Neurological Disorders

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

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.

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.

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.

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

Funding Type: 
Tools and Technologies II
Grant Number: 
RT2-01927
ICOC Funds Committed: 
$1 816 157
Disease Focus: 
Alzheimer's Disease
Neurological Disorders
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
Elucidating how genetic variation contributes to disease susceptibility and drug response requires human Induced Pluripotent Stem Cell (hIPSC) lines from many human patients. Yet, current methods of hIPSC generation are labor-intensive and expensive. Thus, a cost-effective, non-labor intensive set of methods for hIPSC generation and characterization is essential to bring the translational potential of hIPSC to disease modeling, drug discovery, genomic analysis, etc. Our project combines technology development and scaling methods to increase throughput and reduce cost of hiPSC generation at least 10-fold, enabling the demonstration, and criterion for success, that we can generate 300 useful hiPSC lines (6 independent lines each for 50 individuals) by the end of this project. Thus, we propose to develop an efficient, cost effective, and minimally labor-intensive pipeline of methods for hIPSC identification and characterization that will enable routine generation of tens to hundreds of independent hIPSC lines from human patients. We will achieve this goal by adapting two simple and high throughput methods to enable analysis of many candidate hIPSC lines in large pools. These methods are already working in our labs and are called "fluorescence cell barcoding" (FCB) and expression cell barcoding (ECB). To reach a goal of generating 6 high quality hIPSC lines from one patient, as many as 60 candidate hIPSC colonies must be expanded and evaluated individually using labor and cost intensive methods. By improving culturing protocols, and implementing suitable pooled analysis strategies, we propose to increase throughput at least 10-fold with a substantial drop in cost. In outline, the pipeline we propose to develop will begin with the generation of 60 candidate hIPSC lines per patient directly in 96 well plates. All 60 will be analyzed for diagnostic hIPSC markers by FCB in 1 pooled sample. The 10 best candidates per patient will then be picked for expression and multilineage differentiation analyses with the goal of finding the best 6 from each patient for digital karyotype analyses. Success at 10-fold scaleup as proposed here may be the first step towards further scaleup once these methods are fully developed. Aim 1: To develop a cost-effective and minimally labor-intensive set of methods/pipeline for the generation and characterization high quality hIPSC lines from large numbers of human patients. We will test suitability/develop a set of methods that allow inexpensive and rapid characterization of 60 candidate hIPSC lines per patient at a time. Aim 2: To demonstrate/test/evaluate the success and cost-effectiveness of our pipeline by generating 6 high quality hIPSC lines from each of 50 human patients from [REDACTED]. We will obtain skin biopsies and expand fibroblasts from 50 patients, and generate and analyze a total of 6 independent hIPSC lines from each using the methods developed in Aim 1.
Statement of Benefit to California: 
Many Californians suffer from diseases whose origin is poorly understood, and which are not treatable in an effective or economically advantageous manner. Part of solving this problem relies upon elucidating how genetic variation contributes to disease susceptibility and drug response and better understanding disease mechanism. Achieving these goals can be accelerated through the use of human Induced Pluripotent Stem Cell (hIPSC) lines from many human patients. Yet, current methods of hIPSC generation are labor-intensive and expensive. Thus, a cost-effective, non-labor intensive set of methods for hIPSC generation and characterization is essential to bring the translational potential of hIPSC to disease modeling, drug discovery, genomic analysis, etc. If successful, our project will lead to breakthroughs in understanding of disease, development of better therapies, and economic development in California as businesses that use our methods are launched. In addition, new therapies will bring cost-savings in healthcare to Californians, stimulate employment since Californians will be employed in businesses that develop and sell these therapies, and relieve much suffering from the burdens of chronic disease.
Progress Report: 
  • An important problem in stem cell and regenerative medicine research has been the ability to quickly and cheaply generate and characterize reprogrammed stem cells from defined human patients. The primary goal of our project is to address this need by developing new technologies that allow stem cell lines to be characterized in large mixed pools as opposed to one by one. Our new methods use flow cytometry and highly sensitive methods for detecting the activity of genes in the cell lines. We made excellent progress in the first year and reduced flow cytometry methods to practice taking advantage of a method called fluorescence cell barcoding. Methods for analyzing activity of genes and chromosome number are in progress and being tested. Our ultimate goal is to reduce cost tenfold and increase speed by about tenfold and our methods development is on track to accomplish this aim.
  • A key bottleneck in reprogramming technology to make induced pluripotent stem (IPS) cell lines is the ability to make large numbers of lines from large numbers of patients in a way that is cost effective and minimizes labor. Our project has focused primarily on dropping the cost of characterization of candidate lines. We have made a number of discoveries about the behavior of candidate reprogrammed lines that allow us to drop cost and labor needed for candidate reprogrammed line characterization. We measured the frequency of candidate lines that were well-behaved in a large retroviral reprogramming experiment, which allows us to rigorously estimate how many candidate lines must be picked and analyzed if 4-6 high-quality lines are to be generated for every patient fibroblast sample subjected to typical retroviral reprogramming technology. We then continued our work on developing a combination of different array and microfluidic chip technologies to measure the chromosome number in each candidate line and the ability of each line to be pluripotent, i.e., to be able to generate many different type of cells similar to embryonic stem cells. We are optimistic that our work will simplify and drop the cost of the characterization process so that it costs far less than before our work was initiated.

