Neurological Disorders

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

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.
  • Over the last decade, the importance of the stem cells for cell replacement therapy, human disease modeling and drug toxicity/therapy screenings has been greatly appreciated by both the general public and the scientific community. In our application utilizing human embryonic stem cells and induced pluripotent stem cells (iPSCs), 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. While we increased our knowledge about the electrophysiological characteristics of RTT neurons during Year 2 funding, we mainly focused on the transplantation of the normal and diseased cells, as well as the molecular signatures of transplanted cells at a single cell level, during Year 3 of the funding period. Following our initial transplantation experiments, we observed clustering of the transplanted cells at the injection site, even though there were number of cells integrating into the host brains. In order to circumvent this problem and answer our original questions, we developed an alternative approach. Specifically, we adopted the “transparent brain” methodology to better visualize the integration and the projections of the transplanted cells, as well as the circuitries that they participate, in the host environment to reveal the connectivity of wild type and RTT neurons with the host circuits. With this method, we’re able to follow the transplanted RTT neurons at a higher resolution -without the limitations of the conventional approaches- for studying human iPSC-derived RTT neurons integrated into mouse brains. As part of our last Specific Aim, we’ve performed single neuron gene expression profiling coupled with electrophysiological recordings both in vitro and in vivo. Specifically, we implemented electrophysiological recordings from the transplanted RTT iPSC-derived neurons and isolated the genomic material from the same cell to perform transcriptome analyses. We collected significant amount of data from RNA sequencing experiments and have been performing relevant bioinformatic analyses. In order to complete the gene expression profiling analysis, we obtained a no-cost-extension of the project, and upon completion of the no-cost-extension period, the relevant report will be filed outlining the outcomes of the single neuron transcriptome analysis coupled with electrophysiology. Collectively, our findings provide mechanistic insights into RTT disease pathophysiology, which will facilitate the development of novel therapies for RTT. Lastly, our approach is applicable for studying other neurological disorders in addition to RTT.

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.

Development and preclinical testing of new devices for cell transplantation to the brain.

Funding Type: 
Tools and Technologies II
Grant Number: 
RT2-01975
ICOC Funds Committed: 
$1 831 723
Disease Focus: 
Neurological Disorders
Parkinson's Disease
oldStatus: 
Active
Public Abstract: 
The surgical tools currently available to transplant cells to the human brain are crude and underdeveloped. In current clinical trials, a syringe and needle device has been used to inject living cells into the brain. Because cells do not spread through the brain tissue after implantation, multiple brain penetrations (more than ten separate needle insertions in some patients) have been required to distribute cells in the diseased brain region. Every separate brain penetration carries a significant risk of bleeding and brain injury. Furthermore, this approach does not result in effective distribution of cells. Thus, our lack of appropriate surgical tools and techniques for clinical cell transplantation represents a significant roadblock to the treatment of brain diseases with stem cell based therapies. A more ideal device would be one that can distribute cells to large brain areas through a single initial brain penetration. In rodents, cell transplantation has successfully treated a great number of different brain disorders such as Parkinson’s disease, epilepsy, traumatic brain injury, multiple sclerosis, and stroke. However, the human brain is about 500 times larger than the mouse brain. While the syringe and needle transplantation technique works well in mice and rats, using this approach may not succeed in the much larger human brain, and this may result in failure of clinical trials for technical reasons. We believe that the poor design of current surgical tools used for cell delivery is from inadequate interactions between basic stem cell scientists, medical device engineers, and neurosurgeons. Using a multidisciplinary approach, we will first use standard engineering principles to design, fabricate, refine, and validate an innovative cell delivery device that can transplant cells to a large region of the human brain through a single brain penetration. We will then test this new prototype in a large animal brain to ensure that the device is safe and effective. Furthermore, we will create a document containing engineering drawings, manufacturing instructions, surgical details, and preclinical data to ensure that this device is readily available for inclusion in future clinical trials. By improving the safety and efficacy of cell delivery to the brain, the development of a superior device for cell transplantation may be a crucial step on the road to stem cell therapies for a wide range of brain diseases. In addition, devices and surgical techniques developed here may also be advantageous for use in other diseased organs.
