Neurological Disorders

Coding Dimension ID: 
303
Coding Dimension path name: 
Neurological Disorders
Funding Type: 
Basic Biology III
Grant Number: 
RB3-05229
Investigator: 
Type: 
PI
ICOC Funds Committed: 
$1 391 400
Disease Focus: 
Autism
Neurological Disorders
Pediatrics
Stem Cell Use: 
iPS Cell
oldStatus: 
Closed
Public Abstract: 

Autism spectrum disorders (ASD) are a group of neurodevelopmental diseases that occur in as many as 1 in 150 children in the United States. Three hallmarks of autism are dysfunctional communication, impaired social interaction, and restricted and repetitive interests and activities. Even though no single genetic defect has been ascribed to having a causative role in the majority of ASD cases, twin concordance studies and rare familial forms of the disease strongly support a genetic malfunction and a combinatorial effect of genetic risk factors may contribute to the variability in the symptoms. One major obstacle to ASD research is the difficulty in obtaining human neural tissue to model the disease in vitro. Mouse models of ASD are limited since only rare genetic mutations have been identified so far, and single mutations in those genes cannot fully reproduce the range of critical behaviors characteristic of ASD. Direct reprogramming of patient tissues to induced pluripotent stem (iPS) cells and derivation of forebrain neurons from them will provide much needed insight into the molecular mechanism of neuronal dysfunction in diverse individuals on the autism spectrum. The use of patient-derived stem cells to characterize cellular defects brings together two investigative approaches. One is the identification of common cellular and molecular mechanisms that are central to deficiencies across diverse populations of patients. The other is quantitative comparison of pathological features that address differences amongst diverse patients. Our major goal is to characterize the synaptic dysfunction using concrete, quantifiable parameters in human neurons that have specific mutations in key synaptic proteins. This approach will give us a handle into the molecular synaptic complexes that may also be altered in sporadic ASD cases and could help us develop drug strategies that can normalize synaptic function. Although several groups are interested in generating iPS cells from autistic patients, these efforts generally do not have genomic information on the patients, and the large diversity of mutations associated with autism could lead to large variation in synaptic phenotypes. By focusing on generating iPS cells from patients carrying mutations in a small number of critical synaptic proteins and characterizing the molecular components of this complex, we are likely to be in a strong position to identify novel molecular defects associated with autistic synapses. Relative biochemical comparisons of wildtype and mutant protein complexes could help us find ways to restore synaptic function in ASD.

Statement of Benefit to California: 

Many children in California are affected by autism spectrum disorders, which include monogenic syndromes such as Fragile X syndrome and Rett syndrome. However, the majority of cases are idiopathic and an interplay of multiple genetic risk factors is suspected. Since no current drug therapies exist for autism and an accurate diagnosis can only be made in early childhood by largely behavioral criteria, the cost of care and social burden for such a disorder is high, not to mention the devastation to the quality of life for the families of affected children. We would like to identify a core set of proteins found in synapses that are disrupted or dysregulated in autism by a biochemical approach. If we succeed in this effort, we may be able to identify novel biomarkers and molecular targets for specific patient profiles, and by cross-correlating the genetic background to specific behavioral traits in specific individuals, we may come up with molecular targets that are able to address particular symptoms, which should greatly aid in therapeutic regimens that complement existing behavioral therapies. Generating iPS neurons with known copy number variations associated with autism would be a major resource for other laboratories in California and in the field in general. The economic benefit to California is manifold, as many pharmaceutical and biotech companies in California will want to exploit these novel cell lines and the therapeutic targets identified through them in order to design better drugs for autism.

Progress Report: 
  • The main goal of this project is to establish a cell culture model human neuronal model of autism spectrum disorders (ASD) by generating induced pluripotent stem (iPS) cell lines from patients harboring mutations in genes associated with autism, differentiating them into forebrain neurons, and characterizing their synaptic defects at the cellular and molecular level. We have successfully obtained iPS cells from two autism spectrum disorders, tuberous sclerosis complex (TSC) and Rett syndrome (RTT). We obtained fibroblasts from patients with mutations in the TSC1 and TSC2 genes through the Coriell biorepository. We then reprogrammed them into several TSC patient-specific iPS cell lines. Furthermore, we have obtained male MECP2 mutant iPS cell lines from the lab of Dr. Alysson Muotri to study in parallel with the TSC lines.
  • We differentiated ASD iPS cell lines into neural progenitor cell (NPCs) and have been examining differences in protein levels and signaling pathways in these cells. Pathway analyses from MECP2 mutant NPCs suggest there may be a marked deficit in several major intracellular signaling pathways, and we are validating those deficits by biochemical analyses and genetic manipulations. Both TSC and RTT forebrain neurons show significant differences in synaptic regulation compared to their respective controls. Alterations in synaptic regulation are being assessed by gene expression analysis, staining for synaptic markers, and electrophysiology. We have made major progress toward realizing our goal of establishing novel iPS cell models for ASD. Furthermore, we obtained very interesting data that should help us elucidate the cell signaling deficits that lead to neuronal dysfunction.
  • We set out to establish an in vitro human neuronal model of autism spectrum disorders (ASD) by generating induced pluripotent stem (iPS) cell lines from patients harboring specific genetic mutations in syndromic forms of autism, such as Rett Syndrome (RTT) and Tuberous sclerosis (TS). We then differentiated them into neural progenitor cells (NPCs) and forebrain neurons, in order to compare their differentiation potential and to characterize mutation-associated deficits at the cellular and molecular level. Previously published data on cellular and animal models indicate that synaptic deficits are a major feature of the pathophysiology of RTT and TS.
  • We employed patient-derived induced pluripotent stem cells (iPSCs) from male RTT patients and gender-matched parental controls to probe for functional and molecular deficits in RTT. A similar approach was taken for TS.
  • As MECP2 is expressed in both the developing and mature central nervous system, we investigated deficits that may arise during early developmental stages (i.e. at the neural progenitor cell or NPC stage), which could then significantly affect neurodevelopmental processes such as neurogenesis and gliogenesis. By quantitative proteomics, we showed that the RTT cells have changes on the molecular level, at both the NPC and neuron stage, compared to their WT control, and that these changes may reflect some of the deficits in the developmental process. We report delays in maturation, such as misregulation of LIN28 at the NPC stage and subsequent deficits in glial differentiation.
  • Taken together, these results provide a framework for identifying novel early pathways that are perturbed in RTT, as well as potential therapeutics to minimize functional deficits. More generally, it will be of interest to see if these pathways and possible therapeutics may carry over to other related forms of neurodevelopmental disorders, in particular, idiopathic autism.
Funding Type: 
Basic Biology III
Grant Number: 
RB3-05232
Investigator: 
Name: 
Type: 
PI
ICOC Funds Committed: 
$1 341 064
Disease Focus: 
Neuropathy
Neurological Disorders
Stem Cell Use: 
iPS Cell
oldStatus: 
Active
Public Abstract: 

Induced pluripotent stem cells (iPSCs) have tremendous potential for patient-specific cell therapies, which bypasses immune rejection issues and ethical concerns for embryonic stem cells (ESCs). However, to fully harness the therapeutic potential of iPSCs, many fundamental issues of cell transplantation remain to be addressed, e.g., how iPSC-derived cells participate in tissue regeneration, which type of cells should be derived for specific therapy, and what kind of matrix is more effective for cell therapies. The goal of this project is to use iPSC-derived neural crest stem cells (NCSCs) and nerve regeneration as a model to address these fundamental issues of stem cell therapies. NCSCs are multipotent and can differentiate into cell types in all three germ layers (including neural, vascular, osteogenic and chondrogenic cells), which makes NCSC a valuable model to study stem cell differentiation and tissue regeneration. Peripheral nerve injuries and demyelinating diseases (e.g., multiple sclerosis, familial dysautonomia) affect millions of people. Stem cell therapy is a promising approach to cure these diseases, which will have broad impact on healthcare.

This project will advance our understanding of how extracellular microenvironment (native or engineered) regulates stem cell fate and behavior during tissue regeneration, and whether stem cells such as iPSC-NCSCs and differentiated cells such as iPSC-Schwann cells have different therapeutic effects. The results from this project will provide insights that will facilitate the translation of stem cell technologies into therapies for nerve injuries, demyelinating diseases and many other disorders that may be treated with iPSC-NCSCs.

Statement of Benefit to California: 

Induced pluripotent stem cells (iPSCs), especially iPSCs without the integration of reprogramming factors into the genome, are valuable to model disease and to generate autologous cells for therapies. Understanding the role and differentiation of iPSC-derived cells in tissue regeneration will facilitate the translation of stem cell technologies into clinical applications.

iPSC-derived neural crest stem cells (NCSCs) can differentiate into a variety of cell types, and hold promise for the therapies of diseases such as nerve injuries, demyelinating diseases, spina bifida, vascular diseases, osteoporosis and arthritis. The isolation and characterization of iPSC-NCSCs will provide a basis for their broad applications in tissue regeneration and disease modeling.

This project will use peripheral nerve regeneration as a model to address the fundamental issues of using iPSC-NCSCs for therapies. Peripheral nerve injuries (over 800,000 cases in the United States every year) are very common following traumatic injuries and major surgeries (e.g., removing tumor), which often require surgical repair. Stem cell therapies can accelerate nerve regeneration and avoid the degeneration of muscle and other tissues lack of innervation. Since iPSC-NCSCs can promote the myelination of axons, the therapies for nerve injuries could also be adopted to treat demyelinating diseases.

In many cases of stem cell therapies, matrix and scaffold materials are needed to enhance cell survival and achieve local delivery. The studies on appropriate matrix for stem cell delivery will provide a rational basis for designing and optimizing materials for stem cell therapies.

The fundamental issues addressed in this project, such as the differentiation and signaling of transplanted cells, the therapeutic effects of cells at the different stages of differentiation and the roles of delivery matrix/materials, will have implications for stem cell therapies in many other tissues.

Overall, the results from this project will advance our knowledge on stem cell differentiation and function during tissue regeneration, help us translate the knowledge into clinical applications, and benefit the health care in California and our society.

