death and the leading cause of permanent disability in the United States, costing over $50 billion dollars annually, as approximately 150,000 chronic stroke patients survive the acute event and are left with permanent, severe motor and/or sensory deficits. While much less common, multiple sclerosis (MS) is the primary non-traumatic cause of neurologic disability in young adults. Most patients are diagnosed in their 20s-40s and live for many decades after diagnosis with increasing needs for expensive services, medications and ultimately long-term care. Existing strategies for stem cell based therapies include both strategies to replace lost cells and to augment regeneration after injury, but most of these efforts have emphasized the role of undifferentiated stem cells in treatment despite the realization that the main nexus of injury in both diseases is frequently a differentiated cell type – the oligodendrocyte. This project will use new insights into the development of oligodendrocytes from the laboratories of the investigators to find ways to improve production of oligodendrocytes from human ES cells and human neural stem cells, test whether these cells can improve the clinical outcome in rodent models of stroke and MS after transplantation and search for new molecular treatments that would augment the regeneration of oligodendrocytes from resident brain stem cells after injury. This is the first step to translating the basic fundamental understanding of oligodendrocyte development into viable therapies for important human diseases that are major burdens on the citizens of California.
Progress Report: 
  • Over the last year we have succeeded in generating nearly pure cultures of human ES cell derived oligodendrocyte precursors from two different human ES cell lines. We are now also testing whether manipulation of transcription factors or morphogenic signaling pathways regulates the ability of these cells to differentiate into oligodendrocytes that produce myelin. We are testing these cells in a rodent stroke model to determine if they survive in the region of the stroke. If they survive, we will test whether they help to treat the strokes. We are also testing cells in transplantation into a developmental ischemia model and a model for genetic failure to produce myelin.
  • Our proposal centers on developing novel effective methods to generate oligodendrocytes from human ES cells. We focus on identifying signaling pathways (using studies in rodent neural stem cells) that can be adapted to human ES cells and used to regulate the efficiency of oligodendrocyte specification and differentiation from human ES cells. We then hope to use these human ES cell derived oligodendrocytes to determine whether transplantation of these cells is feasible in well characterized animal models associated with damage to oligodendrocytes. Over the last year we have made major progress toward these goals.
  • First, we have completed and submitted for publication two studies identifying the roles of Wnts and Sox10 in regulating the development of oligodendrocytes both during brain development and during stem cell differentiation in vitro. One of these papers is in the final stages of consideration after revision and the other is submitted awaiting reviews.
  • Second, we have developed a novel method for culturing human ES cell derived oligodendrocyte precursors. This is based on modifications of published methods but leads to greatly enhanced purity of final oligodendrocytes in our cultures (about 80% oligodendrocytes and 20% astrocytes). We have used this culture approach to address the role of sonic hedgehog in the differentiation of oligodendrocytes from human oligodendrocyte progenitors and have identified sonic hedgehog as a major regulator of oligodendrocyte differentiation and myelin production. This is quite distinct from rodent neural cells where sonic hedgehog doesn't appear to have this function. This will provide a novel therapeutic target to affect oligodendrocyte maturation and regeneration in disease models and will be of great utility for studying the function of mature human oligodendrocytes. This work is in preparation for submission.
  • Third, we have made some significant progress in our transplantation studies. We completed studies transplanting human ES derived oligodendrocyte progenitors into a rodent model of focal stroke and found that at 1 week post stroke and 2 weeks post stroke the survival of oligodendrocytes from these transplants is very minimal. Thus, we have discontinued this work because of this feasibility issue. We have moved on to examine studies of transplantation into newborn rodents with hypoxic injury and with dysmyelination becahse of the shiverer mutation. The progress here is good. The hypoxia model we are using is a chronic (up to 1 week) exposure to low oxygen tension of P2 mice, which is known to cause oligodendrocyte injury. We are initially characterizing the injury to oligodendrocytes at various durations of hypoxic exposure so that we can identify the best time point to transplant our cells into the brains. We are using immunodeficient mice to decrease the chances of rejection of the transplanted cells. In addition, we are generating a mouse colony with the shiverer allele combined with an immunodeficiency allele in order to be able to transplant cells in this model. In the meantime, we are determining the survival of transplanted cells into newborn mice to identify technical factors that will need to be overcome to allow efficient transplantation and to determine if our human cells participate in differentiation in these mice. Preliminarily we have found good survival of oligodendrocyte lineage cells after transplantation into P2 mice and the expression of myelin antigens after an appropriate period of development in vivo. This is very encouraging.
  • In the last year we have continued our efforts to transplant oligodendrocyte progenitors obtained by differentiation of human ES cells. Our progress in this area has been mixed because of substantial technical hurdles in consistent production of the oligodendrocyte progenitors from frozen stocks of cells. This will necessitate a no-cost extension for a small portion of the work to allow completion of the analysis of already transplanted animals.
  • We have made substantial progress as well in showing that these cells are capable of myelinating axons effectively in vitro. In addition, we've found that the human ES derived oligodendrocytes are capable of myelinating artificial nanofibers in vitro as well. This may serve as a useful platform in the future for drug discovery or other high throughput studies.
  • We have also identified an important novel molecular regulator of oligodendrocyte number and development and this work will continue into the future.
  • In this NCE period we were completing studies with animals that had received neonatal ischemic injury and were implanted with human ES cell derived cells of the oligodendrocyte lineage. These experiments showed that the cells survive and have oligodendrocyte lineage markers for three weeks post injection. Longer survival experiments are still ongoing.

