Neurological Disorders

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

Evaluation of Safety and Preliminary Efficacy of Escalating Doses of GRNOPC1 in Subacute Spinal Cord Injury

Funding Type: 
Targeted Clinical Development
Grant Number: 
CT1-05168
ICOC Funds Committed: 
$24 846 856
Disease Focus: 
Spinal Cord Injury
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
The proposed project is designed to assess the safety and preliminary activity of escalating doses of human embryonic stem cell (hESC) derived oligodendrocyte progenitor cells for treatment of spinal cord injury. Oligodendrocyte progenitor cells have two important functions: they produce neurotrophic factors which stimulate the survival and growth of neurons (nerve cells) after injury, and they mature in the spinal cord to produce myelin, the insulation which envelops neuronal axons (nerve cell bodies responsible for conduction) and facilitates unimpeded nerve impulse conduction. After extensive efficacy and safety testing, clinical testing of this product was initiated in 2010. Clinical testing is being initiated in paraplegic patients with neurologically complete thoracic injuries (i.e., those in which no motor or sensory function remains below the level of the injury). In the first cohort, a dose equivalent to the lowest efficacious dose observed in preclinical rodent studies is being administered. During the course of the proposed program, clinical safety studies testing increasing doses will be conducted. Upon demonstration of safety, clinical testing will be expanded to tetraplegic patients (complete cervical injuries) and to patients with incomplete thoracic injuries for additional safety testing. In each of the proposed studies, preliminary evidence of activity will be monitored using measures of improved neurological function and performance of daily living activities. The project plan also includes the manufacture of cells to be used in the clinical trials and additional supporting activities. By completion of the proposed project, we expect to have accumulated substantial safety data and preliminary efficacy data in three different patient subpopulations. This data will provide key information to inform the design and execution of advanced efficacy studies.
Statement of Benefit to California: 
The proposed project has the potential to benefit the state of California through 1) providing improved medical outcomes for patients with spinal cord injury and their families, 2) increasing California’s leadership in the emerging field of stem cell research, and 3) preserving and creating high quality, high paying jobs for Californians. Over 12,000 Americans suffer spinal cord injuries each year, and approximately 1.3 million people in the US are estimated to be living with spinal cord injuries. Although specific estimates for the state of California are not available, it is known that the majority of spinal cord injuries result from motor vehicle accidents, falls, acts of violence and recreational sporting activities, all of which are prevalent in California. Spinal cord injury affects not only the patient but family members, friends, healthcare workers and employers. It is estimated that one year after injury, only 11.6% of spinal cord injury patients are employed, and that spinal cord injuries cost $40.5 billion annually in the US. As the most populous state, California is disproportionately affected, negatively impacting our productivity, healthcare system and public finances. There are currently no approved therapies for the treatment of spinal cord injury. The product described in this application has initiated phase 1 clinical testing in patients with complete thoracic spinal cord injury. Even partial correction of any of the debilitating consequences of spinal cord injury could potentially enhance activities of daily living and increase employment while decreasing reliance on attendant care and subsequent medical interventions. California has a history of leadership in biotechnology, and is emerging as a leader in the development of stem cell therapeutics. Cutting edge stem cell research, in many cases funded by CIRM, is already underway in academic research laboratories and biotechnology companies throughout the state. The proposed project has the potential to further increase California’s leadership in the field of stem cell therapeutics through the performance of the first clinical testing of an hESC-derived therapy. The applicant has been located in California since its inception, and currently employs 182 full-time employees at its California headquarters with more than 50% of employees holding an advanced degree. These positions are highly skilled positions, offering competitive salaries and comprehensive benefits. The successful performance of the proposed project would enable significant additional jobs creation in preparation for pivotal trials and product registration.