Progress Report: 
  • Induced pluripotent stem cells (iPSCs) have tremendous potential for regenerative medicine applications. Here we use peripheral nerve regeneration as a model to address the fundamental issues of using iPSCs and their derivatives for therapies. Specifically, we used integration-free iPSCs for our studies because this type of iPSCs has potential for clinical applications. We derived and characterized neural crest stem cells (NCSCs) from integration-free iPSCs, and demonstrated that these NCSCs can differentiate into a variety of cell types, including Schwann cells. We delivered NCSCs into nerve conduits to treat peripheral nerve injuries, and performed functional studies, electrophysiology analysis and histological analysis. Ongoing studies suggest that the transplantation of iPSC-NCSCs accelerate nerve regeneration. To investigate the interactions of transplanted stem cells with endogenous neural progenitors, we isolated and characterized endogenous progenitors from injured nerves, which will be used for mechanistic studies. In addition, we engineered the chemical components and the structure of nerve conduits, and developed and characterized hydrogels that could be used to deliver neurotrophic factors and minimize scar formation. The roles of neurotrophic factors, transplanted/endogenous stem cells and matrix for stem cell delivery will be investigated.
  • We use peripheral nerve regeneration as a model to address the critical issues of using induced pluripotent stem cells (iPSCs) and their derivatives for tissue regeneration. In the past year, we have made progress in all three Specific Aims. We generated 5 new integration-free IPSC lines by using episomal reprogramming. We also tested the methods of using biomaterials and chemical compounds to reprogram cells, in the presence or absence of transcriptional factors. We have derived and characterized additional neural crest stem cell (NCSC) lines from these new iPSC lines, and demonstrated that these NCSCs are multipotent in their differentiation potential. To investigate the mechanisms of how NCSCs enhanced the functional recovery of transected sciatic nerves, we examined the effects of paracrine signaling, cell differentiation and matrix stiffness. In vivo experiments showed that transplanted cells secreted neurotrophic factors to promote axon regeneration. In addition, NCSCs differentiated into Schwann cells to enhance myelination. The stiffness of extracellular matrix (ECM) indeed has effect on NCSC differentiation.
  • Here we use peripheral nerve regeneration as a model to address the critical issues of using induced pluripotent stem cells (iPSCs) and their derivatives for tissue regeneration. In the past year, we have made progress in all three Specific Aims, as detailed below. In Specific Aim 1, we generated 5 new integration-free IPSC lines by using episomal reprogramming. We also optimized the protocol to derive neural crest stem cells (NCSCs) from integration-free human iPSCs, and fully characterized the derived cells. Transplantation of selected NCSC lines significantly improved the functional recovery of peripheral nerve following injury. In addition, transplanted NCSCs differentiated into Schwann cells around regenerated axons. Nerve growth factor (NGF) appeared to be a major neurotrophic factor expressed by NCSCs, which was involved in nerve regeneration. In Specific Aim 2, we derived and characterized Schwann cells from NCSCs. Transplantation of NCSCs or Schwann cells showed that NCSC transplantation had better functional recovery than Schwann cell transplantation, suggesting that the differentiation stage of transplanted cells is critical for stem cell therapies. In Specific Aim 3, we demonstrated that the soft matrix worked much better than stiffer matrix for NCSC delivery and the functional recovery of damaged nerve. A new direction for this Specific Aim is a ground-breaking finding that matrix stiffness regulates the direct reprograming of fibroblasts into neurons, which has applications in generating neurons for drug discovery and disease modeling. Overall, our findings underline the importance of stem cell differentiation stage and biomaterials property in stem cell therapies, and will have broad impact on using stem cells for nerve regeneration and many other regenerative medicine applications.
Funding Type: 
Basic Biology III
Grant Number: 
RB3-02161
Investigator: 
Institution: 
Type: 
PI
ICOC Funds Committed: 
$1 268 868
Disease Focus: 
Spinal Muscular Atrophy
Neurological Disorders
Pediatrics
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 

Spinal muscular atrophy (SMA) is one of the most common autosomal recessive disorders that cause infant mortality. SMA is caused by loss of the Survival of Motor Neuron (SMN) protein, resulting in motor neuron (MN) degeneration in the spinal cord. Although SMN protein plays diverse roles in RNA metabolism and is expressed in all cells, it is unclear why a deficiency in SMN only causes MN degeneration. Since patient samples are rarely available, most knowledge in SMA is gained from animal model studies. While these studies have provided important information concerning the cause and mechanism of SMA, they are limited by complicated genetic manipulation. Results from different models are also not always consistent. These problems can be resolved if SMA patient’s MNs become readily available. Recent progress in the generation of induced pluripotent stem (iPS) cells from differentiated adult cells provides an opportunity to establish human cell-based models for neurodegenerative diseases. These cells, due to their self-renewal property, can provide an unlimited supply of the affected cell type for disease study in vitro. In this regard, SMA iPS cells may represent an ideal candidate for disease modeling as SMA is an early onset monogenic disease: the likelihood to generate disease-specific phenotypes is therefore higher than iPS cells derived from a late onset disease. In addition, the affected cell type, namely MNs, can readily be generated from iPS cells for the study. For these reasons, we established several SMA iPS cell lines from a type 1 patient and showed specific deficits in MNs derived from these iPS cells. Whether MNs derived from these iPS cell lines can recapitulate a whole spectrum of SMA pathology in animals and patients remains unclear. An answer to this question can ensure the suitability of using the iPS cell approach to study SMA pathogenesis in cell culture. We propose to examine cellular and functional deficits in MNs derived from these SMA iPS cells in Aim 1. The availability of these iPS cells also provides an opportunity to explore the mechanisms of selective MN degeneration in SMA. Dysregulation of some cellular genes has been implicated in SMA pathogenesis. We propose to use these iPS cell lines to address how one such gene is affected by SMN deficiency (Aim 2) and how a deficit in these genes leads to selective MN degeneration (Aim 3). Our study should provide valuable insights in the understanding of SMA pathogenesis and aid in exploring new molecular targets for drug intervention.

Statement of Benefit to California: 

Spinal muscular atrophy (SMA) is one of the most common autosomal recessive disorders in humans and the most common genetic cause of infant mortality. SMA is caused by loss of the Survival of Motor Neuron (SMN) protein, resulting in motor neuron (MN) degeneration in the spinal cord. SMA has a carrier frequency of approximately 1 in 35 and an incidence of 1 in 6000 in human population. In severe SMA cases, the disease onset initiates before 6 months of age and death within the first 2 years of life. Currently, there is no cure for SMA. Since MN samples from patients are rarely available, most knowledge in SMA is gained from animal model studies. While these studies have provided important information concerning the cause and mechanism of SMA, they are limited by complicated genetic manipulation. Results from different models are not always consistent either. Large-scale drug screening to treat SMA is also hampered by the lack of suitable cell lines for the study. These problems can potentially be resolved if SMA patient’s MNs become readily available. Our effort to derive induced pluripotent stem (iPS) cells from a SMA patient provides an unlimited supply of SMA cells to carry out studies to explore the disease mechanism in vitro. A better understanding in the disease mechanisms would benefit California by the identification of potential cellular targets for drug treatment. The knowledge gained from our study can also facilitate the use of these iPS cells as a platform for large-scale drug screening and validation. Our study should provide valuable insights in the understanding of SMA pathogenesis and aid in exploring new molecular targets for drug intervention.

Progress Report: 
  • During the past fiscal year, we have established in vitro coculture between motoneurons and myocytes. This coculture system will form the basis for the analysis of potential SMA pathogenesis induced by the motoneurons derived from SMA iPS cells. We have also started the analysis of potential cellular targets whose activity is affected by SMN deficiency.
  • Spinal muscular atrophy (SMA) is one of the most common autosomal recessive disorders that cause infant mortality. SMA is caused by loss of the Survival of Motor Neuron (SMN) protein, resulting in motor neuron (MN) degeneration in the spinal cord. Although SMN protein plays diverse roles in RNA metabolism and is expressed in all cells, it is unclear why a deficiency in SMN only causes MN degeneration. Since patient samples are rarely available, most knowledge in SMA is gained from animal model studies. While these studies have provided important insights of the cause and mechanism of SMA, they are limited by complicated genetic manipulation. Results from different models are also not always consistent. These problems can be addressed using induced pluripotent stem cells (iPSCs) derived from patient’s fibroblasts. These cells, due to their self-renewal capacity and their ability to differentiate into neuronal cells, can in theory provide an unlimited supply of the affected MNs for SMA study. We propose to examine cellular and functional deficits in MNs derived from these SMA iPS cells in Aim 1. To increase the yield of MN production, we have tested new strategies to differentiate SMA iPSCs into MNs. The improvement makes it feasible to isolate more pure populations of MNs for the study of SMA pathogenesis in vitro. The availability of these iPSC lines also provides an opportunity to explore the mechanisms of selective MN degeneration in SMA. Dysregulation of some cellular genes has been implicated in SMA pathogenesis. We continue to study the role of one particular cellular gene whose expression is reduced in SMA (Aim 2). We are taking approaches to reveal how SMN deficiency causes this change in gene expression. We are also taking a genomic approach to reveal all the affected genes and the signaling pathways in SMA MNs and understand how a deficit in these genes leads to selective MN degeneration (Aim 3). Our study should provide valuable insights in the understanding of SMA pathogenesis and aid in exploring new molecular targets for drug intervention.
  • Spinal muscular atrophy (SMA) is one of the most common genetic disorders that cause infant mortality. SMA is caused by loss of the Survival of Motor Neuron (SMN) protein, resulting in motor neuron degeneration in the spinal cord. Although SMN protein plays diverse roles in cells and is expressed in all cells, it is unclear why a deficiency in SMN only causes motoneuron degeneration. Since patient samples are rarely available, most knowledge in SMA is gained from animal model studies. While these studies have provided important information concerning the cause and mechanism of SMA, they are limited by complicated genetic manipulation. Results from different models are also not always consistent. These problems can be resolved if SMA patient’s motoneurons become readily available. The progress in the generation of stem cell lines from differentiated adult cells, termed induced pluripotent stem cells (iPSCs), provides an opportunity to establish human cell-based models for neurodegenerative diseases like SMA. We have previously established several SMA iPSC lines from a type 1 patient and showed specific deficits in motoneurons derived from these iPSCs. The availability of these iPSCs provides an opportunity to explore the mechanisms of selective motoneuron degeneration in SMA. We used motoneurons derived from SMA iPSCs to study potential defects in the formation of neuromuscular junctions. We also demonstrated a regulatory gene product affected by SMN deficiency. Several potential downstream targets of the regulatory gene product involved in neuron migration and synaptic transmission were identified. The roles of these genes in selective motoneuron degeneration observed in SMA are currently under study. One technical obstacle of using iPSC-derived motoneurons to study SMA in a dish is that motoneurons generally constitute only a fraction of the resulting cell population. The lack of capacity to isolate motoneurons hampers our study of SMA pathogenesis and the identification of potential downstream targets of SMN. We have employed a new approach, termed gene editing, to mark differentiated motoneurons with a fluorescence protein to facilitate their isolation by cell sorting. A proof-of-principle experiment was carried out and demonstrated the feasibility of this strategy. We are currently applying this strategy to mark motoneurons derived from SMA iPSCs.
Funding Type: 
Basic Biology III
Grant Number: 
RB3-02143
Investigator: 
Type: 
PI
ICOC Funds Committed: 
$1 355 063
Disease Focus: 
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Cell Line Generation: 
iPS Cell
Public Abstract: 

