Neurological Disorders

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

Human iPSC modeling and therapeutics for degenerative peripheral nerve disease

Funding Type: 
New Faculty Physician Scientist
Grant Number: 
RN3-06530
ICOC Funds Committed: 
$3 031 737
Disease Focus: 
Neuropathy
Neurological Disorders
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
The applicant is an MD/PhD trained physician scientist, whose clinical expertise is neuromuscular disorders including peripheral nerve disease. The proposal is aimed at providing a research proposal and career development plan that will allow the applicant to develop an independent research program, which attempts to bring stem cell based therapies to patients with peripheral nerve diseases. The proposal will use “adult stem cells” derived from patients with an inherited nerve disease, correct the genetic abnormality in those cells, and determine the feasibility of transplanting the genetically engineered cells back into peripheral nerve to slow disease progression.
Statement of Benefit to California: 
The proposed research will benefit the State of California as it will support the career development of a uniquely trained physician scientist to establish an innovative translational stem cell research program aimed toward direct clinical application to patients. The cutting edge technologies proposed are directly in line with the fundamental purpose of the California Initiative for Regenerative Medicine. If successful, both scientific and patient advocate organizations would recognize that these advances came directly from the unique efforts of CIRM and the State of California to lead the world in stem cell research. Finally, as a result of funding of this award, further financial investments from private and public funding organizations would directly benefit the State in the years to come.
Progress Report: 
  • During this award period we have made significant progress. We have established induced pluripotent stem cell (iPSC) lines from four patients with Charcot-Marie-Tooth disease type 1A (CMT1A) due to the PMP22 duplication. We have validated our strategy to genetically engineer induced pluripotent stem cells from patients with inherited neuropathy, and have genetically engineered several patient lines. We further have begun to differentiate these iPSCs into Schwann cell precursors, to begin to investigate cell type specific defects that cause peripheral neurodegeneration in patients with CMT1A. Finally we have imported and characterized a transgenic rat model of CMT1A in order to begin to investigate the ability to inject iPSC derived Schwann cell precursors into rodent nerves as a possible neuroprotective strategy.

White matter neuroregeneration after chemotherapy: stem cell therapy for “chemobrain”

Funding Type: 
New Faculty Physician Scientist
Grant Number: 
RN3-06510
ICOC Funds Committed: 
$2 800 536
Disease Focus: 
Neurological Disorders
Brain Cancer
Cancer
Stem Cell Use: 
Adult Stem Cell
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
Chemotherapy for cancer is often life saving, but it also causes a debilitating syndrome of impaired cognition characterized by deficits in attention, concentration, information processing speed, multitasking and memory. As a result, many cancer survivors find themselves unable to return to work or function in their lives as they had before their cancer therapy. These cognitive deficits, colloquially known as "chemobrain" or "chemofog," are long-lasting and sometimes irreversible. For example, breast cancer survivors treated with chemotherapy suffer from cognitive disability even 20 years later. These cognitive problems occur because chemotherapy damages the neural stem and precursor cells necessary for the health of the brain's infrastructure, called white matter. We have discovered a powerful way to recruit the stem/precursor cells required for white matter repair that depends on an interaction between the electrical cells of the brain, neurons, and these white matter stem/precursor cells. In this project, we will determine the key molecules responsible for the regenerative influence of neurons on these white matter stem cells and will develop that molecule (or molecules) into a drug to treat chemotherapy-induced cognitive dysfunction. If successful, this will result in the first effective treatment for a disease that affects at least a million cancer survivors in California.
Statement of Benefit to California: 
Approximately 100,000 Californians are diagnosed with cancer each year, and the majority of these people require chemotherapy. While cancer chemotherapy is often life saving, it also causes a debilitating neurocognitive syndrome characterized by impaired attention, concentration, information processing speed, multitasking and memory. As a result, many cancer survivors find themselves unable to return to work or function in their lives as they had before their cancer therapy. These cognitive deficits, colloquially known as "chemobrain" or "chemofog" are long-lasting; for example, cognitive deficits have been demonstrated in breast cancer survivors treated with chemotherapy even 20 years later. With increasing cancer survival rates, the number of people living with cognitive disability from chemotherapy is growing and includes well over a million Californians. Presently, there is no known therapy for chemotherapy-induced cognitive decline, and physicians can only offer symptomatic treatment with medications such as psychostimulants. The underlying cause of "chemobrain" is damage to neural stem and precursor cell populations. The proposed project may result in an effective regenerative strategy to restore damaged neural precursor cell populations and ameliorate or cure the cognitive syndrome caused by chemotherapy. The benefit to California in terms of improved quality of life for cancer survivors and restored occupational productivity would be immeasurable.
Progress Report: 
  • Cancer chemotherapy can be lifesaving but frequently results in long-term cognitive deficits. This project seeks to establish a regenerative strategy for chemotherapy-induced cognitive dysfunction by harnessing the potential of the interactions between active neurons and glial precursor cells that promote myelin plasticity in the healthy brain. In the first year of this award, we have made on-track progress towards establishing a working experimental model system of chemotherapy-induced neurotoxicity that faithfully models the human disease both in terms of the cellular damage as well as functional deficits in cognition. We have also been able to identify several therapeutic candidate molecules that we will be studying in the coming years of the project to ascertain which of these candidates are sufficient to promote OPC population repletion and neuro-regeneration after chemotherapy exposure.