Statement of Benefit to California: 
The citizens of California have invested generously into stem cell research for the treatment of human diseases. While significant progress has been made in our ability to produce appropriate cell types in clinically relevant numbers for transplantation to the brain, these efforts to cure disease may fail because of our inability to effectively deliver the cells. Our proposed development of a superior device for cell transplantation may thus be a crucial step on the road to stem cell therapies for a wide range of brain disorders, such as Parkinson’s disease, stroke, brain tumors, epilepsy, multiple sclerosis, and traumatic brain injury. Furthermore, devices and surgical techniques developed in our work may also be advantageous for use in other diseased organs. Thus, with successful completion of our proposal, the broad community of stem cell researchers and physician-scientists will gain access to superior surgical tools with which to better leverage our investment into stem cell therapy.
Progress Report: 
  • The surgical tools currently available to transplant cells to the human brain are crude and underdeveloped. In current clinical trials, a syringe and needle device has been used to inject living cells into the brain. Because cells do not spread through the brain tissue after implantation, multiple brain penetrations (more than ten separate needle insertions in some patients) have been required to distribute cells in the diseased brain region. Every separate brain penetration carries a significant risk of bleeding and brain injury. Furthermore, this approach does not result in effective distribution of cells. Thus, our lack of appropriate surgical tools and techniques for clinical cell transplantation represents a significant roadblock to the treatment of brain diseases with stem cell based therapies. A more ideal device would be one that can distribute cells to large brain areas through a single initial brain penetration.
  • In this first year of progress, we have designed, prototyped, and tested a stereotactic neurosurgical device capable of delivering cells to a volumetrically large target region through a single cortical brain penetration. We compared the performance of our device to a currently used cell transplantation implement – a 20G cannula with dual side ports. Through a single initial penetration, our device could transplant materials to a region greater than 4 cubic centimeters. Modeling with neurosurgical planning software indicated that our device could distribute cells within the entire human putamen – a target used in Parkinson’s disease trials – via a single transcortical penetration. While reflux of material along the penetration tract was problematic with the 20G cannula, resulting in nearly 80% loss of cell delivery, our device was resistant to reflux. We also innovated an additional system that facilitates small and precise volumes of injection. Both dilute and highly concentrated neural precursor cell populations tolerated transit through the device with high viability and unaffected developmental potential. Our device design is compatible with currently employed frame-based, frameless, and intraoperative MRI stereotactic neurosurgical targeting systems.
  • The surgical tools currently available to transplant cells to the human brain are crude and underdeveloped. In current clinical trials, a syringe and needle device has been used to inject living cells into the brain. Because cells do not spread through the brain tissue after implantation, multiple brain penetrations (more than ten separate needle insertions in some patients) have been required to distribute cells in the diseased brain region. Every separate brain penetration carries a significant risk of bleeding and brain injury. Furthermore, this approach does not result in effective distribution of cells. Thus, our lack of appropriate surgical tools and techniques for clinical cell transplantation represents a significant roadblock to the treatment of brain diseases with stem cell based therapies. A more ideal device would be one that can distribute cells to large and anatomically complex brain areas through a single initial brain penetration.
  • In the first year of progress, we designed, prototyped, and tested a stereotactic neurosurgical device capable of delivering cells to a volumetrically large target region through a single cortical brain penetration. We compared the performance of our device to a currently used cell transplantation implement – a 20G cannula with dual side ports. Through a single initial penetration, our device could transplant materials to a region greater than 4 cubic centimeters. Modeling with neurosurgical planning software indicated that our device could distribute cells within the entire human putamen – a target used in Parkinson’s disease trials – via a single transcortical penetration. While reflux of material along the penetration tract was problematic with the 20G cannula, resulting in nearly 80% loss of cell delivery, our device was resistant to reflux. We also innovated an additional system that facilitates small and precise volumes of injection. Both dilute and highly concentrated neural precursor cell populations tolerated transit through the device with high viability and unaffected developmental potential. Our device design is compatible with currently employed frame-based, frameless, and intraoperative MRI stereotactic (iMRI) neurosurgical targeting systems.