Understanding the role of LRRK2 in iPSC cell models of Parkinson's Disease

Funding Type: 
Basic Biology III
Grant Number: 
RB3-02221
ICOC Funds Committed: 
$1 482 822
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Stem Cell Use: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
The goal of this research is to utilize novel research tools to investigate the molecular mechanisms that cause Parkinson’s disease (PD). The proposed work builds on previous funding from CIRM that directed the developed patient derived models of PD. The majority of PD patients suffer from sporadic disease with no clear etiology. However some PD patients harbor specific inherited mutations have been shown to cause PD. The most frequently observed form of genetic parkinsonism is caused by the LRRK2 G2019S mutation it the most common. This mutation accounts for approximately 1.5-2% of patients with apparently sporadic PD, increasing to 4-6% of patients with a family history of PD, and even higher in isolated populations. Importantly, LRRK2 induced PD is clinically and pathologically largely indistinguishable from sporadic PD. This proposal focuses on studying the most frequent cause of familial PD and induces disease that is clinically and pathologically identical to sporadic PD cases. It is likely that LRRK2 regulates a pathway(s) that is important in the more common sporadic form of PD as well. Therefore by employing relevant models of PD, we hope to drive the biological understanding of LRRK2 in a direction that facilitates the development of disease therapeutics in the future. We ascertained patients harboring mutations in LRRK2 [heterozygous (+/G2019S) and homozygous (G2019S/G2019S)] as well as sporadic cases and age matched controls. We have successfully derived iPSCs from each genotype and differentiated these to DA neurons. We will use these as a model system to investigate these LRRK2 based models of PD. We will adapt current biochemical assays of LRRK2, which are source material intensive, to the small culture volumes required for the differentiation of iPSCs to DA neurons. This is a crucial necessity for development for utilizing iPSC derived DA neurons as tractable models of LRRK2 based PD. We will then probe the roles of LRRK2 in neuronal cell differentiation and survival. We will also ask whether the mutant LRRK2 induces changes in autophagy, as this has been postulated as a mechanism of LRRK2 induced pathogenesis. By studying wild-type and disease mutant LRRK2, in DA models of PD we hope to provide crucial understanding of the role mutant LRRK2 has in disease.
Statement of Benefit to California: 
It is estimated that by the year 2030, 75,000-120,000 Californians will be affected by Parkinson’s disease. Currently, there is no cure, early detection mechanism, preventative treatment, or effective way to slow disease progression. The increasing disability caused by the progression of disease burdens the patients, their caregivers as well as society in terms of healthcare costs. The majority of PD patients suffer from sporadic disease with no clear etiology, and a in a handful of these patients specific inherited mutations have been shown to cause PD. The most frequently mutated gene is called Leucine Rich Repeat Kinase 2 (LRRK2). Our goal is to study the mutated gene product in patient based models of Parkinson’s disease. In previous CIRM funding, we have developed patient derived induced pluripotent stem cells (iPSCs) from patients harboring mutations in LRRK2. We have been successful in differentiating populations these iPSCs into the neurons that are depleted in PD. The next step is to utilize these cells as models of mutation induced PD ‘in a dish’. We will employ these pertinent disease models to answer basic biology questions that remain about the function of LRRK2. This project brings together scientists previously funded by CIRM with scientists well versed in the study of LRRK2. This multidisciplinary approach to studying the causes of PD is a natural benefit to the State of California and its citizens. By bringing a better understanding of the role of LRRK2 in the cells that are lost in the progression of PD, we will bring more concrete knowledge of PD as a whole, bringing more hope for the development of a therapeutic for disease.
Progress Report: 
  • The overarching goal of this work is to utilize models of Parkinson's disease (PD) that originate from cells of PD affected patients harboring mutations within the LRRK2 gene so that we may discern the role of mutated LRRK2 in disease. Mutations in LRRK2 are the most common cause of familial PD. The disease presentation of patients with LRRK2 mutation is typically clinically indistinguishable from sporadic PD cases, making the onset of disease due to LRRK2 dysfunction clinically relevant. We have employed stem cells derived from these patients to generate neuronal cells in which we can determine the roles of LRRK2 in the PD mutated and the unmutated state. We have focused on a cellular process called autophagy that regulates the cell response to nutrient deprivation and plays a role in the selective degradation of proteins within the cell.
  • In the first year of funding we have analyzed the expression of the protein LRRK2 in induced pluripotent stem cells, neuronal precursor cells and have begun to differentiate the neuronal precursors to dopaminergic cells of the type lost in PD (a difficult task in itself). We have applied a novel method for detection of LRRK2 in situ by marrying the protein detection of antibodies and the sensitivity of nucleic acid amplification. We will continue to develop this methodology for maximum sensitivity to LRRK2. We have established assays to assess the effects of the LRRK2 mutant on autophagy that are relevant to PD and neurological diseases in general. We have met or made great progress on most of our anticipated milestones and are eager to proceed to the next phase of the project.
  • The overarching goal of this work is to utilize stem cell based models of Parkinson's disease (PD) derived from cells of PD affected patients that harbor mutations in the LRRK2 gene so that we may elucidate the deleterious role of mutated LRRK2 in disease. Mutations in LRRK2 are the most common cause of familial PD. The disease presentation for these patients with LRRK2 mutation is typically clinically similar to those with sporadic disease, making the onset of disease due to LRRK2 dysfunction clinically relevant. We have utilized stem cells harboring a mutation in LRRK2 and also daughter cells of that line in which genomic editing techniques have been applied to correct the PD mutation or disrupt the LRRK2 gene. We have generated the same kind of cells in culture that are lost during PD and hope that next, we can determine how these mutations that eventually cause disease disrupt normal neuronal function. We have made great progress in the understanding the expression of LRRK2 in early differentiation of stem cells to neurons and his will inform our future studies on mutation caused dysfunctions.