Spinal ischemic paraplegia: modulation by human embryonic stem cell implant

Funding Type: 
Comprehensive Grant
Grant Number: 
RC1-00131
ICOC Funds Committed: 
$2 445 716
Disease Focus: 
Spinal Cord Injury
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
schemia-induced paraplegia often combined with a qualitatively defined increase in muscle tone (i.e. spasticity and rigidity) is a serious complication associated with a temporary aortic cross-clamping ( a surgical procedure to repair an aortic aneurysm). In addition to spinal ischemic injury-induced spasticity and rigidity a significant population of patients with traumatic spinal injury develop a comparable qualitative deficit i.e. debilitating muscle spasticity. At present there are no effective treatment which would lead to a permanent amelioration of spasticity and rigidity and corresponding improvement in ambulatory function. In recent studies, by using rat model of spinal ischemic injury we have demonstrated that spinal transplantation of rat or human neurons leads to a clinically relevant improvement in motor function and correlates with a long term survival and maturation of grafted cells. More recently we have demonstrated a comparable maturation of human spinal precursors grafted spinally in immunosupressed minipig. In the proposed set of experiments we wish to characterize a therapeutical potential of human blastocyst-derived neuronal precursors when grafted into previously ischemia- injured rat or minipig spinal cord. Defining the potency of spinally grafted hESC-derived neuronal precursors in two in vivo models of spinal ischemic injury serves to delineate the differences and/or uniformity in the cell maturation when cells are transplanted in 2 different animals species and can provide an important data set for future implications of such a therapies in human patients.
Statement of Benefit to California: 
Traumatic or ischemic spinal cord injury affect a significant number of people and in majority of cases can lead to a variable degree of motor dysfunction (such as paraparesis or paraplegia) and often combined with increased muscle tone (i.e. spasticity and rigidity). In contrast to other organ systems the central nervous system and spinal cord in particular has minimal or no neuron-regenerative capacity and therefore if a significant population of spinal cord neurons or fibers is lost the resulting deficit is permanent and irreversible. At present there is no effective therapy which would lead to a clinically relevant neurological improvement in patients with ischemia or trauma-induced paraplegia. Initial experimental data using paraplegic rats show that spinal grafting of rat or human neuronal precursors can provide a significant amelioration of spasticity and lead to improved ambulatory function. In the proposed set of experiments we wish to characterize a therapeutical potential of human blastocyst-derived neuronal precursors when grafted into previously ischemia- injured rat or minipig spinal cord. If proven effective such a treatment can potentially be used in patients with spinal ischemic paraplegia or in patients with other spinal injury-related dysfunction associated with a region-specific neuronal loss.
Progress Report: 
  • Transient spinal cord ischemia is a serious complication associated with aortic cross clamping (a surgical procedure required for the repair of aortic aneurysm). Neurological dysfunction resulting from transient spinal cord ischemia may be clinically expressed as paraparesis, fully-developed spastic paraplegia, or flaccid paraplegia. In spastic paraplegia, the underlying spinal pathology is characterized by a selective loss of inhibitory cells (neurons) in the ischemia-injured spinal cord. That loss of inhibition produces increased muscle tone (i.e. spasticity). While there are some current pharmacological treatments for spasticity that provide a certain degree of functional improvement, there are no effective therapies that lead to clinically-relevant, long-lasting recovery. One of the therapeutic approaches pursued by our group is the characterization of functional changes after spinal cord transplantation of neuronal cells previously generated in culture with the goal of replacing missing inhibitory neurons in the spinal cord. In our recent experiments, we characterized the survival and differentiation of human embryonic stem cell-derived neural precursors that were grafted into the spinal cord of rats with a previous spinal ischemic injury. Our initial data demonstrate that spinal grafting of neural precursors generated from 3 independent human embryonic stem cell lines is associated with long-term cell engraftment of grafted cells. A significant population of the grafted cells displayed neuronal differentiation, progressive maturation, and expression of markers which are typical for mature, functional human neurons. Initial analysis of grafted cells also indicated the development of functional connectivity between transplanted neurons and surviving neurons of the recipient. A significant advancement in our effort to characterize the effect of such a treatment was the use of a sorting technique which permits the generation of large quantities of highly-purified neural precursors. The capacity to generate such large quantities of pure cell populations is particularly important in our large preclinical animal model (minipig), which is essential to move this therapeutic approach to clinic. In addition, we characterized an efficient cell freezing protocol. The sorting and freezing techniques together allow large quantities of identical cell populations to be frozen for future transplantation, ensuring a group of animals receives an identical cell population. Our plan for the next year is to perform long-term functional recovery studies in our minipig model of spinal ischemia.
  • Transient spinal cord ischemia is a serious complication associated with aortic cross clamping, i.e., the procedure required to replace aortic aneurysm. The major neurological deficit resulting from spinal ischemic injury is the loss of motor function in lower extremities, also called paraplegia. The pathological mechanism leading to the loss of function is the result of progressive death of spinal cells (i.e., neurons) in the affected region of the spinal cord. At present there is no effective therapy for spinal ischemia-induced paraplegia.
  • In our previous completed studies, we have characterized the survival and neuronal maturation of human embryonic stem cell derived neural precursors analyzed at 2 weeks to 2 months after spinal transplantation in spinal ischemia-injured rats. A comparable survival and maturation was seen compared to fetal human spinal cord-derived cells. In our next studies, we will define the therapeutic potency of spinally grafted ES-NPCs once cells are grafted into the spinal cord of immunodeficient rats (i.e., animals which do not require immunosuppression) and the effect of cell grafting assessed for up to 4 months after cell transplantation. In subsequent studies, the degree of treatment effect will be studied in continuously immunosuppressed minpigs with previous spinal ischemic injury.
  • Transient spinal cord ischemia is a serious complication associated with aortic cross clamping, i.e., the procedure required to replace aortic aneurysm. The major neurological deficit resulting from spinal ischemic injury is the loss of motor function in the lower extremities, also called paraplegia. The pathological mechanism leading to the loss of function is the result of progressive death of spinal cells (i.e., neurons) in the affected region of the spinal cord. At present there is no effective therapy for spinal ischemia-induced paraplegia. In our previous completed studies, we have characterized the survival and neuronal maturation of human embryonic stem cell-derived neural precursors grafted into the lumbar spinal cord in immunodeficient rats and have demonstrated good tolerability of long-term immunosuppression in rodents and minipigs after using subcutaneously implanted tacrolimus pellets. In our ongoing studies, our goal is to characterize the effect of clonally expanded embryonic stem cell-derived neural precursors after spinal grafting in long-term immunosuppressed rats and minipigs and immunodeficient rats with previous spinal ischemic injury.