A major goal of stem cell research is to generate various functional human cell types that can be used to better understand how these cells work and to use them directly in therapies. There are currently no effective treatments, let alone a cure, for many neurological conditions. Two particular devastating neurological conditions, spinal cord injury and amyotrophic lateral sclerosis (ALS, or Lou Gehrig's disease) share a common element. That is, in both conditions, the corticospinal motor neurons that control skilled voluntary movement are severely damaged, leading to significant loss of motor control. There has been extensive research on spinal cord injury and ALS in recent years. In the field of spinal cord injury, much effort has been devoted to repairing the damaged nerve paths, but this has turned out to be extremely challenging. The work on ALS, on the other hand, has mostly focused on the spinal motor neurons (often referred to as the lower motor neurons in the context of ALS). Our proposed study focuses on the corticospinal motor neurons (or the upper motor neurons) and, more broadly, the subcerebral projection neurons. Taking clues from studies in mice, we aim to understand how the subcerebral projection neurons including the corticospinal motor neurons can be made from human embryonic stem cells. We will focus on the later steps in differentiation that are not well understood, which gave rise to different types of neurons in the cerebral cortex. To aid in this process, we have engineered a fluorescent reporter in human embryonic stem cells, which, when the stem cells are turned into corticospinal motor neurons and related subcerebral projection neurons, will light up – literally. We will probe the molecular control of this process and determine if corticospinal motor neurons made in a culture dish, when introduced back into an organism, can send projections to the spinal cord, as they would normally do during development. Most of our knowledge about the development of corticospinal motor neurons comes from studies with mouse models. As there are likely to be important differences between humans and mice, we will pay special attention to the similarities and differences between mouse and human corticospinal motor neurons. Knowledge gained from this study will pave the way to make better disease-models-in-a-dish for neurological conditions such as ALS and to develop therapies for ALS, spinal cord injury, traumatic brain injury, stroke and other neurological conditions when corticospinal motor neurons are damaged.

Statement of Benefit to California: 

Neurological conditions affect millions of Californians each year. Spinal cord injury is one particularly debilitating neurological condition. The disability, loss of earning power, and loss of personal freedom associated with spinal cord injury is devastating for the injured individual, and creates a financial burden of an estimated $400 million annually for the state of California. Research is the only solution as currently there is no cure for spinal cord injury. A major functional deficit for patients of spinal cord injury is the loss of motor control. Corticospinal motor neurons mediate skilled, voluntary movement in humans and damage to these neurons leads to severe disability. Our proposed study focuses on the understanding of how corticospinal motor neurons and, more broadly, subcerebral projection neurons can be made from human embryonic stem cells under culture conditions, and how they can be introduced back to central nervous system. Understanding this process will allow scientists to design ways to use these cells for transplantation therapies not only for spinal cord injury, but also for other neurological conditions such as amyotrophic lateral sclerosis (ALS, or Lou Gehrig's disease). Effective treatments promoting functional repair will significantly increase personal independence for people with spinal cord injury and decrease the financial burden for the State of California. More importantly, treatments that enhance functional recovery will improve the quality of life for those who are directly or indirectly affected by spinal cord injury, ALS and other neurological conditions.

Progress Report: 
  • A major goal of stem cell research is to generate various functional human cell types to promote repair or replacement in injury or disease. Our lab studies the repair of central nervous system after injury such as a spinal cord injury. We have been utilizing a fluorescent reporter line we developed with CIRM funding to enrich and characterize human corticospinal motor neurons, a neuronal population that is damaged or lost in spinal cord injury and amyotrophic lateral sclerosis (ALS, or Lou Gehrig's disease). These neurons control skilled voluntary movement in humans, the loss or damage of which leads to paralysis and disability. We have made significant progress in this funding period. We validated that our fluorescent reporter works as intended. We found that reporter gene expression represents cells of different developmental stages at different times of differentiation. We have done the first batches of transplantation studies to show that it is possible to use the reporter gene to track the cells and cellular processes in the host central nervous system. In addition, we have developed a separate reporter gene to universally mark all embryonic stem-derived cells, a tool that may be useful to other stem cell researchers. We are now ready to move to the next phase of the project: to characterize corticospinal motor neurons in more detail in vitro and in vivo. Knowledge gained from this study will pave the way to make better disease-models-in-a-dish for neurological conditions such as ALS and to develop therapies for ALS, spinal cord injury, traumatic brain injury, stroke and other neurological conditions when corticospinal motor neurons are damaged of lost.
  • A major goal of stem cell research is to generate various functional human cell types to promote repair or replacement in injury or disease. Our lab studies the repair of central nervous system after injury such as a spinal cord injury. We have been utilizing a fluorescent reporter line we developed with CIRM funding to derive and characterize human corticospinal motor neurons, a neuronal population that is damaged or lost in spinal cord injury and amyotrophic lateral sclerosis (ALS, or Lou Gehrig's disease). These neurons are of paramount importance to skilled voluntary movement in humans, the loss or damage of which leads to paralysis and disability. The goal for making a reporter line is that whenever the cells light up (literally), we will know what they have become the type of cells that we would wish to get. Following last year’s initial progress, we have made significant progress in this funding period. We found that our fluorescent reporter is useful in following the desired cell types throughout cell growth in culture dishes or after we introduce these cells into animal models by transplantation. We have performed experiments to validate the identity and usefulness of these cells. In culture, these cells exhibit the desired signature gene expression pattern, electrophysiological properties and morphologies as well. We will continue to improve our culture condition to maximize efficiency and purity. Meanwhile, we have transplanted these cells into the mouse brain to study them in the complex central nervous system because many of the properties cannot be studied in cell culture such as the connection of nerve cells to other brain area or spinal cord. We were excited to find that these cells, once transplanted, can survive, integrate into the mouse central nervous system, and send out long neuronal processes characteristic of endogenous nerve cells. Some of the projections appear to take the path of the projections of the corticospinal motor neurons, indicating that our approach will likely succeed. Thanks to CIRM’s support, we will continue to investigate the various parameters to improve our transplantation studies. Knowledge gained from this study will pave the way to make better disease-models-in-a-dish for neurological conditions such as ALS and to develop therapies for ALS, spinal cord injury, traumatic brain injury, stroke and other neurological conditions when corticospinal motor neurons are damaged of lost.
  • A major goal of stem cell research is to generate various functional human cell types from stem cells both for developing cell transplantation therapies and for better understanding human biology. Our lab studies the repair of central nervous system after injury and in particular spinal cord injury. To complement our studies of the molecular control of axon regeneration using animal models of spinal cord injury, we have been developing ways to derive human corticospinal motor neurons from human embryonic stem cells through this CIRM funded project. These neurons are of paramount importance to skilled voluntary movement in humans, loss or damage of which leads to paralysis and disability in patients of spinal cord injury and amyotrophic lateral sclerosis (ALS, or Lou Gehrig's disease). We took advantage of a reporter line we developed with a prior CIRM SEED grant to generate human corticospinal motor neurons. This reporter line carries a fluorescent reporter gene under the control of an endogenous gene encoding a molecular marker and determinant of corticospinal motor neurons, Fezf2. The idea was that whenever the cells carrying the reporter gene lights up – literally, we would know the cells are expressing Fezf2. Using this approach, we have learned quite a bit about human cells that express Fezf2. First, there are a large population of human neural stem cells that express Fezf2 early in neural differentiation, which is likely mirrored in human development. Fezf2 positive neural stem cells can become Fezf2 positive neurons, but they can also become Fezf2 negative neurons. On the contrary, Fezf2 negative stem/progenitor cells do not become Fezf2 positive neurons. During neural differentiation in culture starting from human embryonic stem cells, Fezf2 expression is dynamic. Earlier Fezf2-expressing neural stem cells have different properties from the late Fezf2-expressing neural stem cells in that they have different capabilities to turning into differential neuronal types. Particular in this last year of funding, we conducted in-depth characterization of the molecular signature of Fezf2 positive and negative neural stem/progenitor cells, as well as neurons that had been derived from these progenitors, at different times in differentiation. Hierarchical cluster analysis not only provided new insights on the different cell populations in the differentiation culture over time but also on the different molecular markers based on studies in mice. We have also extended our in vivo transplantation studies to determine how well these neural progenitor cells may survive and integrate into the mouse nervous system. The data indicate that neural progenitors that express the fluorescence reporter can survive, integrate into the host nervous system and send out extensive axonal trajectories. Some axons grew along the appropriate paths expected for corticospinal and related subcerebral projection neurons while others appear to wonder off the course. These data indicate that one challenge in future research will be to elucidate mechanisms of control the re-connection of transplanted stem cell derivatives with appropriate host targets when cell transplantation therapies are used to replace lost or damaged neurons in disease or injury.
Funding Type: 
Basic Biology III
Grant Number: 
RB3-05219
Investigator: 
ICOC Funds Committed: 
$1 372 660
Disease Focus: 
Infectious Disease
Neurological Disorders
Pediatrics
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 

Human cytomegalovirus (HCMV) is the major cause of birth defects, almost all of which are neuronal in origin. Approximately 1% of newborns are infected, and of the 13% that are symptomatic at birth, 50% will have severe permanent hearing deficits, vision loss, motor impairment, and mental retardation. At least 14% of asymptomatic infants also will later show disabilities. Much of this effect is likely caused by HCMV affecting neural development in the fetus.

Embryonic stem cells are an excellent source of human progenitors, which are cells that can turn into mature neurons i.e. neural differentiation. We know from published cell culture studies that HCMV affects neural progenitor cells during neural differentiation, but it is unclear as to what are the underlying molecular mechanisms for its effect. A major goal of our research is to understand at a high-resolution how HCMV controls the way neural progenitors become proper neurons. Elucidation of the genes that are affected will serve as a basis for therapeutic strategies to ameliorate the effects of HCMV infection in newborns.

