Neurological Disorders

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

Derivation of Inhibitory Nerve Cells from Human Embryonic Stem Cells

Funding Type: 
Comprehensive Grant
Grant Number: 
RC1-00346
ICOC Funds Committed: 
$2 507 223
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Statement of Benefit to California: 
Progress Report: 
  • Our goal is to develop a novel cell-based therapy to treat patients with epilepsy, Parkinson’s disease and brain injury. The strategy is to use human embryonic stem cells to produce inhibitory nerve cells for transplantation and therapeutic modulation of neural circuits, an approach that may have widespread clinical application. In preliminary studies using inhibitory neuron precursors from embryonic rodent brains, we have demonstrated that this approach can relieve symptoms in animal models of Parkinson’s disease and epilepsy. To turn this approach into a patient therapy we need to develop methods to obtain large numbers of human cells suitable for transplantation. The object of this proposal is to develop methods for producing unlimited numbers of exactly the right type of inhibitory nerve cell using human embryonic stem (ES) cells as the starting material.
  • One strategy to make large numbers of inhibitory neurons would be to convert human ES cells into neural stem (NS) cell lines that could be stably propagated indefinitely, and then to convert the NS cells into inhibitory nerve cells. However, we discovered that NS cell lines do not retain the capacity to generate neurons after extended culture periods but instead produce only glial cells. We have therefore begun to create neurons directly from ES cells, without interrupting the differentiation to amplify cell number at the neural progenitor phase. Using this approach, we have been successful at specifying the right pathway to produce the specific neural progenitor cell we need during the process of differentiation from ES cells. Because there are multiple subytpes of inhibitory neuron, we are testing various cell culture manipulations to enrich for the specific neuron subtype that matches our desired cell type. In addition, we are developing reporter cell lines that will allow us to observe differentiation from ES cell to inhibitory neuron in real time and purify the cells of interest for transplantation. Finally, we are also testing whether artificially expressing key proteins that regulate gene expression and are required for inhibitory neuron production during brain development can more efficiently drive a high percentage of ES cells to differentiate into the desired cell type.
  • With these tools in place, we hope to begin animal transplantation studies using human ES-derived inhibitory nerve cells within the coming year. If successful, this accomplishment will set the stage for studies in primates, and hasten the day when inhibitory nerve cells may be used as patient therapy for a wide variety of debilitating neurological disorders including Parkinson’s disease, epilepsy, and brain injury.
  • This past year, we have made significant strides toward the production of inhibitory nerve cells and precursor (MGE) cells from human embryonic stem (ES) and induced pluripotent stem (iPS) cells. These stem cell-derived MGE progenitor cells appropriately mature into inhibitory neurons upon further culture and following transplantation into the newborn mouse brain. Additionally, human ES cell-derived inhibitory neurons possess active membrane properties by electrophysiology analysis. Work is ongoing to determine their functional potential following transplantation: whether these cells can make connections, or synapses, with each other and with neurons in the host brain in order to elevate inhibitory tone in the transplanted animals. Following successful completion of this aim in the coming year, we will be well positioned to examine the therapeutic potential of these cells in pre-clinical epilepsy and Parkinson's disease animal models.
  • Inhibitory nerve cell deficiencies have been implicated in many neurological disorders including epilepsy. The decreased inhibition and/or increased excitation lead to hyper-excitability and brain imbalance. We are pursuing a strategy to re-balance the brain by injecting inhibitory nerve precursor cells. Most inhibitory nerve cells come from the medial ganglionic eminence (MGE) during fetal development. We have previously documented that mouse MGE transplants reduce seizures in animal models of epilepsy and ameliorate motor symptoms in a rat model of Parkinson’s disease. This project aims to develop human MGE cells from human embryonic stem (ES) cells and to investigate their function in animal models of human disease. In the past year, we have successfully developed a robust and reproducible method to generate human ES cell-derived MGE cells and have performed extensive gene expression and functional analyses. The gene expression profiles of these ES-derived MGE cells resemble those of mouse and human fetal MGE. They appropriately mature into inhibitory nerve cells in culture and following injection into rodent brain. Also, the ES-derived inhibitory cells exhibit active electrical properties and establish connections (synapses) with other nerve cells in culture and in the rodent brain. Thus, we have succeeded in deriving inhibitory human MGE cells from human ES cells and are now transplanting these cells into animal models of disease.

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: 
Statement of Benefit to California: 
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: 
Statement of Benefit to 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: 
Statement of Benefit to California: 
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: 
Statement of Benefit to California: 
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: 
Statement of Benefit 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: 
Statement of Benefit to California: 
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: 
Statement of Benefit to California: 
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.