  • In this second year of progress, we have produced and tested the iMRI compatible version of our cell delivery device. The device components are fabricated from materials that are FDA-approved for use in medical devices, and we have assembled the device under Good Manufacturing Practice (GMP) conditions. Our device functions seamlessly with an FDA-approved stereotactic iMRI neurosurgical platform and computer-aided targeting system, and we have demonstrated that this iMRI-compatible system can deliver to the volume and shape of the human putamen through a single initial brain penetration. Thus, by using modern materials and manufacturing techniques, we have produced a neurosurgical device and technique that enables clinicians to “tailor” cell delivery to individual patient anatomical characteristics and specific disease states. This modern and “easy to use” platform technology furthermore allows “real-time” monitoring of cell delivery and unprecedented complication avoidance, increasing patient safety.
  • In this third year of progress, we have made final design refinements to the Radially Branched Deployment (RBD) cell transplantation device, which is fully compatible with currently employed interventional MRI stereotactic (iMRI) neurosurgical targeting systems. These design changes increase the "usability" of the device and enhance patient safety. The iMRI-guided RBD technology advances our ability to properly “tailor” the distribution of cell delivery to larger brain target volumes that vary in size and shape due to individual patient anatomy and different disease states. Furthermore, iMRI-guided RBD may increase patient safety by enabling intraoperative MRI monitoring. Importantly, this platform technology is easy-to-use and has a low barrier to implementation, as it can be performed “inside” essentially any typical diagnostic 1.5T MRI scanner found in most hospitals. We believe that this ease of access to the technology will facilitate the conduct of multi-site clinical trials and the future adoption of successful cellular therapies for patient care worldwide. In summary, by improving intracerebral cell delivery to the human brain, iMRI-guided RBD may have a transformative impact on the safety and efficacy of cellular therapeutics for a wide range of neurological disorders, helping ensure that basic science results are not lost in clinical translation.
  • Working with a California-based medical device manufacturer, we have developed manufacturing and testing procedures that are now being compiled into a design history file, which is a document required for eventual commercial use of the device. We are also working with an FDA regulatory consultant to prepare a 510K application to seek marketing clearance from the FDA.

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.

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.

Site-specific integration of Lmx1a, FoxA2, & Otx2 to optimize dopaminergic differentiation

Funding Type: 
Tools and Technologies II
Grant Number: 
RT2-01880
ICOC Funds Committed: 
$1 619 627
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
The objective of this study is to develop a new, optimized technology to obtain a homogenous population of midbrain dopaminergic (mDA) neurons in a culture dish through neuronal differentiation. Dopaminergic neurons of the midbrain are the main source of dopamine in the mammalian central nervous system. Their loss is associated with one of the most prominent human neurological disorders, Parkinson's disease (PD). There is no cure for PD, or good long-term therapeutics without deleterious side effects. Therefore, there is a great need for novel drugs and therapies to halt or reverse the disease. Recent groundbreaking discoveries allow us to use adult human skin cells, transduce them with specific genes, and generate cells that exhibit virtually all characteristics of embryonic stem cells, termed induced pluripotent stem cells (iPSCs). These cell lines, when derived from PD patient skin cells, can be used as an experimental pre-clinical model to study disease mechanisms unique to PD. These cells will not only serve as an ‘authentic’ model for PD when further differentiated into the specific dopaminergic neurons, but that these cells are actually pathologically affected with PD. All of the current protocols for directed neuronal differentiation from iPSCs are lengthy and suboptimal in terms of efficiency and reproducibility of defined cell populations. This hinders the ability to establish a robust model in-a-dish for the disease of interest, in our case PD-related neurodegeneration. We will use a new, efficient gene integration technology to induce expression of midbrain specific transcription factors in iPSC lines derived from a patient with PD and a sibling control. Forced expression of these midbrain transcription factors will direct iPSCs to differentiate into DA neurons in cell culture. We aim at achieving higher efficiency and reproducibility in generating a homogenous population of midbrain DA neurons, which will lay the foundation for successfully modeling PD and improving hit rates of future drug screening approaches. Our study could also set a milestone towards the establishment of efficient, stable, and reproducible neuronal differentiation using a technology that has proven to be safe and is therefore suitable for cell replacement therapies in human. The absence of cellular models of Parkinson’s disease represents a major bottleneck in the scientific field of Parkinson’s disease, which, if solved, would be instantly translated into a wide range of clinical applications, including drug discovery. This is an essential avenue if we want to offer our patients a new therapeutic approach that can give them a near normal life after being diagnosed with this progressively disabling disease.