MEF2C-Directed Neurogenesis From Human Embryonic Stem Cells

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

Using Human Embryonic Stem Cells to Understand and to Develop New Therapies for Alzheimer's Disease

Funding Type: 
Comprehensive Grant
Grant Number: 
RC1-00116
ICOC Funds Committed: 
$2 512 664
Disease Focus: 
Aging
Alzheimer's Disease
Neurological Disorders
Genetic Disorder
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
Cell Line Generation: 
Embryonic Stem Cell
iPS Cell
oldStatus: 
Closed
Public Abstract: 
Alzheimer’s Disease (AD) is a progressive incurable disease that robs people of their memory and ability to think and reason. It is emotionally, and sometimes financially devastating to families that must cope when a parent or spouse develops AD. Unfortunately, however, we currently lack an understanding of Alzheimer’s Disease (AD) that is sufficient to drive the development of a broad range of therapeutic strategies. Compared to diseases such as cancer or heart disease, which are treated with a variety of therapies, AD lacks even one major effective therapeutic approach. A key problem is that there is a paucity of predictive therapeutic hypotheses driving the development of new therapies. Thus, there is tremendous need to better understand the cellular basis of AD so that effective drug and other therapies can be developed. Several key clues come from rare familial forms of AD (FAD), which identify genes that can cause disease when mutant and which have led to the leading hypotheses for AD development. Recent work on Drosophila and mouse models of Alzheimer’s Disease (AD) has led to a new suggestion that early defects in the physical transport system that is responsible for long-distance movements of vital supplies and information in neurons causes neuronal dysfunction. The type of neuronal failure caused by failures of the transport systems is predicted to initiate an autocatalytic spiral of biochemical events terminating in the classic pathologies, i.e., plaques and tangles, and the cognitive losses characteristic of AD. The problem, however, is how to test this new model and the prevailing “amyloid cascade” model, or indeed any model of human disease developed from studies in animal models, in humans. It is well known that mouse models of AD do not fully recapitulate the human disease, perhaps in part because of human-specific differences that alter the details of the biochemistry and cell biology of human neurons. One powerful approach to this problem is to use human embryonic stem cells to generate human neuronal models of hereditary AD to test rigorously the various hypotheses. These cellular models will also become crucial reagents for finding and testing new drugs for the treatment of AD.
Statement of Benefit to California: 
Alzheimer’s Disease (AD) is emotionally devastating to the families it afflicts as well as causing substantial financial burdens to individuals, to families, and to society as a whole. In California, the burden of Alzheimer’s Disease is substantial, so that progress in the development of therapeutics would make a significant financial impact in the state. Although there are not a great deal of data about the burden of AD in California specifically, the population of California is 12% of that of the United States and most information suggests that California has a “typical” American burden of this disease. For example, information from the Alzheimer’s Association (http://www.alz.org/alzheimers_disease_alzheimer_statistics.asp) reveals: 1) An estimated 4.5 million Americans have Alzheimer’s disease, which has more than doubled since 1980. This creates an estimated nationwide financial burden of direct and indirect annual costs of caring for individuals with AD of at least $100 billion. Thus, a reasonable estimate is that California has more than half a million AD patients with an estimated cost to California of $12 billion per year! 2) One in 10 individuals over 65 and nearly half of those over 85 are affected, which means that as our population ages, we will be facing a tidal wave of AD. Current estimates are that with current rates of growth that the AD patient population will double or triple in the next 4 decades. 