Triplet Repeat Instability in Human iPSCs

Funding Type: 
Basic Biology III
Grant Number: 
RB3-05022
ICOC Funds Committed: 
$1 755 861
Disease Focus: 
Huntington's Disease
Neurological Disorders
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
Over twenty human genetic diseases are caused by expansion of simple DNA sequences composed of repeats of three nucleotides (such as CAG, CTG, CGG and GAA) within essential genes. These repeats can occur within the region of a gene that encodes the protein, generally resulting in proteins with large stretches of repeats of just one amino acid, such as runs of glutamine. These proteins are toxic, cause the death of specific types of brain cells and result in diseases such as Huntington’s disease (HD) and many of the spinocerebellar ataxias (a type of movement disorder). Other repeats can be in regions of genes that do not code for the protein itself, but are copied into messenger RNA, which is a copy of the gene that serves to generate the protein. These RNAs with expanded repeats are also toxic to cells, and sometimes these RNAs sequester essential cellular proteins. One example of this type of disease is Myotonic Dystrophy type 1, a form of muscular dystrophy. Lastly, there are two examples of repeat disorders where the repeats silence the genes harboring these mutations: these are Friedreich’s ataxia (FRDA) and Fragile X syndrome (FXS). One limitation in the development of drugs to treat these diseases is the lack of appropriate cell models that represent the types of cells that are affected in these human diseases. With the advent of the technology to produce induced pluripotent stem cells from patient skin cells, and our ability to turn iPSCs into any cell type, such as neurons (brain cells) that are affected in these triplet repeat diseases, such cellular models are now becoming available. Our laboratories have generated iPSCs from fibroblasts obtained from patients with HD, FXS and FRDA. By comparing cells before and after reprogramming, we found that triplet repeats were expanded in the FRDA iPSCs, but not in HD iPSCs. This application is aimed at the understanding the molecular basis underlying triplet repeat expansion/instability that we have observed during the establishment and propagation of iPSCs from disease-specific fibroblasts. While artificial systems with reporter gene constructs have reproduced triplet repeat expansion in bacteria, yeast and mammalian cells, no cellular models have previously been reported that recapitulate repeat expansions at the endogenous cellular genes involved in these diseases. Therefore, our observations that repeat expansion is found in FRDA iPSCs provides the first opportunity to dissect the mechanisms involved in expansion at the molecular level for the authentic cellular genes in their natural chromatin environment. Repeat expansion is the central basis for these diseases, no matter what the outcome of the expansion (toxic protein or RNA or gene silencing), and a fuller understanding of how repeats expand may lead to new drugs to treat these diseases.
Statement of Benefit to California: 
A major obstacle in the development of new drugs for human diseases is our lack of cell models that represent the tissues or organs that are affected in these diseases. Examples of such diseases are the triplet-repeat neurodegenerative diseases, such as Huntington’s disease, the spinocerebellar ataxias, forms of muscular dystrophy, Fragile X syndrome and Friederich’s ataxia. These diseases, although relatively rare compared to cancer or heart disease, affect thousands of individuals in California. Recent advances in stem cell biology now make it possible to generate cells that reflect the cell types at risk in these diseases (such as brain, heart and muscle cells), starting from patient skin cells. Skin cells can be turned into stem cell-like cells (induced pluripotent stem cells or iPSCs), which can then give rise to just about any cell type in the human body. During the course of our studies, we found that iPSCs derived from Friedreich’s ataxia patient skin cells mimic the behavior of the genetic mutation in this disease. A simple repeat of the DNA sequence GAA is found in the gene encoding an essential protein called frataxin, and this repeat increases in length between generations in human families carrying this mutation. Over a certain threshold, the repeats silence this gene. It is also known that the repeats expand in brain cells in individuals with this disease. With the advent of patient derived iPSCs and neurons, we now have human model systems in which to study the mechanisms responsible for repeat expansion. We have already identified one set of proteins involved in repeat expansion and we now wish to delve more deeply into how the repeats expand. In this way, we may be able to identify new targets for drug development. We will extend our studies to Huntington’s disease and Fragile X syndrome. We have identified two possible therapeutic approaches for Friedreich’s ataxia, and identified molecules that either reactivate the silent gene or block repeat expansion. Our studies in related diseases may provide possible therapeutic strategies for these other disorders as well, which will be of benefit to patients suffering from these diseases, both in California and world-wide.
Progress Report: 