Statement of Benefit to California: 
The proposed research could lead to a robust model in-a-dish for Parkinson’s disease (PD)-related neurodegeneration. This outcome would deliver a variety of benefits to the state of California. First, there would be a profound personal impact on patients and their families if the current inevitable decline of PD patients could be halted or reversed. This would bring great happiness and satisfaction to the tens of thousands of Californians affected directly or indirectly by PD. Progress toward a cure for PD is also likely to accelerate the development of treatments for other degenerative disorders. The technology for PD modeling in-a-dish could be applied to other cell types such as cardiomyocytes (for heart diseases) and beta-cells (for diabetes). The impact would likely stimulate medical progress on a variety of conditions in which stem cell based drug screening and therapy could be beneficial. An effective drug and therapy for PD would also bring economic benefits to the state. Currently, there is a huge burden of costs associated with the care of patients with long-term degenerative disorders like PD, which afflict tens of thousands of patients statewide. If the clinical condition of these patients could be improved, the cost of maintenance would be reduced, saving billions in medical costs. Many of these patients would be more able to contribute to the workforce and pay taxes. Another benefit is the effect of novel, cutting-edge technologies developed in California on the business economy of the state. Such technologies can have a profound effect on the competitiveness of California through the formation of new manufacturing and health care delivery facilities that would employ California citizens and bring new sources of revenue to the state. Therefore, this project has the potential to bring health and economic benefits to California that is highly desirable for the state.
Progress Report: 
  • Dopaminergic (DA) neurons of the midbrain are the main source of dopamine in the mammalian central nervous system. Their loss is associated with a prominent human neurological disorder, Parkinson's disease (PD). There is no cure for PD, nor are there any good long-term therapeutics without deleterious side effects. Therefore, there is a great need for novel therapies to halt or reverse the disease. The objective of this study is to develop a new technology to obtain a purer, more abundant population of midbrain DA neurons in a culture dish. Such cells would be useful for disease modeling, drug screening, and development of cell therapies.
  • Recent discoveries allow us to use adult human skin cells, introduce specific genes into them, and generate cells, termed induced pluripotent stem cells (iPSC), that exhibit the characteristics of embryonic stem cells. These iPSC, when derived from PD patient skin cells, can be used as an experimental model to study disease mechanisms that are unique to PD. When differentiated into DA neurons, and these cells are actually pathologically affected with PD.
  • The current methods for directed DA neuronal differentiation from iPSC are inadequate in terms of efficiency and reproducibility. This situation hinders the ability to establish a robust model for PD-related neurodegeneration. In this study, we use a new, efficient gene integration technology to induce expression of midbrain-specific genes in iPSC lines derived from a patient with PD and a normal sibling. Forced expression of these midbrain transcription factor genes directs iPSC to differentiate into DA neurons in cell culture. A purer population of midbrain DA neurons may lay the foundation for successfully modeling PD and improving hit rates in drug screening approaches.
  • The milestones for the first year of the project were to establish PD-specific iPSC lines that contain genomic “docking” sites, termed “attP” sites. In year 2, these iPSC/attP cell lines will be used to insert midbrain-specific transcription factors with high efficiency, mediated by enzymes called integrases. We previously established an improved, high-efficiency, site-specific DNA integration technology in mice. This technology combines the integrase system with newly identified, actively expressed locations in the genome and ensures efficient, uniform gene expression.