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.

Engineering Defined and Scaleable Systems for Dopaminergic Neuron Differentiation of hPSCs

Funding Type: 
Tools and Technologies II
Grant Number: 
RT2-02022
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.

Development of Single Cell MRI Technology using Genetically-Encoded Iron-Based Reporters

Funding Type: 
Tools and Technologies II
Grant Number: 
RT2-02018
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.

Editing of Parkinson’s disease mutation in patient-derived iPSCs by zinc-finger nucleases

Funding Type: 
Tools and Technologies II
Grant Number: 
RT2-01965
ICOC Funds Committed: 
$1 327 983
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
The goal of this proposal is to establish a novel research tool to explore the molecular basis of Parkinson’s disease (PD) - a critical step toward the development of new therapy. To date, a small handful of specific genes and associated mutations have been causally linked to the development of PD. However, how these mutations provoke the degeneration of specific neurons in the brain remains poorly understood. Moreover, conducting such genotype-phenotype studies has been hampered by two significant experimental problems. First, we have historically lacked the ability to model the relevant human cell types carrying the appropriate gene mutation. Second, the genetic variation between individuals means that the comparison of a cell from a disease-carrier to a cell derived from a normal subject is confounded by the many thousands of genetic changes that normally differentiate two individuals from one another. Here we propose to combine two powerful techniques – one genetic and one cellular – to overcome these barriers and drive a detailed understanding of the molecular basis of PD. Specifically, we propose to use zinc finger nucleases (ZFNs) in patient-derived induced pluripotent stem cells (iPSC) to accelerate the generation of a panel of genetically identical cell lines differing only in the presence or absence of a single disease-linked gene mutation. iPSCs have the potential to differentiate into many cell types – including dopaminergic neurons that become defective in PD. Merging these two technologies will thus allow us to study activity of either the wild-type or the mutant gene product in cells derived from the same individual, which is critical for elucidating the function of these disease-related genes and mutations. We anticipate that the generation of these isogenic cells will accelerate our understanding of the molecular causes of PD, and that such cellular models could become important tools for developing novel therapies.
Statement of Benefit to California: 
Approx. 36,000-60,000 people in the State of California are affected with Parkinson’s disease (PD) – a number that is estimated to double by the year 2030. This debilitating neurodegenerative disease causes a high degree of disability and financial burden for our health care system. Importantly, recent work has identified specific gene mutations that are directly linked to the development of PD. Here we propose to exploit the plasticity of human induced pluripotent stem cells (iPSC) to establish models of diseased and normal tissues relevant to PD. Specifically, we propose to take advantage of recent developments allowing the derivation of stem cells from PD patients carrying specific mutations. Our goal is to establish advanced stem cell models of the disease by literally “correcting” the mutated form of the gene in patient cells, therefore allowing for direct comparison of the mutant cells with its genetically “repaired” yet otherwise identical counterpart. These stem cells will be differentiated into dopaminergic neurons, the cells that degenerate in the brain of PD patients, permitting us to study the effect of correcting the genetic defect in the disease relevant cell type as well as provide a basis for the establishment of curative stem cells therapies. This collaborative project provides substantial benefit to the state of California and its citizens by pioneering a new stem cell based approach for understanding the role of disease causing mutations via “gene repair” technology, which could ultimately lead to advanced stem cell therapies for Parkinson’s disease – an unmet medical need without cure or adequate long-term therapy.