  • Over twenty human genetic diseases are caused by expansion of simple trinucleotide repeat sequences within essential genes, resulting in toxic proteins (as in the polyglutamine expansion diseases, such as Huntington’s disease (HD)), toxic RNAs (as in Myotonic Dystrophy type 1), or gene repression (as in Friedreich’s ataxia (FRDA) and Fragile X syndrome (FXS)). Our laboratories have generated induced pluripotent stem cells (iPSCs) from fibroblasts obtained from patients with Huntington’s disease (HD), Fragile X syndrome (FXS), Myotonic dystrophy type 1 (DM1) and Friedreich’s ataxia (FRDA). By comparing cells before and after reprogramming, we found that triplet repeats were expanded in the FRDA and DM1 iPSCs, but not in HD iPSCs. During growth of the iPSCs in culture, the repeats continue to expand, suggesting that expansion might be linked to DNA replication in these cells. The expansion we observe in iPSCs does not occur in the fibroblast (skin cells) from which the iPSCs were derived. Similarly, on differentiation of the FRDA iPSCs into neurons (brain cells), repeat expansion stops. This observation suggests that some cellular factors necessary for expansion may be selectively expressed in iPSCs, but not in fibroblasts or neurons.
  • Over the past year, our studies have been aimed at the understanding the molecular basis underlying triplet repeat expansion/instability that we have observed during the establishment and propagation of iPSCs from disease-specific fibroblasts. Previous studies have implicated the mismatch repair (MMR) enzymes in repeat expansion in mouse models for HD and DM1. We find that silencing of the MSH2 gene, encoding one of the subunits of the MMR enzymes, impedes repeat expansion in human FRDA iPSCs. We find that components of the human mismatch repair (MMR) system are associated with the disease alleles in the FRDA and DM1 iPSCs, and that silencing of these genes at the level of their messenger RNAs is sufficient to suppress repeat expansion. Moreover, we have monitored the levels of the MMR enzymes in fibroblasts, iPSCs and neurons, and as expected these enzymes are present at higher amounts in the iPSCs, suggesting that it is the availability of these enzymes in iPSCs that may be responsible for repeat expansion.
  • We wish to determine whether it is the DNA structure of triplet-repeats or protein recognition of the repeats that recruits the MMR enzymes to triplet repeats in iPSCs. To this end, we used a series of small molecule probes that can be designed to target particular DNA sequences in the human genome, and we find that a molecule that targets the GAA-TTC repeats in the FRDA frataxin gene displaces MMR enzymes and prevents repeat expansion. We are currently exploring the mechanism whereby this molecule displaces the MMR enzymes. A deeper understanding of the molecular events that lead to repeat expansion at the endogenous cellular genes responsible for these diseases will likely lead to discoveries of new therapeutic strategies for these currently untreatable disorders.
  • Over the past year, our research efforts have focused on the generality of the results we found in human induced pluripotent stem cells derived from patients with the neurodegenerative disease Friedreich's ataxia (FRDA). FRDA is one of the trinucleotide repeat (TNR) diseases, and our major previous finding was that the GAA•TCC trinucleotide repeats that cause FRDA expand during isolation and propagation of FRDA hiPSCs. This expansion was shown to be dependent on enzymes that are involved in the repair of mismatches in the human genome. To extend these studies, we have now focused on hiPSCs from the related TNR diseases myotonic dystrophy, Huntington's disease and Fragile X syndrome. Myotonic dystrophy type 1 (DM1) is an inherited dominant muscular dystrophy caused by expanded CTG•CAG triplet repeats in the 3’ UTR of the DMPK1 gene, which produces a toxic gain-of-function CUG RNA. It has been shown that the severity of disease symptoms, age of onset and progression are related to the length of the triplet repeats. However, the mechanism(s) of CTG•CAG triplet-repeat instability is not fully understood. Human induced pluripotent stem cells (iPSCs) were generated from DM1 and Huntington’s disease (HD) patient fibroblasts. We isolated 41 iPSC clones from DM1 fibroblasts, all showing different CTG•CAG repeat lengths, thus demonstrating somatic instability within the initial fibroblast population. During propagation of the iPSCs, the repeats expanded in a manner analogous to the intergenerational expansion observed in DM1 patient families. The correlation between repeat length and expansion rate identified the interval between 57 and 126 repeats as being an important length threshold where expansion rates dramatically increased. Moreover, longer repeats showed faster triplet-repeat expansion. The relatively short repeats in the gene responsible for Huntington's disease are below this threshold and hence do not expand in the iPSCs. The overall tendency of triplet repeats to expand ceased on differentiation into differentiated embryoid body or neurospheres. The mismatch repair components MSH2, MSH3 and MSH6 were highly expressed in iPSCs compared to fibroblasts, and only occupied the DMPK1 gene harboring longer CTG•CAG triplet repeats. In addition, shRNA silencing of MSH2 impeded CTG•CAG triplet-repeat expansion. We have also generated hiPSC lines from seven male subjects clinically diagnosed with fragile X syndrome. These hiPSCs have been thoroughly characterized with respect to pluripotency, DNA methylation status at the FMR1 gene, CGG repeat length, FMR1 expression and neuronal differentiation. The information gained from these studies provides new insight into a general mechanism of triplet repeat expansion in iPSCs.