The significance of our studies also extends to the serious problem of HCMV infection in immunocompromised individuals, with recipients of allogeneic transplants having a high risk of severe disease and allograft rejection. This potential problem in stem cell therapy has received little attention thus far. The proposed use of stem cell transplantation in treating neuronal injury and neurodegenerative diseases, as well as transplantation of other organ-specific precursors, makes it imperative to understand how disseminated HCMV infection in immunosuppressed recipients will affect the function and differentiation of the cells.

Statement of Benefit to California: 

Human cytomegalovirus (HCMV) is the major viral cause of birth defects. In 2009, there were 526,774 births in California, resulting in congenital HCMV infection in approximately 5,200 newborns, with at least 800 infants expected to have long-lasting disabilities. Congenital cytomegalovirus infection is the most common nongenetic congenital cause of deafness. In contrast, before the development of the rubella vaccine, less than 70 infants per year in the entire US were reported to have congenital rubella syndrome, also associated with deafness. The burden to families and the economic costs to society of congenital cytomegalovirus infection are immense, and there is no vaccine available. Our proposed research serves to form the basis of future therapies to ameliorate, or even reduce this medical burden.

The significance of our studies also extends to the serious problem of HCMV infection in immunocompromised individuals who receive transplants of organs and stem cells from other individuals. Infection in these transplant recipients often results in severe disease and rejection of the transplant. The California Institute for Regenerative Medicine has made a major commitment to provide funding to move stem cell-based therapies to clinical trials. The goal of using stem cell transplantation to treat neuronal injury and neurodegenerative diseases, as well as transplantation of other organ-specific precursors, makes it imperative to understand how disseminated HCMV infection in immunosuppressed recipients will affect the function and differentiation of the cells.

Our research will provide the knowledge base to understand the genes that are changed during HCMV infection of human neural progenitors and neurons. It will also provide a foundation for studies of how other viruses will affect human neurons, and likely, other cell-types. Intellectual property from this work will feed into opportunities for antiviral strategies and increased jobs in biotech for Californians.

Progress Report: 
  • Congenital human cytomegalovirus (HCMV) infection is a major cause of central nervous system structural anomalies and sensory impairments in the newborn. It is likely that the timing of infection as well as the range of susceptible cells at the time of infection will affect the severity of the disease. A major goal of our research is to understand at a high-resolution the effects of HCMV infection on the neural lineage specification and maturation of stem and progenitor cells. Elucidation of the genes and cellular processes that are affected will serve as a basis for therapeutic strategies to ameliorate the effects of HCMV infection in newborns. The significance of our studies also extends to the serious problem of HCMV infection in immunocompromised individuals, with recipients of allogeneic transplants having a high risk of severe disease and allograft rejection. This potential problem in stem cell therapy has received little attention thus far. The proposed use of stem cell transplantation in treating neuronal injury and neurodegenerative diseases, as well as transplantation of other organ-specific precursors, makes it imperative to understand how disseminated HCMV infection in immunosuppressed recipients will affect the function and differentiation of the cells.
  • This past year, we have made significant progress in accomplishing the goals of this project. We used human embryonic stem cells-derived primitive pre-rosette neural stem cells (pNSCs) maintained in chemically defined conditions to study host-HCMV interactions in early neural development. Infection of pNSCs with HCMV was largely inefficient and non-progressive. At low multiplicity of infection (MOI), we observed severe defects with regards to the proportion of cells expressing the major immediate-early proteins (IE) despite an optimal viral entry, thus indicating the existence of a blockade to specific pre-IE events. IE expression, even at high MOI, was found to be restricted to a subset of cells negative for the expression of the forebrain marker FORSE-1. Treatment of pNSCs with the caudalizing agent retinoic acid rescued IE expression, suggesting that the hindbrain microenvironment might be more permissive for the infection. Transactivation of the viral early genes was found to be severely debilitated and expression of the late genes was barely detectable even at high MOI. Differentiation of pNSCs into primitive neural progenitor cells (pNPCs) restored IE expression but not the transactivation of early and late genes. Increasing the number of viral particles bypassed this barrier to early gene expression and thus permitted expression of the late genes in pNPCs. Consequently, viral spread was only observed at high MOI but was largely restricted to one cycle of replication as secondarily infected cells failed to efficiently express early genes. Of note, virions produced in pNPCs and pNSCs were exclusively cell-associated. Finally, we found that viral genomes could persist in pNSCs culture up to a month after infection despite the absence of detectable IE expression by immunofluorescence. Clonogenic expansion of infected pNSCs revealed that the presence of viral DNA and IE proteins were insufficient to block host cell division therefore allowing the survival of viral genomes via cellular division rather than viral replication. These results highlight the complex array of hurdles that HCMV must overcome in order to infect primitive neural stem cells and suggest that these cells might act as a reservoir for the virus. To study in greater depth the molecular basis of the interaction of HCMV with cell of the neural lineage, we also have initiated high-throughput genomics approaches to analyze HCMV microRNAs, alterations in cellular microRNA and gene expression profiles, and global defects in host alternative splicing in infected and uninfected pNSC-derived NPCs. Interestingly, although there are many changes in host cell gene expression in the infected cells, there was only a small overlap with the set of changes we had found in infected human fibroblasts. This highlights the importance of performing these studies in the relevant targets of the virus in the developing fetus.
  • We expect that the results of these studies will provide an unprecedented resolution of the effects on neurogenesis when HCMV infects a newborn, serve as a foundation for future therapeutic efforts in preventing the birth defects due to HCMV, and provide insight into the serious potential problem of disseminated HCMV in immunosuppressed individuals receiving transplanted allogeneic stem cells.
  • Congenital human cytomegalovirus (HCMV) infection is a major cause of central nervous system structural anomalies and sensory impairments in the newborn. A major goal of our research is to understand at a high-resolution the effects of HCMV infection on the neural lineage specification and maturation of stem and progenitor cells. Elucidation of the genes and cellular processes that are affected will serve as a basis for therapeutic strategies to ameliorate the effects of HCMV infection in newborns. The significance of our studies also extends to the serious problem of HCMV infection in immunocompromised individuals, with recipients of allogeneic transplants having a high risk of severe disease and allograft rejection. This potential problem in stem cell therapy has received little attention thus far. The proposed use of stem cell transplantation in treating neuronal injury and neurodegenerative diseases, as well as transplantation of other organ-specific precursors, makes it imperative to understand how disseminated HCMV infection in immunosuppressed recipients will affect the function and differentiation of the cells.
  • This past year, we have made significant progress in accomplishing the goals of this project. We used human embryonic stem cell (hESC)-derived primitive pre-rosette neural stem cells (pNSCs) to study host-HCMV interactions in early neural development and found with several different lines of hESC-derived pNSCs that HCMV infection is inefficient and non-progressive. Differentiation of pNSCs into primitive neural progenitor cells (pNPCs) restored some viral early gene expression but not the transactivation of late genes. Impaired viral gene expression in pNSCs was not a result of inefficient viral entry or nuclear import of viral DNA but correlated with deficient nuclear import of the virion-associated protein UL82, which is believed to play a role in removing barriers to viral RNA synthesis. Additionally, we found that viral genomes could persist in pNSCs culture up to a month after infection despite the absence of detectable viral lytic gene expression, although we could also detect expression of viral latency-associated genes, suggesting that the virus becomes latent in pNSCs.
  • To study in greater depth the molecular basis of the interaction of HCMV with cells of the neural lineage, we have continued high-throughput genomics approaches to analyze HCMV microRNAs, alterations in cellular microRNA and gene expression profiles, and global defects in host alternative splicing in infected and uninfected pNSC-derived NPCs. We found that in infected NPCs, there was specific downregulation of transcripts related to neuron differentiation. These findings demonstrate the capacity of HCMV infection to alter the neural identities of key precursor cells in the developing nervous system. We also analyzed our infected NPC RNA-seq database for differences in host mRNA polyadenylation patterns and found that over a hundred transcripts were significantly altered in terms of their 3' end cleavage site preference, with the majority of these events resulting in shortened 3' UTRs.
  • Our finding that HCMV induces major changes in the transcriptome of NPCs, particularly at the level of neural genes, suggested that the virus might affect these cells functionally. To directly evaluate this, we differentiated pNSCs into midbrain dopaminergic (mDA) neurons and infected these cells at different times of the differentiation process. Seeding pNSCs in differentiation medium for 6 weeks yields a high frequency of mature neurons with long axonal projections. Infection of pNSCs at the start of differentiation greatly reduces generation of beta III-tubulin+ neurons 4 weeks later, and prevents differentiation to mature MAP2+ neurons. Infection after 1 week of differentiation also reduces the number of beta III-tubulin+ neurons and results in massive cell death. Infection at 2 weeks after differentiation start does not reduce the number of βIII-tubulin+ or MAP2+ neurons, but the cells display major anomalies. Since neurons are highly sensitive to oxidative stresses and HCMV infection increases the production of reactive oxygen species in fibroblasts, we investigated whether the same effect occurred in neuronal cultures. When pNSCs were infected at week 4 after differentiation, high levels of ROS were detected. These results suggest that the complex effects of HCMV infection at various stages of neural cell differentiation on both cell survival and maturation may account for the broad range of birth defects.
  • We expect that the results of these studies will provide an unprecedented resolution of the effects on neurogenesis when HCMV infects a newborn, serve as a foundation for future therapeutic efforts in preventing the birth defects due to HCMV, and provide insight into the serious potential problem of disseminated HCMV in immunosuppressed individuals receiving transplanted allogeneic stem cells.
  • Congenital and childhood sensorineural hearing loss (SNHL) is a multifactorial disease that severely impacts quality of life. The single most important etiology of congenital SNHL is prenatal human cytomegalovirus (HCMV) infection, which accounts for 20-30% of all deafness in infants and children. Approximately 1 in 150 children is born with congenital HCMV, and 1 in 5 of these children will be born with or will develop permanent neural disabilities, the most common of which is SNHL. SNHL can be either bilateral or unilateral, and the severity of the hearing loss and its progression varies widely. Although the association of congenital HCMV infection and SNHL has been recognized for 50 years, how infection induces the hearing loss is unknown. Since the target of HCMV is cells of the neural lineage, a major goal of our research is to understand at high-resolution the effects of HCMV infection on neural lineage specification and maturation of stem and progenitor cells. Elucidation of the genes and cellular processes that are affected will serve as a basis for therapeutic strategies to ameliorate the effects of HCMV infection in newborns. The significance of our studies also extends to the problem of HCMV infection in immunocompromised individuals, with recipients of allogeneic transplants having a high risk of severe disease and allograft rejection. The proposed use of stem cell transplantation in treating neuronal injury and neurodegenerative diseases, as well as transplantation of other organ-specific precursors, makes it imperative to understand how disseminated HCMV infection in immunosuppressed recipients will affect the function and differentiation of the cells.
  • This past year, we made significant progress in accomplishing the goals of this project. To study in greater depth the molecular basis of the interaction of HCMV with cells of the neural lineage, we continued high-throughput genomics approaches to analyze HCMV microRNAs, alterations in cellular microRNA and gene expression profiles, and global defects in host alternative splicing in infected and uninfected pNSC-derived NPCs. We also analyzed our infected NPC RNA-seq database for differences in host mRNA polyadenylation patterns and found that over a hundred transcripts were significantly altered in terms of their 3' end cleavage site preference, with the majority of these events resulting in shortened 3' UTRs.
  • One of our most striking findings was that there was strongly enhanced expression of three miRNAs that play a critical role in auditory development. Other genes involved in cochlear development were also dysregulated by infection. These results implicate a novel molecular mechanism of damage to the developing inner ear of congenitally infected children, based on altered developmental gene regulation. These findings coupled with our success in inducing differentiation of human ES into otic progenitors that can be further differentiated to hair cell-like cells and immature auditory neurons have provided a strong foundation to pursue in depth HCMV infection of auditory progenitor cells, with the goal of determining how infection compromises the gene expression and function of human ES-derived inner ear cells and to use this information to develop new strategies for the treatment and prevention of hearing loss in congenitally infected children.
  • Regrettably, we are not able to continue these highly important studies, as this CIRM grant ended and a new proposal to CIRM was not funded. This was very disappointing because there is a gap in the portfolio of grants awarded by CIRM, which is a lack of attention to important problems relating to Child Health. One of the most important areas in stem cells that have been neglected is congenital sensorineural hearing loss. Only 3 grants relating to hearing loss have ever been awarded by CIRM. Yet the incidence of just congenital hearing loss (not including hearing loss from other causes or associated with aging) is 400 in 100,000 newborns. Moreover, the disability will last for life, causing a significant burden to the patient and family and a high economic cost to society. There is no drug or vaccine to prevent congenital hearing loss due to CMV, and thus it is essential develop new therapeutic strategies, including stem cells, gene targeting, and drug discovery. Unfortunately, there is no accepted mechanism by which HCMV infection leads to hearing loss, and a lack of public awareness regarding the serious medical problems resulting from congenital HCMV Infection has made it difficult to garner support for studies to identify an etiology.
  • We appreciate the support CIRM has provided and hope that in the future there will be sufficient public awareness of the devastating effects of congenital HCMV infection to bring pressure upon funding agencies to recognize and support this important research.
Funding Type: 
Basic Biology III
Grant Number: 
RB3-05009
Investigator: 
Name: 
Type: 
PI
ICOC Funds Committed: 
$1 372 660
Disease Focus: 
Amyotrophic Lateral Sclerosis
Neurological Disorders
Dementia
Stem Cell Use: 
Embryonic Stem Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 