  • The PD patient-specific iPSC lines we used were PI-1754, which contains a severe mutation in the SNCA (synuclein alpha) gene, and an unaffected sibling line, PI-1761. The SNCA mutation causes dramatic clinical symptoms of PD, with early-onset progressive disease. We use a homologous recombination-based procedure to place the “docking” site, attP, at well-expressed locations in the SNCA and control iPSC lines (Aim 1.1). We also included a human embryonic stem cell line, H9, to monitor our experimental procedures. The genomic locations we chose for placement of the attP sites included a site on chromosome 22 (Chr22) and a second, backup site on chromosome 19 (Chr19). These two sites were chosen based on mouse studies, in which mouse equivalents of both locations conferred strong gene expression. In order to perform recombination, we constructed targeting vectors, each containing an attP cassette flanked by 5’ and 3’ homologous fragments corresponding to the human genomic location we want to target. For the Chr22 locus, we were able to obtain all 3 targeting constructs for the PI-1754, PI-1761 and H9 cell lines. For technical reasons, we were not able to obtain constructs for the Chr19 location Thus, we decided to focus on the Chr22 locus and move to the next step.
  • We introduced the targeting vectors into the cells and selected for positive clones by both drug selection and green fluorescent protein expression. For the H9 cells, we obtained 110 double positive clones and analyzed 98 of them. We found 8 clones that had targeted the attP site precisely to the Chr22 locus. For the PI-1761 sibling control line, we obtained 44 clones, and 1 of them had the attP site inserted at the Chr22 locus. The PI-1754 SNCA mutant line, on the other hand, grows slowly in cell culture. We are in the process of obtaining enough cells to perform the recombination experiment in that cell line.
  • In summary, we demonstrated that the experimental strategy proposed in the grant indeed worked. We were successful in obtaining iPSC lines with a “docking” site placed in a pre-selected human genomic location. These cell lines are the necessary materials that set the stage for us to fulfill the milestones of year 2.
  • Parkinson's disease (PD) is caused by the loss of dopaminergic (DA) neurons in the midbrain. These DA neurons are the main source of dopamine, an important chemical in the central nervous system. PD is a common neurological disorder, affecting 1% of those at 60 years old and 4% of those over 80. Unfortunately, there is no cure for PD, nor are there any long-term therapeutics without harmful side effects. Therefore, there is a need for new therapies to halt or reverse the disease. The goal of this study is to develop a new technology that helps us obtain a purer, more abundant population of DA neurons in a culture dish and to characterize the resulting cells. These cells will be useful for studying the disease, screening potential drugs, and developing cell therapies.
  • Due to recent discoveries, we can introduce specific genes into adult human skin cells and generate cells similar to embryonic stem cells, termed induced pluripotent stem cells (iPSC). These iPSC, when derived from PD patients, can be used as an experimental model to study disease mechanisms that are unique to PD, because when differentiated into DA neurons, these cells are actually pathologically affected with PD. We are using a PD iPSC line called PI-1754 derived from a patient with a severe mutation in the SNCA gene, which encodes alpha-synuclein. The SNCA mutation causes dramatic clinical symptoms of PD, with early-onset progressive disease. For comparison we are using a normal, unaffected sibling iPSC line PI-1761. We are also using a normal human embryonic stem cell (ESC) line H9 as the gold standard for differentiation.
  • The current methods for differentiating iPSC into DA neurons are not adequate in terms of efficiency and reliability. Our hypothesis is that forced expression of certain midbrain-specific genes called transcription factors will direct iPSC to differentiate more effectively into DA neurons in cell culture. We use transcription factors called Lmx1a, Otx2, and FoxA2, abbreviated L, O, and F. In this project, we have developed a new, efficient gene integration technology that allows us rapidly to introduce and express these transcription factor genes in various combinations, in order to test whether they stimulate the differentiation of iPSC into DA neurons.
  • In the first year of the project, we began establishing iPSC and ESC lines that contained a genomic “landing pad” site for insertion of the transcription factor genes. We carefully chose a location for placement of the genes based on previous work in mouse that suggested that a site on human chromosome 22 would provide strong and constant gene expression. We initially used ordinary homologous recombination to place the landing pad into this site. By the end of year 1 of the project, this method was successful in the normal iPSC and in the ESC, but not in the more difficult-to-grow PD iPSC. To solve this problem, in year 2 we introduced a new and more powerful recombination technology, called TALENs, and were successful in placing the landing pad in the correct position in all three of the lines, including the PD iPSC.