3) The potential benefit of research such as that proposed in this grant application is that finding a treatment that could delay onset by five years could reduce the number of individuals with Alzheimer’s disease by nearly 50 percent after 50 years. This would be significant since a person with Alzheimer’s disease will live an average of eight years and as many as 20 years or more from the onset of symptoms. Finding better treatments will thus have significant financial benefits to California. 4) After diagnosis, people with Alzheimer’s disease survive about half as long as those of similar age without AD or other dementia. 5) In terms of financial impact on California families, the statistics (http://www.alz.org/alzheimers_disease_alzheimer_statistics.asp) are that more than 7 out of 10 people with Alzheimer’s disease live at home. Almost 75 percent of their care is provided by family and friends. The remainder is “paid’ care costing an average of $19,000 per year. Families pay almost all of that out of pocket. The average cost for nursing home care is $42,000 per year but can exceed $70,000 per year in some areas of the country. The average lifetime cost of care for an individual with Alzheimer’s is $174,000. Thus, any progress in developing better therapy for AD will have a substantial positive impact to California.
Progress Report: 
  • We have made significant progress on developing human stem cell based systems to probe the causes and features of Alzheimer's Disease. We are focusing on using human embryonic and human pluripotent stem cell lines carrying genetic changes that cause hereditary Alzheimer's Disease (AD). In one approach, we have made progress by developing iPS cells carrying small genetic changes in the presenilin 1 gene, which cause severe early onset AD. We also made substantial progress on developing methods to measure the distribution within neurons of products linked to Alzheimer's Disease. Finally, we have completed development of a cell sorting method to purify neuronal stem cells, neurons, and glia from human embryonic stem cells and human IPS cells. Together, these methods should allow us to continue making progress on using pluripotent human stem cells to probe the molecular basis for how cellular changes found in neurons in the brain of AD patients are generated. In addition, these methods we are developing are moving us closer to having sources of normal and AD human neurons generated in the laboratory for drug-testing and development.
  • We continue to make significant progress developing human stem cell based disease models to probe the causes of Alzheimer's Disease (AD) and to eventually develop drugs. In the past year we generated and analyzed several new human pluripotent stem cell lines (hIPS) carrying genetic changes that cause hereditary AD or that increase the risk of developing AD. We detected AD related characteristics in neurons with hereditary and in one case of a sporadic genetic type. While considerable confirmatory work needs to be done, our data raise the possibility that AD can be modeled in human neurons made from hIPS cells. In the coming year, we hope to continue making progress on using pluripotent human stem cells to probe the molecular basis for how cellular changes found in neurons in the brain of AD patients are generated. In addition, the methods we are developing are moving us closer to having sources of normal and AD human neurons generated in the laboratory for drug-testing and development.
  • In our final year of funding, we made significant progress developing human stem cell based disease models to probe the causes of Alzheimer's Disease (AD) and to eventually develop drugs. We generated and analyzed several new human pluripotent stem cell lines (hIPS) carrying genetic changes that cause hereditary AD or that increase the risk of developing AD. We detected AD related characteristics in neurons with hereditary and in one case of a sporadic genetic type. While considerable confirmatory work needs to be done, our data raise the possibility that AD can be modeled in human neurons made from hIPS cells. The methods we developed are moving us closer to having sources of normal and AD human neurons generated in the laboratory for drug-testing and development.