Induced Pluripotent Stem Cells for Tissue Regeneration

Funding Type: 
Basic Biology III
Grant Number: 
RB3-05232
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.

Use of human iPS cells to study spinal muscular atrophy

Funding Type: 
Basic Biology III
Grant Number: 
RB3-02161
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.

Generation and characterization of corticospinal neurons from human embryonic stem cells

Funding Type: 
Basic Biology III
Grant Number: 
RB3-02143
ICOC Funds Committed: 
$1 355 063
Disease Focus: 
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
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.

Viral-host interactions affecting neural differentiation of human progenitors

Funding Type: 
Basic Biology III
Grant Number: 
RB3-05219
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.

Studying neurotransmission of normal and diseased human ES cell-derived neurons in vivo

Funding Type: 
Basic Biology III
Grant Number: 
RB3-02129
ICOC Funds Committed: 
$1 382 400
Disease Focus: 
Autism
Neurological Disorders
Rett's Syndrome
Pediatrics
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
Stem cells, including human embryonic stem cells, provide extraordinary new opportunities to model human diseases and may serve as platforms for drug screening and validation. Especially with the ever-improving effective and safe methodologies to produce genetically identical human induced pluripotent stem cells (iPSCs), increasing number of patient-specific iPSCs will be generated, which will enormously facilitate the disease modeling process. Also given the advancement in human genetics in defining human genetic mutations for various disorders, it is becoming possible that one can quickly start with discovery of disease-related genetic mutations to produce patient-specific iPSCs, which can then be differentiated into the right cell type to model for the disease in vitro, followed by setting up the drug screening paradigms using such disease highly relevant cells. In the context of neurological disorders, both synaptic transmission and gene expression can be combined for phenotyping and phenotypic reversal screening and in vitro functional (synaptic transmission) reversal validation. The missing gap for starting with the genetic mutation to pave the way to drug discovery and development is in vivo validation-related preclinical studies. In order to fill this gap, in this application we are proposing to use Rett syndrome as a proof of principle, to establish human cell xenografting paradigm and perform optogenetics and in vivo recording or functional MRI (fMRI), to study the neurotransmission/connectivity characteristics of normal and diseased human neurons. Our approach will be applicable to many other human neurological disease models and will allow for a combination of pharmacokinetic, and in vivo toxicology work together with the in vivo disease phenotypic reversal studies, bridging the gap between cell culture based disease modeling and drug screening to in vivo validation of drug candidates to complete the cycle of preclinical studies, paving the way to clinical trials. A success of this proposed study will have enormous implications to complete the path of using human pluripotent stem cells to build novel paradigms for a complete drug development process.
Statement of Benefit to California: 
Rett Syndrome (RTT) is a progressive neurodevelopmental disorder caused by primarily loss-of-function mutations in the X-linked MeCP2 gene. It mainly affects females with an incidence of about 1 in 10,000 births. After up to 18 months of apparently normal development, children with RTT develop severe neurological symptoms including motor defects, mental retardation, autistic traits, seizures and anxiety. RTT is one of the Autism Spectrum Disorders (ASDs) that affects many children in California. In this application, we propose to use our hESC-based Rett syndrome (RTT) model as a proof-of-principle case to define a set of core transcriptome that can be used for drug screenings. Human embryonic stem cells (hESCs) hold great potential for cell replacement therapy where cells are lost due to disease or injury. For the diseases of the central nervous system, hESC-derived neurons could be used for repair. This approach requires careful characterization of hESCs prior to utilizing their therapeutic potentials. Unfortunately, most of the characterization of hESCs are performed in vitro when disease models are generated using hESC-derived neurons. In this application, using RTT as a proof of principle study, we will bridge the gap and perform in vivo characterization of transplanted normal and RTT human neurons. Our findings will not only benefit RTT and other ASD patients, but also subsequently enable broad applications of this approach in drug discovery using human pluripotent stem cell-based disease models to benefit the citizens of California in a broader spectrum.