Progress Report: 
  • The goal of this proposal was to establish a novel research tool to explore the molecular basis of Parkinson’s disease (PD) - a critical step toward the development of new therapy. To date, a small handful of specific genes and associated mutations have been causally linked to the development of PD. However, how these mutations provoke the degeneration of specific neurons in the brain remains poorly understood.
  • In the first year of the grant, we have successfully modified the LRRK2 G2019S mutation in patient-derived induced pluripotent stem cells (iPSC) using zinc-finger technology. We created several clonal lines with the gene correction and also with a knockdown of the LRRK2 gene.
  • We characterized these lines for pluripotency, karyotype, and differentiation potential and currently, we are testing the lines for functional differences in the next reporting period and will generate iPSCs with specific LRRK2 mutations introduced using zinc-finger technology.
  • Despite the growing number of diseases linked to single gene mutations, determining the molecular mechanisms by which such errors result in disease pathology has proven surprisingly difficult. The ability to correlate disease phenotypes with a specific mutation can be confounded by background of genetic and epigenomic differences between patient and control cells. To address this problem, we employed zinc finger nucleases-based genome editing in combination with a newly developed high-efficiency editing protocol to generate isogenic patient-derived induced pluripotent stem cells (iPSC) differing only at the most common mutation for Parkinson's disease (PD), LRRK2 p.G2019S. We show that correction of the LRRK2 p.G2019S mutation rescues a panel of neuronal cell phenotypes including reduced dopaminergic cell number, impaired neurite outgrowth and mitochondrial dysfunction. These data reveal that PD-relevant cellular pathophysiology can be reversed by genetic repair, thus confirming the causative role of this prevalent mutation – a result with potential translational implications.
  • The goal of this proposal has been to establish a novel research tool to explore the molecular basis of Parkinson’s disease (PD) - a critical step toward the development of new therapies. To date, a small handful of specific genes and associated mutations have been causally linked to the development of PD. However, how these mutations provoke the degeneration of specific neurons in the brain remains poorly understood.
  • Moreover, conducting such genotype-phenotype studies has been hampered by two significant experimental problems. First, we have historically lacked the ability to model the relevant human cell types carrying the appropriate gene mutation. Second, the genetic variation between individuals means that the comparison of a cell from a disease-carrier to a cell derived from a normal subject is confounded by the many thousands of genetic changes that normally differentiate two individuals from one another.
  • We proposed to use zinc finger nucleases (ZFNs) in patient-derived induced pluripotent stem cells (iPSC) to accelerate the generation of a panel of genetically identical cell lines differing only in the presence or absence of a single disease-linked gene mutation.
  • To this end, we have successfully generated a panel of LRRK2 isogenic cell lines that differ only in "one building block" in the genomic DNA of a cell which can cause PD, therefore we genetically 'cured' the cells in the culture dish. These lines are invaluable because they are a set of tools that allow to study the effect of this mutation in the context of neurodegeneration and cell death. We received interest from many outside academic laboratories and industry to distribute these novel tools and these cell lines will hopefully lead to the discovery of new drugs that can halt or even reverse PD.

Developing a method for rapid identification of high-quality disease specific hIPSC lines

Funding Type: 
Tools and Technologies II
Grant Number: 
RT2-01927
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.