MicroRNAs in Human Stem Cell Differentiation and Mental Disorders

Funding Type: 
SEED Grant
Grant Number: 
RS1-00462
ICOC Funds Committed: 
$791 000
Disease Focus: 
Autism
Neurological Disorders
Developmental Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Statement of Benefit to California: 
Progress Report: 
  • Human stem cells, both embryonic and induced pluripotent stem cells, offer exciting opportunities for cell-based therapies in injured or diseased human brains or spinal cords. The clinical efficacy of grafted progenitor cells critically depends on their ability to migrate to the appropriate sites in the adult central nervous system without unwanted proliferation and tumor formation. However, little is known about the cellular behavior of human neural progenitor cells derived from human stem cells or how their proliferation and migration are coordinated. During this reporting period, we continued to study human neural progenitor cells derived from human stem cells, a cell culture system established during the prior reporting period. We focused on microRNAs, a class of small, noncoding RNAs of ~21–23 nucleotides that regulate gene expression at the posttranscriptional level. These small RNAs mostly destabilize target mRNAs or suppress their translation by binding to complementary sequences in the 3' untranslated regions (3'UTRs). Our results obtained during this reporting period indicate that some microRNAs have very interesting functions in human neural progenitors, both in in vitro cell culture system and when transplanted into mouse brains. These new findings may have important implications for stem cell based therapies for neurodegenerative diseases or brain/spinal cord injuries.

Using human embryonic stem cells to treat radiation-induced stem cell loss: Benefits vs cancer risk

Funding Type: 
SEED Grant
Grant Number: 
RS1-00413
ICOC Funds Committed: 
$625 617
Disease Focus: 
Cancer
Neurological Disorders
Skeletal Muscle
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Statement of Benefit to California: 
Progress Report: 
  • We have undertaken an extensive series of studies to delineate the radiation response of human embryonic stem cells (hESCs) and human neural stem cells (hNSCs) both in vitro and in vivo. These studies are important because radiotherapy is a frontline treatment for primary and secondary (metastatic) brain tumors. While radiotherapy is quite beneficial, it is limited by the tolerance of normal tissue to radiation injury. At clinically relevant exposures, patients often develop variable degrees of cognitive dysfunction that manifest as impaired learning and memory, and that have pronounced adverse effects on quality of life. Thus, our studies have been designed to address this serious complication of cranial irradiation.
  • We have now found that transplanted human embryonic stem cells (hESCs) can rescue radiation-induced cognitive impairment in athymic rats, providing the first evidence that such cells can ameliorate radiation-induced normal-tissue damage in the brain. Four months following head-only irradiation and hESC transplantation, the stem cells were found to have migrated toward specific regions of the brain known to support the development of new brain cells throughout life. Cells migrating toward these specialized neural regions were also found to develop into new brain cells. Cognitive analyses of these animals revealed that the rats who had received stem cells performed better in a standard test of brain function which measures the rats’ reactions to novelty. The data suggests that transplanted hESCs can rescue radiation-induced deficits in learning and memory. Additional work is underway to determine whether the rats’ improved cognitive function was due to the functional integration of transplanted stem cells or whether these cells supported and helped repair the rats’ existing brain cells.
  • The application of stem cell therapies to reduce radiation-induced normal tissue damage is still in its infancy. Our finding that transplanted hESCs can rescue radiation-induced cognitive impairment is significant in this regard, and provides evidence that similar types of approaches hold promise for ameliorating normal-tissue damage throughout other target tissues after irradiation.
  • A comprehensive series of studies was undertaken to determine if/how stem cell transplantation could ameliorate the adverse effects of cranial irradiation, both at the cellular and cognitive levels. These studies are important since radiotherapy to the head remains the only tenable option for the control of primary and metastatic brain tumors. Unfortunately, a devastating side-effect of this treatment involves cognitive decline in ~50% of those patients surviving ≥ 18 months. Pediatric patients treated for brain tumors can lose up to 3 IQ points per year, making the use of irradiation particularly problematic for this patient class. Thus, the purpose of these studies was to determine whether cranial transplantation of stem cells could afford some relief from the cognitive declines typical in patients afflicted with brain tumors, and subjected to cranial radiotherapy. Human embryonic (hESCs) and neural (hNSCs) stem cells were implanted into the brain of rats following head only irradiation. At 1 and 4 months later, rats were tested for cognitive performance using a series of specialized tests designed to determine the extent of radiation injury and the extent that transplanted cells ameliorated any radiation-induced cognitive deficits. These cognitive tasks take advantage of the innate tendency of rats to explore novelty. Successful performance of this task has been shown to rely on intact spatial memory function, a brain function known to be adversely impacted by irradiation. Our data shows that irradiation elicits significant deficits in learning and spatial task recognition 1 and 4-months following irradiation. We have now demonstrated conclusively, and for the first time, that irradiated animals receiving targeted transplantation of hESCs or hNSCs 2-days after, show significant recovery of these radiation induced cognitive decrements. In sum, our data shows the capability of 2 stem cell types (hESC and hNSC) to improve radiation-induced cognitive dysfunction at 1 and 4 months post-grafting, and demonstrates that stem cell based therapies can be used to effectively to reduce a serious complication of cranial irradiation.

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