Progress Report: 
  • The potential of stem cells, such as human embryonic stem cells and induced pluripotent stem cells (iPSCs), has been widely recognized for cell replacement therapy, modeling human diseases and serving as a platform for drug screening and validation. In this grant, we proposed to use Rett syndrome as a proof of principle, to establish a human cell xenografting paradigm (i.e., transplanting human cells into mouse/rat embryos) and perform in vivo analyses to study the neurotransmission characteristics of normal and diseased human neurons. We initially determined that it was feasible to use the lentiviral CamKII-ChR2 construct to drive excitatory neuronal-specific expression of ChR2 in mouse hippocampal pyramidal neurons as well as human embryonic stem cell derived neurons. Importantly, we have found that both ChR2 expressing mouse hippocampal neurons and human neurons derived from embryonic stem cells can spike action potentials when stimulated in vitro, indicating that exogenously expressed ChR2 is functional. Furthermore, we successfully transplanted human embryonic stem cell derived neural stem/progenitor cells into fetal rat forebrain at embryonic day 17. Our analysis of the recipient animals at postnatal day 21 showed that approximately 40-50% of the cells survived and began to express neuronal markers, such as NeuN, indicating the neuronal differentiation, as well as the long-term survival, of transplanted human cells in the recipient animals. As originally proposed, we will proceed with the documentation of the in vivo phenotype of Rett syndrome diseased neurons. Our approach will be particularly crucial to not only validate candidate drugs or other therapeutic interventions to treat Rett syndrome using xeno-transplanted human Rett neurons, but also to study the in vivo behavior of those neurons with and without the therapeutic intervention.
  • Stem cells, such as human embryonic stem cells and induced pluripotent stem cells (iPSCs), carry great potentials for cell replacement therapy, human diseases modeling and drug screenings. We proposed to use Rett syndrome (RTT) as a proof of principle, to establish a human cell xenografting paradigm (i.e., transplanting human cells into mouse/rat brains) to study the function of normal and diseased human neurons in vivo. During the 2nd year of funding, we gained new insights into the electrophysiological characteristics of RTT neurons. Specifically, we found that the neurotransmission phenotype of neurons derived from RTT patient-specific iPSCs was highly circuitry-dependent. On the other hand, when cell-intrinsic electrophysiological properties were measured, extremely stable abnormalities in action potential profiles, resting membrane potentials, etc. were observed, indicative of the validity of the culture system. Given that currently scientists have very limited control over the features of neuronal connections formed in culture conditions, our findings make the in vivo assessment of RTT neuronal properties even more desirable, because the circuitry features are more amenable in vivo, with anatomical cues. In light of aforementioned in vitro findings, we focused our attention to both cell-intrinsic electrophysiological characteristics of RTT neurons, as well as their connectivity or neural network properties, after neurons were integrated into host circuits in vivo following xenotransplantation. Our preliminary data demonstrated that the action-potential abnormalities of RTT neurons are preserved in vivo after xenotransplantation. So far we have established a relatively optimized system for studying human iPSC-derived RTT neurons integrated into mouse brains. We are poised to uncover not only the neuronal intrinsic electrophysiological properties but also the connectivity of wild type and RTT neurons with host circuits. Moreover, we have made substantial progress with regards to a novel technology, i.e., single neuron gene expression profiling coupled with electrophysiological recordings both in vitro and in vivo. Up to now, 8 RTT iPSC-derived neurons were profiled via RNA sequencing following electrophysiological recordings, and some interesting clues have already been revealed. Currently we are collecting more neurons and we expect to make unprecedented discoveries with mechanistic insights into RTT disease pathophysiology, which will facilitate the development of novel therapies for RTT. This paradigm is also generally applicable for studying other neurological disorders.