Use of hiPSCs to develop lead compounds for the treatment of genetic diseases

Funding Type: 
Tools and Technologies II
Grant Number: 
RT2-01920
ICOC Funds Committed: 
$1 833 054
Disease Focus: 
Neurological Disorders
Pediatrics
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
This study will use Ataxia-Telangiectasia (A-T), an early-onset inherited neurodegenerative disease of children, as a model to study the mechanisms leading to cerebellar neurodegeneration and to develop a drug that can slow or halt neurodegeneration. We will start with skin cells that were originally grown from biopsies of patients with A-T who specifically carry “nonsense” type of mutations in the ATM gene. We will convert these skin cells to stem cells capable of forming neural cells that are lacking in the brain (cerebellum) of A-T patients; presumably these neural cells need ATM protein to develop normally. We will then test the effects of our most promising new “readthrough compounds” (RTCs) on the newly-developed neural cells. Our lab has been developing the drugs over the past six years. At present, there is no other disease model (animal or in a test tube) for evaluating the effects of RTCs on the nervous system and its development. Nor is there any effective treatment for the children with A-T or other progressively-deteriorating ataxias. Success in this project would open up at least three new areas for understanding and treating neurodegenerative diseases: 1) the laboratory availability of human neural cells with specific disease-causing mutations; 2) a new approach to learning how the human brain develops and 3) a new class of drugs (RTCs) that correct nonsense mutations, even in the brain, and may correct neurodegeneration.
Statement of Benefit to California: 
This project seeks to merge the expertise of two major research cultures: one with long-standing experience in developing a treatment for a progressive childhood-onset disease called Ataxia-telangiectasia and another with recent success in converting skin cells into cells of the nervous system. California citizens will benefit by finding new ways to treat neurodegenerative diseases, like A-T, Parkinson and Alzheimer, and expanding the many possible applications of stem cell technology to medicine. More specifically, we will construct a new “disease in a dish” model for neurodegeneration, and this will enable our scientists to test the positive and negative effects of a new class of drugs for correcting inherited diseases/mutations directly on brain cells. These advances will drastically decrease drug development costs and will stimulate new biotech opportunities and increase tax revenues for California, while also training the next generation of young scientists to deliver these new medical products to physicians and patients within the next five years.
Progress Report: 
  • No effective treatments are available for most neurodegenerative diseases. This study uses Ataxia-Telangiectasia (A-T), an early-onset inherited neurodegenerative disease of children, as a model to study the mechanisms leading to cerebellar neurodegeneration and to develop a drug that can slow or halt neurodegeneration. Aim1 proposed to use “Yamanaka factors” to reprogram A-T patient-derived skin fibroblasts, which carry nonsense mutations that we have shown can be induced by RTCs to express full-length and functional ATM protein, into iPSCs. We have successfully reprogrammed A-T fibroblasts to hiPSCs and teratoma formation shows their pluripotency. Aim2 will use these established iPSCs to model neurodegeneration, focusing on differentiation to cerebellar cells, such as Purkinje cells and granule cells. We have generated the Purkinje cell promoter –driven GFP reporter system and will use this system to examine the differentiation capacity of A-T iPSCs to Purkinje cells. Aim3 will utilize the newly-developed neural cells carrying disease-causing ATM nonsense mutations as targets for evaluating the potential therapeutic effects of leading RTCs. We have already started to test the efficacy and toxicity of our lead RTC compounds on A-T iPSC-derived neural progenitor cells. The continuation of this study will help us to pick up one promising RTC compound for IND application. This project is on the right track towards its objective for the development of disease models with hiPSCs and the test of our lead small molecule compounds for the treatment of A-T or other neurodegenerative diseases.
  • No effective treatments are available for most neurodegenerative diseases. This study uses Ataxia-Telangiectasia (A-T), an early-onset inherited neurodegenerative disease of children, as a model to study the mechanisms leading to cerebellar neurodegeneration and to develop a drug that can slow or halt neurodegeneration. Aim1 proposed to use “Yamanaka factors” to reprogram A-T patient-derived skin fibroblasts, which carry nonsense mutations that we have shown can be induced by RTCs to express full-length and functional ATM protein, into iPSCs. Aim2 will use these established iPSCs to model neurodegeneration, focusing on differentiation to cerebellar cells, such as Purkinje cells and granule cells. Aim3 will utilize the newly-developed neural cells carrying disease-causing ATM nonsense mutations as targets for evaluating the potential therapeutic effects of leading RTCs.
  • During the past two years of this project, we established Ataxia-telangiectasia (A-T) patient-derived iPSC lines from two patients which contain nonsense mutations and splicing mutations. These two lines are currently used for testing the mutation-targeted therapies with small molecule readthrough (SMRT) compounds and antisense morpholino oligonucleotides (AMOs). Manuscript describing this work was recently accepted, showing that SMRT compounds can abrogate phenotypes of A-T iPSC-derived neural cells
  • This is the third year (last year) progress report. During the first two years of this project, we have already established two Ataxia-telangiectasia (A-T) patient-derived iPSC lines which contain nonsense mutations and splicing mutations, respectively. These two lines are currently used for testing the mutation-targeted therapies with small molecule readthrough (SMRT) compounds and antisense morpholino oligonucleotides (AMOs). In the third year, we have formally published our results from the first two years’ research work in Nature Communications (Lee et al., 2013). In the last year, we continue to make progresses in the characterization of A-T iPSCs and their derived neuronal cells as well as developing the mutation-targeted therapies for neurodegeneration diseases