Molecular and Cellular Transitions from ES Cells to Mature Functioning Human Neurons

Funding Type: 
Comprehensive Grant
Grant Number: 
RC1-00115
ICOC Funds Committed: 
$2 879 210
Disease Focus: 
Amyotrophic Lateral Sclerosis
Neurological Disorders
Parkinson's Disease
Genetic Disorder
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Human embryonic stem cells (hESCs) are pluripotent entities, capable of generating a whole-body spectrum of distinct cell types. We have developmental procedures for inducing hESCs to develop into pure populations of human neural stem cells (hNS), a step required for generating authentic mature human neurons. Several protocols have currently been developed to differentiate hESCs to what appear to be differentiated dopaminergic neurons (important in Parkinson’s disease (PD) and cholinergic motor neurons (important in Amyolateral Sclerosis (ALS) in culture dishes. We have developed methods to stably insert new genes in hESC and we have demonstrated that these transgenic cells can become mature neurons in culture dishes. We plan to over express alpha synuclein and other genes associated with PD and superoxide dismutase (a gene mutated in ALS) into hESCs and then differentiate these cells to neurons, and more specifically to dopaminergic neurons and cholinergic neurons using existing protocols. These transgenic cells can be used not only for the discovery of cellular and molecular causes for dopaminergic or cholinergic cell damage and death in these devastating diseases, but also can be used as assays to screen chemical libraries to find novels drugs that may protect against the degenerative process. Until recently the investigation of the differentiation of these human cells has only been observed in culture dishes or during tumor formation. Our recent results show that hESC implanted in the brains of mice can survive and become active functional human neurons that successfully integrate into the adult mouse forebrain. This method of transplantation to generate models of human disease will permit the study of human neural development in a living environment, paving the way for the generation of new models of human neurodegenerative and psychiatric diseases. It also has the potential to speed up the screening process for therapeutic drugs.
Statement of Benefit to California: 
We plan to develop procedures to induce human ES cells into mature functioning neurons that carry genes that cause the debilitating human neurological diseases, Parkinson’s disease and Amyolateral Sclerosis (ALS). We will use the cells to reveal the genes and molecular pathways inside the cells that are responsible for how the mutant genes cause damage to specific types of brain cells. We also will make the cells available to other researchers as well as biotech companies so that other investigators can use these cells to screen small molecule and chemical libraries to discover new drugs that can interfere with the pathology caused by these mutant cells that mimic human disease, in hopes of accelerating the pace of discovery.
Progress Report: 
  • Our research is focused on studying two debilitating diseases of the nervous system: Parkinson’s disease (PD) and Amyotrophic Lateral Sclerosis (ALS) also known as Lou Gehrig’s disease. While the causes and symptoms of these two conditions are very different, they share one aspect in common: patients gradually lose specific types of nerve cells, namely the so-called dopaminergic neurons in PD, and motor neurons in ALS. If we can find ways to protect the neurons from dying, we might be able to slow or even halt disease progression in ALS and PD patients. In the past two years, our lab has developed robust procedures to generate these two classes of neurons from human embryonic stem cells and we have been studying the molecular changes that govern their specialization. Since last year, we have been using neurons to elucidate the molecular mechanisms that underlie the demise of these cells.
  • ALS is one of the most common neuromuscular diseases, afflicting more than 30,000 Americans. Patients rapidly lose their motor neurons – the nerve cells that extend from the brain through the spinal cord to the muscles, thereby controlling their movement. Therapy options are extremely limited and people with ALS usually succumb to respiratory failure or pneumonia within three to five years from the onset of symptoms. Most ALS patients have no family history of ALS and carry no known genetic defects that may help explain why they develop the disease. However, a small number of ALS patients have mutations in the superoxide dismutase 1 (SOD1) gene, which encodes an enzyme that scavenges so-called free radicals – aggressive oxidizing molecules that are by-products of the cells’ normal metabolism. Researchers therefore believe that accumulation of these free radicals may damage motor neurons in ALS and contribute to their death.
  • To test this idea, we introduced the mutated form of the SOD1 gene into astrocytes – cells that provide metabolic and structural support to neurons – and cultured our stem cell-derived motor neurons along with these SOD1-mutant astrocytes. Indeed, while motor neurons grown on ‘normal’ astrocytes were fully viable, we saw widespread death of motor neurons in cocultures with ‘mutant’ astrocytes, along with elevated levels of free radicals. We think that this is due to our mutant astrocytes being causing inflammation, and so our future efforts are focused on understanding the role of the immune system, specifically the function of microglia – the resident immune cells of the brain and spinal cord – in our co-cultures with human motor neurons. We are very excited about these results because they show that our cocultures may be a very useful tool to screen drugs that may counteract the neurotoxicity caused by inflammation and free radicals. We have already begun testing several known antioxidants, and found some of them to be very effective in improving motor neuron survival in the culture dish. Such compounds may ultimately improve the condition of ALS patients.