Cellular tools to study brain diseases affecting synaptic transmission

Funding Type: 
Tools and Technologies II
Grant Number: 
RT2-02061
ICOC Funds Committed: 
$1 906 494
Disease Focus: 
Autism
Neurological Disorders
Rett's Syndrome
Pediatrics
Stem Cell Use: 
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.

Use of iPS cells (iPSCs) to develop novels tools for the treatment of spinal muscular atrophy.

Funding Type: 
Tools and Technologies II
Grant Number: 
RT2-02040
ICOC Funds Committed: 
$1 933 022
Disease Focus: 
Spinal Muscular Atrophy
Neurological Disorders
Pediatrics
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 
Spinal Muscular Atrophy (SMA) is one of the most common lethal genetic diseases in children. One in thirty five people carry a mutation in a gene called survival of motor neurons 1 (SMN1) which is responsible for this disease. If two carriers have children together they have a one in four chance of having a child with SMA. Children with Type I SMA seem fine until around 6 months of age, at which time they begin to show lack of muscular development and slowly develop a "floppy" syndrome over the next 6 months. Following this period, SMA children become less able to move and are eventually paralyzed by the disease by 3 years of age or earlier. We know that this mutation causes the death of motor neurons - which are important for making muscle cells work. Interestingly, there is a second gene which can lessen the severity of the disease process (SMN2). Children with more copies of this modifying gene have less severe symptoms and can live for longer periods of time (designated Type II, III and IV and living longer periods respectively). There is no therapy for SMA at the current time. One of the roadblocks is that there are no human models for this disorder as it is very difficult to make the motor neurons that die in the disease in the laboratory. The researchers in the current proposal have recently created pluripotent stem cells from a patient with Type I SMA (the most severe) and shown that motor neurons grown out from the pluripotent stem cells also die in the culture dish just like they do in children. This is an important model for SMA. The proposed research takes this model of SMA and extends it to Type II and Type III children in order to have a wider range of disease severity in the culture dish (Type IV is very rare and difficult to get samples from). It then develops new technologies to produce very large numbers of motor neurons and perform large scale analysis of their survival profiles. Finally, it will explore whether novel compounds can slow down the degeneration of motor neurons in this model which should lead to the discovery of dew drugs that then may be used to treat the disease.
Statement of Benefit to California: 
The aim of this research is to develop novel drugs to treat a lethal childhood disease - SMA. There would be three immediate benefits to the state of California and its citizens. 1. Children in California would have access to novel drugs to slow or prevent their disease. 2. SMA is a world wide disease. The institutions involved with the research would be able to generate income from any new drugs developed and the profit from this would come back to California. 3. The project will employ a number of research staff in Californian institutions
Progress Report: 
  • This year we have created a large number of new SMA lines, developed ways to differentiate them into motor neurons using high content dishes, and begun to analyze the health of the motor neurons over time. We have also submitted a new paper showing that much of the cell death seen in the dying motor neurons is due to apoptosis - a form of cell death that is treatable with specific types of drug. We are now using these new lines to begin setting up screening runs with drug libraries and should be able to start these in the new year of funding.
  • In this year we have made more induced pluripotent stem (iPSC) cell lines from Spinal Muscular Atrophy patients also using blood cells in addition to skin cells. Blood cells from patients are usually more readi;y accessible. As such, this technique can be used to make larger bank of similar cell lines. We have also rigorously tested all the iPSCs them for their quality. These lines are now available for distribution to other California researchers along with a certificate of analysis.