  • We were now in a position to insert the midbrain-specific transcription factor genes with high efficiency. For this step, we developed a new genome engineering methodology called DICE, for dual integrase cassette exchange. In this technology, we use two site-specific integrase enzymes, called phiC31 and Bxb1, to catalyze precise placement of the transcription factor genes into the desired place in the genome.
  • We constructed gene cassettes carrying all pair-wise combinations of the L, O, and F transcription factors, LO, LF, and OF, and the triple combination, LOF. We successfully demonstrated the power of this technology by rapidly generating a large set of iPSC and ESC that contained all the above combinations of transcription factors, as well as lines that contained no transcription factors, as negative controls for comparison. Two examples of each type of line for the 1754 and 1761 iPSC and the H9 ESC were chosen for differentiation and functional characterization studies. Initial results from these studies have demonstrated correct differentiation of neural stem cells and expression of the introduced transcription factor genes.
  • In summary, we were successful in obtaining ESC and iPSC lines from normal and PD patient cells that carry a landing pad in a pre-selected genomic location chosen and validated for strong gene expression. These lines are valuable reagents. We then modified these lines to add DA-associated transcription factors in four combinations. All these lines are currently undergoing differentiation studies in accordance with the year two and three timelines. During year three of the project, the correlation between expression of various transcription factors and the level of DA differentiation will be established. Furthermore, functional studies with the PD versus normal lines will be carried out.
  • The objective of this project is to develop approaches and technologies that will improve neuronal differentiation of stem cells into midbrain dopaminergic (DA) neurons. DA neurons are of central importance in the project, because they are that cells that are impaired in patients with Parkinson’s disease (PD). Current differentiation methods typically produce low yields of DA neurons. The methods also give variable results, and cell populations contain many types of cells. These impediments have hampered the study of disease mechanisms for PD, as well as other uses for the cells, such as drug screening and cell replacement therapy. Our strategy is to develop a novel method to introduce genes into the genome at a specific place, so we can rapidly add genes that might help in the differentiation of DA neurons. The genes we would like to add are called transcription factors, which are proteins involved differentiation of stem cells into DA neurons. We have placed the genes for three transcription factors into a safe, active position on human chromosome 22 in the cell lines we are studying. These cells, called pluripotent stem cells, have the potential to differentiate into almost any type of cell. We are using embryonic stem cells in our study, as well as induced pluripotent stem cells (iPSC), which are similar, but are derived from adult cells, rather than an embryo. We are using iPSC derived from a PD patient, as well as iPSC from a normal person, for comparison. By forced expression of these neuronal transcription factors, we may achieve more efficient and reproducible generation of DA neurons. The effects of expressing different combinations of the three transcription factors called Lmx1a, FoxA2, and Otx2 on DA neuronal differentiation will be evaluated in the context of embryonic stem cells (ESC) as the gold standard, as well as in iPSC derived from a PD patient with a severe mutation in alpha-synuclein and iPSC derived from a normal control. Comparative functional assays of the resulting DA neurons will complete the analysis.
  • To date, this project has created a novel technology for modifying the genome. The strategy developed out of the one that we originally proposed, but contains several innovations that make it more powerful and useful. The new methodology, called DICE for Dual Integrase Cassette Exchange, allowed us to generate “master” or recipient cell lines for ESC, normal iPSC, and PD iPSC. These recipient cell lines contain a “landing pad” placed into a newly-identified actively-expressed location on human chromosome 22 called H11 that permits robust expression of genes placed into it. We then generated a series of cell lines by "cassette exchange" at the H11 locus. In cassette exchange, the new genes we want to add take the place of the landing pad we originally put into the cells. Cassette exchange is a good way to introduce various genes into the same place in the chromosomes. We created cell lines expressing three neuronal transcription factors suspected to be involved in DA neuronal differentiation, in all pair-wise combinations, including lines with expression of all three factors, and negative control lines with no transcription factors added. This collection of modified human pluripotent stem cell lines is now being used to study neural differentiation. The modified ESC have undergone differentiation into DA neurons and are being evaluated for the effects of the different transcription factor combinations on DA neuronal differentiation. During the final year of the project, this differentiation analysis will be completed, and we will also analyze functional properties of the differentiated DA neurons, with special emphasis on disease-related features of the cells derived from PD iPSC.

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