Human embryonic and patient-specific induced pluripotent stem cells have the remarkable capacity to differentiate into many cell-types, including neurons, thus enabling the modeling of human neurological diseases in vitro, and permit the screening of molecules to correct diseases. Maintaining the pluripotent state of the stem cell, directing the stem cell towards a neuronal lineage, keeping the neuronal progenitor and stem cells alive - these are all maintained by thousands of different proteins in the cell at these different "stages". Thus the levels and types of proteins are highly controlled by gene regulatory mechanisms.

Genes produce pre-messenger RNA (mRNA) transcripts in the nucleus, which undergo a process of refinement called splicing, whereby long (1,000-100,000 bases) stretches of nucleotides are excised, and much shorter pieces (150 bases) are ligated together to form mature messenger RNA to eventually make proteins in the cytoplasm. Strikingly, some pieces of RNA are used in a particular cell-type, but not another, in a process called "alternative splicing". This is the most prevalent form of generating transcriptome diversity in the human genome, and is important for pushing cells from one state to another i.e. stem cells to neurons, maintaining a cell state i.e. keeping a stem cell pluripotent, or a neuron alive and functioning. Alternative splicing is highly controlled by the recognition of even smaller stretches (6-10 bases) of RNA binding sites) by proteins that bind directly to RNA called splicing factors.

The goal of the proposed research is to produce a regulatory map of where these splicing factors bind within pre-mRNAs across the entire human genome with unprecedented resolution using a high-throughput biochemical strategy. Furthermore, using advanced genomic technologies, we will deduce what happens to splicing when these factors do not bind to their binding sites. Finally, using molecular and imaging methods, we will analyze what happens to survival of stem and neuronal cells when these factors are depleted or over-expressed, and if stem cells are induced to make neurons if the levels of these factors are altered. Completion of the proposed research is expected to transform our understanding of the regulatory mechanisms underlying transcriptome complexity important for neurological disease modeling, especially human neurodegeneration, and stem cell biology. In turn, this will facilitate more accurate comparisons of diseased states of neurons from stem-cell models of Amyotrophic Lateral Sclerosis (ALS), Myotonic Dystropy, Spinal Muscular Atrophy (SMA), Parkinson’s and Alzheimer’s to identify mis-spliced genes and the splicing factors responsible for therapeutic intervention.

Statement of Benefit to California: 

Our research provides the foundation for decoding the mechanisms that control the transcriptome complexity of stem cells and neurons derived from stem cells. Our work has direct application in the design of novel strategies to understand the impact of splicing factor misregulation, or mutations within the binding sites for these splicing factors in neurological diseases that heavily impact Californians, such as Amyotrophic Lateral Sclerosis (ALS), Myotonic Dystropy, Spinal Muscular Atrophy (SMA), Parkinson’s and Alzheimer’s. Our research has and will continue to serve as a basis for understanding deviations from "normal" stem and neuronal cells, enabling us to make inroards to understanding neurological disease modeling using neurons differentiated from reprogammed patient-specific lines. Such disease modeling will have great potential for California health care patients, pharmaceutical and biotechnology industries in terms of improved human models for drug discovery and toxicology testing. Our improved knowledge base will support our efforts as well as other Californian researchers to study stem cell models of neurological disease and regenerative medicine, and for the design of new diagnostics and treatments, thereby maintaining California's position as a leader in clinical and biomedical research.

Progress Report: 
  • The overwhelming majority of human genes undergo extensive alternative splicing, but save for several dozens of these regulated splicing events, it is not known which proteins are responsible for controlling these key splicing decisions. Furthermore, mutations in several of these proteins, known as splicing factors, have recently been shown to be causative of neurodegeneration. In this proposal we aim to understand the importance of splicing factor regulation of alternative splicing in controlling pluripotency, fate decision towards the neural lineage and neuronal survival. In our recent publication in Cell Reports, Huelga et al demonstrated that the ubiquitously expressed heterogeneous nuclear ribonucleoproteins (hnRNPs) commonly cooperate and antagonize one another to regulate alternative splicing in a somatic human cell-line. In year one of this grant, we have interrogated several key members of these hnRNP proteins in human neural progenitor and differentiated neurons from embryonic stem cells and induced pluripotent stem cells.
  • The overwhelming majority of human genes undergo extensive alternative splicing, but save for several dozens of these regulated splicing events, it is not known which proteins are responsible for controlling these key splicing decisions. Furthermore, mutations in several of these proteins, known as splicing factors, have recently been shown to be causative of neurodegeneration. In this proposal we aim to understand the importance of splicing factor regulation of alternative splicing in controlling pluripotency, fate decision towards the neural lineage and neuronal survival. In years one and two, we have made significant progress in analyzing the functions of three hnRNP proteins, namely TAF15, EWSR1 and hnRNP A2/B1. All three have been associated with neurological diseases, in particular ALS and FTD. We have also made progress in generating and successfully validating reagents to deplete the larger class of RNA binding proteins in human neural progenitors. Finally, we are making slower but steady progress in depleting RBFOX proteins in human neurons.
  • The overwhelming majority of human genes undergo extensive alternative splicing, but save for several dozens of these regulated splicing events, it is not known which proteins are responsible for controlling these key splicing decisions. Furthermore, mutations in several of these proteins, known as splicing factors, have recently been shown to be causative of neurodegeneration. In this proposal we aim to understand the importance of splicing factor regulation of alternative splicing in controlling pluripotency, fate decision towards the neural lineage and neuronal survival. In year 3 of the proposal, we have completed a deeper analysis of hnRNP A2/B1 which we are preparing for manuscript submission. HnRNP A2/B1 is implicated in neurological diseases such as ALS and FTD.
Funding Type: 
Tools and Technologies II
Grant Number: 
RT2-02061
Investigator: 
Institution: 
Type: 
PI
ICOC Funds Committed: 
$1 906 494
Disease Focus: 
Autism
Neurological Disorders
Rett's Syndrome
Pediatrics
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
Cell Line Generation: 
Embryonic Stem Cell
iPS Cell
oldStatus: 
Active
Public Abstract: 

There is a group of brain diseases that are caused by functional abnormalities. The brains of patients afflicted with these diseases which include autism spectrum disorders, schizophrenia, depression, and mania and other psychiatric diseases have a normal appearance and show no structural changes. Neurons, the cellular units of the brain, function by making connections (or synapses) with each other and exchanging information in form of electric activity. Thus, it is believed that in those diseases many of these connections are not working properly. However, using current technology, there is no way to investigate individual neuronal synapses in the human brain. This is because it is not ethical to biopsy the brain of a living person if it is not for the direct benefit to the patient. Therefore, scientists cannot study synaptic function in psychiatric diseases. Because of the limited knowledge about the functional consequences in the affected brains, there is no cure for these diseases and the few existing therapies are often associated with severe side effects and cannot restore the normal function of the brain. Therefore, it is of great importance to better study the disease processes. A better knowledge on what the defects are on the cellular level will enable us then in a second step to test existing drugs and measure its effect or screen for new therapeutic drugs that can improve the process and hopefully also the disease symptoms.