Development of small molecule screens for autism using patient-derived iPS cells

Funding Type: 
Tools and Technologies II
Grant Number: 
RT2-01906
ICOC Funds Committed: 
$1 884 808
Disease Focus: 
Autism
Neurological Disorders
Pediatrics
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
Autism Spectrum Disorders (ASDs) are a heritable group of neuro-developmental disorders characterized by language impairments, difficulties in social integrations, and the presence of stereotyped and repetitive behaviors. There are no treatments for ASDs, and very few targets for drug development. Recent evidence suggests that some types of ASDs are caused by defects in calcium signaling during development of the nervous system. We have identified cellular defects in neurons derived from induced pluripotent stem cells (iPSCs) from patients with Timothy Syndrome (TS), caused by a rare mutation in a calcium channel that leads to autism. We propose to use cells carrying this mutant calcium channel to identify drugs that act on calcium signaling pathways that are involved in ASDs. Our research project has three aims. First, we will determine whether known channel modulators reverse the cellular defects we observe in cells from TS patients. It is possible that we will find that existing drugs already approved for use in humans might be effective for treating this rare but devastating disorder. Our second aim is to determine whether screens using neuronal cells derived from ASD patients can be used to identify calcium signaling modulators. A bottleneck to therapy development for ASDs has been the lack of appropriate in vitro models for these disorders, and we would like to determine whether our studies could serve as the basis for a new type of screen in human neurons. Our third aim is to identify signaling molecules that might be affected in patients with ASDs, which could be targets for future drug discovery. There is increasing evidence that several types of ASDs are caused by defects in neuronal activity and calcium signaling. More specifically, the CaV1.2 calcium channel that we are studying has been implicated in syndromic and non-syndromic forms of autism, and also in schizophrenia and bipolar disorder. One of the more exciting aspects of our screen of neurons with a mutation in CaV1.2 is that it gives us a tool to explore calcium-mediated signaling pathways that are defective in ASDs. We will try to modify calcium signaling in neurons from ASD patients by changing the expression of proteins that are known to affect calcium signaling in other contexts. These experiments will identify targets that are active in human neurons and that affect cellular phenotypes that are defective in ASD. In summary, the work described in this proposal constitutes a critical step to fulfilling the promise that reprogramming of patient-specific cells offers for the treatment of neuropsychiatric disorders such as autism. Our studies will identify lead compounds that could be tested in the clinic for a rare form of autism, and novel molecular targets for therapeutic development in the future. Importantly, these studies will provide a proof of principle that iPSC-derived cells are valuable for drug discovery for neuropsychiatric disorders.
Statement of Benefit to California: 
Autism Spectrum Disorders (ASDs) affect approximately 1 in 110 children in California. In addition to the devastating effects that ASDs have on the families of affected individuals, treating and educating people with ASDs imposes a heavy economic burden on the state. In 2007, almost 35,000 individuals with autism were receiving services from the California Regional Centers, and the number was expected to rise to 50,000 by last year. Recent estimates suggest that the lifetime cost of caring for an individual with an ASD can exceed $3 million. In spite of their impact on our society, there are currently no effective therapies for ASDs. Our lack of cellular and molecular tools to study these disorders means that there are no good targets for drug screening, so there are very limited prospects for developing effective pharmacological treatments in the near future. New drug discovery paradigms are needed to help develop therapies for these neuropsychiatric conditions. The research described in this proposal could have a dramatic impact on drug discovery methods for ASDs. First, we hope to identify drugs that are effective in treating Timothy Syndrome, a rare form of autism caused by an electrophysiological defect in a calcium channel. Second, we aim to develop new tools to explore calcium-mediated signaling pathways that are defective in ASDs. If successful, our research will identify a family of molecular targets that will be useful for developing therapies for ASDs in the future.
Progress Report: 
  • Autism Spectrum Disorders (ASDs) are a heritable group of neuro-developmental disorders characterized by language impairments, difficulties in social integrations, and the presence of stereotyped and repetitive behaviors. There are no treatments for ASDs, and very few targets for drug development. The goal of this CIRM project is to develop a series of in vitro screens for drugs that might affect the underlying cellular defects in ASDs.
  • Since ASDs are uniquely human, we proposed to design, optimize and conduct high-throughput chemical screens using human neurons derived from induced pluripotent stem cells (iPSCs). Our lab identified cellular defects in neurons derived from patients with Timothy Syndrome (TS), a syndromic disorder often presenting with autism that is caused by a rare mutation in a calcium channel. In our project, we proposed to develop in vitro screening assays for ASDs based on these TS phenotypes, and to screen these assays to identify drugs that might affect behavioral symptoms of autism. In the first year of this award, we conducted preliminary screens and found that certain calcium channel modulators reverse some of the differentiation defects that we observe in these cells. We also extended observations that we had made in mice and showed that TS neurons have defects in the structure and length of their dendrites, measurable features that we can use as the basis for additional drug screens. We have therefore progressed within the aims of the original award.