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.

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.

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.

Role of HLA in neural stem cell rejection using humanized mice

Funding Type: 
Transplantation Immunology
Grant Number: 
RM1-01735
ICOC Funds Committed: 
$1 472 634
Disease Focus: 
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Cell Line Generation: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
One of the key issues in stem cell transplant biology is solving the problem of transplant rejection. Despite over three decades of research in human embryonic stem cells, little is known about the factors governing immune system tolerance to grafts derived from these cells. In order for the promise of embryonic stem cell transplantation for treatment of diseases to be realized, focused efforts must be made to overcome this formidable hurdle. Our proposal will directly address this critically important issue by investigating the importance of matching immune system components known as human leukocyte antigens (HLA). Because mouse and human immune systems are fundamentally different, we will establish cutting-edge mouse models that have human immune systems as suitable hosts within which to conduct our stem cell brain transplant experiments. Such models rely on immunocompromised mice as recipients for human blood-derived stem cells. These mice go on to develop a human immune system, complete with HLAs, and can subsequently be used to engraft embryonic stem cell-derived brain cells that are either HLA matched or mismatched. Due to our collective expertise in the central nervous system and animal transplantation studies for Parkinson’s disease, our specific focus will be on transplanting embryonic stem cell-derived neural stem cells into brains of both healthy and Parkinson's diseased mice. We will then detect: 1) abundance of brain immune cell infiltrates, 2) production of immune molecules, and 3) numbers of brain-engrafted embryonic stem cells. Establishing this important system would allow for a predictive model of human stem cell transplant rejection based on immune system matching. We would then know how similar HLAs need to be in order to allow for acceptance stem cell grafts.
Statement of Benefit to California: 
In this project, we propose to focus on the role of the human immune system in human embryonic stem cell transplant rejection. Specifically, we aim to develop cutting-edge experimental mouse models that possess human immune systems. This will allow us to determine whether immune system match versus mismatch enables embryonic stem cell brain transplant acceptance versus rejection. Further, we will explore this key problem in stem cell transplant biology both in the context of the healthy and diseased brain. Regarding neurological disease, we will focus on neural stem cell transplants for Parkinson's disease, which is one of the most common neurodegenerative diseases, second only to Alzheimer's disease. If successful, our work will pave the way toward embryonic stem cell-based treatment for this devastating neurological disorder for Californians and others. In order to accomplish these goals, we will utilize two of the most common embryonic stem cell types, known as WiCell H1 and WiCell H9 cells. It should be noted that these particular stem cells will likely not be reauthorized for funding by the federal government due to ethical considerations. This makes our research even more important to the State of California, which would not only benefit from our work but is also in a unique position to offer funding outside of the federal government to continue studies such as these on these two important types of human embryonic stem cells.

Induction of immune tolerance after spinal grafting of human ES-derived neural precursors