This proposal aims to develop a technology to overcome this limitation and ultimately provide neurons directly derived from affected patients. This will uniquely allow the study functional neuronal aspects in the patients' own neurons without the need to extract neurons from the brain. Our proposal has two steps, that we want to undertake in parallel with mouse and human cells. First, we want to find ways to optimally generate neurons from skin fibroblasts. Naturally, these artificial neurons will have to exhibit all functional properties that the neurons from the brain have. This includes their ability to form functional connections with each other that serve to exchange information between two cells. In the second step, we will generate such neuronal cells from a genetic form of a psychiatric disease and evaluate whether these cultured neuronal cells indeed exhibit changes in their functional behavior such as the formation of fewer connections or a decreased probability to activate a connection and thus limit the disease cells to communicate with other cells.

Statement of Benefit to California: 

Our proposed research is to develop a cellular tool which will enable the research community to study human brain diseases that are caused by improperly functioning connections between brain cells rather than structural abnormalities of the brain such as degeneration of neurons or developmental abnormalities. These diseases, which are typically classified as psychiatric diseases, include schizophrenia, bipolar diseases (depression, mania) autism spectrum disorders, and others. There are many people in California and world-wide that suffer from these mentally debilitating diseases. Therefore, there is a great need to develop therapies for these diseases. However, currently drug development is largely restricted to animal models and very often drug candidates that are successful in e.g. rodent animals can not be applied to human. It would thus be much better to possess a model that reflects the human disease much closer, ideally using human cells.

We have experimental evidence that we can develop such a model. In particular, we will convert skin cells from patients suffering from psychiatric diseases into stem cells that are "pluripotent", which means they can differentiate into all cell types of the body including neurons. We want to explore whether these patient-derived neurons still contain the disease features that the neurons have in the brain. If we could indeed capture the disease in these cells, our technology would have a major impact on future work in this area. We believe that this approach could be applied to many neurological diseases including neurodegenerative diseases.

Our technology would not only provide a unique experimental basis to begin to understand how these diseases work, but it would allow to then interfere with the identified cellular abnormalities which would secondarily result in the development of new drugs that can counteract the diseases and would hopefully also work for the patients themselves.

Therefore, all those Californians that suffer from one of the above mentioned diseases will benefit from our research project, if it is successful.

Progress Report: 
  • During this first year of our project we have largely focused on testing various methods to directly differentiate human ES cells into neurons. As described in more detail below we were very successful and developed ways to differentiate human stem cell lines into neuronal cells with high purity and good maturation characteristics. For example, we can analyze the electrical currents in these cells which are important functional properties of neurons and we observed that these cells indeed behave just like neurons in the brain. More specifically, the cells were able to generate action potentials which are necessary in the brain to transmit information from one neuron to the other as well as form synapses, which are the structures that connect the different neurons with each other.Because the differentiation of different stem cell lines needs to be robust and reproducible we spent a lot of time optimizing the protocol and tested many different stem cell lines. This revealed a high degree of reproducibility and purity of the stem cell-derived nerve cells and we have tested human embryonic stem cells (i.e. stem cells derived from the embryo) as well as induced pluripotent (iPS) cells (i.e. stem cells reprogrammed from human skin cells). Reassuringly, the same method works in all these cell lines with very similar dynamics and functional properties of the nerve cells.
  • We also have made significant advances to convert human fibroblasts into nerve cells directly and without going through an intermediate iPS cell state. We have identified a neuronal factor called NeuroD1 as critical co-factor that in addition to the three factors that we had identified earlier to work in mouse. Those 4 factors together now allowed the generation of fully functional so called "induced neuronal" (iN) cells from both fetal and early postnatal human foreskin fibroblasts. We have also tested a number of small molecules to attempt to increase the reprogramming efficiency.
  • Finally, we have generated some essential components that will allow us to study Rett Syndrome using these technologies that are being developed at the same time (described above). In the last year we have generated several lines of iPS cells from Rett Syndrome patients and are in the process of fully characterizing them. We plan to soon apply our optimized differentiation protocol to these cells as well as control cells to look for any possible disease trait that distinguishes cells from patients and controls.
  • The generation of human pluripotent stem cells from discarded embryos (embryonic stem cells or ES cells) and directly from skin cells through reprogramming (induced pluripotent stem cells or iPS cells) holds great promise, and may revolutionize the study of human diseases. In particular, the principle possibility to turn these stem cells into fully functional neurons would provide a novel cell platform that provides excellent experimental access to study human neurons that are derived from healthy controls or diseased individuals. However, the goal to actually derive mature neurons from these stem cell populations has not been accomplished yet. While there have been many ways developed how to instruct these stem cells into specific neurons and even neuronal subtypes, these differentiation protocols take many months to complete and are laborious and most importantly, do not yield fully mature neurons. We have recently discovered a way to convert human newborn skin cells directly into functional neurons but the efficiencies were low and also most of these induced neuronal cells were still immature.
  • The goal of this project is to improve these methods and develop tools that actually allow the generation of mature human neurons. We proposed to approach this problem in two different and complementary ways: (1) We proposed to apply the methods that we used to convert human skin cells into neurons to both stem cell populations (ES and iPS cells). (2) We proposed to further improve the direct conversion of skin cells into induced neuronal cells by systematic evaluation of culture conditions and small molecule modulators alone and in combination. Finally, we then proposed to apply our newly derived tools to study one common autism-related childhood disease, called Rett Syndrome, which affects exclusively girls, which undergo normal development and brain maturation but after a period of months to years present with developmental retardation and in some cases severe behavioral and social deficiencies.
  • We are very happy to be able to report that we have made great progress towards the development of our proposed tools and are now beginning already to apply them to the study of Rett Syndrome as proposed. In particular, we have perfected the application of the technique to convert human stem cells into fully functional induced neuronal cells. With this approach we are ready, to investigate the detailed electric connectivity of neural circuits in induced neuronal cells in disease and non-disease condition.
  • We have also made good progress with the second approach and showed that it is possible to improve the conversion efficiencies significantly by using small molecule inhibitors and changing the environmental oxygen concentration. We are currently exploring whether these efficiencies are high enough to enable disease modeling while we continue to optimize the culture methods.
  • We have generated a new tool to study brain function on the cellular level. The differentiation of pluripotent stem cells like embryonic or induced pluripotent stem cells into functional nerve cells (neurons) remains a challenge. We here demonstrated that specific factors that normally regulate brain development can be exploited to "fast forward" the differentiation of human stem cells into neurons. Since these neurons are induced using exogenous factors we call these cells "induced neuronal cells" or in brief "iN" cells. Stem cell-derived iN cells show all principal functional properties of neurons, ie, they can communicate with each other (form synapses) and use electrical signals to convey information (ability to generate action potentials). Within just 2-3 weeks fully functional neuronal networks can be established using these human neurons.
  • We next demonstrated that different factor combinations yields different kind of neurons allowing us to reconstruct complex cell mixtures resembling those of normal neuronal cultures.
  • We also show that iN cells are useful proxies that report disease traits on the cellular level. In particular we demonstrated that a gene mutation that is associated with Schizophrenia leads to a functional defect measurable in human iN cells. This might lead to important new methods to find treatments for these devastating diseases.
Funding Type: 
Tools and Technologies II
Grant Number: 
RT2-02022
Investigator: 
Type: 
PI
ICOC Funds Committed: 
$1 493 928
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
oldStatus: 
Active
Public Abstract: 

Human pluripotent stem cells (hPSC) have the capacity to differentiate into every cell in the adult body, and they are thus a highly promising source of differentiated cells for the investigation and treatment of numerous human diseases. For example, neurodegenerative disorders are an increasing healthcare problem that affect the lives of millions of Americans, and Parkinson's Disease (PD) in particular exacts enormous personal and economic tolls. Expanding hPSCs and directing their differentiation into dopaminergic neurons, the cell type predominantly lost in PD, promises to yield cells that can be used in cell replacement therapies. However, developing technologies to create the enormous numbers of safe and healthy dopaminergic neurons required for clinical development and implementation represents a bottleneck in the field, because the current systems for expanding and differentiating hPSCs face numerous challenges including difficulty in scaling up cell production, concerns with the safety of some materials used in the current cell culture systems, and limited reproducibility of such systems.

An emerging principle in stem cell engineering is that basic advances in stem cell biology can be translated towards the creation of “synthetic stem cell niches” that emulate the properties of natural microenvironments and tissues. We have made considerable progress in engineering bioactive materials to support hESC expansion and dopaminergic differentiation. For example, basic knowledge of how hESCs interact with the matrix that surrounds them has led to progress in synthetic, biomimetic hydrogels that have biochemical and mechanical properties to support hESC expansion. Furthermore, biology often presents biochemical signals that are patterned or structured at the nanometer scale, and our application of materials chemistry has yielded synthetic materials that imitate the nanostructured properties of endogenous ligands and thereby promise to enhance the potency of growth factors and morphogens for cell differentiation.

We propose to build upon this progress to create general platforms for hPSC expansion and differentiation through two specific aims: 1) To determine whether a fully defined, three dimensional (3D) synthetic matrix for expanding immature hPSCs can rapidly and scaleably generate large cell numbers for subsequent differentiation into potentially any cell , and 2) To investigate whether a 3D, synthetic matrix can support differentiation into healthy, implantable human DA neurons in high quantities and yields. This blend of stem cell biology, neurobiology, materials science, and bioengineering to create “synthetic stem cell niche” technologies with broad applicability therefore addresses critical challenges in regenerative medicine.

Statement of Benefit to California: 

This proposal will develop novel tools and capabilities that will strongly enhance the scientific, technological, and economic development of stem cell therapeutics in California. The most important net benefit will be for the treatment of human diseases.

Efficiently expanding immature hPSCs in a scaleable, safe, and economical manner is a greatly enabling capability that would impact many downstream medical applications. The development of platforms for scaleable and safe cell differentiation will benefit therapeutic efforts for Parkinson’s Disease. Furthermore, the technologies developed in this proposal are designed to be tunable, such that they can be readily adapted to numerous downstream applications.

The resulting technologies have strong potential to benefit human health. Furthermore, this proposal directly addresses several research targets of this RFA – the development and validation of stem cell scale-up technologies including
novel cell expansion methods and bioreactors for both human pluripotent cells and differentiated cell types – indicating that CIRM believes that the proposed capabilities are a priority for California’s stem cell effort. While the potential applications of the proposed technology are broad, we will apply it to a specific and urgent biomedical problem: developing systems for generating clinically relevant quantities of dopaminergic neurons from hPSCs, part of a critical path towards developing therapies for Parkinson’s disease. This proposal would therefore work towards developing capabilities that are critical for hPSC-based regenerative medicine applications in the nervous system to clinically succeed.