  • PD is the second most common neurodegenerative disease and develops when neurons in the brain, and in particular, in a part of the brain known as the substantia nigra die. These neurons are called dopaminergic because they produce dopamine, a molecule that is necessary for coordinated body movement. Many dopaminergic neurons are already lost when patients develop PD symptoms, which include trembling, stiffness, and slow movement. Around one million Americans are currently suffering from PD, and 60,000 new cases are diagnosed each year. While several surgical and pharmacological treatment options exist, they cannot slow or halt disease progression and are instead aimed at treating the symptoms. The exact causes for neuron death in PD are unknown but among others inflammation in the affected brain area may play a role in disease progression.
  • In a joint effort with the laboratories of Christopher Glass and Michael Rosenfeld at the University of California, San Diego, we showed using animal experiments that a protein called Nurr1 is crucial for the development and survival of dopaminergic neurons. We found that the Nurr1 gene is turned on by inflammatory signals and suppresses genes that encode neurotoxic factors. Microglia are the major initiators of the neurotoxic response to inflammatory stimuli, which is then amplified by astrocytes. Thus our findings reveal an important role for Nurr1 in microglia and astrocytes to protect dopaminergic neurons from exaggerated production of inflammation-induced neurotoxic mediators. We are now using human embryonic stem cell-derived dopaminergic neurons, cultured along with human atrocytes and microglia to test whether we can demonstrate this positive role of Nurr1 in a culture dish as well.
  • We are investigating the molecular mechanisms underlying two major neurological diseases: Parkinson’s disease (PD) and Amyotrophic Lateral Sclerosis (ALS), also known as Lou Gehrig’s disease. In the past year, we have taken our previously developed human embryonic stem cell (hESC)-based cell culture model for PD and ALS another step further: we have begun building an assay system that may eventually allow both the identification of biomarkers for early diagnosis and the screening of drug candidates for ALS and PD. By transplanting hESC-derived neurons into live animals and brain slices, we have also made first inroads into recapitulating the disease processes in animal model systems.
  • While the causes and symptoms of ALS and PD are very different, they share one aspect in common: in both, patients gradually lose specific types of nerve cells, namely, the so-called dopaminergic neurons in PD, and motor neurons in ALS; it this neuron death that causes both diseases. Previously, we showed with our hESC-based cell culture system that an inflammatory response in astrocytes (the brain cells that provide metabolic and structural support to neurons) is involved in loss of motor neurons. Similarly, we demonstrated that microglia (the brain’s immune cells) and astrocytes together protect dopaminergic neurons from exaggerated production of inflammation-induced neurotoxic mediators. This function of astrocytes and microglia was dependent on a protein called Nurr1: we found that the Nurr1 gene is turned on by inflammatory signals and suppresses genes that encode neurotoxic factors.
  • We have now begun to characterize in depth the specific signaling molecules that communicate the inflammation cue from the glial cells to neurons. To do this, we cultured astrocytes and microglia in the petri dish, induced inflammation and collected cell culture supernatants from the ‘inflamed’ and normal cells. We then measured the levels of specific so-called cytokines, the inflammatory signaling molecules secreted by the glial cells. Once we have obtained a characteristic cytokine ‘signature’ of disease-associated glial cells, we can begin to unravel the molecular pathways that lead to inflammation. Thus our research may lead to the discovery of early diagnostic markers and enable drug screening for compounds that suppress or prevent these neurotoxic inflammatory processes.
  • Our cell culture assays have provided a great deal of insight into the signaling cascades that eventually lead to neuron death. However, they probably cannot fully recapitulate the complex interplay between the neurons and the cellular environment in which they reside within the brain. We have therefore begun to transplant hESC-derived neurons into the brains of mice. Our results indicate that the neurons rapidly extended processes and developed dendritic branches and axons that integrated into the existing neuronal network. In the coming year, we plan to build on these results, using our hESC-derived neuronal models of PD and ALS to better understand mechanisms of dysregulation. Specifically, we will examine alterations in synapse formation, cell survival, and neuron maturation. We will also devise strategies for functional recovery and rescue in the context of the living animal.
  • We are investigating the molecular mechanisms underlying two major neurological diseases: Parkinson’s disease (PD) and Amyotrophic Lateral Sclerosis (ALS), also known as Lou Gehrig’s disease. In the past year, we took our human embryonic stem cell (hESC)-based neural cell culture model for PD and ALS another step further and built sensitive and quantitative assays that can allow for the screening of drug candidates for ALS and PD. We have also consistently improved our transplanting techniques and are now able to detect functional, electrophysiologically active, hESC-derived neurons in live animals. This experiment was crucial to show that, under our culture conditions, human neurons derived from embryonic stem cells were able to integrate and form meaningful connections with other neurons in a given adult brain environment.
  • Moreover, we are now performing an in-depth characterization of the specific signaling molecules that communicate the inflammation cues from the glial cells to neurons in the presence of ALS-causing mutations (SOD1G37R) and PD-causing mutations (recombinant alfa-synuclein). In this report we have explored another functional assay to measure glial function and inflammatory response using astrocytes that express ALS-causing mutations. In addition, we report here that adding PD-causing mutagens to mixed cultures of human neurons and astrocytes results in the death of dopaminergic neurons, the type of neurons affected in PD. We are currently testing new compounds that can decrease the neuronal toxicity observed.
  • Our research may not only lead to the discovery of early diagnostic markers but also enable drug screening for compounds that suppress or prevent these neurotoxic inflammatory processes.