  • For the remainder of the grant, however, we are proposing to broaden the scope of this project to include iPSC-based screens using neurons from patients with more prevalent forms of ASDs. In other research in our lab, we have characterized phenotypes in neurons derived from patients with two other diseases that are more prevalent than TS: DiGeorge Syndrome (DGS) and Phelan-McDermid Syndrome (PMDS), two neurodevelopmental disorders resulting from deletions within chromosome 22 and patients present symptoms that often include autism. We have shown that these cells have defects in the length of their dendrites, in the structure and function of their synapses, and in their ability to transmit electrical impulses. We propose to broaden the scope of our work to develop screens for TS, DGS, and PMDS. These screens will serve as a basis for identifying drugs that lessen or reverse cellular defects in these disorders, and thus may lead to more generalized treatments for ASDs.
  • We believe that this research not only fulfills critical steps in the development of a novel test for potential ASD treatments, but demonstrates the power of iPSC technology for understanding the underlying mechanisms of neurological disorders. Expanding the scope of our original project will help us increase the impact of our studies on therapeutic development and on the understanding of the neurobiology of ASDs.
  • Autism Spectrum Disorders (ASDs) are a heritable group of neurodevelopmental disorders that affect the verbal, social, and behavioral abilities of affected individuals. There are no pharmacological treatments for ASDs, in part because of a lack of validated cellular and animal models for use in drug screens. The goal of this project is to develop and validate a cell-based high throughput screening method that we will use to identify therapies for ASDs.
  • Our laboratory has established methods for collecting skin samples from patients and reprogramming these cells into induced pluripotent stem (iPS) cells, which we then differentiate into neurons. We have characterized neurons from patients with ASDs, and identified cellular phenotypes that are amenable to high-throughput methods to identify drug targets. Our efforts in Year 2 of our CIRM funding have focused on Phelan-McDermid Syndrome (PMDS), an inherited progressive neurodevelopmental disorder characterized by developmental delay, absent or severely impaired speech, and an increased risk of autism. We have discovered that neurons from PMDS patients who have autism have defects in excitatory synaptic transmission caused by the loss of one copy of the gene Shank3. Shank3 lies in the region of Chromosome 22 that is deleted in PMDS, and is important for the development of synapses. Based on our studies, PMDS neurons can be distinguished from their wildtype counterparts by low expression levels of Shank3 measured by quantitative PCR, decreased number of excitatory synapses labeled by immunocytochemistry and imaged with a microscope, and reduced excitatory cellular currents measured electrophysiologically. Each of these phenotypes is amenable to high throughput screening of therapeutic compounds. We tested several candidate therapeutics and found that prolonged treatment with the growth factor IGF-1 partially reverses the defects we have discovered in PMDS neurons. While IGF-1 is highly bioactive and therefore not an ideal drug candidate, it can be used to validate our screening method.
  • We are currently running trials to select the best phenotype and assay for larger-scale screening. In parallel, we have developed protocols to culture large numbers of iPSC-derived neurons for high throughput screens, and we are growing and banking working stocks of PMDS and control neurons. These experiments will help us identify drug candidates for PMDS, and will represent a significant advance in HTS approaches for the testing of ASD therapies using iPSC-based systems.
  • Autism Spectrum Disorders (ASDs) are a heritable group of neurodevelopmental disorders that affect the verbal, social, and behavioral ability of affected in individual. There are no treatments for ASD, in part because the biological basis for the disorders are not know. In addition, there are no methods for screening drugs that may be therapeutic. The goal of this project was to develop screening assays based on stem cells that were derived from individuals with autism.
  • Using skin samples from affected individuals, our laboratory was able to generate induced pluripotent stem cells (iPSC) and use these stem cells to generate neurons. With CIRM support, we have now generated iPSC from many individuals, some of whom carry genetic alterations that cause autism. Work under this award focused on two genetic disorders, Timothy Syndrome (TS) and Phelan-McDermid Syndrome (PMDS). Both are inherited syndromes that affect several body systems and also greatly increase the risk of autism. In each case, we found that neurons from affected individuals displayed changes in the way neurons connect and communicate. The effects were pronounced in PMDS neurons, in part due to the loss of the Shank3 gene that is involved in the function of the excitatory synapse. Work in year 3 has focused on identifying a robust alteration in neuron function that can be used for drug screening.
  • One such phenotype was discovered and involves a change in the way calcium is utilized when neurons communicate by generating an electrical current. Using chemicals that detect calcium, fluorescent assays were developed that show a robust difference in calcium response in PMDS neurons relative to neurons from unaffected individuals. Adapting the fluorescent calcium reporter assay to a high-throughput format also required the invention of new stem cell culture methods for generating neurons that were more efficient and less costly. Ultimately, a novel strategy was developed that now permits the production of very large numbers of neurons that can be assayed in high throughput screens. A limited screen using candidate drugs has confirmed the utility of the assay and future work will utilize these assays in large scale screens for drugs that normalize or augment the synaptic defects.