The principal investigator and co-investigator have a strong record of translating basic science and engineering into practice through interactions with industry, particularly within California. Finally, this collaborative project will focus diverse research groups with many students on an important interdisciplinary project at the interface of science and engineering, thereby training future employees and contributing to the technological and economic development of California.

Progress Report: 
  • Human pluripotent stem cells (hPSC) have the capacity to differentiate into every cell in the adult body, and they are thus a highly promising source of differentiated cells for the investigation and treatment of numerous human diseases. For example, neurodegenerative disorders are an increasing healthcare problem that affect the lives of millions of Americans, and Parkinson's Disease (PD) in particular exacts enormous personal and economic tolls. Expanding hPSCs and directing their differentiation into dopaminergic neurons, the cell type predominantly lost in PD, promises to yield cells that can be used in cell replacement therapies. However, developing technologies to create the enormous numbers of safe and healthy dopaminergic neurons required for clinical development and implementation represents a bottleneck in the field, because the current systems for expanding and differentiating hPSCs face numerous challenges including difficulty in scaling up cell production, concerns with the safety of some materials used in the current cell culture systems, and limited reproducibility of such systems.
  • This project has two central aims: 1) To determine whether a fully defined, three dimensional (3D) synthetic matrix for expanding immature hPSCs can rapidly and scaleably generate large cell numbers for subsequent differentiation into potentially any cell , and 2) To investigate whether a 3D, synthetic matrix can support differentiation into healthy, implantable human DA neurons in high quantities and yields. In the first year of this project, we have made progress in both aims. Specifically, we are conducting high throughput studies to optimize matrix properties in aim 1, and we have developed a material formulation in aim 2 that supports a level of DA differentiation that we are now beginning to optimize with a high throughput approach.
  • This blend of stem cell biology, neurobiology, materials science, and bioengineering to create “synthetic stem cell niche” technologies with broad applicability therefore addresses critical challenges in regenerative medicine.
  • Human pluripotent stem cells (hPSC) have the capacity to differentiate into every cell in the adult body, and they are thus a highly promising source of differentiated cells for the investigation and treatment of numerous human diseases. For example, neurodegenerative disorders are an increasing healthcare problem that affect the lives of millions of Americans, and Parkinson's Disease (PD) in particular exacts enormous personal and economic tolls. Expanding hPSCs and directing their differentiation into dopaminergic neurons, the cell type predominantly lost in PD, promises to yield cells that can be used in cell replacement therapies. However, developing technologies to create the enormous numbers of safe and healthy dopaminergic neurons required for clinical development and implementation represents a bottleneck in the field, because the current systems for expanding and differentiating hPSCs face numerous challenges including difficulty in scaling up cell production, concerns with the safety of some materials used in the current cell culture systems, and limited reproducibility of such systems.
  • This project has two central aims: 1) To determine whether a fully defined, three dimensional (3D) synthetic matrix for expanding immature hPSCs can rapidly and scaleably generate large cell numbers for subsequent differentiation into potentially any cell , and 2) To investigate whether a 3D, synthetic matrix can support differentiation into healthy, implantable human DA neurons in high quantities and yields. In the first year of this project, we have made progress in both aims. Specifically, we are conducting high throughput studies to optimize matrix properties in aim 1, and we have developed a material formulation in aim 2 that supports a level of DA differentiation that we are now beginning to optimize with a high throughput approach.
  • This blend of stem cell biology, neurobiology, materials science, and bioengineering to create “synthetic stem cell niche” technologies with broad applicability therefore addresses critical challenges in regenerative medicine.
  • Human pluripotent stem cells (hPSC) have the capacity to differentiate into every cell in the adult body, and they are thus a highly promising source of differentiated cells for the investigation and treatment of numerous human diseases. For example, neurodegenerative disorders are an increasing healthcare problem that affect the lives of millions of Americans, and Parkinson's Disease (PD) in particular exacts enormous personal and economic tolls. Expanding hPSCs and directing their differentiation into dopaminergic neurons, the cell type predominantly lost in PD, promises to yield cells that can be used in cell replacement therapies. However, developing technologies to create the enormous numbers of safe and healthy dopaminergic neurons required for clinical development and implementation represents a bottleneck in the field, because the current systems for expanding and differentiating hPSCs face numerous challenges including difficulty in scaling up cell production, concerns with the safety of some materials used in the current cell culture systems, and limited reproducibility of such systems.
  • This project has two central aims: 1) To determine whether a fully defined, three dimensional (3D) synthetic matrix for expanding immature hPSCs can rapidly and scaleably generate large cell numbers for subsequent differentiation into potentially any cell , and 2) To investigate whether a 3D, synthetic matrix can support differentiation into healthy, implantable human DA neurons in high quantities and yields. In the first year of this project, we have made progress in both aims. Specifically, we are conducting high throughput studies to optimize matrix properties in aim 1, and we have developed a material formulation in aim 2 that supports a level of DA differentiation that we are now beginning to optimize with a high throughput approach.
  • This blend of stem cell biology, neurobiology, materials science, and bioengineering to create “synthetic stem cell niche” technologies with broad applicability therefore addresses critical challenges in regenerative medicine.
Funding Type: 
Tools and Technologies II
Grant Number: 
RT2-02018
Investigator: 
Name: 
Institution: 
Type: 
PI
ICOC Funds Committed: 
$1 930 608
Disease Focus: 
Stroke
Neurological Disorders
Stem Cell Use: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 

Clinical application of cell transplantation therapy requires a means of non-invasively monitoring these cells in the patient. Several imaging modalities, including MRI, bioluminescence imaging, and positron emission tomography have been used to track stem cells in vivo. For MR imaging, cells are pre-loaded with molecules or particles that substantially alter the image brightness; the most common such labelling strategy employs iron oxide particles. Several studies have shown the ability of MRI to longitudinally track transplanted iron-labeled cells in different animal models, including stroke and cancer. But there are drawbacks to this kind of labeling. Division of cells will result in the dilution of particles and loss of signal. False signal can be detected from dying cells or if the cells of interest are ingested by other cells.

To overcome these roadblocks in the drive toward clinical implementation of stem cell tracking, it is now believed that a genetic labeling approach will be necessary, whereby specific protein expression causes the formation of suitable contrast agents. Such endogenous and persistent generation of cellular contrast would be particularly valuable to the field of stem cell therapy, where the homing ability of transplanted stem cells, long-term viability, and capacity for differentiation are all known to strongly influence therapeutic outcomes. However, genetic labeling or "gene reporter" strategies that permit sensitive detection of rare cells, non-invasively and deep in tissue, have not yet been developed. This is therefore the translational bottleneck that we propose to address in this grant, through the development and validation of a novel high-sensitivity MRI gene reporter technology.

There have been recent reports of gene-mediated cellular production of magnetic iron-oxide nanoparticles of the same composition as the synthetic iron oxide particles used widely in exogenous labeling studies. It is an extension of this strategy, combined with our own strengths in developing high-sensitivity MRI technology, that we propose to apply to the task of single cell tracking of metastatic cancer cells and neural stem cells.

If we are successful with the proposed studies, we will have substantially advanced the field of in vivo cellular imaging, by providing a stable cell tracking technology that could be used to study events occurring at arbitrary depth in tissue (unlike optical methods) and over unlimited time duration and arbitrary number of cell divisions (unlike conventional cellular MRI).
With the ability to track not only the fate (migration, homing and proliferation) but also the viability and function of very small numbers of stem cells will come new knowledge of the behavior of these cells in a far more relevant micro-environment compared with current in vitro models, and yet with far better visualization and cell detection sensitivity compared with other in vivo imaging methods.

Statement of Benefit to California: 

Stem cell therapy has enormous promise to become a viable therapy for a range of illnesses, including stroke, other cardiovascular diseases, and neurological diseases. Progress in the development of these therapies depends on the ability to monitor cell delivery, migration and therapeutic action at the disease site, using imaging and other non-invasive technologies. If breakthroughs could be made along these lines, it would not only be of enormous benefit to the citizens of the state of California, but would also greatly reduce healthcare costs.

From a broader research perspective, the state of California is the front-runner in stem cell research, having gathered not only private investments, as demonstrated by the numerous biotechnology companies that are developing innovative tools, but also extensive public funds that allows the state, through CIRM, to sponsor stem cell research in public and private institutions. In order to preserve the leadership position and encourage research on stem cells, CIRM is calling for research proposals to develop innovative tools and technologies that will overcome current roadblocks in translational stem cell research. This proposal will benefit the state by providing important new technology that will be valuable for both basic and translational stem cell research.

A key bottleneck to the further development and translation of new stem cell therapies is the inability to track stem cells through a human body. It is possible to image stem cells using embedded optical fluorescence labels, but optical imaging does not permit tracking of cells deep in tissue. Other imaging modalities and their associated cellular labels (for example positron emission tomography) have also been used to track cells but do not have the sensitivity to detect rare or single cells. Finally, MRI has been used to track cells deep in tissue, down to the single cell level, but only by pre-loading cells with a non-renewable supply of iron oxide nanoparticles, which prevents long-term tracking and assessment of cell viability and function. We propose here to develop MRI technology and a new form of genetically-encoded, long-term cell labeling technology, to a much more advanced state than available at present. This will make it possible to use MRI to detect and follow cancer and stem cells as they migrate to and proliferate at the site of interest, even starting from the single cell stage. This will provide a technology that will help stem cell researchers, first and foremost in California, to understand stem cell behavior in a realistic in vivo environment. This technology will be translatable to future human stem cell research studies.