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

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

Genetic Encoding Novel Amino Acids in Embryonic Stem Cells for Molecular Understanding of Differentiation to Dopamine Neurons

Funding Type: 
New Faculty I
Grant Number: 
RN1-00577
ICOC Funds Committed: 
$2 626 937
Disease Focus: 
Parkinson's Disease
Neurological Disorders
oldStatus: 
Closed
Public Abstract: 
Embryonic stem cells have the capacity to self-renew and differentiate into other cell types. Understanding how this is regulated on the molecular level would enable us to manipulate the process and guide stem cells to generate specific types of cells for safe transplantation. However, complex networks of intracellular cofactors and external signals from the environment all affect the fate of stem cells. Dissecting these molecular interactions in stem cells is a very challenging task and calls for innovative new strategies. We propose to genetically incorporate novel amino acids into proteins directly in stem cells. Through these amino acids we will be able to introduce new chemical or physical properties selectively into target proteins for precise biological study in stem cells. Nurr1 is a nuclear hormone receptor that has been associated with Parkinson’s disease (PD), which occurs when dopamine (DA) neurons begin to malfunction and die. Overexpression of Nurr1 and other proteins can induce the differentiation of neural stem cells and embryonic stem cells to dopamine (DA) neurons. However, these DA neurons did not survive well in a PD mouse model after transplantation. In addition, it is unclear how Nurr1 regulates the differentiation process and what other cofactors are involved. We propose to genetically introduce a novel amino acid that carries a photocrosslinking group into Nurr1 in stem cells. Upon illumination, molecules interacting with Nurr1 will be permanently linked for identification by mass spectrometry. Using this approach, we aim to identify unknown cofactors that regulate Nurr1 function or are controlled by Nurr1, and to map sites on Nurr1 that can bind agonists. The function of identified cofactors in DA neuron specification and maturation will be tested in mouse and human embryonic stem cells. These cofactors will be varied in combination to search for more efficient ways to induce embryonic stem cells to generate a pure population of DA neurons. The generated DA neurons will be evaluated in a mouse model of PD. Additionally, the identification of the agonist binding site on Nurr1 will facilitate future design and optimization of potent drugs.
Statement of Benefit to California: 
Parkinson’s disease (PD) is the second most common human neurodegenerative disorder, and primarily results from the selective and progressive degeneration of ventral midbrain dopamine (DA) neurons. Cell transplantation of DA neurons differentiated from neural stem cells or embryonic stem cells raised great hope for an improved treatment for PD patients. However, DA neurons derived using current protocols do not survive well in mouse PD models, and the details of DA neuron development from stem cells are unclear. Our proposed research will identify unknown cofactors that regulate the differentiation of embryonic stem cells to DA neurons, and determine how agonists activate Nurr1, an essential nuclear hormone receptor for DA neuron specification and maturation. This study may yield new drug targets and inspire novel preventive or therapeutic strategies for PD. These discoveries may be exploited by California’s biotech industry and benefit Californians economically. In addition, we will search for more efficient methods to differentiate human embryonic stem cells into DA neurons, and evaluate their therapeutic effects in PD mouse models. Therefore, the proposed research will also directly benefit California residents suffering from PD.
Progress Report: 
  • Patients with Parkinson’s disease have malfunctioning or dying dopaminergic (DA) neurons. Human embryonic stem cells can be differentiated into DA neurons for transplantation with the potential to cure this disease, yet the differentiation mechanism is not very clear. A nuclear hormone receptor named Nurr1 is found to regulate the differentiation process. To study the regulation mechanism, we proposed to genetically incorporate nonnatural amino acids into Nurr1 in stem cells, and use the novel properties of these amino acids to identify the interacting protein partners of Nurr1. Once these partners are discovered, effective protocols can be developed to generate high purity DA neurons for therapeutic purposes. In the past year, we made significant progress in genetically inserting nonnatural amino acids in stem cells. We are in the process of making stem cell lines that have this capacity. We also set up functional assays for studying Nurr1 and its mutants containing nonnatural amino acids. These results paved the way for our future identification of Nurr1 interacting networks in stem cells.