Development of a Hydrogel Matrix for Stem Cell Growth and Neural Repair after Stroke

Funding Type: 
Tools and Technologies II
Grant Number: 
RT2-01881
ICOC Funds Committed: 
$1 825 613
Disease Focus: 
Stroke
Neurological Disorders
Stem Cell Use: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
Stroke is the leading cause of adult disability. Most patients survive their initial stroke, but do not recover fully. Because of incomplete recovery, up to 1/3 of stroke patients are taken from independence to a nursing home or assisted living environment, and most are left with some disability in strength or control of the arms or legs. There is no treatment that promotes brain repair and recovery in this disease. Recent studies have shown that stem cell transplantation into the brain can promote repair and recovery in animal models of stroke. However, a stem cell therapy for stroke has not reached the clinic. There are at least three limitations to the development of a human stroke stem cell therapy: most of the transplanted cells die, most of the cells that survive do not interact with the surrounding brain, and the process of injecting stem cells into the brain may damage the normal brain tissue that is near the stroke site. The studies in this grant develop a novel investigative team and research approach to achieve a solution to these limits. Using the combined expertise of engineering, stem cell biology and stroke scientists the studies in this grant will develop tissue bioengineering systems for a stem cell therapy in stroke. The studies will develop a biopolymer hydrogel that provides a pro-growth and pro-survival environment for stem cells when injected with them into the brain. This approach has three unique aspects. First, the hydrogel system utilizes biological components that mimic the normal brain environment and releases specific growth factors that enhance transplanted stem cell survival. Second, these growth factors will also likely stimulate the normal brain to undergo repair and recovery, providing a dual mechanism for neural repair after stroke. Third, this approach allows targeting of the stroke cavity for a stem cell transplant, and not normal brain. The stroke cavity is an ideal target for a stroke stem cell therapy, as it is a cavity and can receive a stem cell transplant without displacing normal brain, and it lies adjacent to the site in the brain of most recovery in this disease—placing the stem cell transplant near the target brain region for repair in stroke. The progress from stroke stem cell research has identified stem cell transplantation as a promising treatment for stroke. The research in this grant develops a next generation in stem cell therapies for the brain by combining new bioengineering techniques to develop an integrated hydrogel/stem cell system for transplantation, survival and neural repair in this disease.
Statement of Benefit to California: 
Advances in the early treatment of stroke have led to a decline in the death rate from this disease. At the same time, the overall incidence of stroke is projected to substantially increase because of the aging population. These two facts mean that stroke will not be lethal, but instead produce a greater number of disabled survivors. A 2006 estimate placed over half of the annual cost in stroke as committed to disabled stroke survivors, and exceeding $30 billion per year in the United States. The studies in this grant develop a novel stem cell therapy in stroke by focusing on one major bottleneck in this disease: the inability of most stem cell therapies to survive and repair the injured brain. With its large population California accounts for roughly 24% of all stroke hospital discharges in the Unites States. The development of a new stem cell therapy approach for this disease will lead to a direct benefit to the State of California.
Progress Report: 
  • This grant develops a tissue bioengineering approach to stem cell transplantation as a treatment for brain repair and recovery in stroke. Stem cell transplantation has shown promise as a therapy that promotes recovery in stroke. Stem cell transplantation in stroke has been limited by poor survival of the transplanted cells. The studies in this grant utilize a multidisciplinary team of bioengineers, neuroscientists/neurologists and stem cell biologists to develop an approach in which stem or progenitor cells can be transplanted into the site of the stroke within a biopolymer hydrogel that provides an environment which supports cell survival and treatment of the injured brain. These hydrogels need to contain naturally occurring brain molecules, so that they do not release foreign or toxic components when they degrade. Further, the