Funding Type: 
Transplantation Immunology
Grant Number: 
RM1-01720
ICOC Funds Committed: 
$1 387 800
Disease Focus: 
Spinal Cord Injury
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
oldStatus: 
Closed
Public Abstract: 
Previous clinical studies have shown that grafting of human fetal brain tissue into the CNS of adult recipients can be associated with long-term (more then 10 years) graft survival even after immunosuppression is terminated. These clinical data represent in part the scientific base for the CNS to be designated as an immune privilege site, i.e., immune response toward grafted cells is much less pronounced. With rapidly advancing cell sorting technologies which permit effective isolation and expansion of neuronal precursors from human embryonic stem cells, these cells are becoming an attractive source for cell replacement therapies. Accordingly, there is great need to develop drug therapies or other therapeutic manipulations which would permit an effective engraftment of such derived cells with only transient or no immunosuppression. Accordingly, the primary goal in our studies is to test engraftment of 3 different neuronal precursors cell lines of human origin once grafted into spinal cord in transiently immunosuppressed minipigs. In addition, because the degree of cell engraftment can differ if cells are grafted into injured CNS tissue, the survival of cells once grafted into previously injured spinal cord will also be tested. Second, we will test the engraftment of neuronal cells generated from pig skin cells (fibroblasts) after genetic reprogramming (i.e., inducible pluripotent stem cells, iPS). Because these cells will be transplanted back to the fibroblast donor, we expect stable and effective engraftment in the absence of immunosuppression. Jointly by testing the above technologies (transient immunosuppression and iPS-derived neural precursors), our goal is to define the optimal neuronal precursor cell line(s) as well as immunosuppressive (or no) treatment which will lead to stable and permanent engraftment of spinally transplanted cells.
Statement of Benefit to California: 
Brain or spinal cord neurodegenerative disorders, including stroke, amyotrophic lateral sclerosis, multiple sclerosis or spinal trauma, affect many Californians. In the absence of a functionally effective cure, the cost of caring for patients with such diseases is high, in addition to a major personal and family impact. Our major goal is to develop therapeutic manipulations which are well tolerated by patients and which will lead to stable survival of previously spinal cord-grafted cells generated from human embryonic stem cells. If successful, this advance can serve as a guidance tool for CNS cell replacement therapies in general as it will define the optimal immune tolerance-inducing protocols. In addition, the development of this type of therapeutic approach (pharmacological or cell-replacement based) in California will serve as an important proof of principle and stimulate the formation of businesses that seek to develop these types of therapies (providing banks of inducible pluripotent stem cells) in California with consequent economic benefit.
Progress Report: 
  • The use of autologous, induced pluripotent stem cell-derived cell lines in replacement therapies holds great promise in future clinical use. No need for immunosuppression, otherwise required to prevent transplanted cell rejection, would represent a substantial advance in the current clinical utilization of cell replacement therapies. In our recently completed studies we have found that autologous porcine iPSC-derived neural precursors (NPCs) grafted back to the donor animal spinal cord in the absence of immunosuppression was associated with a poor cell survival and extensive inflammation at cell-grafted sites. Our data raises immunological concerns on the use of autologous iPS-cell derivatives for future regenerative medicine in humans.
  • The use of autologous, induced pluripotent stem cell-derived cell lines in replacement therapies holds great promise in future clinical use. No need for immunosuppression, otherwise required to prevent transplanted cell rejection, would represent a substantial advance in the current clinical utilization of cell replacement therapies. In our recently completed studies we have found that autologous porcine iPSC-derived neural precursors (NPCs) grafted back to the donor animal spinal cord in the absence of immunosuppression was associated with a poor cell survival and extensive inflammation at cell-grafted sites. In more recent study we have determined that the same cell population of iPS-NPCs survive and mature once grafted spinally in immunosupressed pigs.The mechanism of the immunogenicity of iPS-NPCs is being currently determined.
  • The use of autologous, induced pluripotent stem cell-derived cell lines in replacement therapies holds great promise in future clinical use. No need for immunosuppression, otherwise required to prevent transplanted cell rejection, would represent a substantial advance in the current clinical utilization of cell replacement therapies. In our recently completed studies, we have found that autologous porcine iPSC-derived neural precursors (NPCs) trigger a positive T-cell mediated reaction in vitro and that this response is not present if autologous T-cells are co-cultured with autologous fibroblasts. These data show that the reprogramming step induces a potent immunogenicity and that extensive screening of clonally-derived iPS-NPCs will be needed to identify clones of autologous NPCs with acceptable immunogenicity profile. Identification of differences in gene activity in differentially derived iPS-NPCs is currently in progress.

Generation of disease models for neurodegenerative disorders in hESCs by gene targeting