  • Patients with Parkinson’s disease have malfunctioning or dying dopaminergic (DA) neurons. Human embryonic stem cells can be differentiated into DA neurons for transplantation with the potential to cure this disease, yet the differentiation mechanism is not very clear. A nuclear hormone receptor named Nurr1 is found to regulate the differentiation process. To study the regulation mechanism, we proposed to genetically incorporate nonnatural amino acids into Nurr1 in stem cells, and use the novel properties of these amino acids to identify the interacting protein partners of Nurr1. Once these partners are discovered, effective protocols can be developed to generate high purity DA neurons for therapeutic purposes. In the past year, we figured out several mechanisms that prevent the efficient incorporation of nonnatural amino acids into proteins in stem cells. We now have developed new strategies to overcome these difficulties. In the meantime, we developed another complementary approach in order to detect unknown proteins that interact with Nurr1 during the differentiation process of stem cells. We are employing these new methods to identify Nurr1 interacting networks in stem cells.
  • Patients with Parkinson’s disease have malfunctioning or dying dopaminergic (DA) neurons. Human embryonic stem cells can be differentiated into DA neurons for transplantation with the potential to cure this disease, yet the differentiation mechanism is not very clear. The differentiation of embryonic stem cells to DA neurons has been found to be regulated by a nuclear hormone receptor Nurr1, but how Nurr1 involves in this complicated process remains unclear - no ligands or protein partners have been uncovered for Nurr1. To understand the regulation mechanism in molecular details, we proposed to incorporate non-natural amino acids into Nurr1 directly in stem cells, and use the novel properties of these amino acids to identify the protein partners of Nurr1. Once these partners are discovered, effective protocols can be developed to generate high purity DA neurons for therapeutic purposes. In the past year, we figured out a right solution for generating stem cell lines capable of incorporating non-natural amino acids. We also created a novel bacterial strain for efficient producing Nurr1 proteins with the non-natural amino acids inserted. With these progresses we are now probing proteins that interact with Nurr1 during the differentiation of stem cells, which should eventually enable us to come up with new strategies for making DA neurons from embryonic stem cells.
  • Patients with Parkinson’s disease have malfunctioning or dying dopaminergic (DA) neurons. Human embryonic stem cells can be differentiated into DA neurons for transplantation with the potential to cure this disease, yet the differentiation mechanism is not very clear. The differentiation of embryonic stem cells to DA neurons has been found to be regulated by a nuclear hormone receptor Nurr1, but how Nurr1 is involved in this complicated process remains unclear - no ligands or protein partners have been uncovered for Nurr1. To understand the regulation mechanism in molecular details, we proposed to incorporate non-natural amino acids into Nurr1 directly in stem cells, and use the novel properties of these amino acids to identify the protein partners of Nurr1. Once these partners are discovered, effective protocols can be developed to generate high purity DA neurons for therapeutic purposes. In the past year, after testing numerous conditions in various cell lines, we discovered that photo-crosslinking is inefficient in capturing proteins interacting with Nurr1, possibly because the affinity between the unknown target protein and Nurr1 is too low. To overcome this challenge, we developed a new strategy of capture interacting proteins based on a novel class of non-natural amino acids, which do not require additional reagents nor external stimuli to function. We demonstrated the ability of these amino acids to crosslink proteins in the process of interacting with other proteins in live cells. We have also generated stable cell lines that are able to incorporate such non-natural amino acids. Using these new methods, we have been probing proteins that interact with Nurr1 during the differentiation of stem cells, which should eventually enable us to come up with new strategies for making DA neurons from embryonic stem cells.

Pages

Subscribe to RSS - Neurological Disorders

© 2013 California Institute for Regenerative Medicine