hydrogels have to remain liquid so that the injection approach can be minimally invasive, and then gel within the brain. In the past year the fundamental properties of the hydrogels have been determined and the optimal physical characteristics, such as elasticity, identified. Hydrogels have been modified to contain molecules which stem or progenitor cells will recognize and support survival, and to contain growth factors that will both immediately release and, using a novel nanoparticle approach, more slowly release. These have been tested in culture systems and advanced to testing in rodent stroke models. This grant also tests the concept that the stem/progenitor cell that is more closely related to the area within the brain that receives the transplant will provide a greater degree of neural repair and recovery. Progress has been made in the past year in differentiating induced pluripotent stem cells along a lineage that more closely resembles the part of the brain injured in this stroke model, the cerebral cortex.
  • This grant determines the effect of a tissue bioengineering approach to stem cell survival and engraftment after stroke, as means of improving functional recovery in this disease. Stem cell transplantation in stroke has been limited by the poor survival of transplanted cells and their lack of differentiation in the brain. These studies use a biopolymer hydrogel, made of naturally occurring molecules, to provide a pro-survival matrix to the transplanted cells. The studies in the past year developed the chemical characteristics of the hydrogel that promote survival of the cells. These characteristics include the modification of the hydrogel so that it contains specific amounts of protein signals which resemble those seen in the normal stem cell environment. Systematic variation of the levels of these protein signals determined an optimal concentration to promote stem cell survival in vitro. Next, the studies identified the chemistry and release characteristics from the hydrogel of stem cell growth factors that normally promotes survival and differentiation of stem cells. Two growth factors have been tested, with the release characteristics more completely defined with one specific growth factor. The studies then progressed to determine which hydrogels supported stem cell survival in vivo in a mouse model of stroke. Tests of several hydrogels determined that some provide poor cell survival, but one that combines the protein signals, or “motifs”, that were studied in vitro provided improved survival in vivo. These hydrogels did not provoke any additional scarring or inflammation in surrounding tissue after stroke. Studies in the coming year will now determine if these stem cell/hydrogel matrices promote recovery of function after stroke, testing both the protein motif hydrogels and those that contain these motifs plus specific growth factors.
  • This grant determines the effect of a tissue bioengineering approach to stem cell survival and engraftment after stroke, as means of improving functional recovery in this disease. Stem cell transplantation in stroke has been limited by the poor survival of transplanted cells and their lack of differentiation in the brain. These studies use a biopolymer hydrogel, made of naturally occurring molecules, to provide a pro-survival matrix to the transplanted cells. The studies in past years developed the two chemical characteristics of hydrogels that contain recognition or signal elements for stem cells: “protein motifs” that resemble molecules in the normal stem cell environment and growth factors that normally communicate to stem cells in the brain. The hydrogels were engineered so that they contain these familiar stem cell protein motifs and growth factors and release the growth factors over a slow and sustained time course. In the past year on this grant, we tested the effects of hydrogels that had the combined characteristics of these protein motifs and growth factors, at varying concentrations, for their effect on induced pluripotent neural precursor cells (iPS-NPCs) in culture. We identified an optimum concentration for cell survival and for differentiation into immature neurons. We then initiated studies of the effects of this optimized hydrogel in vivo in a mouse model of stroke. These studies are ongoing. They will determine the cell biological effect of this hydrogel on adjacent tissue and on the transplanted cells—determining how the hydrogel enhances engraftment of the transplant. The behavioral studies, also under way, will determine if this optimized hydrogel/iPS-NPC transplant enhances recovery of movement, or motor, function after stroke.

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