  • Motor neurons are a type of neuron that control muscle movement and are markedly destroyed in SMA patients. In order for these powerful iPS cells form patients to be useful for discovering new drugs for SMA it is very important that we can make motor neurons from iPSCs in large quantities of millions to billions in number. Only then will testing of thousands to millions of new drugs would be feasible in neurons from SMA patients. To this end, we have created a method for making a predecessor cell type to human motor neurons from human iPSCs in a petri dish. These predecessor cells, known as motor neuron precursor spheres (iMNPS), are grown as clumps of floating spherical balls, each containing thousands such cells that are grown in large numbers repeatedly for long periods of time. We have made these iMNPS now from many SMA patients as well as healthy humans. These spheres can be preserved for long period of time by freezing them at very low temperatures. They are then awoken at a later time making it convenient for testing large numbers of drugs.
  • Since iPSCs have the power to make any cell type in the human body, they can also be contaminated with other unwanted types of cells. Typically such a technique is very difficult to accomplish in pluripotent stem cells such as embryonic and iPSCs. Therefore, we have designed a more efficient scheme to generate iPSC lines from SMA patients that will become fluorescent color (green, red or blue) when then motor neurons are made from iPSCs. These types of cells are known as reporter cell lines. This will aid in picking out the desired cell type from patient iPSCs, in this case a motor neuron, and discard any unwanted cell types. This will enormously simplify testing of new drugs in SMA patient motor neurons.
  • Deficiency of an important protein in SMA patients is one of the key causes to the course of the disease. We have also designed an automated method for identifying new drugs in patient motor neurons that will test for correction of SMN protein levels in motor neurons.
  • In Year 3 we completed making all iPSC lines from Spinal Muscular Atrophy patients. We rigorously tested all the iPSCs for quality. These lines are now available for distribution to other California researchers along with a quality control certificate.
  • Motor neurons are a type of neuron that control muscle movement and are markedly destroyed in SMA patients. In order for these powerful iPS cells form patients to be useful for discovering new drugs for SMA it is very important that we can make motor neurons from iPSCs in billions and repeatedly. Only then will testing of thousands to millions of new drugs would be feasible in neurons from SMA patients.
  • To this end, we have created a method for making a predecessor cell type to human motor neurons from human iPSCs in a petri dish. These predecessor cells, known as motor neuron precursor spheres (iMPS), are grown as clumps of floating spherical balls, each containing thousands such cells that are grown in large numbers repeatedly for long periods of time. We have now tested our method in multiple patient cells and characterized these spheres. The iMPS have now been produced from many SMA patients as well as healthy humans. The next step we have developed is to take the iMPS to make motor neurons that are similar to those that are affected in SMA children. We have then discovered a method for creating them quickly. These aggregate spheres and spinal cord motor neurons from them can be preserved for long period of time by freezing them at very low temperatures. They are then awoken at a later time making it convenient for testing large numbers of drugs.
  • Since iPSCs have the power to make any cell type in the human body, they can also be contaminated with other unwanted types of cells. Typically such a technique is very difficult to accomplish in pluripotent stem cells such as embryonic and iPSCs. Therefore, we have designed a more efficient scheme to generate iPSC lines from SMA patients that will become fluorescent color (green, red or blue) when then motor neurons are made from iPSCs. These types of cells are known as reporter cell lines. This will aid in picking out the desired cell type from patient iPSCs, in this case a motor neuron, and discard any unwanted cell types. This will enormously simplify testing of new drugs in SMA patient motor neurons. Using new technologies that can edit, cut, copy, and paste new DNA in the stem cell genome, we are also developing ways to engineer iPS cell lines that will tag the motor neurons when they are made. This will allow us another method for making pure motor neurons and tracking them in a dish among other types of cells while they are alive.
  • Deficiency of an important SMN protein in SMA patients is one of the key causes to the course of the disease. An automated method has been developed for identifying what causes the SMA neurons to become sick and test new drugs in motor neurons. We are now gearing up to test some ~1400 known compounds on patient motor neurons to determine whether we can raise SMN protein levels in motor neurons.

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