Funding Type: 
Tools and Technologies I
Grant Number: 
RT1-01107
ICOC Funds Committed: 
$869 262
Disease Focus: 
Amyotrophic Lateral Sclerosis
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Cell Line Generation: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
The ability to target a specific locus in the mouse genome and to alter it in a specific fashion has fundamentally changed experimental design and made mice the preeminent model for studying human diseases . However, pathogenesis in humans have unique pathways that may not be revealed by only using mouse or other animal models. An approach that combines the advantages of mouse models with parallel experiments in human embryonic stem cells (hESCs) offers significant advantages over current methodologies. With the large number of hESC lines available, the ability to grow cells in defined media, the development of drug resistant feeders and the reports of strategies to insert DNA with increasing efficiency into hESC, it would only be a matter of time to obtain homologous recombinants in hESCs. In order to provide direct clues to pathogenesis in human tissues, we propose to use homologous recombination to establish in vitro human disease models in hESCs. As a proof of principle, we have chosen Lou Gehrig's disease (or amyotrophic lateral sclerosis, ALS). ALS is a disease that progressively and selectively attacks motoneurons in the brain and the spinal cord. It becomes fatal when motoneurons controlling breathing are affected. Approximately 2% of ALS cases have been identified to be caused by mutations of the the Cu-Zn superoxide dismutase (SOD1) gene in an autosomal dominant trait. Animal models have been established and researchers have been able to propose disease mechanisms which led to potential treatments, although no cure has been offered yet. This in part might be due to lack of human cell based models and varied mutant copy numbers in transgenic animals as well as the random nature of their integration into the genome. Here, we propose to generate hESC lines by gene targeting to harbor point mutations in the SOD1 gene, which recapitulates the genetic defects in SOD1 mutated ALS patients. We will further target these mutations in hESC reporter lines of the two important cell types in ALS: motoneurons and astrocytes. The availability of these SOD1 mutated hESC and hESC reporter lines will allow researchers to obtain purified “diseased” motoneurons and astrocytes, which will facilitate the dissection of ALS pathogenesis. The completion of this proposal will provide (1) a highly efficient protocol for performing homologous recombination in hESCs, (2) a package of motoneuron and astrocyte reporters which are useful for both disease and developmental studies along the neural lineages, and (3) a set of ALS disease platforms of hESC lines to serve as an hESC ALS disease in vitro model, as well as a virtually unlimited source of “diseased” motoneurons and astrocytes. This work not only will provide tools to move pathogenesis research for ALS, but also can be reliably extended into other neural and non-neural lineage diseases, of which genetic defects have been identified, including Huntington's disease (HD) and Parkinson’s disease (PD).
Statement of Benefit to California: 
The overall objectives for this proposal are to create in vitro human neurodegenerative disease models using human embryonic stem cells (hESCs), and as a proof of principle, three point mutations of the SOD1 gene which cause familial amyotrophic lateral sclerosis (FALS) will be tested first. These SOD1 missense mutations, G37R, G85R and G93A, have been identified in FALS patients and widely used in rodent models of FALS. We propose to create SOD1 mutations in hESC lines by gene targeting technology which has been proven to be revolutionary in establishing disease models in animals. In addition, we will use similar protocol to generate motoneuron and astrocyte reporter lines in hESCs, since these two cell types and the interaction between them play the most critical roles in the pathogenesis of ALS. After obtaining the three SOD1 missense mutants in motoneuron and astrocyte reporter lines, we will extend our efforts to characterization of these lines, by examining their growth, survival, cell death and other biochemical properties. We will also perform large scale comparisons for genomic and proteomic profiles of the diseased hESC lines with wild type hESCs, as well as comparing the “diseased” and wild type hESC-derived populations of motoneurons and astrocytes. These experiments will not only provide direct clues for ALS pathogenesis research but also serve as a proof of principle for general disease research using hESCs as a model system. The protocols and reagents developed in this work will be available for Californian researchers and physicians to use for similar neurodegenerative diseases or diseases of other systems. This work will eventually facilitate the scale-up in establishment of human diseases models using human tissues or human cell culture systems for our colleagues in California and around the world.
Progress Report: 
  • The overall objectives for this proposal are to create in vitro human neurodegenerative disease models and to elucidate pathogenesis of amyotrophic lateral sclerosis (ALS), an adult onset fatal motoneuron disease. Using gene targeting and reprogramming technology, we have created ALS disease models in human pluripotent stem cells and are generating neural lineage reporters which will facilitate the downstream efforts on systemic characterization of these diseased cell lines, at undifferentiated stage and after induced lineage differentiation toward motoneurons and astrocytes. These experiments will not only provide direct clues for ALS pathogenesis but also serve as a proof of principle for general disease research using human pluripotent stem cells as a model system. We also aim to provide optimized protocols for easy to access gene targeting which eventually facilitate the development of personalized medicine, the future of regenerative medicine. The novel targeting protocol combined with our experience on directed differentiation along the neural lineage will not only will make tools to move the pathogenesis research for ALS, but also can be reliably extended to other neural and non-neural diseases, of which genetic defects have been identified, including Huntington's disease and Parkinson’s disease.
  • The overall objectives for this proposal are to create in vitro human neurodegenerative disease models for amyotrophic lateral sclerosis (ALS), an adult onset fatal motoneuron disease. Using gene targeting, site-specific integration and reprogramming technology, we have created ALS disease models in human pluripotent stem cells and generated neural lineage reporters which will facilitate the downstream efforts on systemic characterization of these diseased cell lines, at undifferentiated stage and after forced lineage differentiation toward motoneurons and astrocytes. We have optimized protocols for gene targeting using homologous recombination and site-specific integration and insertion. The novel targeting protocol combined with our experience on directed differentiation along the neural lineage are useful tools to pathogenesis research for ALS, as well as to other neural and non-neural diseases, including Huntington's disease and Parkinson’s disease.

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