Progress Report: 
  • We have made good progress in the first year. This project involves four separate scientific teams, brought together for the first time, representing diverse backgrounds ranging from magnetic resonance imaging (MRI) physics and cell tracking (Dr. Rutt), microbiology (Dr. Matin), nano and magnetic characterization (Dr. Moler) and stem cell imaging in stroke models (Dr. Guzman). Substantial progress has been made by all four teams, and we are starting to see important interactions between the teams. An overall summary of progress is that we have evaluated three different bacterial genes (magA, mms6, mamB) in one mammalian cell line (MDA-MB-231BR) and have shown significant iron accumulation in vitro with two of these genes, which is a very positive result implying that these genes may have the required characteristics to act as "reporter genes" for MRI-based tracking of cells labeled with these genes. MR imaging of mouse brain specimens has yielded promising results and in vivo imaging experiments are underway at medium MRI field strength (3 Tesla). At the same time, we are ramping up our higher field, higher sensitivity MR imaging methods and will be ready to evaluate the different variations of our MR reporter gene at 7 Tesla (the highest magnetic field widely available for human MRI) in the near future. Finally, methods to perform quantitative characterization of our reporter cells are being developed, with the goal of being able to characterize magnetic properties down to the single cell level, and also to be able to assess iron loading levels down to the single level in brain tissue slices.
  • We have made good progress in the second year. This project involves four separate scientific teams, brought together for the first time for this project, representing diverse backgrounds ranging from magnetic resonance imaging (MRI) physics and cell tracking (Dr. Rutt), microbiology (Dr. Matin), nano and magnetic characterization (Dr. Moler) and imaging reporter development and testing in small animal models of disease (Dr. Contag). Substantial progress has been made by all four teams, and we are starting to see important interactions between the teams.
  • An overall summary of progress is that we have been evaluating three different bacterial genes (magA, mms6, mamB) in two mammalian cell lines (MDA-MB-231BR and DAOY). In year I we had shown significant iron accumulation in vitro with two of these genes, which was a very positive result implying that these genes may have the required characteristics to act as "reporter genes" for MRI-based tracking of cells labeled with these genes. In year 2, we diversified and intensified the efforts to achieve expression of one or more of the bacterial genes in different cell lines, using different genetic constructs. We began a concerted effort to achieve optical labeling such that we could visualize the gene expression and to identify sub-cellular localization of the report gene products.
  • We obtained promising results from MR imaging of mouse brain. In vivo imaging experiments were accomplished at medium MRI field strength (3 Tesla). At the same time, we ramped up our higher field, higher sensitivity MR imaging methods and began to evaluate the sensitivity gains enabled at the higher magnetic field strength of 7 Tesla (the highest magnetic field widely available for human MRI
  • Finally, methods to perform quantitative characterization of our reporter cells were developed, with the goal of being able to characterize magnetic properties down to the single cell level, and also to be able to assess iron loading levels down to the single level in brain tissue slices.
  • We have made good progress in the third year. This project involves four separate scientific teams, brought together for the first time for this project, representing diverse backgrounds ranging from magnetic resonance imaging (MRI) physics and cell tracking (Dr. Rutt), microbiology (Dr. Matin), nano and magnetic characterization (Dr. Moler) and imaging reporter development and testing in small animal models of disease (Dr. Contag). Substantial progress has been made by all four teams, and we have benefited from important interactions between all teams in this third year.
  • An overall summary of progress is that we evaluated several iron-binding bacterial genes (magA, mamB, mms6, mms13), both singly and doubly, in two mammalian cell lines (MDA-MB-231BR and DAOY). In year 2, we diversified and intensified the efforts to achieve expression of one or more of the bacterial genes in different cell lines, using different genetic constructs. We completed an effort to achieve optical labeling such that we could visualize the gene expression and to identify sub-cellular localization of the report gene products. In year 3, while continuing to face challenges with single gene constructs, we succeeded in finding substantial iron uptake in cells containing unique double gene expression, notably magA and mms13.
  • We completed much of the development of our higher field, higher sensitivity MR imaging methods and evaluated the sensitivity gains enabled at the higher magnetic field strength of 7 Tesla (the highest magnetic field widely available for human MRI).
  • Finally, we demonstrated novel nanomagnetic methods to characterize our reporter cells, able to characterize magnetic properties down to the single cell level.
  • We have made good progress during this 6-month extension period. This project involves four separate scientific teams, brought together for the first time for this project, representing diverse backgrounds ranging from magnetic resonance imaging (MRI) physics and cell tracking (Dr. Rutt), microbiology (Dr. Matin), nano and magnetic characterization (Dr. Moler) and imaging reporter development and testing in small animal models of disease (Dr. Contag). Substantial progress has been made by all four teams, and we have benefited from important interactions between all teams in this third year.
  • An overall summary of progress is that we evaluated several iron-binding bacterial genes (magA, mamB, mms6, mms13), singly, doubly and triply, in several mammalian cell lines (MDA-MB-231BR, DAOY, COS1, 293FT). In year 3 as well as through the extension period, we succeeded in finding substantial iron uptake in cells containing certain expressed genes, notably mms13 by itself, as well as combinations of mms13 with mms6 and mamB.
  • We completed the development of our higher field, higher sensitivity MR imaging methods and evaluated the sensitivity gains enabled at the higher magnetic field strength of 7 Tesla (the highest magnetic field widely available for human MRI).
  • Finally, we demonstrated novel nanomagnetic methods to characterize our reporter cells, able to characterize magnetic properties down to the single cell level.
Funding Type: 
Tools and Technologies II
Grant Number: 
RT2-01927
Investigator: 
ICOC Funds Committed: 
$1 816 157
Disease Focus: 
Alzheimer's Disease
Neurological Disorders
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 

Elucidating how genetic variation contributes to disease susceptibility and drug response requires human Induced Pluripotent Stem Cell (hIPSC) lines from many human patients. Yet, current methods of hIPSC generation are labor-intensive and expensive. Thus, a cost-effective, non-labor intensive set of methods for hIPSC generation and characterization is essential to bring the translational potential of hIPSC to disease modeling, drug discovery, genomic analysis, etc.

Our project combines technology development and scaling methods to increase throughput and reduce cost of hiPSC generation at least 10-fold, enabling the demonstration, and criterion for success, that we can generate 300 useful hiPSC lines (6 independent lines each for 50 individuals) by the end of this project. Thus, we propose to develop an efficient, cost effective, and minimally labor-intensive pipeline of methods for hIPSC identification and characterization that will enable routine generation of tens to hundreds of independent hIPSC lines from human patients. We will achieve this goal by adapting two simple and high throughput methods to enable analysis of many candidate hIPSC lines in large pools. These methods are already working in our labs and are called "fluorescence cell barcoding" (FCB) and expression cell barcoding (ECB).

To reach a goal of generating 6 high quality hIPSC lines from one patient, as many as 60 candidate hIPSC colonies must be expanded and evaluated individually using labor and cost intensive methods. By improving culturing protocols, and implementing suitable pooled analysis strategies, we propose to increase throughput at least 10-fold with a substantial drop in cost. In outline, the pipeline we propose to develop will begin with the generation of 60 candidate hIPSC lines per patient directly in 96 well plates. All 60 will be analyzed for diagnostic hIPSC markers by FCB in 1 pooled sample. The 10 best candidates per patient will then be picked for expression and multilineage differentiation analyses with the goal of finding the best 6 from each patient for digital karyotype analyses. Success at 10-fold scaleup as proposed here may be the first step towards further scaleup once these methods are fully developed.

Aim 1: To develop a cost-effective and minimally labor-intensive set of methods/pipeline for the generation and characterization high quality hIPSC lines from large numbers of human patients. We will test suitability/develop a set of methods that allow inexpensive and rapid characterization of 60 candidate hIPSC lines per patient at a time.

Aim 2: To demonstrate/test/evaluate the success and cost-effectiveness of our pipeline by generating 6 high quality hIPSC lines from each of 50 human patients from [REDACTED]. We will obtain skin biopsies and expand fibroblasts from 50 patients, and generate and analyze a total of 6 independent hIPSC lines from each using the methods developed in Aim 1.

Statement of Benefit to California: 

Many Californians suffer from diseases whose origin is poorly understood, and which are not treatable in an effective or economically advantageous manner. Part of solving this problem relies upon elucidating how genetic variation contributes to disease susceptibility and drug response and better understanding disease mechanism. Achieving these goals can be accelerated through the use of human Induced Pluripotent Stem Cell (hIPSC) lines from many human patients. Yet, current methods of hIPSC generation are labor-intensive and expensive. Thus, a cost-effective, non-labor intensive set of methods for hIPSC generation and characterization is essential to bring the translational potential of hIPSC to disease modeling, drug discovery, genomic analysis, etc.

If successful, our project will lead to breakthroughs in understanding of disease, development of better therapies, and economic development in California as businesses that use our methods are launched. In addition, new therapies will bring cost-savings in healthcare to Californians, stimulate employment since Californians will be employed in businesses that develop and sell these therapies, and relieve much suffering from the burdens of chronic disease.

Progress Report: 
  • An important problem in stem cell and regenerative medicine research has been the ability to quickly and cheaply generate and characterize reprogrammed stem cells from defined human patients. The primary goal of our project is to address this need by developing new technologies that allow stem cell lines to be characterized in large mixed pools as opposed to one by one. Our new methods use flow cytometry and highly sensitive methods for detecting the activity of genes in the cell lines. We made excellent progress in the first year and reduced flow cytometry methods to practice taking advantage of a method called fluorescence cell barcoding. Methods for analyzing activity of genes and chromosome number are in progress and being tested. Our ultimate goal is to reduce cost tenfold and increase speed by about tenfold and our methods development is on track to accomplish this aim.
  • A key bottleneck in reprogramming technology to make induced pluripotent stem (IPS) cell lines is the ability to make large numbers of lines from large numbers of patients in a way that is cost effective and minimizes labor. Our project has focused primarily on dropping the cost of characterization of candidate lines. We have made a number of discoveries about the behavior of candidate reprogrammed lines that allow us to drop cost and labor needed for candidate reprogrammed line characterization. We measured the frequency of candidate lines that were well-behaved in a large retroviral reprogramming experiment, which allows us to rigorously estimate how many candidate lines must be picked and analyzed if 4-6 high-quality lines are to be generated for every patient fibroblast sample subjected to typical retroviral reprogramming technology. We then continued our work on developing a combination of different array and microfluidic chip technologies to measure the chromosome number in each candidate line and the ability of each line to be pluripotent, i.e., to be able to generate many different type of cells similar to embryonic stem cells. We are optimistic that our work will simplify and drop the cost of the characterization process so that it costs far less than before our work was initiated.
  • Reprogrammed stem cell lines, i.e., induced pluripotent stem cell lines, have the potential to revolutionize research into causes of disease and genetic contributions to the causes of disease. One key limitation, however, is the ability to generate large numbers of different stem cell lines from different people to sample the range of genetic variation in the human population as it relates to disease development. A key bottleneck is the speed and cost with which reprogrammed stem cell lines can be generated and validated for usefulness. We have succeeded in developing a streamlined workflow for characterization of reprogrammed stem cell lines that drops the cost for characterization from several thousand dollars to a few hundred dollars and increases the speed and number of lines that can be handled substantially. We take advantage of novel genetic characterization methods to analyze genetic stability and the pattern of gene expression as it reveals the capabilities of the stem cell lines. We are finishing up the loose ends on this project now and should have a high quality publication prepared for submission shortly that describes this simple and inexpensive workflow that we have developed with modern gene characterization methods.

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